WO2024176192A1

WO2024176192A1 - Immunogenic compositions

Info

Publication number: WO2024176192A1
Application number: PCT/IB2024/051778
Authority: WO
Inventors: Ugur Sahin; Alexander Muik; Annette VOGEL; Carla Iris CADIMA COUTO
Original assignee: BioNTech SE
Priority date: 2023-02-24
Filing date: 2024-02-23
Publication date: 2024-08-29

Abstract

Disclosed herein are compositions comprising protein antigens and RNA encoding the same (e.g., compositions comprising protein antigens and RNA encoding antigens) for use in treating and/or preventing various infectious agents. Also disclosed herein are immunogenic compositions and medical preparations comprising the same, and methods of making and using the same. In some embodiments, the technologies provided herein can be used to address and/or overcome immune imprinting in various infectious diseases.

Description

IMMUNOGENIC COMPOSITIONS

Cross-Reference to Related Applications

[0001] The present application claims the benefit of U.S. Prov. Appln. No. 63/448,244 filed February 24, 2023, U.S. Prov. Appln. No. 63/486,953 filed February 24, 2023, U.S. Prov. Appln. No. 63/448,243 filed February 24, 2023, and U.S. Prov. Appln. No. 63/486,958 filed February 24, 2023, the contents of all of which are incorporated herein in their entireties.

Background

[0002] Infectious diseases represent a major threat to human health and well-being. Caused by pathogenic microorganisms, such as bacteria, viruses, parasites or fungi, infectious diseases, also known as communicable diseases, can be spread directly or indirectly from one person to another. Vaccines, which are pharmaceutical preparations that provide or improve immunity to a particular disease, are useful to protect human subjects from certain infectious diseases.

[0003] Since the initial discovery of SARS-CoV-2, a number of variants have arisen around the world. The emergence of these novel circulating variants of SARS-CoV-2 has raised significant concerns about the temporal efficacy of vaccine interventions. The emergence of Omicron (B.1.1.529) variants, which comprise a number of mutations in the S protein, has been of particular concern.

Summary

[0004] In some embodiments, the present disclosure provides technologies (e.g., compositions and methods) that can be used to induce an immune response against an infectious agent (e.g., a virus (e.g., SARS-CoV-2), bacteria, or eukaryotic infectious agent). Technologies provided herein include immunogenic compositions (e.g., RNA compositions), methods of inducing an immune response, and methods of manufacturing immunogenic compositions, among others. In some embodiments, an immunogenic composition delivers an infectious agent antigen (e.g., comprises an infectious agent antigen or a nucleic acid encoding an infectious agent antigen). In some embodiments, an immunogenic composition delivers a SARS-CoV-2 antigen (e.g., comprises a SARS-CoV-2 antigen or a nucleic acid encoding a SARS-CoV-2 antigen). In some embodiments, an immunogenic composition delivers an infectious agent antigen, or an immunogenic portion thereof. In some embodiments, an immunogenic composition delivers a SARS-CoV-2 S protein, or an immunogenic portion thereof. In some embodiments, technologies described herein can produce an immune response characterized by an increased naive immune response, a de novo immune response, and/or a decreased memory B cell response. In some embodiments, technologies provided herein can provide an improved immune response (e.g., higher neutralization antibody titers, increased naive B cell activation, and/or higher titers of antibodies recognizing an epitope unique to a variant of concern (relative to a reference antigen)) against one or more variants of concern (e.g., one or more SARS-CoV-2 variants of concern) (e.g., variants of concern against which current vaccine technologies produce a weak neutralization response). In some embodiments, technologies provided herein can partially or fully address and/or overcome an immune imprinting effect.

[0005] Immune imprinting is a phenomenon in which a previous (e.g., initial) exposure to a first strain or variant of an infectious agent (or one or more antigens thereof) impedes development of an immune response against subsequent strains or variants of an infectious agent (e.g., by interfering with generation of antibodies that bind epitopes unique to the subsequent strain or variant). Immune imprinting can be a particular concern for infectious agents that can acquire a high number or density of mutations in neutralization sensitive region (e.g., SARS- CoV-2).

[0006] A schematic illustrating the immune imprinting phenomenon is shown in Fig. 1. Subjects administered a vaccine that delivers a wild-type (WT) antigen produce antibodies and form memory B cells recognizing the WT antigen. As new Variants of Concern (VOC) arise that evade the immune response induced by the first vaccine, VOC-adapted booster shots are developed and administered to subjects. VOCs often evade the immune system by acquiring mutations at neutralization sensitive epitopes (regions prone to mutation shown in different colors in Fig. 1). Subjects exposed to a VOC-adapted vaccine have a predisposition to activate memory B cells that were formed in response to the initial WT vaccine rather than activate naive B cells. As a result, administering a VOC-adapted vaccine induces production of antibodies that recognize both the WT virus and the VOC but few or no antibodies that are specific to the VOC. So long as the VOC retains some neutralization epitopes from the WT virus, a neutralization response against the VOC can still be induced. As new VOCs continue to lose neutralization epitopes from the WT strain, however, the immune response induced by a VOC-adapted vaccine become less and less effective. Further discussion of the imprinting phenomenon in the SARS- CoV-2 context can be found in Wheatley et al., Trends Immunol, 2021, the contents of which are incorporated by reference herein in their entirety. Immune imprinting is expected to be a particular concern for V OC-adapted vaccines that encode an antigen that comprises a number of mutations at neutralization sensitive sites, (e.g., variants that exhibit close to no conserved neutralizing epitopes).

[0007] Immune imprinting can have important implications for vaccine development. As shown in Fig. 2, a single exposure to Omicron BA.l, BA.2, or BA.4/5 variants has not been found to induce neutralization of Omicron XBB. Without wishing to be bound by theory, this failure to cross neutralize the XBB variant may be due to its low retention of neutralizing B-cell epitopes relative to the original Wuhan strain (see Fig. 5(B)). In short, exposure to BA.l, BA.2, and BA.4/5 may be activating memory B cells that recognize epitopes in both Wuhan and these Omicron variants, and not generating immune responses that recognize features that are unique to these variants. If true, these results suggest that variant adapted vaccines may not produce effective immune responses to variants that retain few neutralization epitopes from the original SARS-CoV-2 variant.

[0008] Among other things, the present disclosure provides important insights for addressing and overcoming immune imprinting in the context of various infectious agents (e.g., in SARS-CoV-2). Without wishing to be bound by theory, the present disclosure provides an insight that immune imprinting can be caused by the retention of memory B cell epitopes in a variant antigen relative to a reference antigen (e.g., an antigen that a subject was first or previously exposed to). Previous strategies have sought to overcome immune imprinting by identifying certain antigen regions that are conserved and neutralizing. The present disclosure provides an insight that a fundamentally different approach can be used to overcome immune imprinting. Specifically, rather than identifying and retaining certain conserved neutralization epitopes, the present disclosure provides an insight that immune imprinting can be addressed and a de novo response induced by removing all memory B cell epitopes from a reference antigen.

[0009] Among other things, the present disclosure also provides certain insights as to how to design antigens that avoid immune imprinting, induce less of a memory B cell response, and/or induce more of a de novo immune response. In particular, the present disclosure provides an insight that such effects can be achieved by administering immunogenic portions of an antigen, and also provides insights in regards to (i) which portion(s) of an antigen can be removed to provide an improved immune response and (ii) which portions of an antigen are more likely to induce a de novo response. In particular, the present disclosure provides an insight that receptor binding domains and/or regions (e.g., of a SARS-CoV-2 S protein) having a high frequency of mutation and a high number of neutralization epitopes can provide improved immune responses (e.g., when administered as a booster to a subject previously administered a vaccine (e.g., a SARS-CoV-2 S protein ) against a given infectious agent).

[0010] Among other things, the present disclosure provides technologies that are useful for increasing the breadth of immune response. In some embodiments, such an immune response is or comprises a B cell immune response. In some embodiments, a B cell immune response is or comprises an antibody response (e.g., neutralizing antibody response) to arisen epitopes in variant polypeptides.

[0011] Among other things, the present disclosure provides an insight that it may be particularly desirable, especially for circulating infectious diseases (e.g., for which variants can be expected to arise), to encourage immune responses, specifically including antibody responses (e.g., neutralizing responses) to arisen epitopes. In some embodiments, such circulating infectious disease is a bacterial infectious disease. In some embodiments, such circulating infectious disease is a parasitic infectious disease. An exemplary parasitic infectious disease is malaria. In some embodiments, such circulating infectious disease is a viral infectious disease. In some embodiments, a viral infectious disease is associated with an RNA virus. Exemplary viral infectious diseases include, but are not limited to coronavirus, ebolavirus, influenza viruses, norovirus, rotavirus, respiratory syncytial virus, alphaherpesvirus, etc. In particular, the present disclosure, among other things, provides an insight that it may be desirable for SARS-CoV-2 infection (e.g., for which variants can be expected to arise), to encourage immune responses, specifically including antibody responses (e.g., neutralizing responses) to arisen epitopes.

[0012] Among other things, and without wishing to be bound by any particular theory, the present disclosure provides an insight that, where an antigen (e.g., S protein of SARS-CoV-2) includes one or more “memory epitopes”, such presence may bias an immune response to the antigen toward activation of memory B cells, in at least some instances to the detriment of developing a sufficiently effective antibody response (e.g., a neutralizing antibody response) to arisen epitope(s).

[0013] In some embodiments, the present disclosure, among other things, provides technologies for modulating the balance of immune response toward de no priming response to arisen epitopes in variant polypeptides (e.g., in some embodiments XBB variant of SARS-CoV- 2). In some embodiments, the present disclosure, among other things, provides technologies for increasing activation of naive B cell immune response to at least one of the arisen epitopes. In some embodiments, such arisen epitopes are neutralizing epitopes. In some embodiments, the present disclosure, among other things, provides technologies for inducing a priming-favorable cytokine milieu, for example, in lymphoid tissues. In some embodiments, the present disclosure, among other things, provides technologies for inducing a priming-favorable cytokine milieu, for example, in lymphoid tissues. Without wishing to be bound by a particular theory, in some embodiments, induction of a priming-favorable cytokine milieu can be mediated through interferon alpha (IFNa). Without wishing to be bound by a particular theory, in some embodiments, induction of a priming-favorable cytokine milieu can be mediated through a CD4+ T cell immune response.

[0014] In some embodiments, technologies provided herein may be particularly useful to subjects who have been previously exposed (e.g., via infection and/or vaccination) to a reference antigen (e.g., SARS-CoV-2) of an infectious agent and are receiving an immunogenic composition that delivers a variant polypeptide of the reference antigen (e.g., polypeptide of a prior circulating SARS-CoV-2 strain), or an immunogenic portion thereof. In some embodiments, such a variant polypeptide comprises arisen epitopes. In some embodiments, such arisen epitopes are or comprise neutralizing epitopes (e.g., neutralizing antibody epitopes). In some embodiments, technologies provided herein may be particularly used to induce activation of naive B cell immune response (e.g., in some embodiments antibody response, e.g., neutralizing antibody response) to at least one of the arisen epitopes (e.g., in some embodiments at least one of the neutralizing epitopes).

[0015] The present disclosure exemplifies certain aspects of provided technologies through administering a combination of a modified RNA vaccine that delivers a variant polypeptide of a reference antigen of an infectious agent, e.g., a vaccine that delivers a variant of a coronavirus S protein or an immunogenic portion thereof, and a particular interferon-alpha (IFNa)-inducing agent, e.g., a non-modified RNA. In some embodiments, such a non-modified RNA encodes at least one or more T cell epitopes. In some embodiments, such a non-modified RNA encodes at least one or more B cell epitopes. A skilled person, having read the disclosure, will appreciate that such strategies utilized in coronavirus vaccines may be also useful in other infectious diseases, e.g., circulating infectious diseases.

[0016] In some embodiments, disclosed herein is an RNA comprising a nucleotide sequence that encodes a polypeptide comprising or consisting of a variant polypeptide of a reference antigen of an infectious agent (e.g., a SARS-CoV-2 Spike (S) protein), or an immunogenic portion thereof, wherein a B cell memory immune response has been established to the reference antigen (e.g., SARS-CoV-2 S protein), and wherein the variant polypeptide (e.g., SARS-CoV-2 S protein variant) or immunogenic portion thereof has an amino acid sequence that differs from that of the reference antigen (e.g., reference SARS-CoV-2 S protein) in that it has been engineered to reduce the variant’s activation of the B cell memory immune response relative to the reference antigen (e.g., reference SARS-CoV-2 S protein).

[0017] In some embodiments, an antigen of an infectious agent (or portion thereof) comprises an engineered amino acid sequence so that at least one B cell memory epitope present in a reference antigen of the infectious agent (e.g., a SARS-CoV-2 S protein) is modified so that the memory activation potency of a reference antigen (or portion thereof) (e.g., a SARS-CoV-2 S protein protein) is reduced.

[0018] In some embodiments, an amino acid sequence encoded by an RNA is at least 80% identical to the corresponding portion of the reference antigen (e.g., a SARS-CoV-2 S protein).

[0019] In some embodiments, a SARS-CoV-2 S protein variant (or immunogenic portion thereof) has an amino acid sequence that is at least 80% identical to that of a reference SARS-CoV-2 S protein (or an amino acid sequence of the corresponding portion of a reference SARS-CoV-2 S protein).

[0020] In some embodiments, an amino acid sequence encoded by an RNA comprises no more than 50% of the B cell memory epitopes present in a reference antigen. [0021] In some embodiments, a SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises no more than 50% of the memory B cell epitopes present in a reference SARS-CoV-2 S protein.

[0022] In some embodiments, an RNA comprises a nucleotide sequence that encodes an antigen of an infectious agent (or a portion thereof), wherein the amino acid sequence of the antigen was engineered by a process comprising a step of removing memory B cell epitopes of a reference antigen or an immunogenic portion thereof.

[0023] In some embodiments, an RNA comprises a nucleotide sequence that encodes a SARS-CoV-2 Spike (S) protein variant (or an immunogenic portion thereof) whose amino acid sequence is engineered so that at least one memory B cell epitope present in a reference SARS- CoV-2 S protein has been modified so that memory B cell activation potency of the SARS-CoV- 2 S protein variant (or immunogenic portion thereof) has been reduced relative to the reference SARS-CoV-2 S protein.In some embodiments, an RNA comprises a nucleotide sequence that encodes a SARS-CoV-2 Spike (S) protein variant (or an immunogenic portion thereof), wherein the amino acid sequence of the S protein variant or immunogenic portion thereof was engineered by a process comprising a step of removing memory B cell epitopes present in a reference SARS-CoV-2 S protein.

[0024] In some embodiments, a variant SARS-CoV-2 S protein (or immunogenic portion thereof) comprises few memory B cell epitopes of a reference SARS-CoV-2 S protein.

[0025] In some embodiments, one or more memory B cell epitopes in a reference SARS- CoV-2 S protein have been identified by antibody-binding studies (e.g., studies characterizing antibodies produced by subjects administered a vaccine that delivers the reference SARS-CoV-2 S protein and/or infected with a virus comprises the reference SARS-CoV-2 S protein).

[0026] In some embodiments, an antigen of an infectious agent or immunogenic portion thereof is engineered so as to lack regions of a reference antigen comprising a high number (or density) of conserved B cell epitopes.

[0027] In some embodiments, conserved B cell epitopes are non-neutralizing epitopes.

[0028] In some embodiments, one or more memory B cell epitopes comprise or consist of non-neutralizing epitopes and neutralizing epitopes. [0029] In some embodiments, an antigen of an infectious agent or immunogenic portion thereof is engineered so as to lack conserved neutralizing B cell epitopes and conserved non- neutralizing B cell epitopes.

[0030] In some embodiments, an infectious agent has a high mutation rate.

[0031] In some embodiments, an infectious agent has a high number of variants or species.

[0032] In some embodiments, an infectious agent has a large number of variants or species, many of which are immune escaping.

[0033] In some embodiments, an infectious agent is a virus, bacteria, or Plasmodium.

[0034] In some embodiments, an infectious agent is a virus. In some embodiments, a virus is a respiratory virus. In some embodiments, a virus is an influenza virus, RSV, a norovirus, or HIV.

[0035] In some embodiments, an infectious agent is a coronavirus. In some embodiments, a coronavirus is a betacoronavirus. In some embodiments, a coronavirus is a MERS, SARS, or SARS-CoV-2 virus.

[0036] In some embodiments, a plasmodium is P. falciparum, P. vivax, P. ovale, or P. malariae.

[0037] In some embodiments, a variant polypeptide lacks regions that are not mutated frequently in immune-escaping variants of the infectious agent.

[0038] In some embodiments, a variant polypeptide comprises an immunogenic portion of a coronavirus S protein that lacks sequences corresponding to regions outside of the S 1 domain or the receptor binding domain (RBD).

[0039] In some embodiments, a SARS-CoV-2 S protein variant or immunogenic portion thereof is engineered so as to lack regions of a reference SARS-CoV-2 S protein that comprise a high number or density of conserved memory B cell epitopes.

[0040] In some embodiments, conserved memory B cell epitopes are non-neutralizing epitopes. [0041] In some embodiments, a variant SARS-CoV-2 S protein or immunogenic portion thereof is engineered so as to lack regions that are not mutated frequently in immune-escaping SARS-CoV-2 variants.

[0042] In some embodiments, an immunogenic portion of a coronavirus S protein does not comprise an S2 domain.

[0043] In some embodiments, an immunogenic portion of a SARS-CoV-2 S protein variant comprises or consists of the SI domain or the receptor binding domain (RBD).

[0044] In some embodiments, an immunogenic portion of a coronavirus S protein does not comprise an N-terminal domain (NTD).

[0045] In some embodiments, an immunogenic portion of a SARS-CoV-2 S protein variant comprises or consists of the RBD.

[0046] In some embodiments, a reference SARS-CoV-2 S protein is from a strain or variant that was previously prevalent or is currently prevalent in a relevant population of subjects.

[0047] In some embodiments, a reference SARS-CoV-2 S protein was previously delivered by a vaccine. In some embodiment, the vaccine is a commercially approved vaccine, a protein-based vaccine, an RNA vaccine, or any combination thereof.

[0048] In some embodiments, a reference SARS-CoV-2 S protein is a Wuhan S protein.

[0049] In some embodiments, a reference SARS-CoV-2 S protein is an Omicron BA.4/5

S protein.

[0050] In some embodiments, a SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises one or more mutations associated with a SARS-CoV-2 variant that has a high immune escape potential (e.g., a variant of concern).

[0051] In some embodiments, a SARS-CoV-2 variant has been determined to have a high immune escape potential using an in vitro assay (e.g., a viral neutralization assay), in silico analysis (e.g., sequence analysis and/or molecular dynamic simulations), and/or based on infection rates and/or growth rates. [0052] In some embodiments, a SARS-CoV-2 variant with a high immune escape potential is an Omicron variant.

[0053] In some embodiments, a SARS-CoV-2 variant with a high immune escape potential is an XBB variant (e.g., an XBB.l or XBB.1.5 variant) or a BQ.l variant.

[0054] In some embodiments, one or more mutations associated with an XBB.1.5 variant are T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K, where the positions of the one or more mutations are indicated relative to SEQ ID NO: 1.

[0055] In some embodiments, one or more mutations associated with an XBB.1.5 RBD are G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, and Y505H, where the positions of the one or more mutations are indicated relative to SEQ ID NO: 1.

[0056] In some embodiments, one or more mutations associated with an XBB.1.5 SI domain are T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, and P681H, where the positions of the one or more mutations are indicated relative to SEQ ID NO: 1.

[0057] In some embodiments, one or more mutations associated with an XBB.1.5 variant are T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, where the positions of the one or more mutations are indicated relative to SEQ ID NO: 1.

[0058] In some embodiments, one or more mutations associated with an XBB.1.5 SI are T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, where the positions of the one or more mutations are indicated relative to SEQ ID NO: 1.

[0059] In some embodiments, an RNA comprises a nucleotide sequence that encodes an immunogenic portion of a SARS-CoV-2 S protein variant comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 3.

[0060] In some embodiments, an RNA comprises a nucleotide sequence that encodes an immunogenic portion of the SARS-CoV-2 S protein variant comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 5.

[0061] In some embodiments, a variant polypeptide comprises a secretion signal. In some embodiments, a secretion signal is a homologous secretion signal. In some embodiments, a secretion signal is a heterologous secretion signal.

[0062] In some embodiments, a secretion signal is present in the N-terminal portion of a polypeptide (e.g., at the N-terminus).

[0063] In some embodiments, a secretion signal is a SARS-CoV-2 S protein secretion signal, a gD2 secretion signal, a gDl secretion signal, a gBl secretion signal, a gI2 secretion signal, a gE2 secretion signal, an Eboz secretion signal, or an HLA-DR secretion signal.

[0064] In some embodiments, an antigen of an infectious agent or immunogenic portion thereof encoded comprises a hypervariable domain.

[0065] In some embodiments, a hypervariable domain is a region of an antigen that has a high mutation frequency.

[0066] In some embodiments, a hypervariable domain has a high density of neutralization epitopes.

[0067] In some embodiments, a hypervariable domain is a region that is frequently mutated in variants of the infectious agent that have a high immune escape potential.

[0068] In some embodiments, a hypervariable domain is a receptor binding domain (RBD).

[0069] In some embodiments, a hypervariable domain comprises or consists of an RBD or S 1 domain of a coronavirus S protein. [0070] In some embodiments, a reference antigen is: (i) a surface protein or surface glycoprotein of an infectious agent strain or variant that was previously and/or is currently prevalent; and/or (ii) a surface protein or surface glycoprotein of an infectious agent that has been previously delivered in a vaccine (e.g., a commercially available vaccine, an RNA vaccine, or a protein-based vaccine).

[0071] In some embodiments, a surface protein or surface glycoprotein is a coronavirus S protein.

[0072] In some embodiments, a variant polypeptide has been engineered to eliminate one or more memory B cell epitopes of a reference antigen.

[0073] In some embodiments, one or more memory B cell epitopes have previously been determined to be bound by antibodies and/or B cells produced by a subject exposed to the reference antigen (e.g., via a vaccine that delivers the reference antigen and/or infection with a virus that comprises the reference antigen).

[0074] In some embodiments, one or more memory B cell epitopes comprise or consist of non-neutralizing epitopes.

[0075] In some embodiments, one or more memory B cell epitopes comprise or consist of non-neutralizing epitopes and neutralizing epitopes.

[0076] In some embodiments, a variant polypeptide comprises few intact memory B cell epitopes of the reference antigen.

[0077] In some embodiments, a variant polypeptide comprises one or more mutations associated with an infectious agent variant that has a high immune escape potential.

[0078] In some embodiments, an infectious agent variant has been determined to have a high immune escape potential using an in vitro assay (e.g., a viral neutralization assay), via in silico analysis (e.g., sequence analysis and/or molecular dynamic simulations), and/or based on infection rates in subjects in a relevant population.

[0079] In some embodiments, a variant polypeptide comprises few conserved memory B-cell epitopes relative to: (i) a reference antigen of a strain or variant that was previously or is currently prevalent in a relevant population, and/or (ii) one or more reference antigens that have previously been delivered in a vaccine (e.g., a commercially available vaccine and/or a vaccine previously administered to a subject).

[0080] In some embodiments, a variant polypeptide comprises 10 or fewer (e.g., 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, one or less, or no) conserved memory B cell epitopes.

[0081] In some embodiments, a variant polypeptide comprises a secretion signal.

[0082] In some embodiments, a secretion signal is a homologous secretion signal. In some embodiments, a secretion signal is a heterologous secretion signal.

[0083] In some embodiments, a secretion signal is present in the N-terminal portion of the polypeptide (e.g., at the N-terminus of the polypeptide).

[0084] In some embodiments, a secretion signal is a SARS-CoV-2 S protein secretion signal, a gD2 secretion signal, a gDl secretion signal, a gBl secretion signal, a gI2 secretion signal, a gE2 secretion signal, an Eboz secretion signal, or an HLA-DR secretion signal.

[0085] In some embodiments, a SARS-CoV-2 S protein secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 15.

[0086] In some embodiments, a SARS-CoV-2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 9 or 16.

[0087] In some embodiments, a gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 8. In some embodiments, a gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 13. In some embodiments, a gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 33.

[0088] In some embodiments, a gDl secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 12.

[0089] In some embodiments, a gBl secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 37.

[0090] In some embodiments, a gC2 polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 35. In some embodiments, a gC2 polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 32. [0091] In some embodiments, a gI2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 10 or 11.

[0092] In some embodiments, a gE2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 38.

[0093] In some embodiments, an EboZ secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 39.

[0094] In some embodiments, an HLA-DR secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 40.

[0095] In some embodiments, a variant polypeptide (e.g., a SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises a multimerization domain.

[0096] In some embodiments a multimerization domain is in the C-terminal region of a SARS-CoV-2 variant protein or an immunogenic portion thereof (e.g., at the C-terminus).

[0097] In some embodiments, a polypeptide comprises a multimerization domain that is C-terminal to the variant polypeptide.

[0098] In some embodiments, a multimerization domain is a fibritin domain.

[0099] In some embodiments, a fibritin domain comprises a sequence that is at least

80% identical to SEQ ID NO: 95. In some embodiments, a fibritin domain comprises a sequence that is at least 80% identical to SEQ ID NO: 96.

[0100] In some embodiments, a SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises a transmembrane (TM) domain.

[0101] In some embodiments, a variant polypeptide comprises a transmembrane (TM) domain. In some embodiments, a TM domain is a homologous TM domain. In some embodiments, a TM domain is a heterologous TM domain. In some embodiments, a TM domain is present in the C-terminal portion of the polypeptide (e.g., at the C-terminus).

[0102] In some embodiments, a variant polypeptide (e.g., SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises a multimerization domain and a TM domain in the C-terminal portion of the polypeptide, wherein the TM domain is C-terminal to the multimerization domain (e.g., the TM domain is at the C-terminus of the variant polypeptide and the multimerization domain is adjacent to the TM domain (e.g., directly adjacent to the TM domain and/or connected to the TM domain via a GS linker)).

[0103] In some embodiments, a TM domain is a SARS-CoV-2 S protein TM domain, or an influenza TM domain. In some embodiments, a SARS-CoV-2 TM domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 89. In some embodiments, a SARS-CoV-2 TM domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 90.

[0104] In some embodiments, an RNA comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 120.

[0105] In some embodiments, an immunogenic portion of a SARS-CoV-2 S protein variant comprises a sequence that is at least 80% identical to SEQ ID NO: 130.

[0106] In some embodiments, an RNA comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 135.

[0107] In some embodiments, an RNA comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 145.

[0108] In some embodiments, an RNA comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 150.

[0109] In some embodiments, a nucleotide sequence that encodes a SARS-CoV-2 S protein variant (or immunogenic portion thereof) is codon-optimized for expression in mammalian subjects.

[0110] In some embodiments, a nucleotide sequence that encodes a SARS-CoV-2 S protein variant (or immunogenic portion thereof) is codon-optimized for expression in human subjects.

[0111] In some embodiments, a nucleotide sequence encoding a SARS-CoV-2 S protein variant (or immunogenic portion thereof) has an enriched G/C content relative to wild-type sequence.

[0112] In some embodiments, a nucleotide sequence that encodes a variant polypeptide or the polypeptide is codon-optimized for expression in mammalian subjects. [0113] In some embodiments, a nucleotide sequence that encodes a variant polypeptide or an immunogenic portion thereof has been codon-optimized for expression in human subjects.

[0114] In some embodiments, a nucleotide sequence encoding a variant polypeptide or a portion thereof has an enriched G/C content relative to wild-type sequence.

[0115] In some embodiments, G/C content has been increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%.

[0116] In some embodiments, an RNA comprises a heterologous 3’ UTR or 5’UTR.

[0117] In some embodiments, a heterologous 5' UTR comprises or consists of a modified human alpha-globin 5 '-UTR.

[0118] In some embodiments, a heterologous 3’ UTR comprises or consists of a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA.

[0119] In some embodiments, an RNA comprises a poly(A) sequence.

[0120] In some embodiments, a poly(A) sequence has a length of about 100-150 nucleotides.

[0121] In some embodiments, a poly(A) sequence is a disrupted poly(A) sequence.

[0122] In some embodiments, an RNA comprises a 5' cap.

[0123] In some embodiments, an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 122 or 124.

[0124] In some embodiments, an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 131 or 133.

[0125] In some embodiments, an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 136 or 138.

[0126] In some embodiments, an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 146 or 148. [0127] In some embodiments, an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 151 or 153.

[0128] In some embodiments, an RNA is unmodified RNA.

[0129] In some embodiments, an RNA comprises one or more modified nucleotides.

[0130] In some embodiments, a modified nucleotide is pseudouridine (e.g., N1 -methyl- pseudouridine).

[0131] In some embodiments, an RNA comprises a modified nucleotide in place of each uridine.

[0132] In some embodiments, an RNA is an mRNA, a self-amplifying RNA or a trans- amplifying RNA.

[0133] In some embodiments, a composition comprises an RNA described herein, wherein an RNA is fully or partially encapsulated within lipid nanoparticles (LNP), polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes.

[0134] In some embodiments, an RNA is fully or partially encapsulated within LNP.

[0135] In some embodiments, an LNP comprises a cationically ionizable lipid, a neutral lipid, a sterol and a lipid conjugate.

[0136] In some embodiments, an LNP comprises from about 40 to about 50 mol percent of the cationically ionizable lipid; from about 5 to about 15 mol percent of the neutral lipid; from about 35 to about 45 mol percent of the sterol; and from about 1 to about 10 mol percent of the PEG-lipid.

[0137] In some embodiments, provided herein is a method of inducing an immune response, comprising administering an RNA described herein or a composition described herein to a subject.

[0138] In some embodiments, described herein is a method of inducing an immune response in a subject who has previously been exposed to a reference antigen of an infectious agent (e.g., SARS-CoV-2), the method comprising: delivering a variant polypeptide of the reference antigen (e.g., SARS-CoV-2) or an immunogenic portion thereof to the subject, wherein a B cell memory immune response has been established to the reference antigen (e.g., SARS- CoV-2) , and wherein the variant polypeptide (e.g., SARS-CoV-2 variant) has an amino acid sequence that differs from that of the reference antigen (e.g., SARS-CoV-2) in that it has been engineered to reduce the variant polypeptide’s activation of the B cell memory immune response.

[0139] In some embodiments, an infectious agent is an influenza virus, RSV, norovirus, HIV, coronavirus, or a plasmodium.

[0140] In some embodiments, a subject has previously been administered one or more doses of one or more vaccines that deliver the reference antigen (e.g., SARS-CoV-2) .

[0141] In some embodiments, a reference SARS-CoV-2 S protein is a Wuhan SARS- CoV-2 S protein.

[0142] In some embodiments, an immune response comprises a naive B cell immune response.

[0143] In some embodiments, an immune response comprises a reduced memory B cell immune response or an immune response does not comprise a memory B cell immune response.

[0144] In some embodiments, provided herein is a method of manufacturing an immunogenic composition comprising: (a) providing a reference antigen of an infectious agent (e.g., SARS-CoV-2) , wherein the reference antigen is from a strain or variant (e.g., a strain or variant that has previously been prevalent and/or that has previously been delivered as a vaccine) of the infectious agent (e.g, SARS-CoV-2), (b) determining a variant polypeptide of the reference antigen (e.g, SARS-CoV-2 variant) that comprises fewer memory B cell epitopes relative to the reference antigen (e.g, SARS-CoV-2); and (c) producing an immunogenic composition that delivers the variant polypeptide (e.g, SARS-CoV-2 variant) .

[0145] In some embodiments, a variant polypeptide comprises a sequence that corresponds to an immunogenic portion of a reference antigen.

[0146] In some embodiments, a variant polypeptide (e.g, SARS-CoV-2 variant) comprises one or mutations at one or more B cell epitopes of the reference antigen (e.g, SARS- CoV-2) .

[0147] In some embodiments, provided herein is a method of assessing, predicting, or characterizing the ability of an immunogenic composition that delivers an antigen of an infectious agent (e.g, SARS-CoV-2) to induce activation of memory B cells in a subject or a population of subjects, the method comprising determining the number of memory B cell epitopes present in the antigen (e.g, SARS-CoV-2) relative to a reference antigen.

[0148] In some embodiments, a reference antigen (e.g, SARS-CoV-2) is from a strain or variant of an infectious agent that a subject was exposed to and/or that a large portion of a population was exposed to.

[0149] In some embodiments, provided herein is a method of producing a personalized vaccine (e.g, SARS-CoV-2 vaccine) for a subject against an infectious agent, the method comprising steps of: (a) determining a reference antigen of the infectious agent (e.g, SARS-CoV- 2) that a subject has previously been exposed to; (b) determining a variant polypeptide of the reference antigen (e.g, SARS-CoV-2 variant) that comprises fewer memory B cell epitopes relative to the reference antigen (e.g, SARS-CoV-2) ; and (c) producing an immunogenic composition that delivers the variant polypeptide (e.g, SARS-CoV-2 variant).

[0150] In some embodiments, a reference antigen is from a strain or variant of the infectious agent that the subject was first exposed to and/or that was first prevalent in the population of subjects.

[0151] In some embodiments, a reference SARS-CoV-2 S protein is a Wuhan SARS- CoV-2 S protein or an Omicron BA.4/5 SARS-CoV-2 S protein.

[0152] In some embodiments, a reference antigen is from an infectious agent (e.g, SARS-CoV-2) that a subject has previously been vaccinated against or is delivered by one or more vaccines that a significant proportion of the population (e.g., at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least abut 45%, at least about 50%, at least about 55%, or at least about 60%) has previously been administered.

[0153] In some embodiments, a vaccine previously administered to a subject or a large portion of a population was a first generation vaccine.

[0154] In some embodiments, a reference antigen is from an infectious agent (e.g, SARS-CoV-2) that was previously prevalent or is currently prevalent in a relevant geographic region. [0155] In some embodiments, a reference antigen is from an infectious agent variant (e.g, SARS-CoV-2) that first became prevalent in a relevant jurisdiction.

[0156] In some embodiments, antigens (or immunogenic portions thereof) described herein can be engineered to comprise mutations or sequences from two or more antigens of a given infectious agent, where the two or more antigens are from different variants, strains, lineages, etc., of the infectious agent. For example, in some such embodiments, a mutation or sequence in an antigen of a first variant of an infectious agent can be introduced into the corresponding sequence of an antigen of a second variant (or an immunogenic portion thereof). This process can be repeated multiple times, and can be useful, e.g., for removing B cell epitopes (e.g., one or more conserved B cell epitopes). In certain embodiments, antigens described herein can be engineered to incorporate sequences and/or mutations from two or more SARS-COV-2 variants (e.g., epitopes from RBDs, S proteins, and/or SI domains from two or more SARS- CoV-2 variants). For example, in some such embodiments, mutations of a one or more SARS- CoV-2 variants can be introduced in conserved epitopes of a variant SARS-CoV-2 S protein, or an immunogenic portion thereof (e.g., an SI domain or an RBD). Exemplary approaches and methods for introducing mutations or sequences from an antigen of one or more infectious agent variants into a sequence of a first antigen (or an immunogenic portion thereof) are described, e.g., in the U.S. provisional application entitled “Systems and Methods for Engineering Antigens to Promote Tailored Immune Responses”, filed February 24, 2023, and having U.S. Provisional Application No. 63/448,215 (inter alia). Said application describes, among other things, technologies directed to in-silico design of custom, engineered, antigens (e.g., including engineered versions of SARS-CoV 2 variant proteins and portions thereof) for reducing an extent to which a memory immune response is triggered.

[0157] In one aspect, the present disclosure provides a combination comprising: (i) a modified RNA molecule encoding a polypeptide comprising or consisting of a variant polypeptide (e.g., SARS-CoV-2 variant) of a reference antigen of an infectious agent (e.g., SARS-CoV-2), or an immunogenic portion thereof, wherein the variant polypeptide (e.g., SARS- CoV-2 variant) comprises neutralizing epitopes that are absent in the reference antigen (e.g., SARS-CoV-2); and (ii) an agent that induces a priming-favorable cytokine milieu in lymphoid tissues, wherein the agent is present at a dose that is effective to increase activation of naive B cell immune response to at least one of the neutralizing epitopes. [0158] In some embodiments, an agent that induces a priming-favorable cytokine milieu in lymphoid tissues is or comprises interferon alpha (IFN ^) or an IFN ^-inducing agent. In some embodiments, an agent that induces a priming-favorable cytokine milieu in lymphoid tissues is or comprises a CD4+ T cell response inducing agent. [0159] In some embodiments, a reference antigen is: (i) a surface protein or surface glycoprotein of an infectious agent strain or variant (e.g., SARS-CoV-2 strain) that was previously and/or is currently prevalent; and/or (ii) a surface protein or surface glycoprotein of an infectious agent (e.g., SARS-CoV-2) that has been previously delivered in a vaccine (e.g., a commercially available vaccine, an RNA vaccine, or a protein-based vaccine). In some embodiments, a reference antigen is a SARS-CoV-2 S protein of a Wuhan strain or an Omicron BA.4/5 strain. In some embodiments, a reference antigen is a SARS-CoV-2 S protein of a XBB strain (e.g., XBB1, XBB1.5). [0160] In some embodiments, a modified RNA molecule and an agent are co-delivered. [0161] In some embodiments, an IFN ^-inducing agent is or comprises an unmodified RNA molecule. In some embodiments, the amount ratio of the modified RNA molecule to the unmodified RNA molecule is at least or greater than 1:1. In some embodiments, the ratio of modified ribonucleotides to unmodified ribonucleotides in the immunogenic composition is about 1:2 to about 1:10. In some embodiments, a modified ribonucleotide is 1- methylpseudouridine and an unmodified ribonucleotide is uridine. [0162] In some embodiments, an unmodified RNA molecule encodes a polypeptide comprising an antigen of an infectious agent (e.g., SARS-CoV-2). In some embodiments, an antigen is a B-cell antigen. In some embodiments, an antigen is a T-cell antigen. [0163] In some embodiments, an antigen is or comprises one or more T cell epitopes from at least one of an M protein, an N protein, and an ORF1ab protein of SARS-CoV-2. In some embodiments, an antigen is or comprises one or more T cell epitopes from at least two of an M protein, an N protein, and an ORF1ab protein of SARS-CoV-2. [0164] In some embodiments, a modified RNA molecule and the unmodified RNA molecule are separately or co-formulated in lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes. [0165] In some embodiments, an IFNoc-inducing agent is or comprises a self-amplifying RNA molecule or a trans-amplifying RNA molecule. In some embodiments, a self-amplifying RNA molecule or the trans-amplifying RNA molecule is an unmodified RNA molecule. In some embodiments, the amount ratio of the modified RNA molecule to the self-amplifying RNA molecule or the trans-amplifying RNA molecule is greater than 1:5. In some embodiments, a modified RNA molecule and the self- amplifying RNA molecule or trans-amplifying RNA molecule are separately or co-formulated in lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes.

[0166] In some embodiments, a modified RNA molecule comprises modified uridines. In some embodiments, a modified RNA molecule comprises a modified uridine in lieu of each uridine. In some embodiments, modified uridines are or comprise 1 -methyl pseudouridine.

[0167] In some embodiments, a modified RNA molecule encodes a polypeptide comprising an antigen of the infectious agent. In some embodiments, an antigen is a B-cell antigen. In some embodiments, an antigen is a T-cell antigen.

[0168] In another aspect, the present disclosure provides a combination comprising: (i) a composition that comprises or delivers polypeptide comprising or consisting of a variant polypeptide of a reference antigen of an infectious agent, or an immunogenic portion thereof, wherein the variant polypeptide comprises neutralizing epitopes that are absent in the reference antigen; and (ii) an agent that induces a priming-favorable cytokine milieu in lymphoid tissues, wherein the agent is present at a dose that is effective to increase activation of naive B cell immune response to at least one of the neutralizing epitopes, and wherein the agent is or comprises (i) an unmodified RNA molecule or (ii) a self-amplifying RNA molecule or a trans- amplifying RNA molecule, and wherein the RNA molecule is formulated in lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes. In some embodiments, an agent encodes a polypeptide comprising an antigen of the infectious agent (e.g., SARS-CoV-2) . In some embodiments, an antigen is a B-cell antigen. In some embodiments, an antigen is a T-cell antigen.

[0169] In some embodiments, the amount ratio (by mass or moles) of the polypeptide to the agent is within a range of about 1 : 1 to about 20: 1. [0170] In some embodiments, the present disclosure provides an RNA molecule comprising a nucleotide sequence that includes modified ribonucleotides and corresponding unmodified ribonucleotides, wherein the ratio of the modified ribonucleotides to the corresponding unmodified ribonucleotides is within a range of about 1: 10 to about 1: 1; and wherein the nucleotide sequence encodes an antigen of an infectious agent (e.g., SARS-CoV-2).

[0171] In some embodiments, a nucleotide sequence comprises a first domain and a second domain, wherein at least one of the first domain and the second domain comprises modified ribonucleotides and the other domain comprises no modified ribonucleotides. In some embodiments, modified ribonucleotides are 1 -methylpseudouridine and the corresponding unmodified ribonucleotides are uridine.

[0172] In one aspect, the present disclosure provides a method of inducing a priming immune response by: administering to a subject one or both of: (i) a composition that comprises or delivers a polypeptide antigen (e.g., SARS-CoV-2) ; and (ii) an agent that induces a priming- favorable cytokine milieu in lymphoid tissues, wherein the agent is present at a dose that is effective to increase activation of naive B cell immune response to at least one of the neutralizing epitopes.

[0173] In another aspect, the present disclosure provides a method of inducing or supporting a priming immune response to an antigen in a subject by exposing the subject to the antigen under immune priming conditions.

[0174] In some embodiments, a subject has previously been exposed to a variant of the antigen (e.g., SARS-CoV-2).

[0175] In some embodiments, a step of exposing comprises administering a composition that comprises or delivers the antigen. In some embodiments, an antigen is a polypeptide antigen. In some embodiments, a step of exposing comprises administering a “priming adjuvant” to a subject who is or will soon be exposed to the antigen.

[0176] In one aspect, the present disclosure provides a method of inducing an immune response in a subject in need thereof, comprising administering to the subject a first RNA molecule encoding a first antigen (e.g., SARS-CoV-2 antigen) and a second RNA molecule encoding a second antigen, wherein the first RNA molecule is a modified RNA molecule and the second RNA molecule (i) does not comprise a modified ribonucleotide or (ii) is a self-amplifying RNA molecule or a trans-amplifying RNA molecule.

[0177] In one aspect, the present disclosure provides a method of inducing an immune response in a subject in need thereof, comprising administering to the subject a composition comprising a first plurality of RNA molecules encoding first antigens (e.g., SARS-CoV-2) and a second plurality of RNA molecules encoding second antigens, wherein at least 10% (including, e.g., at least 20%, at least 30, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) of the first plurality of RNA molecules are modified RNA molecules, and at least 10% (including, e.g., at least 20%, at least 30, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) of the second plurality of RNA molecules (i) do not comprise a modified ribonucleotide or (ii) are self-amplifying RNA molecules or trans- amplifying RNA molecules.

[0178] In one aspect, the present disclosure provides a method of inducing an immune response in a subject in need thereof, comprising administering to the subject a first dose of a composition comprising a first RNA molecule encoding a first antigen (e.g., SARS-CoV-2) , and a second dose of a composition comprising a second RNA molecule encoding a second antigen, wherein the first RNA molecule is a modified RNA molecule, and the second RNA molecule (i) does not comprise a modified ribonucleotide or (ii) is a self-amplifying RNA molecule or trans- amplifying RNA molecule.

[0179] In some embodiments of various aspects described herein, a first RNA molecule comprises modified uridines. In some embodiments, modified uridines are in place of all uridines. In some embodiments, a second RNA molecule does not comprise a modified ribonucleotide. In some embodiments, a first antigen is or comprises a B cell antigen of an infectious agent and a second antigen is or comprises a T cell antigen. In some embodiments, a B cell antigen is a CoV-2 S antigen or immunogenic portion thereof. In some embodiments, a T cell antigen is from the same infectious agent. In some embodiments, a T cell antigen is a T string epitope. In some embodiments of certain aspects described herein, a T cell antigen is from SARS-CoV-2. In some embodiments, a B cell antigen of SARS-CoV-2 is SARS-CoV-2 S antigen or immunogenic portion thereof. [0180] In some embodiments of various aspects described herein, a first RNA molecule and a second RNA molecule are co-administered. In some embodiments, a first RNA molecule and a second RNA molecule are separately or co-formulated in lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes. In some embodiments, a first RNA molecule and a second RNA molecule are separately administered. In some embodiments, a subject has previously been administered one or more doses of one or more vaccines directed to a reference antigen of an infectious agent, wherein the reference antigen is from an earlier strain or lineage of the infectious agent, and wherein a B cell memory immune response has been established to the reference antigen.

[0181] In some embodiments of certain aspects described herein, a reference antigen is a SARS-CoV-2 S protein of a Wuhan strain or an Omicron BA.4/5 strain. In some embodiments, a reference antigen is a SARS-CoV-2 S protein of a XBB strain. In some embodiments, a XBB strain is a XBBl or XBB 1.5.

[0182] In another aspect, the present disclosure provides a method of inducing an immune response in a subject who was previously exposed to a first SARS-CoV-2 Spike (S) protein, the method comprising a step of delivering a polypeptide comprising a fragment of a second SARS-CoV-2 S protein to the subject, wherein the fragment of the second SARS-CoV-2 S protein comprises or consists a Receptor Binding Domain (RBD) or an S 1 domain of the second SARS-CoV-2 S protein, and wherein the fragment of the second SARS-CoV-2 S protein comprises one or more mutations of one or more SARS-CoV-2 variants.

[0183] In some embodiments, the first SARS-CoV-2 S protein is from a strain or variant that was previously prevalent or is currently prevalent in a relevant jurisdiction.

[0184] In some embodiments, the subject was previously exposed to the first SARS- CoV-2 S protein by: (a) administration of one or more doses of one or more vaccines that deliver the first SARS-CoV-2 S protein, previous infection by a SARS-CoV-2 virus comprising the first SARS-CoV-2 S protein, and/or presence in a jurisdiction where a SARS-CoV-2 strain or variant comprising the first SARS-CoV-2 S protein was prevalent.

[0185] In some embodiments, the fragment of the second SARS-CoV-2 S protein does not comprise one or more regions of a SARS-CoV-2 S protein that are infrequently mutated in SARS-CoV-2 variants. In some embodiments, the fragment of the second SARS-CoV-2 S protein does not comprise an S2 domain. In some embodiments, the fragment of the second SARS-CoV-2 S protein does not comprise an N-terminal domain (NTD). In some embodiments, the fragment of the second SARS-CoV-2 S protein comprises or consists of the RBD. In some embodiments, the fragment of the second SARS-CoV-2 S protein comprises or consists of the SI domain.

[0186] In some embodiments, the fragment of the second SARS-CoV-2 S protein comprises one or mutations associated with a SARS-CoV-2 variant that is prevalent, predicted to be prevalent, predicted to continue to be prevalent, and/or predicted to increase in prevalence in a relevant jurisdiction. In some embodiments, the fragment of the second SARS-CoV-2 S protein comprises one or more mutations associated with a SARS-CoV-2 variant that has a high immune escape potential. In some embodiments, the SARS-CoV-2 variant has been determined to have a high immune escape potential using an in vitro assay (e.g., a viral neutralization assay), in silico analysis (e.g., sequence analysis and/or molecular dynamic simulations), in vivo studies (e.g., mouse or rat studies), and/or based on an infection rate and/or growth rate in a human population.

[0187] In some embodiments, the SARS-CoV-2 variant is an Omicron variant. In some embodiments, the Omicron variant is an XBB variant (e.g., an XBB.l or XBB.1.5 variant), a BQ.l variant, a BA.2.86 variant, or a JN variant. In some embodiments, the one or more mutations associated with an XBB.1.5 variant are T19I, A24-26, A27S, V83A, G142D, A145, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, or N969K, or a combination thereof, where the positions of the one or more mutations are indicated relative to SEQ ID NO: 1.

[0188] In some embodiments, the fragment of the second SARS-CoV-2 S protein comprises or consists of an RBD of an XBB.1.5 SARS-CoV-2 variant, and wherein the RBD comprises one or more of the following mutations relative to SEQ ID NO: 1: G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, or Y505H, or any combination thereof. [0189] In some embodiments, the fragment of the second SARS-CoV-2 S protein comprises or consists of an S 1 domain, and wherein the one or more mutations associated with an XBB.1.5 variant are selected from: T19I, A24-26, A27S, V83A, G142D, Al 44, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, and P681H, or any combination thereof, wherein the positions of the one or more mutations are shown relative to SEQ ID NO: 1.

[0190] In some embodiments, the polypeptide comprising the fragment of the second SARS-CoV-2 S protein is delivered by administering an RNA that comprises a nucleotide sequence encoding the fragment of the second SARS-CoV-2 protein.

[0191] In some embodiments, the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 3. In some embodiments, the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 5.

[0192] In some embodiments, the polypeptide comprises a secretion signal. In some embodiments, the secretion signal is a homologous secretion signal. In some embodiments, the secretion signal is a heterologous secretion signal. In some embodiments, the secretion signal is present at or near the N-terminus of the polypeptide.

[0193] In some embodiments, the secretion signal is a SARS-CoV-2 S protein secretion signal, a gD2 secretion signal, a gDl secretion signal, a gBl secretion signal, a gI2 secretion signal, a gE2 secretion signal, an Eboz secretion signal, or an HLA-DR secretion signal. In some embodiments, the SARS-CoV-2 S protein secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 15. In some embodiments, the SARS-CoV-2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 9. In some embodiments, the SARS-CoV-2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 16. In some embodiments, the gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 8. In some embodiments, the gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 13. In some embodiments, the gDl secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 12. In some embodiments, the gBl secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 37. In some embodiments, the gC2 polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 35. In some embodiments, the gI2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 11. In some embodiments, the gE2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 38. In some embodiments, the EboZ secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 39. In some embodiments, the HLA-DR secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 40.

[0194] In some embodiments, the polypeptide further a multimerization domain. In some embodiments, the multimerization domain in the C-terminal region (e.g., at the C- terminus). In some embodiments, the multimerization domain is a fibritin domain. In some embodiments, the fibritin domain comprises a sequence that is at least 80% identical to SEQ ID NO: 95. In some embodiments, the fibritin domain comprises a sequence that is at least 80% identical to SEQ ID NO: 96.

[0195] In some embodiments, the polypeptide comprises a transmembrane (TM) domain. In some embodiments, the TM domain is a homologous TM domain. In some embodiments, the TM domain is a heterologous TM domain.

[0196] In some embodiments, the TM domain is present in the C-terminal portion of the SARS-CoV-2 S protein variant or immunogenic portion thereof (e.g., at the C-terminus).

[0197] In some embodiments, the polypeptide comprises a multimerization domain and a TM domain at or near the C-terminus. In some embodiments, the TM domain is C-terminal to the multimerization domain. In some embodiments, the multimerization domain is directly adjacent to the fragment of the second SARS-CoV-2 protein or connected to the fragment of the second SARS-CoV-2 protein via a flexible linker, and/or the TM domain is directly adjacent to the multimerization domain or connected to the multimerization domain via a flexible linker.

[0198] In some embodiments, the TM domain is a SARS-CoV-2 S protein TM domain or an influenza TM domain. In some embodiments, the SARS-CoV-2 TM domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 89. In some embodiments, the SARS-CoV-2 TM domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 90. [0199] In some embodiments, the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 120. In some embodiments, the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 130. In some embodiments, the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 135. In some embodiments, the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 145. In some embodiments, the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 150.

[0200] In some embodiments, the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein has been codon-optimized for expression in mammalian subjects. In some embodiments, the nucleotide sequence encoding the fragment of the second SARS- CoV-2 S protein has been codon-optimized for expression in human subjects.

[0201] In some embodiments, the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein has an enriched G/C content relative to wild-type sequence. In some embodiments, the G/C content has been increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%.

[0202] In some embodiments, the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein comprises a heterologous 3’ UTR or 5’UTR. In some embodiments, the heterologous 5' UTR comprises or consists of a modified human alpha-globin 5'-UTR. In some embodiments, the heterologous 3’ UTR comprises or consists of a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA.

[0203] In some embodiments, the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein comprises a poly(A) sequence. In some embodiments, the poly(A) sequence has a length of about 100-150 nucleotides. In some embodiments, the poly(A) sequence is a disrupted poly(A) sequence. [0204] In some embodiments, the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein comprises a 5' cap.

[0205] In some embodiments, the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 122 or 124. In some embodiments, the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 131 or 133. In some embodiments, the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 136 or 138. In some embodiments, the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 146 or 148. In some embodiments, the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 151 or 153.

[0206] In some embodiments, the RNA is unmodified RNA.

[0207] In some embodiments, the RNA comprises one or more modified nucleotides. In some embodiments, the modified nucleotide is pseudouridine (e.g., Nl-methyl-pseudouridine). In some embodiments, the RNA comprises a modified nucleotide in place of each uridine.

[0208] In some embodiments, the RNA is an self-amplifying RNA or trans-amplifying RNA.

[0209] In some embodiments, the RNA is fully or partially encapsulated within lipid nanoparticles (LNP), polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes. In some embodiments, the RNA is fully or partially encapsulated within LNP. In some embodiments, the LNP comprise a cationically ionizable lipid, a neutral lipid, a sterol and a lipid conjugate.

[0210] In some embodiments, the first SARS-CoV-2 S protein is from a strain or variant that the subject was first exposed to and/or that was first prevalent in a population of subjects.

[0211] In some embodiments, the first SARS-CoV-2 S protein is a Wuhan SARS-CoV-2 S protein or an Omicron BA.4/5 SARS-CoV-2 S protein.

[0212] In some embodiments, the first SARS-CoV-2 S protein is from a strain or variant that the subject has previously been vaccinated against or is delivered by one or more vaccines that a significant proportion of the population (e.g., at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least abut 45%, at least about 50%, at least about 55%, or at least about 60%) has previously been administered.

[0213] In some embodiments, the vaccine previously administered to the subject or a significant proportion of the population was a first generation vaccine. In some embodiments, the first SARS-CoV-2 S protein is from a SARS-CoV-2 strain or variant that was previously prevalent or is currently prevalent in a relevant jurisdiction. In some embodiments, the first SARS-CoV-2 S protein is from a variant that first became prevalent in a relevant jurisdiction.

[0214] In some embodiments, the immune response comprises a B cell immune response. In some embodiments, the immune response comprises a naive B cell immune response.

[0215] In some embodiments, (a) the immune response comprises a reduced memory B cell immune response as compared to an immune response induced by administering the full length sequence of the second SARS-CoV-2 S protein, (b) the immune response comprises an increased naive B cell immune response as compared to an immune response induced by administering the full length sequence of the second SARS-CoV-2 S protein, and/or (c) the ratio of the naive B cell immune response to the memory B cell immune response is increased. In some embodiments, (a) the memory B cell immune response is reduced by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% as compared to the immune response induced by a full length sequence of the second SARS-CoV-2 protein; (b) the memory B cell immune response is increased by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% as compared to the immune response induced by a full length sequence of the second SARS-CoV-2 protein; and/or (c) the ratio of the naive immune response to the memory B cell immune response is increased by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% as compared to the immune response induced by a full length sequence of the second SARS-CoV-2 protein.

Brief description of the Figures

[0216] Fig. 1 illustrates an immune imprinting phenomenon observed in various infectious diseases. SARS-CoV-2 is shown as a representative disease, but a similar process is thought to occur in a number of infectious diseases (in particular, in diseases caused by infectious agents having high mutation rates in antigen regions that comprise a high number of neutralization epitopes). Subjects administered a vaccine that delivers a wild-type (WT) antigen produce antibodies and form memory B cells. As new Variants of Concern (VOC) arise, VOC- adapted booster shots are administered. Certain VOCs have high immune escape potential and comprise mutations at neutralization epitopes in hypervariable domains (represented by the portions of the antigen with different colors in the Figure). Subjects exposed to a VOC-adapted vaccine have a predisposition to activate memory B cells formed in response to the initial WT vaccine rather than produce de novo responses that recognize epitopes unique to the VOC (i.e., memory B cells that recognize conserved epitopes in the VOC antigen are more likely to be activated and naive B cells recognizing unique epitopes are less likely to be activated). So long as at least some of the neutralization epitopes in the WT antigen are preserved, administering a VOC-adapted vaccine will increase induction of neutralization antibodies against the VOC. As VOCs continue to evolve, however, and acquire further mutations at neutralization epitopes, neutralization responses induced by VOC-adapted vaccines become less efficacious. Further discussion of the imprinting phenomenon in the SARS-CoV-2 context can be found in Wheatley et al., Trends Immunol, 2021, the contents of which are incorporated by reference herein in their entirety.

[0217] Fig. 2: Exemplary characterization tools for assessing memory B cell response, which can be useful to evaluate immune imprinting phenomenon. (A), (B) Schematic of one- dimensial flow-cytometry analysis of memory B cell (BMEM) phenotyping using fluorochrome- labeled antigens (in Figure 2, a SARS-CoV-2 Spike protein is shown for illustrative purposes; in general any antigen delivered as part of a vaccine (or a subdomain of such an antigen) may be used as a label to characterize BMEM cells). BMEM specificity can be assessed by labelling with antigens (or subdomains) of different infectious agent variants. (C) Serological analysis after depletion of immune serum with antigen (or subdomain) bait (in the Figure, SARS-CoV-2 Spike protein is shown for illustrative purposes). Serum samples collected from a subject exposed to an antigen (e.g., via a prior infection and/or previous vaccination) are incubated with an antigen or subdomain thereof (e.g., RBD or SI domain in the case of a SARS-CoV-2 S protein) immobilized on a support (e.g., a magnetic bead as shown in the figure). Isolation of the bead removes antibodies that bind the bait, and the remaining serum is analyzed to determine the specificity of antibodies in the serum sample (e.g., in the Figure, sera samples are incubated with Wild-Type (Wuhan) Spike immobilized on magnetic beads, the magenetic beads are removed, and any antibodies remaining in the sample that bind a variant are variant-specific antibodies). Beads lacking bait can be used as a negative control (e.g., as shown in the Figure).

[0218] Fig. 3. Experimental design for assessing impact of immune imprinting. For illustrative purposes, the Figure shows an experiment designed to assess the impact of immune imprinting in SARS-CoV-2, but one of skill in the art will recognize that the depicted experiment can be readily adpated to characterize immune imprinting in any infectious disease context. Sera samples were collected from subjects administered 2 or 3 doses of a vaccine delivering a SARS- CoV-2 S protein (e.g., BNT162b2) and (i) infected with an Omicron BA.l SARS-CoV-2 variant, (ii) infected with an Omicron BA.l variant and subsequently adminsitered an Omicron BA.l- adapted vaccine (BNT162b2(omi)), or (iii) two doses of an Omicron BA.l -adapted vaccine.

[0219] Fig. 4. Variant-induced broad neutralization can be mediated by expansion of responses against conserved epitopes (i.e. recall responses). Provided is data demonstrating that, while exposure to a new variant of a infectious agent (Omicron BA.l in the Figure) can induce a broad immune response, in some embodiments, that broad immune response is driven by the activation of memory B cells and recognition of conserved epitopes, rather than generation of new antibodies that recognize epitopes unique to the new variant. A person of skill in the art will recognize that while the data depicted in the Figure pertain to SARS-CoV-2, the results demonstrate that similar effects could be observed in other infectious diseasese and/or that similar experiments could be performed to characterize immune imprinting in other infectious dieases. Pseudovirus neutralization assays and FACS analysis of BMEM cells using fluorochrome-labeled Spike or RBD tetramers were performed on sera samples collected from the patient groups summarized in Figure 3. (A) Shows pseudo virus neutralization assay results. Pseudovirus neutralization titers (pVNso) were collected for pseudoviruses comprising S proteins of various coronavirus variants and strains (variants and strains indicated along X-axis). Assay results demonstrate that Omicron BA.l infection augments broadly neutralizing activity against Omicron variants, especially against BA.l. (B) Shows representative FACS plots, using flourescently labeled S proteins or RBDs of a full length S protein or RBD. FACS results show that a majority of memory B cells bind epitopes that are common to the Wuhan S protein and the Omicron BA.l S protein, or that are unique to the Wuhan S protein, but very few are specific to Omicron BA.l. These results suggest that a first exposure to wild-type S protein has imprinted against novel BMEM responses recognizing BA.l specific epitopes. BNT162b2³ corresponds to sera samples collected from subjects administered three doses of BNT162b2 and who showed no evidence of subsequent SARS-CoV-2 infection. BNT162b2² + Omi corresponds to sera samples collected from patients administered two doses of BNT162b2 and who subsequenctly experienced a breakthrough SARS-CoV-2 infection at a time of high Omicron BA.l prevalence. BNT162b2³ + Omi corresponds to sera samples collected from patients administered three doses of BNT162b2 and who subsequenctly experineced a breakthrough infection at a time of high Omicron BA.l prevalence. Blood drawn 1 month after last vaccination (SARS-CoV-2 naive) or infection (BA.l breakthrough). Data also shown and described in Quandt and Muik et al., 2022, the contents of which are hereby incorporated by reference in their entirety.

[0220] Fig. 5. Immune imprinting can interfere with generation of a de novo response, resulting in poor cross-neutralization of new infectious agent variants. SARS-CoV-2 was chosen as an exemplary infectious agent. One of skill in the art will recognize that the data establishes that immune imprinting can interfere with the generation of effective immune responses in general, and in particular, for infectious diseases having a high concentration of mutations in neutralization sensitive regions of antigens. Sera samples were collected from subjects (i) administered three doses of BNT162b2 (“BNT162b2³”) and showing no evidence of prior SARS-CoV-2 infection, (ii) administered four doses of BNT162b2 (“BNT162b2⁴”) and showing no evidence of prior SARS-CoV-2 infection, (iii) administered three doses of an RNA vaccine and who experienced a subsequent Omicron BA.l breakthrough infection (“mRNA-Vax3 + BA.1”), (iv) administered three doses of an RNA vaccine and who experienced a subsequent Omicron BA.2 breakthrough infection (“mRNA-Vax³ + BA.2”), or (v) administered three doses of an RNA vaccine and who experienced a subsequent Omicron BA.4/5 breakthrough infection (“mRNA-Vax³ + BA.4/5”). (A) Shows pseudovirus neutralization titers. As shown in the figure, sera from mRNA-Vax experienced individuals with BA.l, BA.2 or BA.4/5 breakthrough infection showed limited neutralizing activity against the current, most immune-escaping variants like XBB (highlighted in red boxes). (B) Shows percent conservation of HLA class I and class II T-cell epitopes and neutralizing B-cell epitopes for a number of SARS-CoV-2 variants. XBB displayed the lowest conservation of neutralizing B-cell epitopes across VOCs characterized. Data reproduced from Muik, Alexander, et al. "Progressive loss of conserved spike protein neutralizing antibody sites in Omicron sublineages is balanced by preserved T-cell recognition epitopes." bioRxiv (2022): 2022-12, the contents of which are incorporated by reference herein in their entirety

[0221] Fig. 6. Immune imprinting may not be effectively overcome by repeated exposures to an infectious disease variant. Data is shown from subjects exposed to an Omicron BA.l variant. Sera samples were depleted using the indicated bait protein (e.g., using an assay similar to that depicted in Fig.2(B)), and then screened in a pseudovirus neutralization assay comprising a Wuhan Spike protein (Wuhan-pVNT) or an Omicron BA.1 Spike protein (Omicron BA.l-pVNT). Of the 13 individuals screened, only one showed an Omicron BA.l -specific neutralization response (indicated in red).

[0222] Fig. 7. Imprinting limits build-up of unique epitope-specific B cell memory even after two subsequent exposures to a variant of an infectious agent (Omicron BA.l). Sera samples from subjects administered a booster dose of an RNA vaccine encoding a Omicron BA.l S protein (Omi BA.l Booster), an RNA vaccine encoding a SARS-CoV-2 Wuhan strain (BNT162b2 Booster), or no booster were collected, memory B cells isolated, and analyzed via depletion assays. Sera samples were collected on the day a booster dose was administered (VI), 7 days after a booster dose was administered (V2), and 1 month after a booster dose (V3). Memory B cells (BMEM) were stained for Spike binding, RBD binding, or NTD (N terminal domain) binding for each of Wuhan and Omicron BA.l. Indicated are the percent of screened B cells positive for the indicated probe. BMEM cells binding BA.l specific epitopes in the RBD were not observed. A small population of BMEM cells specific to the BA.1 NTD were observed 1 week after administering an Omicron BA.l -adapted booster. The slight increase of full-length BA.l Spike binding BMEM cells in the BA.l adapted vaccine group most likely represents NTD- binders.

[0223] Fig. 8. Exemplary strategies of addressing immune imprinting. An exemplary approach is to deliver a hypervariable domain of an antigen in an vaccine without other portions of the antigen that contain a large number of non-neutralization epitopes that are shared with a prior-exposure antigen. Shown are novel antigen designs (comprising the SI and RBD- subdomains of a SARS-CoV-2 S protein) for imprint-resistant SARS-CoV-2 vaccines. Mutation density in new SARS-CoV-2 variants of concern (e.g., XBB) is highest in the Sl-fragment and especially in the RBD. Hence, omitting the highly conserved S2 fragment can result in more efficient priming (e.g., by removing conserved epitopes that can activate BMEM cells and/or prevent activation of naive B cells). Shown are certain exemplary antigen designs, including (1) an RBD of an VOC attached to a trimerization domain (e.g., an RBD of XBB.1.5 attached to a T4 foldon domain), (2) an SI domain of an VOC attached to a trimerization domain (e.g., an SI of XBB.1.5 attached to a T4 foldon domain), (3) an RBD of an VOC attached to a trimerization domain and a transmembrane (TM) domain (e.g., an RBD of XBB.1.5 attached to a T4 foldon domain and a TM domain of a SARS-CoV-2 S protein), and (4) an SI domain of an VOC attached to a trimerization domain and a transmembrane domain (e.g., an SI of XBB.1.5 attached to a T4 foldon domain and a TM domain of a SARS-CoV-2 S protein). Constructs (1) and (2) are soluble and secreted, whereas constructs (3) and (4) are TM-anchored. Similar strategies can be used to design imprint-resistant vaccines against other infectious diseases (in particular, diseases caused by infectious agents that comprise regions neutralization-sensitive regions with high rates of mutation).

[0224] Fig. 9. Immunogenicity study in vaccine-experienced mice. Mice are administered two doses of BNT162b2 (encoding an S protein of a Wuhan variant), or a composition comprising a first RNA that encodes a SARS-CoV-2 S protein of a Wuhan variant and a second RNA encoding a full length S protein of an Omicron BA.4/5 variant (Bivalent b2 + BA.4/5), followed by a third and fourth dose of a candidate vaccine. Third and fourth doses include RNA encoding full length Spike protein of a Wuhan strain (BNT162b2); RNA encoding a full length S protein of an XBB.1.5 variant (BNT162b2 (XBB.1.5)); RNA encoding an RBD of an XBB.1.5 S protein comprising a secretory signal and a timerization domain (RBD (XBB.1.5)); RNA encoding an SI domain of an XBB.1.5 S protein comprising a timerization domain (SI (XBB.1.5)); RNA encoding an SI domain of an XBB.1.5 S protein comprising a timerization domain and a transmembrane domain (Sl-TM (XBB.1.5)); and RNA encoding an RBD of an XBB.1.5 S protein comprising a secretory signal, a timerization domain, and a transmembrane domain (RBD-TM (XBB.1.5)). The third dose includes one of the vaccine candidate disclosed in Example 2 (Table 16). Yellow-filled cells indicate days on which sera sample will be collected, gray-filled cells indicate days on which vaccines will be administered, and green-filled cells indicate days on which mice are sacrificed and final samples collected. [0225] Fig. 10. Immunogenicity study in vaccine-experienced mice - sample characterization. Summary of spleen sample, lymph nodes are collected and analyzed as shown in the Figure. Figure also summarizes analysis of blood samples collected throughout the study.

[0226] Fig. 11. Immunogenicity study in vaccine-naive mice. Mice are administered two doses of RNA encoding (i) a full length Spike protein of a Wuhan strain (BNT162b2); (ii) RNA encoding a full length S protein of an XBB.1.5 variant (BNT162b2 (XBB.1.5)); (iii) RNA encoding a full length S protein of an XBB.1.5 variant and comprising a 19 amino acid C- terminal truncation (BNT162b2 (XBB.1.5) Cdl9); (iv) RNA encoding an RBD of an XBB.1.5 S protein comprising a secretory signal (SP19) and a timerization domain (RBD (XBB.1.5) (SP19)); (v) RNA encoding an SI domain of an XBB.1.5 S protein comprising a trimerization domain (SI (XBB.1.5)); (vi) RNA encoding an RBD of an XBB.1.5 S protein comprising a secretory signal (SP19), a timerization domain, and a transmembrane domain (RBD-TM (XBB.1.5) (SP19)); (vii) RNA encoding an SI domain of an XBB.1.5 S protein comprising a timerization domain and a transmembrane domain (Sl-TM (XBB.1.5); and (viii) RNA encoding an RBD of an XBB.1.5 S protein comprising a secretory signal (SP16) and a timerization domain (RBD (XBB.1.5) (SP16)). Yellow-filled cells indicate days on which sera sample will be collected, gray-filled cells indicate days on which vaccines will be administered, and green-filled cells indicate days on which mice are sacrificed and final samples collected.

[0227] Fig. 12. Immunogenicity study in vaccine-naive mice - sample characterization. On the final day study day, spleen samples are collected and analyzed as shown in the figure.

[0228] Fig. 13. Design of an experiment to test immunogenecity of vaccine candidates in vaccine-experienced mice. Top row lists number of mice in each group ("size"), and days on which vaccines were administered (days 0, 21, 126, and 238 post dose 1), and samples were collected (days 0, 21, 35, 63, 91, 119. 126, 133, 154, 182, 210, 238, 245, 259, and 273. Subsequence rows indicate vaccines administered, where doses 1, 2, 3, and 4 are listed from left to right. Mice were split into 12 groups, 6 of which were administered a first dose and a second dose of a monovalent vaccine comprising RNA encoding a full-length S protein of a Wuhan strain ("BNT162b2"), and 6 of which were administered two doses of a bivalent vaccine comprising (i) an RNA encoding a SARS-CoV-2 S protein of a Wuhan strain and (ii) an RNA encoding a SARS-CoV-2 S protein of an Omicron BA.4/5 variant ("Bivalent b2 + BA.4/5"). For all groups, the second dose was administered about 21 days after the first dose. Each group was administered a third dose and a fourth dose of a vaccine candidate. "BNT162b2" refers to a vaccine comprising RNA that encodes a full length S protein; "Bl RBD" refers to a vaccine comprising an RNA that encodes a soluble RBD (does not comprise a transmembrane domain); "B3-RBD-TM" refers to a vaccine comprising an RNA encoding a membrane-anchored RBD (comprises a transmembrane domain); "Bl -like SI" refers a vaccine comprising RNA encoding a soluble SI domain; "B3-like Sl-TM" refers to a vaccine comprising an RNA encoding a membrane- anchored SI domain; "T cell string" refers to RNA encoding T cell epitopes of a SARS-CoV-2 virus; "uRNA" refers to unmodified RNA (comprising unmodified nucleotides, aside from the 5' cap) and "modRNA" refers to RNA comprising modified uridines. If a group lists "XBB.1.5" the RBD, SI, or S protein comprises mutations characteristics of an XBB.1.5 variant; otherwise, encoded amino acid sequence corresponds to that of the original Wuhan strain.

[0229] Fig. 14. Neutralization titers prior to administering vaccine candidates. Shown are geometric mean neutralization titers in mice at day 63 (panel (A)) and 91 (panel (B)) post dose 1, where the mice were vaccinated per the protocol summarized in Fig. 10, and described in Example 4. Shown immediately below the x-axis of each plot is the strain against which neutralization titers were collected. Also below the strains against which neutralization titers were collected, and below the line, is the vaccines administered. "BNT162b2²" refers to mice administered two doses of BNT162b2. "(Bivalent b2+BA.4/5)²" refers to mice administered two doses of a bivalent compisition comprising (i) RNA encoding a SARS-CoV-2 S protein of a Wuhan strain, and (ii) RNA encoding a SARS-CoV-2 S protein of an Omicron BA.4/5 variant. Indicated above each bar is the geometric mean of the neutralizing titers. "d42PD2" stands for 42 days post dose 2, and "d70PD2" stands for 70 days post-dose 2. "EEOD" stands for Eower Eimit of Detection. As shown in the Figure, mice administered two doses of a bivalent vaccine exhibited much higher neutralizing titers against SARS-CoV-2 variants than mice administered two doses of a monovalent composition delivering a full length S protein of a Wuhan strain. (C) Neutralization titers collected in an experiment in which mice were administered a first dose of a BNT162b2, and a second dose of a bivalent vaccine comprising RNA encoding an S protein of a Wuhan strain and RNA encoding an S protein of an Omicron BA.4/5 variant. As shown if the figure, and in contrast to (B), neutralization titers were much lower following dose 2, indicating that two doses of a bivalent vaccine induce higher neutralization titers as compared to a mixed dosing regimen in vaccine naive mice.

[0230] Fig. 15. Neutralization titers induced by vaccine candidates in vaccine- experienced mice. Vaccine candidate abbreviations are the same as those used in Fig. 10, except "B3-like" is used in place of "B3-like Sl-TM" and "B3-RBD" is used in place of "B3-RBD-TM". Shown are neutralization titers in mice administered BNT162b2 as a first and second dose. (A) shows geometric mean neutralization titers against an XBB.1.5-adapted pseudovirus, in mice administered the vaccine canididates indicated in the table, at the time points indicated in the table. Timing corresponds to that shown in Fig. 10. (B) is a plot of the data shown in (A). (C) shows the geometric fold increase for neutralization titers at day 154 vs day 126. (D) provides a bar chart summarizing the values provided in (C). (E) shows geometric mean neutralization titers against a Wuhan-adapted pseudovirus, in mice administered the vaccines indicated in each column, at the time points indicated in the table. (F) provides a plot of the values shown in (E). As shown in the figure, membrane-anchored RBD provided the highest neutralization titers after a single boost, with neturalization titers increased by about 64-fold on day 238 as compared to day 126. In general, shorter constructs delivering only the RBD (either membrane anchored or soluble) were more efficient in generating higher neutralization titers as compared to other constructs.

[0231] Figure 16: Representative FACS Data. Shown is representative FACS data, obtained using baits comprising (i) the full length S protein of the Wuhan strain, and (ii) the full length S protein of the XBB.1.5 SARS-CoV-2 variant attached to different fluorescent labels. Each point in the plot corresponds to a B cell. Indicated to the left of each row is the vaccine candidate administered. Each plot corresponds to cells obtained from individual mice. Each point in a plot corresponds to a B cell. Cells were classified into two groups: (i) Wuhan binders (Wuhan signal above background signal) and (ii) XBB.1.5 binders (XBB.1.5 signal above background signal). Wuhan-binders (black) and XBB.1.5 binders were overlayed on top of the entire B cell population (light grey). An increase in the number of cells located on the y-axis of each (medium grey events, not on the diagonal) indicating XBB.1.5-specific binding represents an improved naive response. [0232] Figure 17: Transmembrane-anchored RBD induces a more-consistent immune response against SARS-CoV-2 variants in vaccine-experienced mice. Shown is a cumulative analysis of FACS data which was representatively show cased in Fig. 13. Spike specific B cell were categorized via a Boolean Gating approach into (i) Wuhan-specific, (ii) shared (binding both XBB.1.5 and Wuhan S protein), and (iii) XBB.1.5-specific B cells. Shown along the x-axis is the vaccine candidate administered. Shown along the y-axis is the percentage of CD19⁺ cells positive for being labeled with the indicated S protein. As shown in the figure, constructs delivering a membrane anchored RBD (“b3”) provided the most consistent induction of B cell immune response, as indicated by the significantly reduced intragroup variability in neutralization titers.

[0233] Figure 18: Activation of memory B cells in vaccinated mice. B cells separated according to their ability to bind different S proteins were stained for CD95 (CD95⁺ indicates B cell activation, serving as a proxy to show that they recently undergo Germinal Center (GM) reaction).

[0234] Figure 19: Example of an experimental protocol for sequencing individual B cells collected from splenocytes. As shown in the figure, splenocytes were harvested from each mouse at the end of the experiment, B -cells were isolated and blocked, and then all B cells were pooled and sorted by FACS using fluorescently labeled full length S protein (Wuhan or XBB.1.5, representative FACS plots for sorting are shown). After sorting cells were subjected to single cell BCR sequencing employing the 10X Genomics technology.

[0235] Figure 20: Sequencing summary statistics. Shown are summary statistics collected using the experimental protocol depicted in Figure 16. As shown in the figure, sequences of VH and VL regions were obtained, as well as a number of clonotypes for each sorted group.

[0236] Figure 21. Fraction and number of CD19⁺ B cells binding a Wuhan S protein, an XBB.1.5 S protein, or both. (A) shows the number of CD19⁺ B cells, and (B) shows the number of clonotypes of CD19⁺ B cells that bind Wuhan Spike, XBB.1.5 Spike, or both. As shown in the figure, constructs encoding an RBD of an S protein produced the highest number of cells and clonotypes that are specific to XBB.1.5, which membrane anchored RBD in particular producing the highest numbers. [0237] Figure 22. Ig isotypes in B cells with different binding specificities. As shown in the figure, following vaccination with different vaccine candidates, no significant difference was seen between them in the relative proportion of different Ig isotypes between B cells displaying different Ig isotypes.

[0238] Figure 23. Comparison of clonotypes across cohorts. Clonotypes that were shared in at least two cohorts were identified and compared across cohorts to determine whether certain clonotypes were characteristic of an individual construct design (e.g., if particular clonotypes were associated with RBD or full-length S protein constructs). B cell responses were found to be mostly private. Any shared clonotypes detected appeared to occur due to background binding, rather than S protein specific binding.

Certain Definitions

[0239] In general, terminology used herein is in accordance with its understood meaning in the art, unless clearly indicated otherwise. Explicit definitions of certain terms are provided below; meanings of these and other terms in particular instances throughout this specification will be clear to those skilled in the art from context.

[0240] In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0241] About. The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0242] Agent'. As used herein, the term “agent”, may refer to a physical entity or phenomenon. In some embodiments, an agent may be characterized by a particular feature and/or effect. In some embodiments, an agent may be a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof. In some embodiments, the term “agent” may refer to a compound, molecule, or entity that comprises a polymer. In some embodiments, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound, molecule, or entity that is substantially free of a particular polymer or polymeric moiety. In some embodiments, the term may refer to a compound, molecule, or entity that lacks or is substantially free of any polymer or polymeric moiety.

[0243] Amino acid: In its broadest sense, as used herein, the term “amino acid” refers to a compound and/or substance that can be, is, or has been incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally- occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L- amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and/or amino- terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and/or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.

[0244] Antibody agent. As used herein, the term “antibody agent” refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses a polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding. For example, in some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some embodiments an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and/or at least one light chain CDR) that is substantially identical to one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1 -5 amino acid substitutions as compared with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1 -5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody agent in or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art to correspond to CDRsl, 2, and 3 of an antibody variable domain; in some such embodiments, an antibody agent in or comprises a polypeptide or set of polypeptides whose amino acid sequence(s) together include structural elements recognized by those skilled in the art to correspond to both heavy chain and light chain variable region CDRs, e.g., heavy chain CDRs 1, 2, and/or 3 and light chain CDRs 1, 2, and/or 3. In some embodiments, an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain. In some embodiments, an antibody agent may be or comprise a polyclonal antibody preparation. In some embodiments, an antibody agent may be or comprise a monoclonal antibody preparation. In some embodiments, an antibody agent may include one or more constant region sequences that are characteristic of a particular organism, such as a camel, human, mouse, primate, rabbit, rat; in many embodiments, an antibody agent may include one or more constant region sequences that are characteristic of a human. In some embodiments, an antibody agent may include one or more sequence elements that would be recognized by one skilled in the art as a humanized sequence, a primatized sequence, a chimeric sequence, etc. In some embodiments, an antibody agent may be a canonical antibody (e.g., may comprise two heavy chains and two light chains). In some embodiments, an antibody agent may be in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide- Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPs™ ); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®;

Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group (e.g., poly-ethylene glycol, etc.)).

[0245] Antigen-. Those skilled in the art, reading the present specification, will appreciate that the term “antigen” refers to a molecule that is recognized by the immune system, e.g., in particular embodiments, the adaptive immune system, such that it elicits an antigen- specific immune response. In some embodiments, an antigen-specific immune response may be or comprise generation of antibodies and/or antigen-specific T cells. In some embodiments, an antigen is a peptide or polypeptide that comprises at least one epitope against which an immune response can be generated. In one embodiment, an antigen is presented by cells of the immune system such as antigen presenting cells like dendritic cells or macrophages. In one embodiments, an antigen or a processed product thereof such as a T-cell antigen is bound by a T- or B-cell receptor, or by an immunoglobulin molecule such as an antibody. Accordingly, an antigen or a processed product thereof may react specifically with antibodies or T lymphocytes (T cells). In one embodiment, an antigen is a parasitic antigen. In accordance with the present disclosure, in some embodiments, an antigen may be delivered by RNA molecules as described herein. In some embodiments, a peptide or polypeptide antigen can be 2-100 amino acids, including for example, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids in length. In some embodiments, a peptide or polypeptide antigen can be greater than 50 amino acids. In some embodiments, a peptide or polypeptide antigen can be greater than 100 amino acids. In some embodiments, an antigen is recognized by an immune effector cell. In some embodiments, an antigen, if recognized by an immune effector cell, is able to induce in the presence of appropriate co- stimulatory signals, stimulation, priming and/or expansion of the immune effector cell carrying an antigen receptor recognizing the antigen. In the context of the embodiments of the present disclosure, in some embodiments, an antigen can be presented or present on the surface of a cell, e.g., an antigen presenting cell. In one embodiment, an antigen is presented by a diseased cell such as a virus-infected cell. In one embodiment, an antigen receptor is a TCR which binds to an epitope of an antigen presented in the context of MHC. In one embodiment, binding of a TCR when expressed by T cells and/or present on T cells to an antigen presented by cells such as antigen presenting cells results in stimulation, priming and/or expansion of said T cells. In one embodiment, binding of a TCR when expressed by T cells and/or present on T cells to an antigen presented on diseased cells results in cytolysis and/or apoptosis of the diseased cells, wherein said T cells preferably release cytotoxic factors, e.g. perforins and granzymes.

[0246] Associated. Two events or entities are “associated” with one another, as that term is used herein, if the presence, level, degree, type and/or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and/or form correlates with incidence of, susceptibility to, severity of, stage of, etc. the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0247] Binding-. Those skilled in the art, reading the present specification, will appreciate that the term “binding” typically refers to a non-covalent association between or among entities or moieties. In some embodiments, binding data are expressed in terms of “IC50”. As is understood in the art, IC50 is the concentration of an assessed agent in a binding assay at which 50% inhibition of binding of reference agent known to bind the relevant binding partner is observed. In some embodiments, assays are run under conditions in which (e.g., limiting binding target and reference concentrations), IC50 values approximate KD values. Assays for determining binding are well known in the art and are described in detail, for example, in PCT publications WO 94/20127 and WO 94/03205, and other publications such Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); and Sette, et al., Mol. Immunol. 31:813 (1994). Alternatively, binding can be expressed relative to binding by a reference standard peptide. For example, can be based on its IC50, relative to the IC50 of a reference standard peptide. Binding can also be determined using other assay systems including those using: live cells (e.g., Ceppellini et al., Nature 339:392 (1989); Christnick et al., Nature 352:67 (1991); Busch et al., Int. Immunol. 2:443 (1990); Hill et al., J. Immunol. 147: 189 (1991); del Guercio et al., J. Immunol. 154:685 (1995)), cell free systems using detergent lysates (e.g., Cerundolo et al., J. Immunol 21:2069 (1991)), immobilized purified MHC (e.g., Hill et al., J. Immunol. 152, 2890 (1994); Marshall et al., J. Immunol. 152:4946 (1994)), ELISA systems (e.g., Reay et al., EMBO J. 11:2829 (1992)), surface plasmon resonance (e.g., Khilko et al., J. Biol.

Chem. 268: 15425 (1993)); high flux soluble phase assays (Hammer et al., J. Exp. Med. 180:2353 (1994)), and measurement of class I MHC stabilization or assembly (e.g., Ljunggren et al., Nature 346:476 (1990); Schumacher et al., Cell 62:563 (1990); Townsend et al., Cell 62:285 (1990); Parker et al., J. Immunol. 149:1896 (1992)).

[0248] Cap. As used herein, the term “cap” refers to a structure comprising or essentially consisting of a nucleoside-5 '-triphosphate that is typically joined to a 5'-end of an uncapped RNA (e.g., an uncapped RNA having a 5'- diphosphate). In some embodiments, a cap is or comprises a guanine nucleotide. In some embodiments, a cap is or comprises a naturally- occurring RNA 5’ cap, including, e.g., but not limited to a 7- methylguanosine cap, which has a structure designated as “m7G.” In some embodiments, a cap is or comprises a synthetic cap analog that resembles an RNA cap structure and possesses the ability to stabilize RNA if attached thereto, including, e.g., but not limited to anti -reverse cap analogs (ARC As) known in the art). Those skilled in the art will appreciate that methods for joining a cap to a 5’ end of an RNA are known in the art. For example, in some embodiments, a capped RNA may be obtained by in vitro capping of RNA that has a 5' triphosphate group or RNA that has a 5' diphosphate group with a capping enzyme system (including, e.g., but not limited to vaccinia capping enzyme system or Saccharomyces cerevisiae capping enzyme system). Alternatively, a capped RNA can be obtained by in vitro transcription (IVT) of a single-stranded DNA template in the presence of a dinucleotide or trinucleotide cap analog.

[0249] Cell-mediated, immunity. “Cell-mediated immunity,” “cellular immunity,” “cellular immune response,” or similar terms are meant to include a cellular response directed to cells characterized by expression of an antigen, in particular characterized by presentation of an antigen with class I or class II MHC. A cellular response relates to immune effector cells, in particular to T cells or T lymphocytes which act as either “helpers” or “killers.” The helper T cells (also termed CD4⁺ T cells or CD4 T cells) play a central role by regulating the immune response and the killer cells (also termed cytotoxic T cells, cytolytic T cells, CD8⁺ T cells, CD8 T cells, or CTLs) kill diseased cells such as virus -infected cells, preventing the production of more diseased cells.

[0250] Co-administration. As used herein, the term “co-administration” refers to use of a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) described herein and an additional therapeutic agent. The combined use of a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) described herein and an additional therapeutic agent may be performed concurrently or separately (e.g., sequentially in any order). In some embodiments, a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) described herein and an additional therapeutic agent may be combined in one pharmaceutically- acceptable carrier, or they may be placed in separate carriers and delivered to a target cell or administered to a subject at different times. Each of these situations is contemplated as falling within the meaning of “co-administration” or “combination,” provided that a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) described herein and an additional therapeutic agent are delivered or administered sufficiently close in time that there is at least some temporal overlap in biological effect(s) generated by each on a target cell or a subject being treated.

[0251] Codon-optimized.'. As used herein, the term “codon-optimized” refers to alteration of codons in a coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism without preferably altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, in some embodiments coding regions are codon-optimized for optimal expression in a subject to be treated using the RNA molecules described herein. In some embodiments, codon-optimization may be performed such that codons for which frequently occurring tRNAs are available are inserted in place of “rare codons.” In some embodiments, codon-optimization may include increasing guanosine/cytosine (G/C) content of a coding region of RNA described herein as compared to the G/C content of the corresponding coding sequence of a wild type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence.

[0252] Conserved Epitope: As used herein, a “conserved epitope” refers to an epitope that is retained in a variant polypeptide relative to a reference polypeptide. An epitope can be determined and/or inferred using methods that are well known in the art, including, e.g., antibody binding studies, B cell binding studies, structural analysis, and infection rates, among others.

[0253] Combination therapy. As used herein, the term “combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, “administration” of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition.

[0254] Comparable: As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0255] Corresponding to : As used herein, the term “corresponding to” refers to a relationship between two or more entities. For example, the term “corresponding to” may be used to designate the position/identity of a structural element in a compound or composition relative to another compound or composition (e.g., to an appropriate reference compound or composition). For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, those of ordinary skill will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid “corresponding to” a residue at position 190, for example, need not actually be the 190^th amino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify “corresponding” amino acids. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, HHpred/HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHLLS, SWIMM, or SWIPE that can be utilized, for example, to identify “corresponding” residues in polypeptides and/or nucleic acids in accordance with the present disclosure. Those of skill in the art will also appreciate that, in some instances, the term “corresponding to” may be used to describe an event or entity that shares a relevant similarity with another event or entity (e.g., an appropriate reference event or entity). To give but one example, a gene or protein in one organism may be described as “corresponding to” a gene or protein from another organism in order to indicate, in some embodiments, that it plays an analogous role or performs an analogous function and/or that it shows a particular degree of sequence identity or homology, or shares a particular characteristic sequence element.

[0256] Derived. '. In the context of an amino acid sequence (peptide or polypeptide)

“derived from” a designated amino acid sequence (peptide or polypeptide), refers to a structural analogue of a designated amino acid sequence. In some embodiments, an amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof. Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof. For example, it will be understood by one of ordinary skill in the art that the antigens suitable for use herein may be altered such that they vary in sequence from the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences.

[0257] Designed: As used herein, the term “designed” refers to an agent (i) whose structure is or was selected by the hand of man; (ii) that is produced by a process requiring the hand of man; and/or (iii) that is distinct from natural substances and other known agents. [0258] Dosing regimen -. Those skilled in the art will appreciate that the term “dosing regimen” may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (z.e., is a therapeutic dosing regimen).

[0259] Encode. As used herein, the term “encode” or “encoding” refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e.g., mRNA) or a defined sequence of amino acids. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a gene, a cDNA, or an RNA molecule (e.g., an mRNA) encodes a polypeptide if transcription and translation of mRNA corresponding to that gene produces the polypeptide in a cell or other biological system. In some embodiments, a coding region of an RNA molecule encoding a target antigen refers to a coding strand, the nucleotide sequence of which is identical to the mRNA sequence of such a target antigen. In some embodiments, a coding region of an RNA molecule encoding a target antigen refers to a non-coding strand of such a target antigen, which may be used as a template for transcription of a gene or cDNA.

[0260] Engineered: In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and/or when a particular residue in a polynucleotide is non-naturally occurring and/or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature.

[0261] Epitope. As used herein, the term “epitope” refers to a moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. For example, an epitope may be recognized by a T cell, a B cell, or an antibody. In some embodiments, an epitope is comprised of a plurality of chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen adopts a relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically near to each other in space when the antigen adopts such a conformation. In some embodiments, at least some such chemical atoms are groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized). Accordingly, in some embodiments, an epitope of an antigen may include a continuous or discontinuous fragment of the antigen. In some embodiments, an epitope is or comprises a T cell epitope. In some embodiments, an epitope may have a length of about 5 to about 30 amino acids, or about 10 to about 25 amino acids, or about 5 to about 15 amino acids, or about 5 to 12 amino acids, or about 6 to about 9 amino acids.

[0262] Expression: As used herein, the term “expression” of a nucleic acid sequence refers to the generation of a gene product from the nucleic acid sequence. In some embodiments, a gene product can be a transcript. In some embodiments, a gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.

[0263] Five prime untranslated region . As used herein, the terms “five prime untranslated region” or “5' UTR” refer to a sequence of an mRNA molecule between a transcription start site and a start codon of a coding region of an RNA. In some embodiments, “5’ UTR” refers to a sequence of an mRNA molecule that begins at a transcription start site and ends one nucleotide (nt) before a start codon (usually AUG) of a coding region of an RNA molecule, e.g., in its natural context.

[0264] Fragment. The term “fragment” as used herein in the context of a nucleic acid sequence (e.g. RNA sequence) or an amino acid sequence may typically be a fragment of a reference sequence. In some embodiments, a reference sequence is a full-length sequence of e.g. a nucleic acid sequence or an amino acid sequence. Accordingly, a fragment, typically, refers to a sequence that is identical to a corresponding stretch within a reference sequence. In some embodiments, a fragment comprises a continuous stretch of nucleotides or amino acid residues that corresponds to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the total length of a reference sequence from which the fragment is derived. In some embodiments, the term “fragment", with reference to an amino acid sequence (peptide or polypeptide), relates to a part of an amino acid sequence, e.g., a sequence which represents the amino acid sequence shortened at the N-terminus and/or C-terminus. In some embodiments, a fragment of an amino acid sequence comprises at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from an amino acid sequence.

[0265] Homology: As used herein, the term “homology” or “homolog” refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or polypeptide molecules are considered to be “homologous” to one another if their sequences are at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or polypeptide molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions). For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as similar to one another as “hydrophobic” or “hydrophilic” amino acids, and/or as having “polar” or “non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a “homologous” substitution. [0266] Humoral immunity: As used herein, the term “humoral immunity” or “humoral immune response” refers to antibody production and the accessory processes that accompany it, including: Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell generation. It also refers to the effector functions of antibodies, which include pathogen neutralization, classical complement activation, and opsonin promotion of phagocytosis and pathogen elimination.

[0267] Identity: As used herein, the term “identity” refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller, 1989, which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.

[0268] Immunologically equivalent: The term “immunologically equivalent” means that an immunologically equivalent molecule such as the immunologically equivalent amino acid sequence exhibits the same or essentially the same immunological properties and/or exerts the same or essentially the same immunological effects, e.g., with respect to the type of the immunological effect. In the context of the present disclosure, in some embodiments, the term “immunologically equivalent” is used with respect to the immunological effects or properties of antigens or antigen variants used for immunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if said amino acid sequence when exposed to the immune system of a subject induces an immune reaction having a specificity of reacting with the reference amino acid sequence.

[0269] In one embodiment, an antigen receptor is an antibody or B cell receptor which binds to an epitope of an antigen. In one embodiment, an antibody or B cell receptor binds to native epitopes of an antigen.

[0270] As used herein, “immune escaping” refers to a variant or strain of an infectious agent that can fully or partially evade an immune response (e.g., a B cell immune response).

[0271] As used here, “immune escape potential” refers to a likelihood of a given variant being able to evade previously developed immune responses. A skilled artisan is aware of various methods for assessing the immune escape potential of a given infectious agent. For example, in some embodiments, immune escape potential can be determined experimentally based on infection rates in a relevant population (e.g., infection rates in subjects previously infected and/or vaccinated with a previous variant of the infectious agent). In some embodiments, an immune escape potential can be determined using one or more in vitro assay(s) (e.g., neutralization assays as described herein). In some embodiments, an immune escape potential can be predicted using the sequence of the variant (e.g., predicted by in silico analysis, location of epitopes relative to previously determined neutralization epitopes, etc.).

[0272] Increased, Induced, or Reduced: As used herein, these terms or grammatically comparable comparative terms, indicate values that are relative to a comparable reference measurement. For example, in some embodiments, an assessed value achieved with a provided pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) may be “increased” relative to that obtained with a comparable reference pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine). Alternatively or additionally, in some embodiments, an assessed value achieved in a subject may be “increased” relative to that obtained in the same subject under different conditions (e.g., prior to or after an event; or presence or absence of an event such as administration of a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) as described herein, or in a different, comparable subject (e.g., in a comparable subject that differs from the subject of interest in prior exposure to a condition, e.g., absence of administration of a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) as described herein.). In some embodiments, comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and/or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and/or prevalence of difference that is required or sufficient to achieve such statistical significance. In some embodiments, the term “reduced” or equivalent terms refers to a reduction in the level of an assessed value by at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or higher, as compared to a comparable reference. In some embodiments, the term “reduced” or equivalent terms refers to a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero. In some embodiments, the term “increased” or “induced” refers to an increase in the level of an assessed value by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or higher, as compared to a comparable reference.

[0273] Ionizable The term “ionizable” refers to a compound or group or atom that is charged at a certain pH. In the context of an ionizable amino lipid, such a lipid or a function group or atom thereof bears a positive charge at a certain pH. In some embodiments, an ionizable amino lipid is positively charged at an acidic pH. In some embodiments, an ionizable amino lipid is predominately neutral at physiological pH values, e.g., in some embodiments about 7.0-7.4, but becomes positively charged at lower pH values. In some embodiments, an ionizable amino lipid may have a pKa within a range of about 5 to about 7.

[0274] Isolated: The term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated”, but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated”. An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0275] Lipid. As used herein, the terms “lipid” and “lipid-like material” are broadly defined as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also typically denoted as amphiphiles.

[0276] Modified: As used herein, the term “modified”, in the context of an amino acid or nucleotide sequence, refers to a change relative to a reference sequence. In some embodiments, a modified amino acid or nucleotide sequence comprises a deletion (e.g., a deletion of a single residue, a short stretch of residues (e.g., 1 to 10 residues), a particular region (e.g., a particular region of a polypeptide, or a nucleotide sequence encoding said region), or a particular domain (e.g., a particular domain of a polypeptide or a nucleotide sequence encoding said domain). In some embodiments, a modified amino acid or nucleotide sequence comprises an insertion (e.g., an insertion of a single residue, a short stretch of residues (e.g., 1 to 10 residues), a particular region (e.g., a particular region of a polypeptide, or a nucleotide sequence encoding said region), or a particular domain (e.g., a particular domain of a polypeptide or a nucleotide sequence encoding said domain). In some embodiments a modified amino acid or nucleotide sequence comprises a substitution.

[0277] RNA lipid nanoparticle . As used herein, the term “RNA lipid nanoparticle” refers to a nanoparticle comprising at least one lipid and RNA molecule(s). In some embodiments, an RNA lipid nanoparticle comprises at least one ionizable amino lipid. In some embodiments, an RNA lipid nanoparticle comprises at least one ionizable amino lipid, at least one helper lipid, and at least one polymer-conjugated lipid (e.g., PEG-conjugated lipid). In various embodiments, RNA lipid nanoparticles as described herein can have an average size (e.g., Z-average) of about 100 nm to 1000 nm, or about 200 nm to 900 nm, or about 200 nm to 800 nm, or about 250 nm to about 700 nm. In some embodiments of the present disclosure, RNA lipid nanoparticles can have a particle size (e.g., Z-average) of about 30 nm to about 200 nm, or about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, an average size of lipid nanoparticles is determined by measuring the particle diameter. In some embodiments, RNA lipid nanoparticles may be prepared by mixing lipids with RNA molecules described herein.

[0278] Lipidoid: As used herein, a “lipidoid” refers to a lipid-like molecule. In some embodiments, a lipoid is an amphiphilic molecule with one or more lipid-like physical properties. In the context of the present disclosure, the term lipid is considered to encompass lipidoids.

[0279] Nanoparticle : As used herein, the term “nanoparticle” refers to a particle having an average size suitable for parenteral administration. In some embodiments, a nanoparticle has a longest dimension (e.g., a diameter) of less than 1,000 nanometers (nm). In some embodiments, a nanoparticle may be characterized by a longest dimension (e.g., a diameter) of less than 300 nm. In some embodiments, a nanoparticle may be characterized by a longest dimension (e.g., a diameter) of less than 100 nm. In many embodiments, a nanoparticle may be characterized by a longest dimension between about 1 nm and about 100 nm, or between about 1 pm and about 500 nm, or between about 1 nm and 1,000 nm. In many embodiments, a population of nanoparticles is characterized by an average size (e.g., longest dimension) that is below about 1,000 nm, about 500 nm, about 100 nm, about 50 nm, about 40 nm, about 30 nm, about 20 nm, or about 10 nm and often above about 1 nm. In many embodiments, a nanoparticle may be substantially spherical so that its longest dimension may be its diameter. In some embodiments, a nanoparticle has a diameter of less than 100 nm as defined by the National Institutes of Health.

[0280] Naturally occurring: The term “naturally occurring” as used herein refers to an entity that can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.

[0281] Neutralization: As used herein, the term “neutralization” refers to an event in which binding agents such as antibodies bind to a biological active site of a virus such as a receptor binding protein, thereby inhibiting the parasitic infection of cells. In some embodiments, the term “neutralization” refers to an event in which binding agents eliminate or significantly reduce ability of infecting cells. [0282] As used herein, a “neutralization epitope” or “neutralization sensitive epitope” refers to an epitope that can be bound by a neutralizing antibody. Neutralization epitopes can be determined using methods that are well known in the art, including, e.g., neutralization assays and antibody binding studies, among other techniques.

[0283] Nucleic acid particle . A “nucleic acid particle” can be used to deliver nucleic acid to a target site of interest (e.g., cell, tissue, organ, and the like). A nucleic acid particle may comprise at least one cationic or cationically ionizable lipid or lipid-like material, at least one cationic polymer such as protamine, or a mixture thereof and nucleic acid. In some embodiments, a nucleic acid particle is a lipid nanoparticle. In some embodiments, a nucleic acid particle is a lipoplex particle.

[0284] Nucleic acid/ Polynucleotide. As used herein, the term “nucleic acid” refers to a polymer of at least 10 nucleotides or more. In some embodiments, a nucleic acid is or comprises DNA. In some embodiments, a nucleic acid is or comprises RNA. In some embodiments, a nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, a nucleic acid is or comprises a single stranded nucleic acid. In some embodiments, a nucleic acid is or comprises a double-stranded nucleic acid. In some embodiments, a nucleic acid comprises both single and double-stranded fragments. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N-phosphoramidite linkages and/or one or more peptide bonds, e.g., as in a “peptide nucleic acid”. In some embodiments, a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxy thymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises on or more, or all, non-natural residues. In some embodiments, a non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 - methyl adenosine, 5 -methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2- aminoadenosine, C5-bromouridine, C5 -fluorouridine, C5 -iodouridine, C5-propynyl-uridine, C5 - propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that comprises one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides long.

[0285] Nucleotide: As used herein, the term “nucleotide” refers to its art-recognized meaning. When a number of nucleotides is used as an indication of size, e.g., of a polynucleotide, a certain number of nucleotides refers to the number of nucleotides on a single strand, e.g., of a polynucleotide.

[0286] Patient: As used herein, the term “patient” refers to any organism who is suffering or at risk of a disease or disorder or condition. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, a patient is a human. In some embodiments, a patient is suffering from or susceptible to one or more diseases or disorders or conditions. In some embodiments, a patient displays one or more symptoms of a disease or disorder or condition. In some embodiments, a patient has been diagnosed with one or more diseases or disorders or conditions. In some embodiments, a disease or disorder or condition that is amenable to provided technologies is or includes a HSV infection. In some embodiments, a patient is receiving or has received certain therapy to diagnose and/or to treat a disease, disorder, or condition. In some embodiments, a patient is a patient suffering from or susceptible to a HSV infection.

[0287] PEG-conjugated lipid. The term “PEG-conjugated lipid" refers to a molecule comprising a lipid portion and a polyethylene glycol portion. [0288] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for parenteral administration, for example, by subcutaneous, intramuscular, or intravenous injection as, for example, a sterile solution or suspension formulation.

[0289] Pharmaceutically effective amount: The term “pharmaceutically effective amount” or “therapeutically effective amount” refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of the treatment of a particular disease, a desired reaction in some embodiments relates to inhibition of the course of the disease. In some embodiments, such inhibition may comprise slowing down the progress of a disease and/or interrupting or reversing the progress of the disease. In some embodiments, a desired reaction in a treatment of a disease may be or comprise delay or prevention of the onset of a disease or a condition. An effective amount of pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) described herein will depend, for example, on a disease or condition to be treated, the severity of such a disease or condition, individual parameters of the patient, including, e.g., age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, doses of pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[0290] Poly(A) sequence: As used herein, the term “poly(A) sequence” or “poly-A tail” refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3 '-end of an RNA molecule. Poly(A) sequences are known to those of skill in the art and may follow the 3’-UTR in the RNAs described herein. An uninterrupted poly(A) sequence is characterized by consecutive adenylate residues. In nature, an uninterrupted poly(A) sequence is typical. RNAs disclosed herein can have a poly(A) sequence attached to the free 3'-end of the RNA by a template-independent RNA polymerase after transcription or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0291] Polypeptide: As used herein, the term “polypeptide” refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L- amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications comprise acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and/or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and/or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and/or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and/or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and/or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide.

[0292] Prevent: As used herein, the term “prevent” or “prevention” when used in connection with the occurrence of a disease, disorder, and/or condition, refers to reducing the risk of developing the disease, disorder and/or condition and/or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time.

[0293] Receptor Binding Domain: As used herein, a “receptor binding domain” (RBD), when used in the context of a generic infectious agent, refers to a region of an infectious agent polypeptide that plays a role in binding a host cell receptor, where the RBD is the region that binds the host cell receptor. In the context of a particular infectious agent, RBD can sometimes refer to a specific region of a protein (e.g., in the context of SARS-CoV-2, RBD refers to a particular region of the S protein).

[0294] Recombinant: The term “recombinant” in the context of the present disclosure means “made through genetic engineering”. In some embodiments, a “recombinant” entity such as a recombinant nucleic acid in the context of the present disclosure is not naturally occurring.

[0295] Reference: As used herein, the term “reference” describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.

[0296] In some embodiments, a reference antigen is an antigen that a subject has previously encountered or has a high likelihood of having previously encountered. For example, in some embodiments, a reference antigen is an antigen delivered by a vaccine that was previously administered to a subject. In some embodiments, a reference antigen is present in a strain or variant of an infectious agent that a subject was previously infected with and/or that was prevalent at a time and region in which the subject was previously infected. In some embodiments, a reference antigen is an antigen of the first strain or variant of an infectious agent that a subject encountered. In some embodiments, a reference antigen is an antigen of a strain or variant of an infectious agent that first became prevalent. In some embodiments, a reference antigen is an antigen delivered by one of the first vaccines that became widely available against a given infectious agent (e.g., for SARS-CoV-2, one of the first commercially approved vaccines delivering a Wuhan Spike protein). In some embodiments, a reference antigen is a Wuhan Spike protein, or an immunogenic portion thereof. In some embodiments, a reference antigen is a Spike protein of an Omicron variant (e.g., a BA.4/5 Omicron variant), or an immunogenic portion thereof.

[0297] Ribonucleic acid (RNA): As used herein, the term “RNA” refers to a polymer of ribonucleotides. In some embodiments, an RNA is single stranded. In some embodiments, an RNA is double stranded. In some embodiments, an RNA comprises both single and double stranded fragments. In some embodiments, an RNA can comprise a backbone structure as described in the definition of “Nucleic acid / Polynucleotide” above. An RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA). In some embodiments where an RNA is a mRNA. In some embodiments where an RNA is a mRNA, a RNA typically comprises at its 3’ end a poly(A) region. In some embodiments where an RNA is a mRNA, an RNA typically comprises at its 5’ end an art-recognized cap structure, e.g., for recognizing and attachment of a mRNA to a ribosome to initiate translation. In some embodiments, a RNA is a synthetic RNA. Synthetic RNAs include RNAs that are synthesized in vitro (e.g., by enzymatic synthesis methods and/or by chemical synthesis methods).

[0298] Ribonucleotide: As used herein, the term “ribonucleotide” encompasses unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides may include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g. , replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, and (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. The term “ribonucleotide” also encompasses ribonucleotide triphosphates including modified and non-modified ribonucleotide triphosphates.

[0299] Risk: As will be understood from context, “risk” of a disease, disorder, and/or condition refers to a likelihood that a particular individual will develop the disease, disorder, and/or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%. In some embodiments risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples. In some embodiments, a reference sample or group of reference samples have a known risk of a disease, disorder, condition and/or event. In some embodiments a reference sample or group of reference samples are from individuals comparable to a particular individual. In some embodiments, relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In some embodiments, risk may reflect one or more genetic attributes, e.g., which may predispose an individual toward development (or not) of a particular disease, disorder and/or condition. In some embodiments, risk may reflect one or more epigenetic events or attributes and/or one or more lifestyle or environmental events or attributes.

[0300] RNA lipoplex particle. As used herein, the term “RNA lipoplex particle” refers to a complex comprising liposomes, in particular cationic liposomes, and RNA molecules. Without wishing to bound by a particular theory, electrostatic interactions between positively charged liposomes and negatively charged RNA results in complexation and spontaneous formation of RNA lipoplex particles. In some embodiments, positively charged liposomes may comprise a cationic lipid, such as in some embodiments DOTMA, and additional lipids, such as in some embodiments DOPE. In one embodiment, a RNA lipoplex particle is a nanoparticle.

[0301] Selective or specific: The term “selective” or “specific”, when used herein in reference to an agent having an activity, is understood by those skilled in the art to mean that the agent discriminates between potential target entities, states, or cells. For example, in some embodiments, an agent is said to bind “specifically” to its target if it binds preferentially with that target in the presence of one or more competing alternative targets. In many embodiments, specific interaction is dependent upon the presence of a particular structural feature of the target entity (e.g., an epitope, a cleft, a binding site). It is to be understood that specificity need not be absolute. In some embodiments, specificity may be evaluated relative to that of a target -binding moiety for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to that of a reference specific binding moiety. In some embodiments, specificity is evaluated relative to that of a reference non-specific binding moiety.

[0302] Stable: As used herein, the term “stable” in the context of the present disclosure refers to a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) as a whole and/or components thereof meeting or exceeding pre-determined acceptance criteria. For example, in some embodiments, a stable pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) exhibits no unacceptable levels of microbial growth, and substantially no or no breakdown or degradation of the active biological molecule component(s). In some embodiments, a stable pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) refers to the integrity of RNA molecules being maintained at least above 90% or more. In some embodiments, a stable pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) refers to at least 90% or more (including, e.g., at least 95%, at least 96%, at least 97%, or more) of RNA molecules being maintained to be encapsulated within lipid nanoparticles. In some embodiments, a stable pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) refers to a formulation that remains capable of eliciting a desired immunologic response when administered to a subject. In some embodiments, a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) remains stable for a specified period of time under certain conditions.

[0303] Subject. As used herein, the term “subject” refers to an organism to be administered with a composition described herein, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans. In some embodiments, a subject is a human subject. In some embodiments, a subject is suffering from a disease, disorder, or condition (e.g., a HSV infection). In some embodiments, a subject is susceptible to a disease, disorder, or condition (e.g., a HSV infection). In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder, or condition (e.g., a HSV infection). In some embodiments, a subject displays one or more non-specific symptoms of a disease, disorder, or condition (e.g., a HSV infection). In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition (e.g., a HSV infection). In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition (e.g., a HSV infection). In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.

[0304] Suffering from. An individual who is “suffering from” a disease, disorder, and/or condition has been diagnosed with and/or displays one or more symptoms of a disease, disorder, and/or condition.

[0305] Susceptible to . An individual who is “susceptible to” a disease, disorder, and/or condition is one who has a higher risk of developing the disease, disorder, and/or condition than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and/or condition may not have been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.

[0306] Synthetic: As used herein, the term “synthetic” refers to an entity that is artificial, or that is made with human intervention, or that results from synthesis rather than naturally occurring. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule that is chemically synthesized, e.g., in some embodiments by solid-phase synthesis. In some embodiments, the term “synthetic” refers to an entity that is made outside of biological cells. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule (e.g., an RNA) that is produced by in vitro transcription using a template.

[0307] Therapy: The term “therapy” refers to an administration or delivery of an agent or intervention that has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect (e.g., has been demonstrated to be statistically likely to have such effect when administered to a relevant population). In some embodiments, a therapeutic agent or therapy is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition. In some embodiments, a therapeutic agent or therapy is a medical intervention (e.g., surgery, radiation, phototherapy) that can be performed to alleviate, relieve, inhibit, present, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.

[0308] Three prime untranslated region : As used herein, the terms “three prime untranslated region” or “3' UTR” refer to a sequence of an mRNA molecule that begins following a stop codon of a coding region of an open reading frame sequence. In some embodiments, the 3' UTR begins immediately after a stop codon of a coding region of an open reading frame sequence, e.g., in its natural context. In other embodiments, the 3' UTR does not begin immediately after stop codon of the coding region of an open reading frame sequence, e.g., in its natural context.

[0309] Threshold level (e.g., acceptance criteria) : As used herein, the term “threshold level” refers to a level that are used as a reference to attain information on and/or classify the results of a measurement, for example, the results of a measurement attained in an assay. For example, in some embodiments, a threshold level means a value measured in an assay that defines the dividing line between two subsets of a population (e.g. a batch that satisfy quality control criteria vs. a batch that does not satisfy quality control criteria). Thus, a value that is equal to or higher than the threshold level defines one subset of the population, and a value that is lower than the threshold level defines the other subset of the population. A threshold level can be determined based on one or more control samples or across a population of control samples. A threshold level can be determined prior to, concurrently with, or after the measurement of interest is taken. In some embodiments, a threshold level can be a range of values.

[0310] Treat: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and/or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject at a later-stage of disease, disorder, and/or condition.

[0311] Vaccination. As used herein, the term “vaccination” refers to the administration of a composition intended to generate an immune response, for example to a disease-associated (e.g., disease-causing) agent. In some embodiments, vaccination can be administered before, during, and/or after exposure to a disease-associated agent, and in certain embodiments, before, during, and/or shortly after exposure to the agent. In some embodiments, vaccination includes multiple administrations, appropriately spaced in time, of a vaccine composition. In some embodiments, vaccination generates an immune response to an infectious agent.

[0312] Vaccine: As used herein, the term “vaccine” refers to a composition that induces an immune response upon administration to a subject. In some embodiments, an induced immune response provides protective immunity.

[0313] Variant: As used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions. Typically, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in a variant are substituted, inserted, or deleted, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues as compared to a reference. Often, a variant polypeptide or nucleic acid comprises a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional residues (i.e., residues that participate in a particular biological activity) relative to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises not more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly fewer than about 5, about 4, about 3, or about 2 additions or deletions as compared to the reference. In some embodiments, a reference polypeptide or nucleic acid is one found in nature.

[0314] Vector, as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” In some embodiments, known techniques may be used, for example, for generation or manipulation of recombinant DNA, for oligonucleotide synthesis, and for tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), which is incorporated herein by reference for any purpose. [0315] All literature and similar material cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, and web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.

Detailed Description

[0316] In some embodiments, the present provides technologies (e.g., compositions, pharmaceutical compositions, immunogenic compositions, vaccines, and methods) that can be used to induce an immune response against an infectious agent. In some embodiments, technologies provided in the present disclosure can be used to mitigate immune imprinting effects and/or induce a stronger de novo immune response (e.g., as compared to other vaccination approaches).

[0317] Infectious agents have evolved various means of evading or subverting host defenses. One way in which an infectious agent can evade immune surveillance is by altering its antigens (e.g., its epitopes); this is particularly important for extracellular pathogens, against which a principal defense is the production of antibody against their surface proteins and/or glyco proteins.

[0318] Among other things, the present disclosure provides technologies that are useful for increasing the breadth of immune response. In some embodiments, such an immune response is or comprises a B cell immune response. In some embodiments, a B cell immune response is or comprises an antibody response (e.g., neutralizing antibody response) to arisen epitopes in variant polypeptides. In some embodiments, variant polypeptides are from various infectious agents.

Natural Evolution of Infectious Agents

[0319] Infectious agents remain a serious public health threat throughout the world. Vaccines and antivirals are available that can provide protection from infection. However, new strains e.g., viral strains, or bacterial strains, etc. emerge continuously because of the plasticity of their genome allowing them to adapt to changing conditions. Infectious agents can hereby be associates with circulating diseases. One notable feature of RNA viruses is their high mutation rate. Unlike DNA viruses which utilize the host replication machinery to detect and repair base- pairing errors during replication, RNA viruses use RNA-dependent RNA polymerases that lack proofreading ability and, therefore, are intrinsically error prone. This may necessitate reformulation of vaccine antigens, and resistance to antivirals can appear rapidly and become entrenched in circulating virus populations. [0320] Infectious agents, such as viruses, display a wide diversity of sizes and shapes. A complete virus particle, known as a virion, comprises a nucleic acid surrounded by a protective coat of protein called a capsid and sometimes an outer envelope that comprises proteins, such as surface proteins, and phospholipid membranes derived from the host cell. Viruses may also contain additional proteins, such as enzymes, within the capsid or attached to the viral genome. Viruses can undergo genetic change by several mechanisms. These include a process called antigenic drift where individual bases in the DNA or RNA mutate to other bases. Most of these point mutations are "silent" — they do not change the protein that the gene encodes — but others can confer evolutionary advantages such as resistance to antiviral drugs. Antigenic shift occurs when there is a major change in the genome of the virus. This can be a result of recombination or reassortment. When this happens pandemics might result. In particular, human immunodeficiency virus (HIV) evades the immune system by constantly changing the amino acid sequence of the proteins on the surface of the virion. This is known as “escape mutation” as the viral epitopes escape recognition by the host immune response.

[0321] Infectious agent surface proteins and/or surface glycoproteins (e.g., antigens) can be immunodominant antigens that are targeted for antibody-mediated neutralization by the humoral immune response by the host. These surface proteins and/or surface glycoproteins (e.g., antigens) present numerous surfaces known as epitopes which are recognized by antibodies that are generated by the host immune system to specifically bind to these virus epitopes via the antibody’s functional ‘paratope’ domain in an epitope-paratope interaction (EPI). EPIs are key aspects of the dynamic interplay between the virus and the host immune response to neutralize the virus. Subsequently, the immune system retains a ‘memory’ of the antigen(s), along with the ability to produce the particular antibodies that target it, in the form of memory B and T cells.

[0322] Host antibody responses upon infectious agent infection vary widely depending on the infectious agent and the host’ s exposure history to the infectious agent, homologous infectious agent, and vaccines. Hosts that have been previously infected or vaccinated typically possess neutralizing antibodies (nAbs) against vulnerable epitopes (e.g., virus epitopes) which protect the host from infection upon infectious agent exposure, with the nAb titer often correlating with the degree of protection against future infections. However, for certain infectious agents pre-existing antibodies resulting from infections of different sub-types may recognize but not effectively neutralize the infectious agent, which may result in paradoxically worse disease in a mechanism known as antibody-dependent enhancement.

[0323] In some embodiments, poorly-neutralizing antibodies are undesirable as they may not protect a host from future exposures and thus lead to reinfection, though non- neutralizing antibodies can still play key roles in protection via Fc function. This neutralization ‘escape’ dynamic occurs, for example, in the case of influenza A strains and SARS-CoV-2 variants featuring mutations in vulnerable epitopes resulting in reinfection of hosts whose antibodies developed during prior infection or vaccination no longer effectively recognize the mutated epitopes. Antibody escape may be more or less pronounced depending on the host’s exposure history, with certain viruses tending to leave an imprint on the host antibody response based on the host’s first exposure to the virus in a mechanism known as original antigenic sin/seniority, which can occur divergently for antibodies (Abs) generated via vaccination versus infection as in the case of SARS-CoV-2 mRNA vaccines.

[0324] In this way, viruses experience continued pressure to evolve mutations in vulnerable epitopes toward acquiring the ability to escape existing antibodies and re-infect hosts. Likewise, hosts continually evolve new (in response to reinfection or additional vaccination) or matured (resulting from accumulation of somatic mutations within memory B cells) antibodies to neutralize viruses bearing mutated or homologous epitopes, wherein these responses are modulated by antigenic exposure history.

Immune escape prone variants

[0325] Infectious agents have developed various means of evading or subverting host defenses.

Antigenic variation

[0326] One way in which an infectious agent can evade immune surveillance is by altering its antigens (e.g., its epitopes); this is particularly important for extracellular pathogens, against which the principal defense is the production of antibody against their surface proteins and/or glyco proteins. Exemplary Infectious Agents

[0327] In some embodiments, an infectious agent is a virus, a bacteria, or a eukaryotic cell (e.g., a plasmodium).

[0328] In some embodiments, an infectious agent is a respiratory virus. In some embodiments, an infectious agent is an RNA virus. In some embodiments, an infectious agent is a coronavirus (e.g., MERS, SARS, or SARS-CoV-2). In some embodiments, an infectious agent is HIV. In some embodiments, an infectious agent is HSV (e.g., HSV-1 or HSV-2). In some embodiments, an infectious agent is RSV. In some embodiments, an infectious agent is a norovirus. In some embodiments, an infectious agent is an influenza virus. In some embodiments, an infectious agent is P. falciparum. In some embodiments, an antigen described herein is an antigen from a virus in the genus Orthopoxvirus. There are 12 species in this genus. Diseases associated with this genus include, but are not limited to smallpox, cowpox, horsepox, camelpox, and monkeypox.

[0329] In some embodiments, an infectious agent is a bacterium. In some embodiments, the bacterium is Mycobacterium. In some embodiments, the bacterium is selected from Haemophilus influenzae, Chlamydophila pneumoniae, Mycoplasma pneumonia, Staphylococcus aureus, Moraxella catarrhalis, Legionella pneumophila, and Streptococcus pneumonia. In some embodiments, the bacterium is Streptococcus pneumonia.

[0330] In some embodiments, an infectious agent is an RNA virus. Compositions provided herein may provide a particular advantage in providing an immune response against RNA viruses, which have a relatively high mutation rate (high relative to other infectious agents).

[0331] In some embodiments, an infectious agent comprises a large number of strains, variants, or lineages. In some embodiments, an infectious agent has a relatively high mutation rate (e.g., relative to other infectious agents).

[0332] In some embodiments, an infectious agent is prone to immune escape.

[0333] In some embodiments, an infectious agent is one for which seasonal, variant- adapted booster shots are regularly provided. [0334] In some embodiments, an infection agent is associated with a circulating infectious disease (e.g., for which variants can be expected to arise).

[0335] Exemplary viral infectious diseases include, but are not limited to coronavirus, ebolavirus, influenza viruses, norovirus, rotavirus, respiratory syncytial virus, alphaherpesvirus, etc.

[0336] In some embodiments, an antigen described herein is or comprises a B cell antigen. In some embodiments, such a B cell antigen comprises one or more antibody epitopes. In some embodiments, such epitopes are antibody binding epitopes. In some embodiments, such epitopes are antibody neutralizing epitopes.

Exemplary Antigens

[0337] In some embodiments, an antigen described herein is or comprises an antigen of an infectious agent. In some embodiments, an infection agent is associated with a circulating infectious disease (e.g., for which variants can be expected to arise). In some embodiments, such circulating infectious disease is a bacterial infectious disease. In some embodiments, such circulating infectious disease is a parasitic infectious disease. An exemplary parasitic infectious disease is malaria. In some embodiments, such circulating infectious disease is a viral infectious disease. In some embodiments, a viral infectious disease is associated with an RNA virus. Exemplary viral infectious diseases include, but are not limited to coronavirus, ebolavirus, influenza viruses, norovirus, rotavirus, respiratory syncytial virus, alphaherpesvirus, etc.

[0338] In some embodiments, an antigen (e.g., SARS-CoV-2) described herein is or comprises a B cell antigen. In some embodiments, such a B cell antigen comprises one or more antibody epitopes. In some embodiments, such epitopes are antibody binding epitopes. In some embodiments, such epitopes are antibody neutralizing epitopes.

[0339] In some embodiments, an antigen described herein is or comprises a T cell antigen. In some embodiments, such a T cell antigen comprises one or more CD4 T cell and/or one or more CD8 T cell epitopes.

[0340] In some embodiments, an antigen described herein includes one or more variant sequences relative to a relevant reference antigen. For example, in some embodiments, a protease cleavage site is removed or blocked; alternatively or additionally, in some embodiments, a terminally truncated antigen is utilized, and/or one or more mutations associated with a viral variant is present in the antigen.

[0341] In some embodiments, utilized sequences may comprise one or more mutations associated with a viral variant (e.g., SARS-CoV-2) (e.g., a variant that prevalent and/or that is predicted to be highly immune escaping). In some embodiments, utilized sequences comprise one or more mutations associated with a variant of concern (e.g., a variant of concern identified by WHO). In some embodiments, utilized sequences comprise one or more mutations associated with a viral variant that has been determined to be or has been predicted to be highly immune escaping (e.g., highly immune escaping relative to an immune response developed in subjects administered a previously approved vaccine and/or a previously prevalent viral variant).

[0342] In some embodiments, an antigen described herein is or comprises a surface protein or a surface glycoprotein of an infectious agent (e.g., SARS-CoV-2) . In some embodiments, an antigen described herein is a surface protein or a surface glycoprotein of an infectious agent strain or variant (e.g., SARS-CoV-2) that was previously and/or is currently prevalent. In some embodiments, an antigen described herein is or comprises a surface protein or surface glycoprotein of an infectious agent (e.g., SARS-CoV-2) that has been previously delivered in a vaccine (e.g., a commercially available vaccine, an RNA vaccine, or a protein- based vaccine).

[0343] In some embodiments, an infectious agent antigen is solvent exposed on the surface of the infectious agent. In some embodiments, an infectious agent antigen is a glyocoprotein. In some embodiments, an infectious agent antigen is involved in host cell recognition. In some embodiments, an infectious agent antigen is involved in host cell entry. In some embodiments, an infectious agent antigen comprises one or more B cell epitopes (e.g., one or more neutralization epitopes).

[0344] In some embodiments, an antigen described herein is or comprises a full-length polypeptide antigen of an infectious agent. In some embodiments, an antigen described herein is or comprises an immunogenic fragment, portion, or domain of a polypeptide antigen of an infectious agent.

[0345] In some embodiments, a composition delivers a hypervariable domain. As used herein, a “hypervariable domain” refers to a domain or region having a high frequency of mutation. In some embodiments, a hypervariable domain comprises a high number of mutations relative to other polypeptide encoded by the infectious agent. In some embodiments, a hypervariable domain comprises a higher frequency of mutations relative to other regions in the antigen (e.g., higher by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% or more). In some embodiments, a hypervariable domain comprises a higher number or density of neutralization-sensitive epitopes (e.g., as compared to other antigens, other antigens encoded by an infectious agent, and/or other regions of the antigen). In some embodiments, a hypervariable domain corresponds to a region in an infectious agent antigen that has an increased frequency of mutation in variants or strains of an infectious agent that have an increased immune escape potential. In some embodiments, a hypervariable domain is a region or domain within a polypeptide (e.g., a viral polypeptide) that binds a host cell receptor and helps mediate cell entry.

[0346] In some embodiments, an antigen described herein is an antigen from a coronavirus. In some embodiments, an antigen described herein is from an alphacoronavirus. In some embodiments, an antigen described herein is from a betacoronavirus. In some embodiments, an antigen described herein is from a gammacoronavirus. In some embodiments, an antigen described herein is from a deltacoronavirus. Exemplary antigens from coronavirus include, but are not limited to spike (S) protein or immunogenic fragments or portions thereof (including, e.g., but not limited to receptor binding domain (RBD), N-terminal domain (NTD)), as well as membrane (M) protein, envelope (E) protein, nucleocapsid protein, or combinations thereof. In some embodiments, an exemplary antigen described herein is a SARS-CoV-2 S protein or an immunogenic fragment or portion thereof (including, e.g., but not limited to RBD or NTD). For example, in some embodiments, such a SARS-CoV-2 S protein or an immunogenic fragment or portion thereof (including, e.g., but not limited to RBD or NTD) is from a Wuhan strain or an Omicron BA.4/5 strain. In some embodiments, such a SARS-CoV-2 S protein or an immunogenic fragment or portion thereof (including, e.g., but not limited to RBD or NTD) is from a XBB strain (e.g., XBB1, XBB1.5 or sublineages thereof).

[0347] In some embodiments, an antigen described herein is an antigen from an influenza virus. In some embodiments, an antigen described herein is from influenza A virus, including, e.g., but not limited to A(H1N1), A(H3N2), etc. In some embodiments, an antigen described herein is from influenza B virus, including, e.g., B(Victoria), B(Yamagata), etc. In some embodiments, an antigen described herein is from influenza C virus. In some embodiments, an antigen described herein is from influenza D virus. Exemplary antigens from influenza viruses include, but are not limited to hemagglutinin (HA), neuraminidase (NA), or immunogenic portions or fragments thereof, or combinations thereof.

[0348] In some embodiments, an antigen described herein is an antigen from a respiratory syncytial virus (RSV), e.g., as described herein.

[0349] In some embodiments, an antigen described herein is an antigen from a norovirus. Noroviruses are members of the Caliciviridae family of small, non-enveloped, positive-stranded RNA viruses. The Norovirus genus includes both human and animal (e.g., murine and canine) noroviruses. Exemplary antigens from noroviruses include, but are not limited to Viral Protein 1 (VP1), Viral Protein 2 (VP2), S domain, P domain, Pl, P2, non- structural proteins, N-terminal proteins (NS 1-2, p48), NTPase (NS3), P22(NS4), VPg (NS5), Protease (NS6), Polymerase (NS7), or immunogenic portions or fragments thereof, or combinations thereof. In some embodiments, a norovirus antigen that is useful in accordance with the present disclosure is a norovirus antigen described in the International Patent Application No. PCT/US22/46799, the relevant content of which is incorporated herein by reference for the purposes described herein.

[0350] In some embodiments, an antigen described herein is an antigen from malarial polypeptide (e.g., as described herein).

[0351] In some embodiments, an antigen described herein is an antigen from a virus in the genus Orthopoxvirus. There are 12 species in this genus. Diseases associated with this genus include, but are not limited to smallpox, cowpox, horsepox, camelpox, and monkeypox.

[0352] In some embodiments, an antigen described herein is an antigen from Herpes simplex virus (e.g., HSV-1 and HSV-2), for example, as described herein.

[0353] In some embodiments, an antigen described herein is useful as a reference antigen of an infectious agent.

[0354] In some embodiments, an antigen described herein is or comprises a variant polypeptide of a reference antigen of an infectious agent, or an immunogenic portion thereof. [0355] In some embodiments, an antigen described herein is or comprises a full-length polypeptide antigen of an infectious agent. In some embodiments, an antigen described herein is or comprises an immunogenic fragment, portion, or domain of a polypeptide antigen of an infectious agent.

[0356] In some embodiments, an antigen that is useful in accordance with the present disclosure is an antigen described in a U.S. Provisional Application entitled “Immunogenic Compositions” and filed February 24, 2023, the entire content of which is incorporated herein by reference for the purposes described herein. In some embodiments, an antigen that is useful in accordance with the present disclosure is an antigen described in a U.S. Provisional Application entitled “SARS-CoV-2-specific Immunogenic Compositions” and filed February 24, 2023, the entire content of which is incorporated herein by reference for the purposes described herein.

[0357] In some embodiments, an antigen described herein is an engineered antigen. For example, in some embodiments, an engineered antigen is designed to promote tailored immune responses. In some embodiments, an engineered antigen is designed using systems and methods as described in US Provisional Application No. 63/448215, the entire content of which is incorporated herein by reference for the purposes described herein.

Coronavirus Overview

[0358] Coronaviruses are enveloped, positive-sense, single-stranded RNA ((+) ssRNA) viruses. They have the largest genomes (26-32 kb) among known RNA viruses and are phylogenetically divided into four genera (a, 0, y, and 5), with betacoronaviruses further subdivided into four lineages (A, B, C, and D). Coronaviruses infect a wide range of avian and mammalian species, including humans. Some human coronaviruses generally cause mild respiratory diseases, although severity can be greater in infants, the elderly, and the immunocompromised. Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV), belonging to betacoronavirus lineages C and B, respectively, are highly pathogenic. Both viruses emerged into the human population from animal reservoirs within the last 15 years and caused outbreaks with high case-fatality rates. The outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that causes atypical pneumonia (coronavirus disease 2019; CO VID- 19) has raged in China since mid- December 2019, and has developed to be a public health emergency of international concern. SARS-CoV-2 (MN908947.3) belongs to betacoronavirus lineage B. It has at least 70% sequence similarity to SARS-CoV.

[0359] In general, coronaviruses have four structural proteins, namely, envelope (E), membrane (M), nucleocapsid (N), and spike (S). The E and M proteins have important functions in the viral assembly, and the N protein is necessary for viral RNA synthesis. The S glycoprotein is responsible for virus binding and entry into target cells. WT S protein is synthesized as a single-chain inactive precursor that is cleaved by furin-like host proteases in the producing cell into two noncovalently associated subunits, SI and S2. SI contains a receptor-binding domain (RBD), which recognizes host-cell receptors. S2 contains a fusion peptide, two heptad repeats, and a transmembrane domain, all of which play a role in mediating fusion of viral and host-cell membranes by undergoing a large conformational rearrangement. S 1 and S2 trimerize to form a large prefusion spike complex.

[0360] In some embodiments, an antigen described herein is an antigen from a coronavirus. In some embodiments, an antigen described herein is from an alphacoronavirus. In some embodiments, an antigen described herein is from a betacoronavirus. In some embodiments, an antigen described herein is from a gammacoronavirus. In some embodiments, an antigen described herein is from a deltacoronavirus.

[0361] SARS-CoV-2 Spike (S) protein can be proteolytically cleaved into SI (685 aa) and S2 (588 aa) subunits. SI of SARS-CoV-2 comprises a receptor-binding domain (RBD), which mediates virus entry into host cells through the host angiotensin-converting enzyme 2 (ACE2) receptor.

[0362] The presentation of COVID-19 is generally with cough and fever, with chest radiography showing ground-glass opacities or patchy shadowing. However, many patients present without fever or radiographic changes, and infections may be asymptomatic which is relevant to controlling transmission. For symptomatic subjects, progression of disease may lead to acute respiratory distress syndrome requiring ventilation and subsequent multi-organ failure and death. Common symptoms in hospitalized patients (in order of highest to lowest frequency) include fever, dry cough, shortness of breath, fatigue, myalgias, nausea/vomiting or diarrhoea, headache, weakness, and rhinorrhoea. Anosmia (loss of smell) or ageusia (loss of taste) may be the sole presenting symptom in approximately 3% of individuals who have COVID-19. [0363] All ages may present with the disease, but notably case fatality rates (CFR) are elevated in persons >60 years of age. Comorbidities are also associated with increased CFR, including cardiovascular disease, diabetes, hypertension, and chronic respiratory disease.

Healthcare workers are overrepresented among CO VID- 19 patients due to occupational exposure to infected patients.

[0364] In most situations, a molecular test is used to detect SARS-CoV-2 and confirm infection. The reverse transcription polymerase chain reaction (RT-PCR) test methods targeting SARS-CoV-2 viral RNA is one method for diagnosing suspected cases of CO VID-19. Samples to be tested are collected from the nose and/or throat with a swab.

SARS-CoV-2 Variants

[0365] Since the initial discovery of SARS-CoV-2, a number of variants have arisen around the world. The emergence of these novel circulating variants of SARS-CoV-2 has raised significant concerns about geographic and temporal efficacy of vaccine interventions. The emergence of Omicron (B.1.1.529) variants, which comprise a number of mutations in the S protein, has been of particular concern.

[0366] In some embodiments, the present disclosure refers to a SARS-CoV-2 variant that is prevalent and/or rapidly spreading in a relevant jurisdiction. In some embodiments, such variants may be identified based on publicly available data (e.g., data provided in the GISAID Initiative database: https://www.gisaid.org, and/or data provided by the World Health Organization WHO (e.g., as provided at https://www.who.int/activities/tracking-SARS-CoV-2- variants). In some embodiments, such a variant refers to a variant disclosed herein.

[0367] The Omicron BA.l variant was first reported to WHO on 24 November 24, 2021, and was detected in South Africa. Omicron and its sublineages have had a major impact on the epidemiological landscape of the COVID-19 pandemic since their initial emergence (WHO Technical Advisory Group on SARS-CoV-2 Virus Evolution (TAG-VE): Classification of Omicron (B.1.1.259): SARS-CoV-2 Variant of Concern (2021); WHO Headquarters (HQ), WHO Health Emergencies Programme, Enhancing Response to Omicron SARS-CoV-2 variant: Technical brief and priority actions for Member States (2022)). Significant alterations in the spike (S) glycoprotein of the first Omicron variant BA.l resulted in the loss of many neutralizing antibody epitopes (M. Hoffmann et al., “The Omicron variant is highly resistant against antibody mediated neutralization: Implications for control of the COVID- 19 pandemic”, Cell 185, 447- 456.el 1 (2022)) and rendered BA.l capable of partially escaping previously established SARS- CoV-2 wild-type strain (Wuhan-Hu- l)-based immunity (V. Servellita, et al., “Neutralizing immunity in vaccine breakthrough infections from the SARS-CoV-2 Omicron and Delta variants”, Cell 185, 1539-1548.e5 (2022); Y. Cao et al., “Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies”, Nature 602, 657-663 (2022)).

[0368] As a result, breakthrough infection of vaccinated individuals with Omicron is more common than with previous Variants of Concern (VOCs). While Omicron BA.l was displaced by the BA.2 variant in many countries around the globe, other variants such as BA.1.1 and BA.3 temporarily and/or locally gained momentum but did not become globally dominant (S. Xia et al., “Origin, virological features, immune evasion and intervention of SARS-CoV-2 Omicron sublineages. Signal Transduct. Target. Ther. 7, 241 (2022); H. Gruell et al., “SARS- CoV-2 Omicron sublineages exhibit distinct antibody escape patterns, Cell Host Microbe 7, 241 (2022).). Omicron BA.2.12.1 subsequently displaced BA.2 to become dominant in the United States, whereas BA.4 and BA.5 displaced BA.2 in Europe, parts of Africa, and Asia/Pacific (H. Gruell et al., “SARS-CoV-2 Omicron sublineages exhibit distinct antibody escape patterns,” Cell Host Microbe 7, 241 (2022); European Centre for Disease Prevention and Control, Weekly

CO VID-19 country overview -Country overview report: Week 31 2022 (2022); J. Hadfield et al., “Nextstrain: Real-time tracking of pathogen evolution,” Bioinformatics 34, 4121 4123 (2018)). Currently, Omicron BA.5 is dominant globally, including in the United States (Centers for Disease Control and Prevention. CO VID Data Tracker. Atlanta, GA: US Department of Health and Human Services, CDC; 2022, August 12. https://covid.cdc.gov/coviddata-tracker (2022)).

[0369] Omicron has acquired numerous alterations (amino acid exchanges, insertions, or deletions) in the S glycoprotein, among which some are shared between all Omicron VOCs while others are specific to one or more Omicron sublineages. Antigenically, BA.2.12.1 exhibits high similarity with BA.2 but not BA.l, whereas BA.4 and BA.5 differ considerably from their ancestor BA.2 and even more so from BA.l, in line with their genealogy (A. Z. Mykytyn et al., “Antigenic cartography of SARS-CoV-2 reveals that Omicron BA.l and BA.2 are antigenically distinct,” Sci. Immunol. 7, eabq4450 (2022).). Major differences of BA.l from the remaining Omicron VOCs include A143-145, L212I, or ins214EPE in the S glycoprotein N-terminal domain and G446S or G496S in the receptor binding domain (RBD). Amino acid changes T376A, D405N, and R408S in the RBD are in turn common to BA.2 and its descendants but not found in BA.l. In addition, some alterations are specific for individual BA.2-descendant VOCs, including L452Q for BA.2.12.1 or L452R and F486V for BA.4 and BAA (BA.4 and BAA encode for the same S sequence). Most of these shared and VOC-specific alterations were shown to play an important role in immune escape from monoclonal antibodies and polyclonal sera raised against the wild-type S glycoprotein. In particular, the BA.4/BA.5-specific alterations are strongly implicated in immune escape of these VOCs (P. Wang et al., “Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7. Nature 593, 130-135 (2021); Q. Wang et al., “Antibody evasion by SARS-CoV-2 Omicron subvariants BA.2.12.1, BA.4, & BAA. Nature 608, 603-608 (2022)).

[0370] Among other things, described herein are certain SARS-CoV-2 antigens for use in inducing an immunogenic response. In some embodiments, a SARS-CoV-2 antigen comprise immunogenic portions of a full-length SARS-CoV-2 polypeptide (e.g., an SI domain of a SARS- COV-2 S protein and/or an RBD of a SARS-CoV-2 S protein). In some embodiments, such antigens are delivered as protein antigens to induce an immunogenic response. In some embodiments, such antigens are delivered using RNA (e.g., modRNA encoding an SI domain and/or RBD of a SARS-CoV-2 S protein and formulated in LNP particles) to induce an immunogenic response.

Exemplary Coronavirus Antigens

[0371] As used herein, a full length SARS-CoV-2 S protein comprising a “Wild-Type” or “Wuhan” sequence has a sequence corresponding to that of the first detected SARS-CoV-2 strain, consisting of 1273 amino acids and having an amino acid sequence according to SEQ ID

NO: 1:

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTK RFDNPVLPFNDGVYFASTEKSNI IRGWI FGTTLDSKTQSLLIVNNATNWIKVCEFQFCNDPFLGVYYHKNNKSWME SEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNI DGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPI GINITRFQTLLALHRSYLTPGDS SSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTV EKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRI SNCVADYSVLYNSAS FSTFKCYGVS PT KLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLK PFERDI STEP YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVCGPKKSTNLVKNKCV NFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEI LDITPCSFGGVSVITPGTNTSNQVAVLYQDVN CTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNS YECDI PIGAGICASYQTQTNSPRRARSVASQSI I AYTMSLGAENSVAYSNNSIAI PTNFTI SVTTEI LPVSMTKTSVDCTMYI CGDSTECSNLLLQYGSFCTQLNRALTGI AVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRS FIEDLLFNKVTLADAGFIKQYGDCLGDIAAR DLI CAQKFNGLTVLPPLLTDEMI AQYTSAL LAGTIT SGWT EGA GAALQI PFAMQMAYRFNGIGVTQNVLYENQKLI A NQFNSAIGKIQDSLSSTASALGKLQDWNQNAQALNTLVKQLS SNFGAI SSVLNDI LSRLDKVEAEVQI DRLITGRL QSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTA PAI CHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQI ITTDNTFVSGNCDWIGIVNNTVYDPLQPELDS FKEELDKY FKNHTS PDVDLGDI SGINASWNIQKEIDRLNEVAKNLNESLI DLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTI MLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT ( SEQ ID NO : 1 )

[0372] Unless otherwise indicated, position numberings in a SARS-CoV-2 S protein given herein are in relation to the amino acid sequence of SEQ ID NO: 1. One of skill in the art reading the present disclosure will understand and be able to determine corresponding positions in a SARS-CoV-2 S protein variant sequence from locations of positions provided relative to the amino acid sequence of SEQ ID NO: 1 (i.e., a person of skill in the art provided positions relative to SEQ ID NO: 1, or another variant, will be able to determine corresponding positions in the S protein sequence of another SARS-CoV-2 variant or a fragment thereof).

[0373] In specific embodiments, a spike (S) protein described herein can be modified in such a way that the prototypical prefusion conformation is stabilized. Certain mutations that stabilize a prefusion confirmation are known in the art, e.g., as disclosed in WO 2021243122 A2 and Hsieh, Ching-Lin, et al. ("Structure-based design of prefusion-stabilized SARS-CoV-2 spikes," Science 369.6510 (2020): 1501-1505), the contents of each which are incorporated by reference herein in their entirety. In some embodiments, a SARS-CoV-2 S protein may be stabilized by introducing one or more proline mutations. In some embodiments, a SARS-CoV-2

S protein comprises a proline substitution at positions corresponding to residues 986 and/or 987 of SEQ ID NO: 1. In some embodiments, a SARS-CoV-2 S protein comprises a proline substitution at one or more positions corresponding to residues 817, 892, 899, and 942 of SEQ

ID NO: 1. In some embodiments, a SARS-CoV-2 S protein comprises a proline substitution at positions corresponding to each of residues 817, 892, 899, and 942 of SEQ ID NO: 1. In some embodiments, a SARS-CoV-2 S protein comprises a proline substitution at positions corresponding to each of residues 817, 892, 899, 942, 986, and 987 of SEQ ID NO: 1.

[0374] In some embodiments, stabilization of the prototypical prefusion conformation of a SARS-CoV-2 S protein may be obtained by introducing two consecutive proline substitutions at residues 986 and 987. Specifically, spike (S) protein stabilized protein variants are obtained in a way that the amino acid residue at position 986 is exchanged to proline and the amino acid residue at position 987 is also exchanged to proline. In one embodiment, a SARS-CoV-2 S protein variant wherein the prototypical prefusion conformation is stabilized comprises the amino acid sequence shown in SEQ ID NO: 2:

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTK RFDNPVLPFNDGVYFASTEKSNI IRGWI FGTTLDSKTQSLLIVNNATNWIKVCEFQFCNDPFLGVYYHKNNKSWME SEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNI DGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPI GINITRFQTLLALHRSYLTPGDS SSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTV EKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRI SNCVADYSVLYNSASFSTFKCYGVSPT KLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLK PFERDI STEP YQAGSTPCNGVEGFNCYFPLQS YGFQPTNGVGYQPYRVWLS FELLHAPATVCGPKKSTNLVKNKCV NFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEI EDIT PCS FGGVSVITPGTNTSNQVAVLYQDVN CTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNS YECDI PIGAGICASYQTQTNSPRRARSVASQSI I AYTMSLGAENSVAYSNNSIAI PTNFTI SVTTEI LPVSMTKTSVDCTMYICGDSTECSNLLLQYGS FCTQLNRALTGI AVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRS FIEDLLFNKVTLADAGFIKQYGDCLGDIAAR DLI CAQKFNGLTVLPPLLTDEMI AQYTSAL LAGTIT SGWT EGA GAALQI PFAMQMAYRFNGIGVTQNVLYENQKLI A NQFNSAIGKIQDSLSSTASALGKLQDWNQNAQALNTLVKQLS SNFGAI SSVLNDI LSRLDPPEAEVQI DRLITGRL QSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTA PAI CHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQI ITTDNTFVSGNCDWIGIVNNTVYDPLQPELDS FKEELDKY FKNHTS PDVDLGDI SGINASWNIQKEIDRLNEVAKNLNESLI DLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTI MLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

( SEQ I D NO : 2 )

[0375] Those skilled in the art are aware of various SARS-COV-2 Spike variants, and/or resources that document them. For example, the following strains, their SARS-CoV-2 S protein amino acid sequences and, in particular, modifications thereof compared to wildtype SARS-

CoV-2 S protein amino acid sequence, e.g., as compared to SEQ ID NO: 1, are useful herein.

B.1.1.7 ("Variant of Concern 202012/01" (VOC-202012/01) [0376] B.1.1.7 (“alpha variant”) is a SARS-CoV-2 variant that was first detected in October 2020 in the United Kingdom from a sample taken the previous month, and quickly began to spread by mid-December. It is correlated with a significant increase in the rate of COVID-19 infection; this increase is thought to be at least partly due to a change of N501Y inside the spike glycoprotein's receptor-binding domain, which is needed for binding to ACE2 in human cells. B.1.1.7 is defined by 23 mutations: 13 non-synonymous mutations, 4 deletions, and 6 synonymous mutations (z.e., there are 17 mutations that change proteins and six that do not). Spike protein changes in B.1.1.7 include deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

B.1.351 (501.V2)

[0377] B.1.351 lineage ( “Beta variant”), colloquially known as South African COVID-

19 variant, has increased transmissibility relative to the original Wuhan strain. The B.1.351 variant is defined by multiple spike protein changes including: L18F, D80A, D215G, deletion 242-244, R246I, K417N, E484K, N501Y, D614G and A701V. There are three mutations of particular interest in the spike region of the B.1.351 genome: K417N, E484K, N501Y.

B.1.1.298 (Cluster 5)

[0378] B .1.1.298 was discovered in North Jutland, Denmark, and is believed to have been spread from minks to humans via mink farms. Several different mutations in the spike protein of the virus have been confirmed. The specific mutations include deletion 69-70, Y453F, D614G, I692V, Ml 2291, and optionally S1147L.

P.l (B.1.1.248)

[0379] Lineage B .1.1.248 (the “gamma variant”), known as the Brazil(ian) variant, is one of the variants of SARS-CoV-2 which has been named P.l lineage. P.l has a number of S- protein modifications (L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, VI 176F) and is similar in certain key RBD positions (K417, E484, N501) to variant B.1.351 from South Africa.

B.1.427/B.1.429 (CAL.20C)

[0380] Lineage B.1.427/B.1.429 (the “epsilon variant”), also known as CAL.20C, is defined by the following modifications in the S-protein: SI 31, W152C, L452R, and D614G, of which the L452R modification is of particular concern. CDC has listed B.1.427/B.1.429 as a "variant of concern".

B.1.525

[0381] B.1.525 ( “eta variant”) carries the same E484K modification as found in the P.l, and B.1.351 variants, and also carries the same AH69/AV70 deletion as found in B.1.1.7, and B.1.1.298. It also carries the modifications D614G, Q677H and F888L.

B.1.526

[0382] B.1.526 ( “iota variant”) was detected as an emerging lineage of viral isolates in the New York region that shares mutations with previously reported variants. The most common sets of spike mutations in this lineage are L5F, T95I, D253G, E484K, D614G, and A701V.

B.l.1.529

[0383] B .1.529 (“Omicron variant”) was first detected in South Africa in November

2021. Omicron multiplies around 70 times faster than Delta variants, and quickly became the dominant strain of SARS-CoV-2 worldwide. Since its initial detection, a number of Omicron sublineages have arisen. Listed below are the current Omicron variants of concern, along with certain characteristic mutations associated with the S protein of each. The S protein of B A.4 and BA.5 have the same set of characteristic mutations, which is why the below table has a single row for “BA.4 or BA.5”, and why the present disclosure refers to a “BA.4/5” S protein in some embodiments. Similarly, the S proteins of the BA.4.6 and BF.7 Omicron variants have the same set of characteristic mutations, which is why the below table has a single row for “BA.4.6 or BF.7”).

Table 1: Omicron Variants of Concern and Characteristic mutations

[0384] In some embodiments, SARS-CoV-2 S proteins described herein comprise one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) characteristic of a certain Omicron variant (e.g., one or more mutations of an Omicron variant listed in Table 1, e.g., each of the mutations associated with a given XBB variant in the above Table 1).

Immunogenic Portions of Coronavirus S Protein

[0385] As noted elsewhere in the present disclosure, in some embodiments, compositions described herein deliver an immunogenic portion of a full length coronavirus S protein (e.g., SARS-CoV-2 S protein).

[0386] In some embodiments, an immunogenic portion of a coronavirus (e.g., SARS- CoV-2) S protein lacks certain features that are in the full length polypeptide (e.g., features that have been shown or predicted to interfere with induction of a naive immune response). For example, in some embodiments, an immunogenic portion of a coronavirus (e.g., SARS-CoV-2) S protein lacks regions that have (i) a low number or density of B cell neutralization epitopes and/or (ii) a high number or density of B cell epitopes not associated with neutralization. For example, in some embodiments, an immunogenic portion of a coronavirus (e.g., SARS-CoV-2) S protein lacks a full S2 domain. In some embodiments, a coronavirus (e.g., SARS-CoV-2) S protein lacking a full S2 domain lacks regions of S2 that have (i) a low number or density of B cell epitopes associated with neutralization or (ii) a high number of B cell epitopes not associated with neutralization, but retains other portions of S2. In some embodiments, an immunogenic portion of a coronavirus (e.g., SARS-CoV-2) S protein lacks the entire S2 domain. In some embodiments, an immunogenic portion of a coronavirus (e.g., SARS-CoV-2) S protein lacks a full S2 domain, but comprises certain sequences that can improve immunogenicity and/or stability of an immunogenic portion (e.g., in some embodiments, an immunogenic portion lacks a full S2 domain but retains a TM sequence).

[0387] A person of skill in the art reading the present disclosure will be able to identify B cell epitopes in a coronavirus (e.g., SARS-CoV-2) S protein and determine which epitopes are or are not associated with neutralization. For example, a person of skill in the art will be aware of numerous studies that have identified such regions using antibody binding studies (e.g., studies characterizing antibodies produced in subjects infected with or vaccinated against SARS- CoV-2).

[0388] In some embodiments, an immunogenic portion of a coronavirus (e.g., SARS- CoV-2) S protein comprises certain regions that have been determined to have a high number or density of neutralization epitopes and optionally a high mutation rate. In some embodiments, an immunogenic portion of a coronavirus (e.g., SARS-CoV-2) S protein comprises an N-terminal domain (NTD) of the S protein. In some embodiments, an immunogenic portion of a coronavirus (e.g., SARS-CoV-2) protein comprises a receptor binding domain (RBD) of the S protein. In some embodiments, an immunogenic portion of a coronavirus (e.g., SARS-CoV-2) S protein comprises an S 1 domain of the S protein.

[0389] In some embodiments, an immunogenic portion of a coronavirus (e.g., SARS- CoV-2) S protein comprises an RBD and an NTD and omits other features of the SI domain.

[0390] Coronavirus (e.g., SARS-CoV-2) S proteins are well characterized, and a person of skill in the art will be able to determine which portions of an S protein sequence correspond to immunogenic portions discussed herein (e.g., which portions of an S protein sequence correspond to the NTD, the RBD, the SI, and the S2 domains). In some embodiments, an RBD of a coronavirus (e.g., SARS-CoV-2) S protein comprises residues 327 to 528 of SEQ ID NO: 1 or a corresponding region.

[0391] In some embodiments, an RBD of a coronavirus (e.g., SARS-CoV-2) S protein comprises the amino acid sequence: VRFPNITNECPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTK LNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKP SGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVG HQPYRVVVLSFELLHAPATVCGPK (SEQ ID NO: 3), or a corresponding region.

[0392] In some embodiments, an RBD of a coronavirus (e.g., SARS-CoV-2) S protein comprises the amino acid sequence:

VRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTK LNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKV GGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVG YQPYRVVVLSFELLHAPATVCGPK (SEQ ID NO: 4), or a correspond.

[0393] In some embodiments, an SI domain of a coronavirus (e.g., SARS-CoV-2) S protein comprises amino acids 1 to 678 of SEQ ID NO: 1, or a corresponding region in an S protein of a SARS-CoV-2 variant. In some embodiments, an SI domain of a SARS-CoV-2 S protein comprises amino acids 1 to 683 of SEQ ID NO: 1, or a corresponding region in an S protein of a SARS-CoV-2 variant. In some embodiments, an SI domain of a SARS-CoV-2 S protein comprises amino acids 1 to 685 of SEQ ID NO: 1, or a corresponding region in an S protein of a SARS-CoV-2 variant.

[0394] In some embodiments, an SI domain of a SARS-CoV-2 S protein comprises the amino acid sequence:

MFVFLVLLPLVSSQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVT WFHAIHVSGTNGTKRFDNPALPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNA TNVVIKVCEFQFCNDPFLDVYQKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGK EGNFKNLREFVFKNIDGYFKIYS KHTPINLERDLPQGFS ALEPL VDLPIGINITRFQTLLAL HRSYLTPVDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTL KSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVAD YSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYK LPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGV AGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF NFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGT NTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSY ECDIPIGAGICASYQTQT (SEQ ID NO: 5) [0395] In some embodiments, an SI domain of a SARS-CoV-2 S protein comprises the amino acid sequence:

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIV NNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMD LEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQT LLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSET KCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISN CVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIA DYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGST PCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKN KCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVS VITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEH VNNSYECDIPIGAGICASYQTQT (SEQ ID NO: 6).

[0396] In some embodiments, an S2 domain of a SARS-CoV-2 S protein comprises amino acids 679 to 1273 of SEQ ID NO: 1, or a corresponding region in an S protein of a SARS- CoV-2 variant. In some embodiments, an SI domain of a SARS-CoV-2 S protein comprises amino acids 684 to 1273 of SEQ ID NO: 1, or a corresponding region in an S protein of a SARS- CoV-2 variant. In some embodiments, an SI domain of a SARS-CoV-2 S protein comprises amino acids 686 to 1273 of SEQ ID NO: 1, or a corresponding region in an S protein of a SARS- CoV-2 variant.

[0397] In some embodiments, compositions described herein deliver an immunogenic portion of an S protein of a SARS-CoV-2 variant. In some embodiments, the variant is a variant of concern (e.g., a variant that has been predicted to and/or has been shown to spread rapidly in a relevant jurisdiction, e.g., as identified by certain public health agencies, e.g., the Center for Disease Control and Prevention (CDC), Public Health England and the COVID-19 Genomics UK Consortium for the UK, the Canadian CO VID Genomics Network (CanCOGeN), and/or the World Health Organization (WHO)). In some embodiments, a variant has been predicted to have a highly likelihood of becoming a variant of concern (e.g., using sequence-based algorithms that predict the ability of a variant to escape previously developed immune responses and/or measure the “fitness” of a given variant, such as described, e.g., in WO2022/235847 and WO2022/235853, the contents of each of which are incorporated by reference herein in their entirety).

[0398] In some embodiments, an RBD comprises mutations associated with a variant described herein. A person of skill in the art will be able to identify which portions of a given variant correspond to immunogenic portions described herein.

[0399] In some embodiments, a polypeptide comprises two or more SARS-CoV-2 subdomains (e.g., two or more SI domains or RBDs). In some embodiments, a polypeptide comprises two or more receptor binding domains linked in tandem, e.g., as described in Dai, Lianpan, et al. "A universal design of betacoronavirus vaccines against CO VID- 19, MERS, and SARS," Cell 182.3 (2020): 722-733, and Han, Yuxuan, et al. "mRNA vaccines expressing homo-prototype/Omicron and hetero-chimeric RBD-dimers against SARS-CoV-2," Cell Research 32.11 (2022): 1022-1025, the contents of each of which are incorporated by reference herein in their entirety. In some embodiments, the two or more subdomains are from the same SARS-CoV-2 variant (e.g., a variant described herein). In some embodiments, at least two of the two or more subdomains are from different SARS-CoV-2 variants (e.g., from different variants of concern, different Omicron variants, an Omicron variant and a non-Omicron variant, or a Wuhan strain and an Omicron variant).

Malaria Overview

[0400] Malaria is a mosquito-borne infectious disease caused by single-celled eukaryotic Plasmodium parasites that are transmitted by the bite of Anopheles spp. mosquitoes (Phillips, M., et al. Malaria. Nat Rev Dis Primers 3, 17050 (2017), which is incorporated herein by reference in its entirety). Mosquitoes that transmit malaria must have been infected through a previous blood meal taken from an infected subject (e.g., a human). When a mosquito bites an infected subject a small amount of blood is taken in containing Malaria parasites. The infected mosquito can then subsequently bite a non-infected subject, infecting the subject.

[0401] Malaria remains one of the most serious infectious diseases, causing approximately 200 million clinical cases and 500,000-600,000 deaths annually. Although significant effort has been invested in developing therapeutic treatments for malaria, many malaria parasites have developed resistance to available therapeutics. According to Malaria Eradication Research Agenda Initiative, malaria eradication will only be achievable through effective vaccination.

[0402] In 2015, the European Medicines Agency gave a positive review to a malaria vaccine candidate known as “RTS,S”, a milestone in malaria vaccine development. In 2019, the World Health Organization launched pilot programs that provide RTS,S to children at least 5 months of age in parts of three sub-Saharan African countries. RTS,S/AS01 is an adjuvanted protein subunit vaccine that consists of a portion of the major repeat region and the C-terminus of CSP from Plasmodium falciparum fused to the Hepatitis B surface antigen (HBsAg). The vaccine is a mix of this PfCSP-HBsAg compound with HBsAg that forms virus-like particles (RTS,S/AS01; Mosquirix™). RTS,S is administered according to a regimen that requires four doses: an initial 3-dose schedule given at least 1 month apart, and a 4th dose 15-18 months after dose 3 (see, for example, Vandoolaeghe & Schuerman Expert Rev Vaccines. 15: 1481, 2016; PATH_MVI_RTSS_Fact Sheet_042019, each of which is incorporated herein by reference in its entirety). Reports indicate that RTS,S protects approximately 30% to 50% of children from clinical disease over 18 months. RTS,S has been reported to induce protective antibody and CD4+ T-cell responses, but only negligible CD8+ T cell responses (see, for example, Moris et al. Hum Vaccin Immunother 14:17, 2018, which is incorporated herein by reference in its entirety). Phase III studies of RTS, S delivered as a three-dose series with a booster after 1 yr (year) showed moderate vaccine efficacy in children aged 5 to 17 months preventing 36% of clinical malaria cases over the full study period with a median follow-up of 4 yrs, with a range of 20% in high to 66% in low transmission settings. Furthermore, published literature suggests that protection wanes over time including reports of potential negative efficacy after 5 yrs in children with high malaria exposure (Olotu et al. 2016, N. Engl. J. Med. 374:2519-29, which is incorporated herein by reference in its entirety). Thus, an effective malaria vaccine remains an unmet medical need of critical importance for global health.

A. Lifecycle

[0403] During a blood meal, infected mosquitos inject, along with their anticoagulating saliva, sporozoites known as the liver stage of Plasmodium spp. Sporozoites journey through the skin to the lymphatics and into hepatocytes of the liver. This journey happens very quickly; it can be complete within only a few minutes (Sinnis et al., Parasitol Int. 2007 Sep; 56(3): 171-8, which is herein incorporated by reference in its entirety). This is a time known to be a bottle kneck of Malaria infection most favorable for therapeutic intervention, as only a small number (thought to be a few hundred at maximum) of sporozoites are injected by the mosquito, with only fraction of that number establishing infection in the liver and developing into mature live-stage parasites (Flores-Garcia et al., mBio. 2018 Nov 20; 9(6):e02194-18, which is herein incorporated by reference in its entirety). Thus, a subject whose immune system is primed to clear sporozoites before they enter hepatocytes can efficiently clear an infection.

[0404] One particular challenge associated with clearing a malarial infection during this bottle neck is that the most abundant and immunogenic protein on the sporozoite surface, the circumsporozoite protein (CSP), is only exposed to the immune system in small quantities and for short duration of time due to the variably low inoculum from the mosquito and the kinetics of hepatocyte infection after inoculation. After liver infection is established, the parasite differentiates into a stage which no longer expresses CSP and instead has a different mosaic of surface antigens. Furthermore, due to the density and close proximity of neighboring CSPs on the surface of the parasite coupled with the bi-valency of antibodies, binding of antibodies to CSP can produce a phenomenon referred to as CSP precipitation reaction, whereby antibodies can crosslink neighboring CSP and cause them to precipitate and shed from the parasite surface, leaving a trail of precipitated antibody bound CSP that the parasite can replace through its normal CSP translocation process (Livingstone et al., Sci Rep 11, 5318 (2021); Steward et al., J Protozool. 1991 Jul-Aug; 38(4):411-21, which are herein incorporated by reference in their entirety).

[0405] When moving from an inoculation site in the skin to the liver, sporozoites traverse host cells (Mota et al., Science 2001 Jan 5;291(5501): 141-4, which is herein incorporated by reference in its entirety). Sporozoites traverse different types of host cells at the dermis, including fibroblasts and phagocytes (Amino et al., Cell Host Microbe. 2008 Feb 14;3(2): 88-96, which is herein incorporated by reference in its entirety), and the liver sinusoidal barrier, containing liver endothelial cellsand Kupffer cells (Frevert et al., PLoS Biol 3(6): el92. 2005, which is herein incorporated by reference in its entirety) and sinusoidal endothelial cells (Tavares et al., J Exp Med 2013 May 6;210(5):905- 15, which is herein incorporated by reference in its entirety), in order to gain access to hepatocytes. Sporozoites preferentially traverse cells with low-sulfated heparin sulfate proteoglycans (HSPGs) but preferentially invade cells with high-sulfated HSPGs (Coppi et al., Cell Host & Microbe 2, 316-327, November 2007, which is herein incorporated by reference in its entirety).

[0406] Cell traversal was first observed as non-phagocytic entry of P. berghei sporozoites into macrophages followed by “escape” from these cells (Vanderberg et al., J. Euk. Microbiol. 37:528-536, 1990, which is herein incorporated by reference in its entirety). The biochemical, biophysical, and stepwise processes of traversal are still being explored. However, it has been suggested by electron microscopy that host cell rupture occurs upon entry and exit from the host cell (Mota et al., 2001; Tavares et al., 2013, which is herein incorporated by reference in its entirety). It has also been shown that P. yoelii sporozoites can enter hepatocytes via a transient vacuole and that host membrane rupture occurs upon cell exit rather than cell entry (Risco-Castillo et al., Cell Host Microbe 2015 Nov 11; 18(5):593-603, which is herein incorporated by reference in its entirety).

[0407] Sporozoites also traverse hepatocytes before establishing a productive hepatocyte infection (Mota et al., 2001, which is herein incorporated by reference in its entirety). Several possibilities emerged as to why this occurs. The first hypothesis suggested that migration through hepatocytes primes parasites for invasion by activating apical exocytosis (Mota et al., Nat Med 2002 Nov; 8(11): 1318-22, which is herein incorporated by reference in its entirety). The second theory suggested that traversal releases hepatocyte growth factor (HGF), making neighboring hepatocytes more susceptible to infection (Carrolo et al., Nat Med. 2003 Nov;9(l l): 1363-9, which is herein incorporated by reference in its entirety). Lastly, other studies suggest that it takes some time for sporozoites to switch off the machinery for traversal and activate invasion machinery (Amino et al., 2008, Coppi et al., 2007, which are herein incorporated by reference in their entirety), and that traversal primarily functions to penetrate cell barriers and avoid phagocytosis en route to the liver (Amino et al., 2008, Coppi et al., 2007, Tavares et al., 2013, which are herein incorporated by reference in their entirety).

[0408] Although it has been shown that sporozoites traverse human cells (Behet et al., Malar J 2014 Apr 5; 13: 136; Cha et al., J Exp Med 2015 Aug 24; 212(9): 1391-403; Dumoulin et al., PLoS One 2015 Jun 12;10(6):e0129623; van Schaijk et al., PLoS ONE, 3 (10). e3549 2008, which are herein incorporated by reference in their entirety), the molecular basis for the traversal process is largely unstudied. Antibodies against circumsporozoite protein (CSP) impair traversal (Dumoulin et al., 2015, which is herein incorporated by reference in its entirety), but this is likely due to inhibition of motility rather than a direct effect (Cha et al., J Exp Med 2016 Sep 19; 213(10):2099-l 12, which is herein incorporated by reference in its entirety). Furthermore, antibodies induced by chloroquine prophylaxis with sporozoites interfere with cell traversal, and these may also target CSP (Behet et al., 2014). Recently it was shown that glyceraldehyde 3- phosphate dehydrogenase (GAPDH) on the parasite surface interacts with CD68 on Kupffer cells during traversal (Cha et al., 2015, Cha et al., 2016, which are herein incorporated by reference in their entirety).

[0409] In rodent malaria parasites such as P. berghei, two sporozoite microneme proteins have been identified that appear to be essential for cell traversal (sporozoite microneme protein essential for cell traversal [SPECT1; Ishino et al., PLoS Biol., 2 (2004), pp. 77-84] and SPECT2 [Ishino et al., Cell. Microbiol., 7 (2005), pp. 199-208], also called perforin-like protein 1 [PLP1] [Kaiser et al., Mol. Biochem. Parasitol., 133 (2004), pp. 15-26], which are herein incorporated by reference in their entirety). Even though genetic disruption of SPECT1 or SPECT2 rendered sporozoites unable to traverse murine cells, they still invaded hepatocytes in vitro (Ishino et al., 2004, Ishino et al., 2005, which are herein incorporated by reference in their entirety). When injected into rodents, sporozoites lacking SPECT1 or SPECT2 were impaired for liver infection, but a small number of sporozoites could still establish liver infection that resulted in subsequent patency. However, depletion of Kupffer cells allowed mutants to establish liver infection at levels comparable with wild-type parasites (Ishino et al., 2004, Ishino et al., 2005, which are herein incorporated by reference in their entirety). This data suggests that traversal by rodent-infecting sporozoites is important for navigating through the sinusoidal layer, but not for hepatocyte invasion, malarial exoerythrocytic forms development, or growth within erythrocytes (Ishino et al., 2004, Ishino et al., 2005, which are herein incorporated by reference in their entirety).

[0410] The ortholog of SPECT2 in P. yoelii, PLP1, has been shown to play a role in cell traversal. Although this protein is not required for hepatocyte entry, it plays a role in egress from transient vacuoles during traversal (Risco-Castillo et al., 2015, which are herein incorporated by reference in their entirety). Thus, sporozoites that infect rodents can traverse host cells by generating a vacuole at the entry step and use a perforin-like protein (e.g., SPECT2/PLP1) to escape from this compartment and/or a host cell, during cell exit. [0411] Once sporozoites have invaded liver cells, they differentiate into merozoites, a replicative form of the parasite capable of lysing hepatocytes after multiple rounds of replication. Within a few days, a few hundred sporozoites can become hundreds of thousands of merozoites. When infected liver cells rupture, they release the merozoites into the bloodstream, where they invade red blood cells and begin the asexual reproductive stage, which is the symptomatic stage of the disease. Within a small number of days, millions of merozoites can be present in blood.

[0412] Malaria symptoms typically develop 4-8 days after initial red blood cell invasion. Replication cycle of merozoites within the red blood cells continues for 36-72 hours, until hemolysis, releasing the merozoites for another round of red blood cell infection. Thus, in synchronous infections (infections that originate from a single infectious bite), fever occurs every 36-72 hours, when infected red blood cells lyse and release endotoxins en masse.

[0413] Plasmodium spp. parasites gain entry into red blood cells through specific ligand-receptor interactions mediated by proteins on the surface of the parasite that interact with receptors on the host erythrocyte (mature red blood cell) or reticulocyte (immature red blood cell), whereas P. falciparum can invade and replicate in erythrocytes and reticulocytes, P. vivax and other species predominantly invade reticulocytes, which are less abundant than erythrocytes. Most of the erythrocyte -binding proteins or reticulocyte -binding proteins that have been associated with invasion are redundant or are expressed as a family of variant forms; however, for P. falciparum, two essential red blood cell receptors (basigin and complement decay-accelerating factor (also known as CD55)) have been identified.

[0414] Plasmodium vivax and Plasmodium ovale can also enter a dormant state in the liver, the hypnozoite.

[0415] Merozoites released from red blood cells can invade other red blood cells and continue to replicate, or in some cases, they differentiate into male or female gametocytes. Gametocytes concentrate in skin capillaries and are then taken up by the mosquito vector in another blood meal. In the gut of the mosquito, each male gametocyte produces eight microgametes after three rounds of mitosis; the female gametocyte matures into a macrogamete. Male microgametes are motile forms with flagellae and seek the female macrogamete. The male and female gametocytes fuse, forming a diploid zygote, which elongates into an ookinete; this motile form secretes a chitinase in order to enter the peritrophic membrane and traverse the midgut epithelium to the basal lateral side of the midgut, establishing itself in the basal lamina as an oocyst Oocysts mature over 14-15 days, undergoing cycles of replication to form sporozoites that are ultimately liberated into the hemocoel, an environment rich in sugars and subtrates beneficial to the parasite’s survival. Thousands of sporozoites can form from a single oocyst and become randomly distributed throughout the hemocoel. These sporozoites are motile and rapidly destroy the hemolymph, with only approximately 20% successfully invading the salivary gland. Following invasion of the salivary gland, sporozoites are re -programmed via an unknown mechanism to prepare for liver invasion. Evidence of this reprogramming has been demonstrated by the inability of midgut sporzoites (directly from oocysts) to invade hepatocytes, and also by the fact that sporzoites which have successfully invaded a salivary gland are unable to do re- invade another salivary gland if presented one. Salivary gland sporozoites alter mosquito behavior and salivary gland function, as less saliva is produced resulting in an increase in mosquito probing behavior, increasing the chances of transmission to a human host via a mosquito bite.

[0416] Some drugs that prevent Plasmodium spp. invasion or proliferation in the liver have prophylactic activity, drugs that block the red blood cell stage are required for the treatment of the symptomatic phase of the disease, and compounds that inhibit the formation of gametocytes or their development in the mosquito (including drugs that kill mosquitoes feeding on blood) are transmission-blocking agents (Phillips, et al. Malaria. Nat Rev Dis Primers 3, 17050 (2017), which is incorporated herein by reference in its entirety).

B. Genome

[0417] Since completion of the first sequence of P. falciparum 3D7 genome in 2002, genomic research on malaria parasites has rapidly advanced. Except for a short diploid phase after fertilization in the mosquito midgut, Plasmodium parasites are haploid throughout their life cycle. The genomes of different species range from 20 to 35 megabases, contain 14 chromosomes, a circular plastid genome of approximately 35 kilobases, and multiple copies of a 6 kilobase mitochondrial DNA. Comparison of genomes from different species showed that homologous genes are often found in synthetic blocks arranged in different orders among different chromosomes. [0418] The adenine-thymine (AT) content of Plasmodium spp. can also be very different, e.g., ~80% AT in P. falciparum, P. reichenowi, and P. gallinaceum; ~75% AT in rodent malaria parasites; and ~60% AT in P. vivax, P. knowlesi, and P. cynomolgi. AT content is often higher in introns and intergenic noncoding regions than in protein-coding exons, with an average of 80.6% AT for the whole P. falciparum genome versus 86.5% for noncoding sequences. The high AT content of P. falciparum reflects large numbers of low-complexity regions, simple sequence repeats, and microsatellites, as well as a highly skewed codon usage bias. Polymorphisms of AT-rich repeats provide abundant markers for linkage mapping of drug resistance genes and for tracing the evolution and structure of parasite populations.

[0419] Malaria parasite genomes carry multigene families that serve important roles in parasite interactions with their hosts, including, for example, antigenic variation, signaling, protein trafficking, and adhesion. Among the gene families, genes encoding P. falciparum erythrocyte membrane protein 1 (PfEMPl) have been studied most extensively. Each individual P. falciparum parasite carries a unique set of 50 to 150 copies of the var gene in its genome, where switches of gene expression can produce antigenic variation. PfEMPl plays an important role in the pathogenesis of clinical developments such as in cerebral and placental malaria, in which it mediates the cytoadherence of infected red blood cells (iRBCs; infected erythrocytes) in the deep tissues. Different PfEMPl molecules bind to various host molecules, including a2- macroglobulin, CD36, chondroitin sulfate A (CSA), complement Iq, CR1, E-selectins and P- selectins, endothelial protein C receptor (EPCR), heparan sulfate, ICAM1, IgM, IgG, PECAM1, thrombospondin (TSP), and VCAM1. Such binding leads to activation of various host inflammatory responses. Hemoglobinopathies, including the hemoglobin C and hemoglobin S trait conditions, interfere with PfEMPl display in knob structures of the iRBCs. This poor display of PfEMPl on the host cell surface offers protection against malaria by reducing the cytoadherence and activation of inflammatory processes that promote the development of severe disease.

[0420] Members of the large Plasmodium interspersed repeat (pir) multigene family are named differently by parasite species, for example, yir in P. yoelii, bir in P. berghei, vir in P. vivax. Several P. falciparum gene families (stevor, rif, and PfMC-2TM) are classified with pir by their similar gene structures, which characteristically include a short first exon, a long second exon, and a third exon encoding a transmembrane domain. In a recent study, the pir genes from P. chabaudi (cir) were shown to be expressed in different cellular locations, within and on the surface of iRBCs, and in merozoites. Malaria parasites devote large portions of their genomes to gene families that ensure evasion of host immune defenses and protection of molecular processes essential to infection. These families emphasize the importance of research on their roles in parasite-host interactions and virulence, despite the difficulties inherent to their investigation.

[0421] An additional, exemplary polymorphic gene family comprises a group of 14 genes encoding proteins with six cysteines (6-Cys). These proteins often localize on the parasite surface interacting with host proteins and are expressed at different parasite developmental stages. 6-Cys proteins also demonstrate diverse functions and have been shown to play roles in, for example, parasite fertilization, mating interactions, evasion of immune responses, and invasion of hepatocytes. The proteins expressed in asexual stages are generally polymorphic and/or under selection, suggesting that they could be targets of the host immune response; however, their functions in parasite development remain largely unknown.

[0422] Plasmodium genomes can be highly polymorphic. Early studies demonstrated polymorphisms involving tens to hundreds of kilobases and that the chromosome structure in P. falciparum is largely conserved in central regions but extensively polymorphic is both length and sequence near the telomeres. Much of the subtelomeric variation was explained by recombination within blocks of repetitive sequences and families of genes.

[0423] The frequency of simple sequence repeats (microsatellites) in P. falciparum is estimated to be approximately one polymorphic microsatellite per kb DNA. Without wishing to be bound by any one theory, this high rate may reflect the AT-rich nature of the genome.

Microsatellites seem to be less frequent in other Plasmodium species that have genomes with lower AT contents. In addition to the highly polymorphic and repetitive structure of Plasmodium genomes, there are also large numbers of Single Nucleotide Polymorphisms (SNPs) and Copy Number Variations (CNVs) (Su et al., Plasmodium Genomics and Genetics: New Insights into Malaria Pathogenesis, Drug Resistance, Epidemiology, and Evolution. Clin Microbiol Rev. 2019 Jul 31;32(4), which is incorporated herein by reference in its entirety).

C. Malarial Proteins [0424] Plasmodium parasites are known to express various proteins at different stages of their lifecycles. Exemplary malarial proteins are described below, and exemplary amino acid sequences are provided in Table 2.

[0425] Circumsporozoite protein (CSP) is a multifunctional protein that is involved in Plasmodium life cycle, as it is required for the formation of sporozoites in the mosquito midgut, the release of sporozoites from the oocyst, invasion of salivary glands, attachment of sporozoites to hepatocytes in the liver, and sporozoite invasion of hepatocytes (see, e.g., Zhao et al. (2016) PLoS ONE 11(8): e0161607). CSP is present in all Plasmodium species, and although variation exists in the amino acid sequence across species, the overall domain structure of a central repeat region and nonrepeat flanking regions is well conserved (see, e.g., Zhao et al. (2016) PLoS ONE 11(8): e0161607; Wahl et al. (2022) J. Exp. Med. 219: e20201313, which are herein incorporated by reference in their entirety). CSP sequences are known (see, e.g., UniProt accession numbers A0A2L1CF52, AOA2L,1CF88, C6FGZ3, C6FH2,7 C6FHG7, M1V060, M1V0A3, M1V0B0, M1V0C4, M1V0E0, M1V9I4, M1VFN9, M1VKZ2, P02893, Q5EIJ9, Q5EIK2, Q5EIK8, Q5EIL3, Q5EIL5, Q5EIL8, Q5R2L2, Q7K740, Q8I9G5, Q8I9J3, Q8I9J4), and Table 1 includes exemplary sequences for CSP P. falciparum isolates from Asia, South America and Africa.

[0426] Table 1A: Exemplary Sequences for CSP P. falciparum isolates from Asia, South America and Africa

[0427] Exemplary CSP amino acid sequence is provided in Table 2A.

[0428] RH5 is found in Plasmodium falciparum (P . falciparum) and not found in the other species of Plasmodium that infect humans. RH5 orthologues are also found in other species belonging to the Lavarenia subgenus, which includes parasites that infect chimpanzees and gorillas, indicating a unique role in P. falciparum invasion of human erythrocytes. See, e.g., Ragotte, et al. Trends Parasitol. 36(6) 2020, which is incorporated herein by reference in its entirety. RH5 is expressed during the mature schizont stages and can complex with Cysteine -rich Protective Antigen (CyRPA) and RH5 -interacting Protein (Ripr) to form an elongated protein trimer on the merozoite surface that binds to erythrocyte surface protein basigin. See, e.g., Ragotte Trends Parasitol 2020 Jun;36(6):545-559, which is herein incorporated by reference in its entirety).

[0429] In humans, RH5 binding to basigin plays an essential role in invasion, acting downstream of membrane deformation. Binding of RH5 to basigin is required for the induction of a spike in calcium within the erythrocyte, which is blocked when merozoites attempt to invade in the presence of anti-RH5, anti-Ripr, or anti-basigin antibodies or soluble basigin. See, e.g., Ragotte (2020).

[0430] RH5 is a 63 kDa protein expressed during the mature schizont stage. It is processed and cleaved to a 45 kDa form which is shed by the parasite. The structure of PfRH5 reveals a kite-like architecture formed from the coming together of two three -helical bundles. See, e.g., Ragotte (2020). [0431] RH5 sequences are known (see, e.g., UniProt accession numbers A0A159SK44, A0A159SK99, A0A159SKS8, A0A159SKW8, A0A159SL23, A0A159SL78, A0A159SL96, A0A159SLM7, A0A159SMC8, A0A159SMR9, A0A161FQT0, A0A1B1UZE2, A0A1B1UZE4, A0A1B1UZE5, A0A346RCI1, A0A346RCJ0, A0A346RCJ2, A0A346RCJ3, A0A346RCJ4, A0A346RCK4, A0A346RCK5, A0A346RCK6, A0A346RCK9, B2L3N7, Q8IFM5), and exemplary RH5 amino acid sequence is provided in Table 2A.

[0432] Pl 13 is a glycosylphosphatidylinositol (GPI)-linked protein that interacts directly with the N terminus of unprocessed RH5, providing a mechanism by which the RH5 invasion complex is tethered to the merozoite surface. See, e.g., Ragotte (2020). Pl 13 orthologues are found in all Plasmodium species sequenced thus far, suggestive of a common and conserved function(s) (Bullen et al. (2022) Molecular Microbiology 117:1245-1262, which is herein incorporated by reference in its entirety). Despite this, in rodent model of malaria, P. berghei, pl 13 knockout parasites were viable indicating the protein was not essential for asexual blood stage growth and invasion. The knockout parasites do, however, display defects in natural sporozoite transmission, leading to delayed patency in infected mice (Offeddu et al. (2014) Mol. Biochem. Parasitology 193: 101-109, which is herein incorporated by reference in its entirety).

[0433] Plasmodium Pl 13 sequences are known (see, e.g., Uniprot accession number Q8ILP3). Exemplary Pl 13 amino acid sequence is provided in Table 2A.

[0434] Cysteine-Rich Protective Antigen (CyRPA) is a 43 kDa protein with a predicted N-terminal secretion signal. CyRPA is part of a multi-protein complex, including RH5 and Ripr, important for triggering Ca²⁺ release and establishment of tight junctions. PfCyRPA is highly conserved, with only a single SNP above 5% prevalence, is essential for invasion (as conditional knockdown causes the loss of invasion activity), and has poor sero-reactivity from natural exposure (See, e.g., Ragotte (2020)).

[0435] Plasmodium CyRPA sequences are known (see, e.g., Uniprot accession number A0A2S1Q7P0, A0A2S1Q7P5, A0A2S1Q7Q4, Q8IFM8). Exemplary CyRPA amino acid sequence is provided in Table 2A.

[0436] RH5 -interacting Protein (Ripr) is an approximately 120 kDa protein and localized to micronemes during the schizont stage of the P. falciparum life cycle. The full-length 120 kDa protein is processed into two fragments of similar size, an N-terminal fragment (including EGF domains 1 and 2) and a C-terminal fragment (including EGF domains 3-10). Ripr colocalizes with RH5 and CyRPA during parasite invasion at the junction between merozoites and erythrocyte. Parasites with conditional knockouts of PfRipr induce membrane deformation, but cannot complete invasion (See, e.g., Ragotte (2020)).

[0437] Plasmodium Ripr sequences are known (see, e.g., UniProt accession numbers A0A193PDI9, A0A193PDK3, A0A193PDK8, A0A193PDL3, A0A193PDL9, A0A193PDP4, A0A193PDQ8, A0A193PE01, A0A193PE05, A0A193PE07, 097302, A0A193PE17). Exemplary Ripr amino acid sequence is provided in Table 2A.

[0438] El 40 is found in every Plasmodium species for which genomic sequence is available, and is well conserved, with amino acid identity ranging from 34-92% among species. See, e.g., Smith , et al. PLoS one 15.5 (2020): e0232234; http://doi: 10.1371/journal.pone.023223; and U.S. Patent Publication No. US 2019/0117752; which are incorporated herein by reference in their entirety. E140 is also highly conserved (95-99%) in P. falciparum strains isolated from different locations around the world, and exhibits a low mutation frequency. E140 is expressed at different life stages of malaria parasites (specifically, E140 has been detected in sporozoites, liver, and blood stage parasites).

[0439] Protein structure algorithms predict that the El 40 protein has five transmembrane domains, presumable spanning a parasite or host-derived membrane. El 40 displays distinct patterns of protein expression in mature sporozoites, late liver, and late schizont stages. It traffics to the anterior and posterior ends of the sporozoite, the parasitophorous vacuole space of the late liver stage and around developing merozoites in the late schizont stage. It is also known to be expressed in mature salivary gland sporozoites as well as oocyst-derived sporozoites and oocysts.

[0440] E140 sequences are known (see, e.g., UniProt accession numbers A0A650D649,

A0A650D653, A0A650D672, A0A650D687, A0A650D690, A0A650D694, A0A650D6A3, A0A650D6B8, A0A650D6L3, A0A650D6L7, Q8I299), and exemplary E140 amino acid sequence is provided in Table 2A.

[0441] CelTOS is required for sporozoite traversal through Kupfer cells during the liver invasion process. CelTOS forms a pore from within the cell, allowing for sporozoite egress into the liver. Antibody epitopes have been characterized from immunized mice and infected human populations (Pf and Pv). In mouse studies, immunization with CelTOS has been shown to provide protection and against challenge. Vaccination with CelTOS may generate antibodies that can bind the extracellular domain of the pore-forming complex, blocking complete formation of the pore and preventing sporozoite traversal into the liver. See, e.g., Jimah et al., Elife 2016 Dec 1; 5:e20621. doi: 10.7554/eLife.20621, which is incorporated herein by reference in its entirety.

[0442] Plasmodium CelTOS sequences are known (see, e.g., Uniprot accession number M1ETJ8, Q53UB7, A0A2R4QLA5, A0A2R4QLI0, A0A2R4QLI5, A0A2R4QLJ1, A0A2R4QLJ4, M1ETJ8, Q53UB8, Q8I5P1). Exemplary CelTOS amino acid sequence is provided in Table 2A.

[0443] SPECT1 and SPECT2 (the latter also sometimes referred to as perforin-like protein 1 (PLP1)) are essential Plasmodium proteins that may play a role in cell traversal. See Yang et al., Cell Rep. 2017 Mar 28; 18(13):3105-3116. doi: 10.1016/j.celrep.2017.03.017, which is incorporated herein by reference in its entirety. Targeted disruption of P . falciparum SPECT1 or SPECT2 has been shown to reduce infectivity of sporozoites in liver-stage development in humanized mice. However, mechanisms of cell traversal of these two proteins are yet to be defined in P. falciparum. See Y ang et al.

[0444] SPECT1 and SPECT2 are considered attractive pre-erythrocytic immune targets due to the key role they are thought to play in the crossing of the malaria parasite across the dermis and the liver sinusoidal wall, prior to invasion of hepatocytes. Recombinant P. falciparum SPECT2 has been shown to cause lysis of red blood cells in a Ca²⁺-dependent manner, as has the MACPF/CDC domain of PfSPECT2. PfSPECT2 has also been implicated in the Ca2+- dependent egress of P. falciparum merozoites from red blood cells.

[0445] Plasmodium SPECT1 and SPECT2 sequences are known (see, e.g., UniProt accession numbers Q8IDR4 and Q9U0J9), and exemplary amino acid sequence is provided in Table 2A.

[0446] Exported protein 1 (EXP1) is a single pass transmembrane protein with an N- terminal signal peptide expressed during intraerythrocytic stage and liver stage (see, e.g., Spielmann et al., Int J Med Microbiol. 2012 Oct; 302(4-5): 179-86, which is herein incorporated by reference in its entirety). EXP1 was shown to initially localize to dense granules in merozoites and then be transported to parasitophorous vacuolar membrane (PVM) after invasion (see, e.g., Iriko et al., Parasitol Int. 2018 Oct; 67(5):637-639, which is herein incorporated by reference in its entirety). Once localized to the PVM, EXP1 forms homo-oligomers with a N-terminus that is exposed to the parasitophorous vacuolar lumen and a C-terminus that is exposed to the red blood cell cytosol (see, e.g., Mesen-Rarmrez et al., PLoS Biol. 2019 Sep 30;17(9):e3000473, which is herein incorporated by reference in its entirety).

[0447] EXP1 has been demonstrated to possess glutathione S-transferase (GST) activity that may protect Plasmodium from oxidative damage (see, e.g., Mesen-Rarmrez et al., PLoS Biol 17(9) 2019 Sep 30; 17(9):e3000473, which is herein incorporated by reference in its entirety). Recently, it was demonstrated that EXP1 is important for Plasmodium survival by maintaining correct localization of EXP2, a nutrient-permeable channel in the PVM (see, e.g., Mesen- Rarmrez et al., 2020).

[0448] P. falciparum EXP1 polypeptide sequences are known (see, e.g., UniProt accession number Q8IIF0, W7JTD3, Q25840, Q548U2, Q5VKK2, Q5VKK5, Q5WRH8, Q6V9G4, Q6V9G6, Q6V9G9, Q6V9H1, Q6V9H2, Q9U590, P04923, P04926). Exemplary EXP1 amino acid sequence is provided in Table 2A.

[0449] Upregulated in infective sporozoites gene 3 (UIS3) is a membrane -bound protein localized to sporozoite parasitophorous vacuolar membrane (PVM) in infected hepatocytes.

UIS3 was shown to interact with liver fatty acid-binding protein (L-FABP) and be involved in fatty acid and/or lipid import during phases of Plasmodium growth (see, e.g., Sharma et al., J Biol Chem. 2008 Aug 29; 283(35): 24077-24088; Mikolajczak et al., Int J Parasitol. 2007 Apr;37(5):483-9, which are herein incorporated by reference in their entirety).

[0450] After sporozoite invasion of host liver cells, there is synthesis of vital

Plasmodium structural features (e.g., parasitophorous vacuolar membrane). During hepatocytic stages, the Plasmodium relies on host fatty acids for rapid synthesis of its membranes (see, e.g., Sharma et al., J Biol Chem. 2008 Aug 29; 283(35): 24077-24088, which is herein incorporated by reference in its entirety). UIS3 insertion in the PVM provides Plasmodium a method to import essential fatty acids and/or lipids during rapid sporozoites growth phases (see, e.g., Sharma et al., 2008).

[0451] Immunization with UIS 3 -deficient Plasmodium berghei sporozoites protected against malaria in rodent malaria model (see, e.g., Mueller et al., Nature. 2005 Jan 13; 433(7022): 164-7, which is herein incorporated by reference in its entirety). UIS 3 -deficient Plasmodium berghei can start the transformation process in the liver; however, they show severe defects during transformation into trophozoites (see, e.g., Mueller et al., 2005). UIS 3 -deficient Plasmodium berghei are also unable to develop into mature liver schizonts and therefore abort malaria infection within the liver itself (see, e.g., Mueller et al., 2005). Further, it was previously demonstrated that UIS3 derived from Plasmodium berghei and UIS3 derived from Plasmodium falciparum exhibited a low (i.e. 34%) amino acid sequence identity (see, e.g., Mueller et al., 2005).

[0452] Plasmodium UIS3 sequences are known (see, e.g., UniProt accession number A0A509ARS3, A0A1C6YLP3, Q8IEU1, A0A384KLI1, A0A1G4H423, A0A077YB01, Q9NFU4). Exemplary UIS3 amino acid sequence is provided in Table 2A.

[0453] Upregulated in infective sporozoites gene 4 (UIS4) contains a single transmembrane domain and localizes to secretory organelles of sporozoites and to the parasitophorous vacuole membrane (PVM) of liver stages. UIS4 is not expressed in blood stages or early sporozoites that are produced in oocysts (see, e.g., Mackellar et al., Eukaryot Cell. 2010 May; 9(5): 784-794, which is herein incorporated by reference in its entirety).

[0454] Deletion of UIS4 gene is associated with arrest of early liver stage development (see, e.g., Vaughan and Kappe, Cold Spring Harb Perspect Med. 2017 Jun 1; 7(6):a025486, which is herein incorporated by reference in its entirety). Recently, UIS4 was demonstrated to be involved in Plasmodium berghei survival by eluding host actin structures deployed as part of host cytosolic defense (see, e.g., Bana et al., iScience. 2022 Apr 22;25(5): 104281. doi: 10.1016/j.isci.2022.104281. eCollection 2022 May 20, which is herein incorporated by reference in its entirety). P. falciparum has an ortholog to UIS4 named ETRAMP10.3 which is not able serve as a functional compliment to P. yoelii UIS4, indicating it likely serves a different function in P. falciparum ’s life cycle (see Mackellar et al., Eukaryot. Cell 9:784-94 (2010), which is herein incorporated by reference in its entirety).

[0455] Plasmodium UIS4 sequences are known (see, e.g., UniProt accession number Q8IJM9). Exemplary UIS4 amino acid sequence is provided in Table 2A.

[0456] Liver specific protein 1 (LISP-1) is expressed during Plasmodium development in hepatocytes and localized to the parasitophorous vacuolar membrane (PVM) (see, e.g., Ishino et al., Cell Microbiol. 2009 Sep; 11(9): 1329-1339). LISP-1 was shown to be expressed at high levels during late liver stages development and to be involved in PVM breakdown and subsequent merozoite release (see, e.g., Ishino et al., Cell Microbiol. 2009 Sep; 11(9): 1329- 1339, which is herein incorporated by reference in its entirety).

[0457] Intracellular Plasmodium deficient in LISP- 1 develop into hepatic merozoites and display normal infectivity to erythrocytes (see, e.g., Ishino et al., Cell Microbiol. 2009 Sep;

11(9): 1329-1339, which is herein incorporated by reference in its entirety). However, LISP1- deficient liver-stage Plasmodium do not rupture PVM and remain trapped inside hepatocytes (see, e.g., Ishino et al., 2009).

[0458] Plasmodium LISP-1 sequences are known (see, e.g., UniProt accession number A0A2I0C2X6, Q8ILR5). Exemplary LISP-1 amino acid sequence is provided in Table 2.

[0459] Liver specific protein 2 (LISP-2) contains a modified 6-cys domain and is expressed during Plasmodium development in hepatocytes (see, e.g., Orito et al., Mol Microbiol. 2013 Jan; 87(l):66-79, which is herein incorporated by reference in its entirety). LISP-2 was shown to be expressed by liver stages Plasmodium, exported to hepatocytes, and be distributed throughout the host cell, including the nucleus (see, e.g., Orito et al., 2013).

[0460] Intracellular Plasmodium deficient in LISP2 do not mature effectively during merozoites development (see, e.g., Orito et al., 2013).

[0461] Plasmodium LISP-2 sequences are known (see, e.g., UniProt accession number A0A2I0BZR4, Q8I1X6, Q9U0D4). Exemplary LISP-2 amino acid sequence is provided in Table 2A.

[0462] Thrombospondin-related adhesion protein (TRAP) contains an N-terminal domain that is commonly referred to as von Willebrand factor A domain, although it is most similar to an integrin I domain because it contains a metal ion-dependent adhesion site (MIDAS) with a bound Mg²⁺ ion that is required for sporozoite motility in vitro and infection in vivo (see, e.g., Lu et al., PLoS One. 2020; 15(1): e0216260, which is herein incorporated by reference in its entirety). The I domain is inserted in an extensible P-ribbon and followed by a thrombospondin repeat (TSR) domain, a proline -rich segment at the C-terminus, a single-pass transmembrane domain, and a cytoplasmic domain (see, e.g., Lu et al., 2020). Sequence analysis of the proline- rich segment revealed the presence of SH3 -domain binding PxxP motifs in Plasmodium TRAPs (Akhouri et al., Malar J. 2008 Apr 22; 7:63. doi: 10.1186/1475-2875-7-63, which is herein incorporated by reference in its entirety).

[0463] TRAP is stored in the micronemes and becomes surface exposed at the sporozoite anterior tip when parasite comes in contact with host cells (Akhouri et al., Malar J. 2008 Apr 22;7:63. doi: 10.1186/1475-2875-7-63, which is herein incorporated by reference in its entirety). TRAP also plays an important role in liver cell invasion of sporozoites by helping sporozoites in gliding motility and in recognition of host receptors on the mosquito salivary gland and hepatocytes (Akhouri et al., Malar J. 2008 Apr 22;7:63. doi: 10.1186/1475-2875-7-63, which is herein incorporated by reference in its entirety).

[0464] Plasmodium TRAP sequences are known (see, e.g., UniProt accession numbers A0A5Q2EXK8, A0A5Q2EZD7, A0A5Q2F1F6, A0A5Q2F2B8, A0A5Q2F2H6, A0A5Q2F4G9, 076110, P16893, Q01507, Q26020, Q76NM2, W8VNB6), and exemplary TRAP amino acid sequence is provided in Table 2A.

[0465] Liver-stage-associated protein (LS AP- 1 ) has been shown to be found mainly at the periphery of the intracellular hepatic parasite throughout its development, but not in blood stage parasites and possibly in minor quantities in salivary gland sporozoites (see, e.g., Siau et al., PLoS Pathog. 2008 Aug 8;4(8):el000121, which is herein incorporated by reference in its entirety). LSAP-1 is among the most abundant transcripts in the salivary gland transcriptome but has not been detected in proteomic surveys of sporozoites. Rather, expression has only been detected only in liver stages (see, e.g., Siau et al., 2008).

[0466] Plasmodium LSAP-1 sequences are known (see, e.g., UniProt accession number Q8I632, W7JR53). Exemplary LSAP-1 amino acid sequence is provided in Table 2A.

[0467] Like LS AP- 1 , LS AP-2 is also among the most abundant transcripts in the salivary gland transcriptome but has not been detected in proteomic surveys of sporozoites. LSAP-2 has shown some efficacy as a vaccine when combined with other antigens. See, e.g., Halbroth et al., Infect Immun. 2020 Jan 22; 88(2):e00573-19. doi: 10.1128/IAI.00573-19. Print 2020 Jan 22, which is incorporated herein by reference in its entirety. [0468] Plasmodium LSAP-2 sequences are known (see, e.g., UniProt accession number Q8I632, W7JR53). Exemplary LSAP-2 amino acid sequence is provided in Table 2.

[0469] Liver-Stage Antigen 1 (LSA-1) is expressed after Plasmodium have invaded hepatocytes and antigen accumulates in the parasitophorous vacuole (see, e.g., Tucker, K. et al., 2016, 'Pre-Erythrocytic Vaccine Candidates in Malaria', in A. J. Rodriguez-Morales (ed.), Current Topics in Malaria, IntechOpen, London. 10.5772/65592, which is herein incorporated by reference in its entirety). The function of LSA-1 remains currently not known (see, e.g., Tucker, K. et al., 2016).

[0470] LS A- 1 is a 230 kDa preerythrocytic stage protein containing a large central region consisting of over eighty 17 amino acid residue repeat units flanked by highly conserved C- and N-terminal regions (Richie, T.L. and Parekh, L.K. (2009) Malaria. In Vaccines for Biodefense and Emerging and Neglected Diseases (Barrett, A.D.T. and Stanberry L.R., eds), pp. 1309-1364, Elsevier, which is herein incorporated by reference in its entirety). LSA1 is expressed only by liver stage Plasmodium and not by sporozoites (Richie, T.L. and Parekh, L.K. (2009) Malaria, which is herein incorporated by reference in its entirety). In Vaccines for Biodefense and Emerging and Neglected Diseases (Barrett, A.D.T. and Stanberry L.R., eds, pp. 1309-1364, Elsevier, which is herein incorporated by reference in its entirety). The repeat region results in significant variation of the protein between strains of Plasmodium falciparum (see, e.g., Tucker, K. et al., 2016).

[0471] Plasmodium LSA-1 sequences are known (see, e.g., UniProt accession number Q25886, Q25887, Q25893, Q26028, Q9GTX5, 096125). Exemplary LSA-1 amino acid sequence is provided in Table 2A.

[0472] Liver stage antigen 3 (LSA-3) is a 200-kDa protein that is composed of three nonrepeating regions (NR- A, NR-B, and NR-C) flanking two short repeat regions and one long repeat region (see, e.g., Tucker, K. et al., 2016). The nonrepeat regions are well conserved across geographically diverse strains of Plasmodium falciparum (see, e.g., Tucker, K. et al., 2016). The most significant variation is in the repeating regions due to organization and number of repeating subunits rather than composition of the repeating regions (see, e.g., Tucker, K. et al., 2016).

[0473] Recently, in vitro data has shown that antibodies against LSA-3 (in particular, the C-terminal portion of LSA-3) may provide some protection (see, e.g., Morita et al, Sci Rep. 2017 Apr 5; 7:46086. doi: 10.1038/srep46086, which is herein incorporated by reference in its entirety).

[0474] Plasmodium LSA-3 sequences are known (see, e.g., UniProt accession number C7DU21, C7DU22, C7DU23, C7DU24, C7DU25, C7DU26, C7DU27, C7DU28, C7DU29, C7DU32, C7DU33, C7DU34, C7DU36, C7DU37, C7DU38, C7DU39, C7DU40, Q8I042, Q8I0A5, Q8I0D0, Q8IFR1, Q8IFR2, Q8IFR3, Q8IFR4, Q8IFR5, Q8IFR6, Q8IFR7, Q8IFR8, Q8IFR9, Q8IFS0, Q8IFS1, Q8IFS2, Q8IFS3, Q8IFS4, Q8IFS5, Q8IFS6, Q8IFS7, Q8IFS8, Q8IFS9, Q8IFT0, Q8IFT1, Q8IFT2, Q8IFT3, Q8IFT4, Q9U0N9, Q9U0P0, A0A2I0BVD6, A0PFM9, 096275). Exemplary LSA-3 amino acid sequence is provided in Table 2A.

[0475] Glutamic acid-rich protein (GARP) is a 80kDA protein which derives its name from its glutamic rich amino acid sequence which comprises 24% of all its residues. GARP is predominantly expressed in ring stages and trophozoites and has been shown to be a non- essential gene in cell culture but highly immunogenic in animal models (Hon et al., Trends Parasitol. 2020 Aug; 36(8):653-655, which is herein incorporated by reference in its entirety). Although GARP is non-essential in cell culture, its localization to the periphery of infected erythrocytes may indicate a role in the sequestration of infected erythrocytes. GARP’s involvement in sequestration has been proposed to occur by way of binding with a chloride/bicarbonate anion exchanger (Lau et al., PLoS Pathog. 10, el004135. 2014, which is herein incorporated by reference in its entirety). Antibodies against GARP have been proposed to serve as signatures of protection against severe malaria and have shown efficacy in experimental trials in monkeys. See, e.g., Hon et al, Trends in Paras 2020 Aug; 36(8):653-655. doi: 10.1016/j.pt.2020.05.012 and Laue et al, Pios Path. 2014 10, el004135, which are herein incorporated by reference in their entirety. GARP sequences are known (see, e.g., UniProt accession number, Q9GTW3, Q9U0N1), and exemplary GARP amino acid sequence is provided in Table 2A.

[0476] Parasite-infected erythrocyte specific protein 2 (PIESP2) (see, e.g., UniProt accession number Q8I488) is a highly immunogenic protein first expressed in the trophozoite stage and believed to be important for the clinical progression of cerebral malaria. Although this protein is predominantly found within erythrocytes, it has been shown to be present on the surface of erythrocytes, allowing them to adhere to endothelial cells in the vasculature of the brain. Antibodies against PIESP2 have been shown to prevent vascular adherence of plasmodium and could prove valuable in preventing the preventing inflammatory response in the brain and impairment of the blood-brain barrier during cerebral malaria progression (see, e.g., Liu et al, Int J Biol Macromol. 2021 Apr 30;177:535-547. doi: 10.1016, (j.ijbiomac.2021.02.145, which is herein incorporated by reference in its entirety). PIESP2 sequences are known (see, e.g., UniProt accession number Q8I488), and exemplary PIESP2 amino acid sequence is provided in Table 2A.

[0477] Shizont egress antigen- 1 (SEA1) is a large 244 kDA protein lacking transmembrane domains or known targeting signals. The function of SEA1 is not known; however, it has been shown to be effective in rodent vaccine studies and has even been proposed as a target of protective antibodies found in children. SEA1 received its name after it was reported that antibodies agasint this protein inhibited egress of plasmodium merizoites. SEA1 localizes closely to centromers during nuclear division, implicating its role in the essential process of replication. To date, various studies have proposed a role for SEA1 in egress, but also in mitotic division of nuclei during replication, (see, e.g., Perrin et al, mBio. 2021 Mar 9;12(2):e03377-20. doi: 10.1128/mBio.03377-20, which is herein incorporated by reference in its entirety). SEA1 sequences are known (see, e.g., UniProt accession number A0A143ZXM2), and exemplary SEA1 amino acid sequence is provided in Table 2A.

D. Embodiments of Malarial Sequences

[0478] An exemplary full length CSP polypeptide amino sequence from Plasmidum falciparum isolate 3D7 is presented in Table 2A as SEQ ID NO:A, and includes the following: a secretory signal (amino acids 1-18); an N-terminal domain (amino acids 19-104); a junction region (amino acids 93-104), a central domain (amino acids 105-272); and a C-terminal domain (amino acids 273-397). In exemplary SEQ ID NO: A, the N-terminal domain includes an N- terminal region (amino acids 19-80); an N-terminal end region (amino acids 81-92); and a junction region (amino acids 93-104). In exemplary SEQ ID NO:A, the junction region includes an R1 region (amino acids 93-97) and amino acids ADGNPDP (SEQ ID NO: B) at positions 98- 104. In exemplary SEQ ID NO:A, the central domain includes a minor repeat region (amino acids 105-128) and a major repeat region (amino acids 129-272). In exemplary SEQ ID NO:1, the minor repeat region includes three repeats of the amino acid sequence NANPNVDP (SEQ ID NO:C). In exemplary SEQ ID NO: 1, the major repeat region includes 35 repeats of the amino acid sequence NANP (SEQ ID NO: D), wherein 35 repeats of the amino acid sequence NANP are separated into two contiguous stretches, and wherein one stretch includes 17 repeats of the amino acid sequence NANP and one includes 18 repeats of the amino acid sequence NANP which flank an amino acid sequence of NVDP (SEQ ID NO: E). The major repeat region includes the amino acid sequences NPNANP (SEQ ID NOT) and NANPNA (SEQ ID NO:G). In exemplary SEQ ID NO:A, the C-terminal domain includes a C-terminal region (amino acids 273-375) and a transmembrane domain (amino acids 376-397). In exemplary SEQ ID NO:A, the C-terminal region includes a Th2R region (amino acids 314-327) and a Th3R region (amino acids 352-363).

[0479] Table 2A: Exemplary amino acid sequences

Influenza Overview

[0480] Influenza illness is caused by influenza viruses, of which there are four types: A, B, C, and D. Types A and B are responsible for the seasonal epidemics that occur every winter in the United States (also known as flu season). Type A viruses are the only type to date that have caused a pandemic (i.e., a global epidemic). Type C viruses generally cause mild illness and are not thought to cause human epidemics, while type D viruses primarily affect cattle, and are not known to infect or cause illness in humans.

[0481] Influenza A viruses are divided into subtypes based on two surface proteins: hemagglutinin (HA) and neuraminidase (NA). 18 HA subtypes and 11 different NA subtypes are known to exist, and more than 130 influenza A subtype combinations have been observed, although many more subtype combinations are possible, given the virus’s propensity for “reassortment” (i.e., the process in which influenza viruses swap gene segments, which can occur when two viruses infect a host at the same time). Subtypes H1N1 and H3N2 are the type A viruses that are currently common in humans. Subtypes can be further broken down into “clades” and “sub-clades” (also known as “groups” and “sub-groups”, respectively), which are organized based on HA gene sequences.

[0482] Clades and sub-clades may be genetically distinct from one another while not being antigenically distinct. For example, it may be possible for two viruses to have distinct HA gene sequences, and thus be genetically distinct, and yet still be bound and neutralized by a given antibody, and thus not antigenically distinct.

[0483] Currently circulating influenza A (H1N1) viruses are related to the 2009 H1N1 virus that emerged in the spring of 2009 and caused the flu pandemic of that year. These viruses, also called A(HlNl)pdm09 viruses or “2009 HINT’, have continued to circulate seasonally since first being discovered, and have undergone several changes both genetically and antigenically.

[0484] Influenza A (H3N2) viruses also comprise many separate, genetically different clades in recent years that contine to circulate.

[0485] Influenza B viruses are classified by lineage rather than subtype. Two lineages of influenza B viruses exist: B/Yamagata and B/Victoria, each of which can be further divided into clades and sub-clades. Influenza B viruses generally change more slowly than influenza A viruses, both genetically and antigenetically. In recent years, both B/Yamagata and B/Victoria have been in co-circulation, although the proportion from each lineage can vary depending on location and season.

[0486] Influenza virus names usually indicate type (A, B, C, D), host of origin (although for humans, the host of origin is usually not indicated), geographical origin, strain number, and year of collection. For influenza A viruses, HA and NA descriptions are provided in parenthesis.

Seasonal flu vaccines are typically formulated to provide protection against multiple influenza viruses that are known to cause epidemics. In recent years, vaccines have been formulated as tetravalent vaccines, to provide antigens against H1N1, H3N2, B/Victoria, and B/Yamagata viruses. In some embodiments, an influenza vaccine can protect both against the viruses that the vaccine comprises or delivers antigens from, and antigenically similar viruses.

Norovirus Overview [0487] Noroviruses are members of the Caliciviridae family of small, non-enveloped, positive-stranded RNA viruses. The Norovirus genus includes both human and animal (e.g., murine and canine) noroviruses.

[0488] Noroviruses typically have a 24-48 hour incubation period between infection and development of symptoms. Symptoms typically persist for 12-72 hours, but reports have indicated that viral shedding can continue long after symptoms have resolved. It is believed that viral shedding can continue for several days or even 1-2 weeks after symptoms have resolved; immunocompromised individuals may continue shedding virus even longer, up to several (e.g., 3, 4, 5, 6, 7, 8 or more) months after infection..

[0489] Noroviruses are highly infectious; it has been reported that doses as low as 20 viral particles may be sufficient to establish infection. Exposure is typically via inhalation or ingestion (e.g., commonly by oral exposure, such as by ingestion of contaminated food). Norovirus virions withstand acidic pH and can survive passage through the stomach.

[0490] Given the above-noted low infection dose and long shedding periods, combined with the high levels of shedded virus ( 10⁸- 10¹⁰ copies of RNA per g) often detected in feces, norovirus infections can spread rapidly within communities.

[0491] Norovirus infection can be asymptomatic, particularly in children (see, for example, Robilotti et al., Clin. Microbiol. Rev., 28: 134, 2015 and references cited therein).

Symptomatic infection typically results in acute gastroenteritis, characterized by symptoms such as vomiting and diarrhea, and/or nausea and severe abdominal cramps. Other reported associated conditions include encephalopathy, intravascular coagulation, necrotizing enterocolitis in premature infants, postinfectious irritable bowel syndrome, and benign infantile seizures. Young children, the elderly, and immunocompromised individuals (e.g., transplant patients or other subjects receiving immunosuppressive medication or therapy) are among those most susceptible to development of serious disease.

[0492] Although it has been reported that 20-30% of cases of norovirus infection in humans can be asymptomatic or “mild” (e.g., resolving within a few days) (Qi et al. Am J Infect Control. 43:833, 2015, doi: 10.1016/j.ajic.2015.04.182; Marshall et al., J Med Virol. 69:568, 2003, doi: 10.1002/jmv.10346), norovirus infection remains a significant risk. Mortality may be as high as 3%, and norovirus infections are believed to be responsible for up to 20% of emergency room visits and hospitalizations, even in middle- to high- income countries (Lopman et al., PLoS Med. 13:el001999, 2016, doi: 10.1371/journal.pmed.l001999). Dehydration associated with norovirus infection can be particularly problematic, particularly in the elderly and/or the very young. Furthermore, in some instances (e.g., in immunocompromised subjects including, for example, transplant patients, patients receiving chemotherapy or immunosuppressive therapy, subjects infected with HIV, etc.) (Cardemil et al. Infect Dis Clin North Am. 31:839, 2017, doi: 10.1016/j .idc.2017.07.012), norovirus infection can become chronic, with serious consequences (Bok et al., Oncol Nurs Forum. 40:434, 2013, doi: 10.1188/13. ONF.434-436; Kaufman et al. Antiviral Res. 105C:80, 2014, doi: 10.1016/j.antiviral.2014.02.012; Trivedi et al. Am J Infect Control. 41:654, 2013, doi:

10.1016/j.ajic.2012.08.002). Without wishing to be bound by any particular theory, the present disclosure proposes that a robust T cell immunization, e.g., as may be achieved as described herein (e.g., via administration or delivery of one or more T cell epitopes as described here, for example via string constructs), may be particularly useful or effective to protect against chronic infection, e.g., by facilitating removal of infected cells.

[0493] No commercial vaccines or specific antivirals are currently available to treat or prevent human norovirus infections. Standard of care remains supportive therapy, particularly to address dehydration and/or electrolyte abnormalities. Some reports have suggested that administration of nitazoxanide may be helpful, for example, to reduce the duration of illness (see, for example, Rossignol et al. Aliment Pharmacol Ther 24:1423, 2006, doi.org/10. I l l 1/j.1365-2036.2006.03128.x.). Enteric administration of human immunoglobulin has also been reported to assist in resolution of diarrhea associated with norovirus infection (see, for example, Chagla et al, J Clin Virol 55:306, 2013, doi.org/10.1016/j.jcv.2013.06.009).

[0494] Furthermore no small animal models have been described that mimic human disease; only recently has an in vivo model (in zebrafish larvae) been shown to support norovirus replication (e.g., of GII.3 and GII.4 variants; see Van Dycke et al., PLoS Pathog. 15:el008009, 2019, doi: 10.1371/journal.ppat.1008009). Some in vitro replication models have been described; specifically, some strains (e.g., Gii.4-Sydney) have been shown to replicate in human B cells (see, for example, Lindesmith et al., J Infect Dis. 216: 1227, 2017, doi:

10.1093/infdis/jix385); and some (e.g., some GII.3 and some GII.4 strains) have been shown to replicate in human intestinal enteroid monolayer cultures (see, for example, Ettayebi et al., Science. 353:1387, 2016, doi: 10.1126/science.aaf5211). Also, a monoclonal antibody (NV8812; see White et al. J Virol. 70:6589-97. doi: 10.1128/JVI.70.10.6589, 1996) to the viral VP1 protein that has been reported to bind to the C-terminal region at residues 300-384, has been reported to block binding of virus-like particles (VLPs) comprising the norovirus VP1 protein to relevant human and animal cells.

[0495] An effective norovirus vaccine remains an unmet medical need of critical importance for global health.

Lifecycle

[0496] Infection by a norovirus begins when the viral capsid binds to a host cell surface receptor; histo-blood group antigens (HBGA) have been described as potential receptors or co- receptors. HBGAs are polymorphic glucans found on the surfaces of red blood cells and of certain epithelial cells (de Graaf et al. Nat Rev Microbiol. 14:421, 2016, doi: 10.1038/nrmicro.2016.48; Mallagaray et al. Nat Commun. 10: 1320, 2019, doi: 10.1038/s41467- 019-09251-5, each of which is incorporated herein by reference in its entirety). It has been reported that individuals who do not express fucosyltransferase 2 (Fut2), which generates HBGAs, are not susceptible to norovirus infection (de Graaf et al. Nat Rev Microbiol. 14:421, 2016, doi: 10.1038/nrmicro.2016.48, which is incorporated herein by reference in its entirety). Moreover, studies have shown that noroviruses recognize a determined group of HBGAs (Huang et al. J Virol. (2005) 79:6714, 2005, doi: 10.1128/JVI.79.11.6714-6722.2005, which is incorporated herein by reference in its entirety); at least four different binding patterns of human noroviruses have been described based on ABO blood type, Lewis blood group, and fut2 status (secretor/nonsecretor) (Huang et al. J Infect Dis. 188: 19, 2003, doi: 10.1086/375742, which is incorporated herein by reference in its entirety). Generally, noroviruses that have HBGA type A/B binding patterns recognize the A and/or B and H antigens, but not the Lewis antigens; and noroviruses that have Lewis binding patterns bind only to Lewis antigens and/or the H antigen (Huang et al. J Virol. 79:6714, 2005, doi: 10.1128/JVI.79.11.6714-6722.2005, which is incorporated herein by reference in its entirety). [0497] After binding, virus becomes internalized, uncoated, and disassembled; host factors are recruited to replicate and translate the genome {reviewed in de Graaf et al., Nat Rev Microbiol. 14:421, 2016, which is incorporated herein by reference in its entirety).

[0498] The genomes of noroviruses that infect humans comprise a linear, positive-sense RNA strand about 7.3-8.3 kb long (often about 7.5-7.7 kb). The 5’ end of the norovirus genome is covalently linked to one of the nonstructural proteins (the VPg protein) it encodes; the 3’ end is polyadenylated.

[0499] Upon internalization, the viral genome is released from the VPg protein, which then recruits host translation initiation factors (e.g., eIF3) and initiates assembly of the translation complex.

[0500] As described in more detail below, translation produces three proteins: structural VP1 and VP2 proteins, and a polyprotein that is autocleaved to produce six (6) non-structural viral proteins, via a cascade that first generates three protein precursors, each of which becomes cleaved into two viral proteins.

[0501] Replication proceeds by transcribing the (+-strand) genome to generate (- strand) RNAs that become templates for synthesis of new (+-strand) genomic and subgenomic RNAs. These subgenomic RNAs contain the ORFs for VP1 and VP2, and are translated to produce these proteins. Replicated genomic RNAs are assembled into new virions that are released from the infected host cells.

Genome

[0502] The norovirus genome includes short untranslated regions (UTRs) at either end; these contain evolutionarily conserved structures that are thought to participate in replication, translation, and/or pathogenesis.

[0503] The norovirus genome includes three open reading frames (ORFs 1, 2, and 3) that together encode eight viral proteins (reviewed in, Robilotti et al., Clin Microbiol Rev. 28:134, 2015, which is incorporated herein by reference in its entirety). ORF-2 and ORF-3 encode the structural components of the virion, viral protein 1 (VP1) and VP2, respectively. ORF-1 encodes the above-mentioned polyprotein that is proteolytically processed into the six nonstructural proteins of the virus: p48 (NA1/NS2), NTPase (NS3), p22(NS4), VPg (N5), Pro (NS6), and Pol (NS7; RdRp), these last two being the norovirus protease and RNA-dependent RNA polymerase, respectively. See, review of norovirus proteins in Compillay- Veliz et al. Front Immunol 11:961, 2020, which is incorporated herein by reference in its entirety)

[0504] VP1 is the primary structural protein of the capsid; 90 dimers of VP1 assemble into the icosahedral (T = 3) capsid, with only a few copies of VP2 included. VP1 includes a shell (S) domain and a protruding (P) domain, with Pl and P2 components (see, for example, Prasad et al., Science 286:287, 1999, doi: 10.1126/science.286.5438.287, which is incorporated herein by reference in its entirety). The S domain makes up the core of the capsid, from which the P domain protrudes. The S domain is involved in binding VP2, thereby associating it with the capsid. The P domain, and particularly, P2, mediates binding to host HBGA molecules (see, e.g., Campillay- Veliz el al., Front. Immunol. 11:961, 2020, doi: 10.3389/fimmu.2020.00961, which is incorporated herein by reference in its entirety). The P domain also mediates interactions between VP1 proteins and therefore impacts size and stability of viral capsids.

[0505] The S domain is located in the N-terminal portion of the VP1 protein, for example extending from about residue 225 to the end, according to canonical numbering systems. The Pl domain is typically considered to begin at residue 226 according to canonical numbering systems, and to be interrupted by the P2 domain, so that Pl includes residues 226-278 and 406-52, and P2 includes residues 278-406 according to canonical numbering systems.

[0506] The P2 subdomain is the most variable region of the VP1 protein, and is believed to be surface exposed on the viral capsid. P2 variants have been reported to be associated with particular epidemic outbreaks (see, for example, 22). The Pro protein is responsible for cleaving the polyprotein generated by translation of ORF1, first into p48/NTPase, p22/VPg and Pro/Pol precursor proteins, and ultimately into the six individual proteins.

[0507] The Pol, VPg, NTPase and p48 proteins have all been reported to play roles in viral replication. NTPase has been reported to have helicase, NTP hydrolase, and chaperone activities; p48 has been reported to increase Pol activity, and also disassembly of the trans-Golgi network, resulting in interference with host cell signaling pathways involved in immune response. [0508] P22 has also been reported to contribute to trans-Golgi disassembly (36), and also to facilitate virion release from cells.

[0509] At least ten (10) different genogroups (GI-GX) of noroviruses have been defined {see, for example, Chhabra et al. J Gen Virol. 100: 1393 406, 2019, doi: 10.1099/jgv.0.001318; see also, Campillay- Veliz et al., Front. Immunol, 11:961, 2020, doi: 10.3389/fimmu.2020.00961, each of which is incorporated herein by reference in its entirety) based on similarity of highly- conserved regions of either the Pol (NS7; RdRp) protein or (ii) VP1 {e.g., the amino acidic regions of VP1, such as are found in the S domain); three of these genogroups (specifically, GI, GII, and GIV) infect and cause acute gastroenteritis in humans. Norovirus genogroups have been further subdivided into genotypes, which in turn include strains and variants {e.g., that arise by mutation). Recombination between or among variants also gives rise to new strains {see, for example, Cannon J Virol. 83:5363, 2009, doi: 10.1128/JVI.02518-08; see also Vinje J Infect Dis. 176:1374, 1997, doi: 10.1086/517325, each of which is incorporated herein by reference in its entirety). The GII.4 genotype is the most prevalent worldwide; its Sydney and New Orleans variants are particularly prevalent {see, Glass et al N Engl J Med. 361: 1776, 2009. doi: 10.1056/NEJMra0804575; Vinje et al. J Infect Dis. 176: 1374, 1997, doi: 10.1086/517325; Tamminen et al Viruses. 11:91, 2019 doi: 10.3390/vl 1020091, each of which is incorporated herein by reference in its entirety) and a GII.P16-GII.4 Sydney recombinant strain was responsible for a 2015 pandemic {see, Lindesmith et al. J Infect Dis. 217: 1145, 2017, doi: 10.1093/infdis/jix651, which is incorporated herein by reference in its entirety). Other highly infectious genotypes include GII.17 {see, for example, Lindesmith et al. J Infect Dis. 217:1145, 2017, doi: 10.1093/infdis/jix651; see also, Lindesmith et al. J Infect Dis. 216:1227, 2017, doi: 10.1093/infdis/jix385, each of which is incorporated herein by reference in its entirety).

[0510] To give an example of norovirus genogrouping, a system has been described in which viruses whose VP1 protein sequences differ by less than 14.3% are classified in the same strain; those whose VP1 protein sequences differ by 14.3-43.8% are classified in the same genotype, and those whose VP1 protein sequences differ between 45-61.4% are classified in the same genogroup {see Zheng et al. Virology 346:312, 2006, doi: 10.1016/j.virol.2005.11.015, which is incorporated herein by reference in its entirety). [0511] It has been reported that individuals infected with norovirus of one genogroup do not typically develop immunity to other genogroups (see, e.g., Esposito and Principi, Front. Immunol. 11: 1383, 2020; see also, Atmar et al., Curr. Opin. Infect. Dis. 31: 422 (2018); and Brown et al. J. Clin. Virol. 96:44 (2017), each of which is incorporated herein by reference in its entirety) even when they may develop immunity to other strains or variants within the genogroup with which they were infected. In some embodiments, as described herein, provided technologies administer or deliver (e.g., by administration of an encoding RNA) polypeptides that, together, are or comprise epitopes from multiple genotypes (e.g., GI and GII) and/or multiple clades.

Norovirus Antigens

[0512] In some embodiments, the present disclosure provides certain norovirus antigen constructs particularly useful in effective vaccination.

[0513] Antigens utilized in accordance with the present disclosure are or include norovirus components (e.g., proteins or fragments or epitopes thereof, including epitopes that may comprise non-amino acid, e.g., carbohydrate moieties), which components induce immune responses when administered to humans (or other animals such as rodents and non-human primates susceptible to norovirus infection).

Norovirus Protein Sequences

[0514] In some embodiments, a provided pharmaceutical composition (e.g., immunogenic composition, e.g., norovirus vaccine) comprises or delivers (e.g., causes expression of in a recipient organism, for example by administration of a nucleic acid construct, such as an RNA construct as described herein, that encodes it) an antigen that is or comprises one or more epitopes (e.g., one or more B-cell and/or one or more T-cell epitopes) of a norovirus protein. In some embodiments, a pharmaceutical composition described herein induces a relevant immune response effective against norovirus (e.g., by targeting a norovirus protein).

[0515] In some embodiments a provided pharmaceutical composition (e.g., immunogenic composition, e.g., norovirus vaccine) comprises or delivers an antigen that is or comprises a full-length norovirus protein. In some embodiments, a provided pharmaceutical composition (e.g., immunogenic composition, e.g., norovirus vaccine) comprises or delivers an antigen that is or comprises a portion of a norovirus protein that is less than a full-length norovirus protein. In some embodiments, a provided pharmaceutical composition (e.g., immunogenic composition, e.g., norovirus vaccine) comprises or delivers a chimeric polypeptide that is or comprises part or all of a norovirus protein and one or more heterologous polypeptide elements (e.g., a secretion signal, TM domain, and/or multimerization domain described herein).

[0516] In some embodiments, an antigen that is included in and/or delivered by a provided pharmaceutical composition (e.g., immunogenic composition, e.g., norovirus vaccine) is or comprises one or more peptide fragments of a norovirus antigen; in some such embodiments, each of the one or more peptide fragments includes at least one epitope (e.g., one or more B cell epitopes and/or one or more T cell epitopes), for example as may be predicted, selected, assessed and/or characterized as described herein.

[0517] In some embodiments, a norovirus protein, or fragment or epitope thereof, utilized in an antigen as described herein may include one or more sequence alterations relative to a particular reference norovirus protein, or fragment or epitope thereof. For example, in some embodiments, a utilized antigen may include one or more sequence variations found in circulating strains or predicted to arise, e.g., in light of assessments of sequence conservation and/or evolution of norovirus proteins over time and/or across strains. Alternatively or additionally, in some embodiments, a utilized antigen may include one or more sequence variations selected, for example, to impact stability, folding, processing and/or display of the antigen or any epitope thereof.

[0518] In some embodiments, a utilized antigen induces an immune response that targets a VP protein, such as a VP1 protein (e.g., an S domain and/or a P domain, such as a P2 domain, thereof). In some embodiments, a utilized antigen induces an immune response that targets a VP1 protein from any of genogroups and/or genotypes. In some embodiments, a utilized antigen induces an immune response that targets a VP1 protein from GI or GIL In some such embodiments, an immune response may be or comprise a T cell immune response.

[0519] In some embodiments, a utilized antigen is or comprises one or more norovirus protein sequences (e.g., conserved sequences and/or sequences that are or comprise one or more B cell epitopes and/or one or more CD4 epitopes and/or one or more CD8 epitopes) of an antigen expressed. Certain B cell and T cell epitopes have been described for noroviruses of various genogroups {see, for example, van Loben Seis & Green, Viruses 11:432, 2019, doi: 10.3390/vl 1050432, which is incorporated herein by reference in its entirety).

[0520] In some embodiments, a utilized antigen is or comprises one or more norovirus protein sequences found in a strain that is circulating or has circulated in a relevant region (e.g., where subjects to be vaccinated are or will be present). It is noted, for example that GII.4 viruses have caused the majority of norovirus outbreaks worldwide, although in recent years, non-GII.4 viruses, such as GII.17 and GII.2, have temporarily replaced GII.4 viruses in several Asian countries. Between 2002 and 2012, new GII.4 viruses emerged about every 2 to 4 years, but since 2012, the same virus (GII.4 Sydney) has been the dominant strain worldwide.

[0521] In some embodiments, an antigen utilized in accordance with the present disclosure an antigen is or comprises a norovirus VP protein selected from the group consisting of VP1 and VP2, and variants thereof and/or fragments or epitopes of any of the foregoing, and combinations of any of the foregoing.

[0522] In some embodiments, an antigen utilized in accordance with the present disclosure is or comprises a norovirus protein selected from the group consisting of a NoV VP1, a NoV VP2, a NoV N-terminal protein (NS1 and/or NS2), a NoV NTPase (NS3), a NoV P22 (NS4), a NoV VPg (NS5), a NoV Protease (NS6), a NoV Polymerase (NS7), and variants thereof and/or fragments or epitopes of any of the foregoing, and combinations of any of the foregoing. In some embodiments, an antigen utilized in accordance with the present disclosure is or comprises a norovirus VP1 protein or variant thereof or one or more fragments or epitopes of such VP1 protein or variant thereof (e.g., used individually or in combination (e.g., as part of a multiepitope construct, such as a string construct, as described herein) with one another and/or with one or more other norovirus proteins or fragments or epitopes thereof). In some embodiments, an antigen utilized in accordance with the present disclosure is or comprises a norovirus VP1 protein of norovirus genogroup GI or variant thereof or one or more fragments or epitopes of such VP1 protein or variant thereof (e.g., used individually or in combination (e.g., as part of a multiepitope construct, such as a string construct, as described herein) with one another and/or with one or more other norovirus proteins or fragments or epitopes thereof, for example from the same or different genogroups and/or genotypes). In some embodiments, an antigen utilized in accordance with the present disclosure is or comprises a norovirus VP1 protein of norovirus genogroup GII or variant thereof or one or more fragments or epitopes of such VP1 protein or variant thereof (e.g., used individually or in combination (e.g., as part of a multiepitope construct, such as a string construct, as described herein) with one another and/or with one or more other norovirus proteins or fragments or epitopes thereof, for example from the same or different genogroups and/or genotypes).

Viral Protein 1 (VP1)

[0523] In some embodiments, a provided pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) comprises or delivers a norovirus VP1 protein, or fragment or epitope thereof; the term “VP1 antigen” may be used herein to refer to an antigen that includes at least one VP1 fragment (e.g., an S domain fragment or P domain fragment) or epitope (e.g., B cell or T cell epitope, e.g., an S domain or P domain B cell or T cell epitope).

[0524] In some embodiments, a provided pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) comprises or delivers a full-length VP1 protein or variant thereof. In some embodiments, a provided pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) comprises or delivers a fragment (e.g., a fragment that is or comprises an S domain or a P domain, or a fragment or epitope of either of the foregoing, such as a Pl or P2 subdomain or fragment or epitope thereof) of a VP1 protein or variant thereof. In some embodiments, a provided pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) comprises or delivers a VP1 antigen (e.g., a full length or fragment VP1, or a variant thereof) separately (e.g., from a separate RNA and/or a separate LNP) from at least one other antigen (e.g., a multi-epitope antigen) as described herein.

[0525] In some embodiments, a provided pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) comprises or delivers a polypeptide that is or comprises a P domain such as a P2 domain. In some embodiments, P domain sequences (e.g., P2 domain sequences) are selected that are expected or known to elicit antibodies, e.g., antibodies that interfere with HBGAs interaction.

[0526] In some embodiments, a provided pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) comprises or delivers antigen(s) that is/are or comprise a plurality of P2 domains of different sequences (e.g., in some embodiments representing different viral variants that, for example, may have been detected or expected in a particular region or population and/or according to observed or expected mutation trends, and/or that may have been expected or predicted, together, to induce or support an immune response that includes antibodies and/or T cells that bind to and/or otherwise are effective against (e.g., that block capsid formation and/or viral entry, and/or that target virus-infected cells) a plurality of viral strains or variants.

[0527] In some embodiments, a provided pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) comprises or delivers a polypeptide including a VP1 epitope that is bound by monoclonal antibody NV8812 (see White et al. J Virol. 70:6589-97. doi: 10.1128/JVI.70.10.6589, 1996, which is incorporated herein by reference in its entirety).

[0528] In some embodiments, a provided pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) comprises or delivers a polypeptide a polypeptide including a VP1 epitope from any genogroup and/or genotype of norovirus. In some such embodiments, a VP1 epitope may be from GI genogroup of norovirus. In some embodiments, a VP1 epitope may be from GII genogroup of norovirus.

Viral Protein 2 (VP2)

[0529] VP2 interacts with VP1 via a highly conserved isoleucine residue (S domain residue 52, according to canonical numbering systems) in its IDPWI motif (see, Vongpunsawad et al. J Virol. 87:4818, (2013), doi: 10.1128/JVI.03508-12). VP2 is also reported to interact with host restriction factors (see Cotten et al. J Virol. 88: 11056, 2014, doi: 10.1128/JVI.01333-14, which is incorporated herein by reference in its entirety).

[0530] VP2 is believed to being involved in capsid formation and/or stabilization; it has been reported that absence of VP2 decreases stability and homogeneity of norovirus capsids or virus-like particles, and furthermore that co-expression of VP1 and VP2 increases their expression relative to when they are separately expressed (see, for example, Vongpunsawad et al. J Virol. 87:4818, (2013), doi: 10.1128/JVI.03508-12; Liu et al. Arch Virol.164:1173, (2019) doi: 10.1007/s00705-019-04192-2, each of which is incorporated herein by reference in its entirety).

N-terminal Protein (NS 1-2; p48) [0531] The noroviral p48 protein, which is located at the N-terminus of the viral polyprotein, is characteristic of its genus; sequence comparisons across genogroups have revealed that the HuNoV Sydney p48 shares 42% identity with NV p48 (GI), 36% with Jena p48 (GUI), and 37% with MNV (GV) (Lateef et al., BMC Genomics. 18:39, 2017, doi: 10.1186/sl2864-016-3417-4, which is incorporated herein by reference in its entirety).

Notwithstanding such low overall sequence identity, p48 proteins across strains and genera have been reported to have comparable structures, including:

(1) a disordered region of proline-rich N-terminal, containing the alleged immunogenic regions for MHC-I binding,

(2) a transmembrane hydrophobic domain at the C-terminal end,

(3) H-box and NC sequence motifs of the permutated NlpC/P60 family of circular peptidases that adapt different enzyme capacities within the same structure, improving the stability and the reducing degradation caused by proteases, and

(4) caspase cleavage and phosphorylation sites, which in eukaryotic cells are involved in the regulation of the cell cycle, apoptosis, and activation of the immune system:

[0532] Functionally, the p48 protein has been reported, when expressed in mammalian cells, to interfere in many intracellular pathways, such as those involving the Jak-STAT, MAPK, p53, and PI3K-Akt signaling pathways, and also to interfere with apoptosis, Toll-like receptors (TLR) signaling pathways, and the production of chemokines and cytokines (Lateef et al., BMC Genomics. 18:39, 2017, doi: 10.1186/sl2864-016-3417-4, which is incorporated herein by reference in its entirety).

[0533] The p48 protein thus (i) assists assembly of the replication complex; (ii) hampers certain cellular signaling pathways, and (iii) inhibits activation of the immune response induced by viral infection.

NTPase (NS3)

[0534] The NTPase protein, also known as NS3, is generated by cleavage of the polyprotein, in which it is located between residues 331 and 696, according to canonical numbering systems. [0535] NS3 shows significant homology to the Enterpvirus 2C protein (see, Pfister et al. J Virol. 75: 1611, 2001, doi: 10.1128/JVI.75.4.1611-1619.2001, which is incorporated herein by reference in its entirety). NS3 has been reported to have enzymatic activity including (a) NTP- dependent helicase activity for unrolling RNA helices; (b) NTP-independent chaperone activity for remodeling of RNA structure and facilitating annealing of RNA chains, and (c) support of RNA synthesis by NS7. Co-expression of p48 and/or p22 has been reported to enhance NS3 activity, including specifically apoptotic activity (see, Yen et al. J Virol. 92:17, 2018, doi: 10.1128/JVI.01824-17, which is incorporated herein by reference in its entirety).

P22 (NS4)

[0536] The norovirus p22 protein is another of the polypeptides formed by cleavage of the encoded preprotein. P22 includes a motif (YX(|)ESDG motif (36), which has been reported to mimic an export signal for vesicles that migrate from the rough ER, and prevents these coated vesicles from fusing with the trans-Golgi network (36). P22 has therefore been proposed to interefer with protein protein secretion and post-translational edification pathways. This motif is highly conserved among different genotypes of the GI and the GII genogroups (Sharp et al. PLoS ONE, 5:el3130, 2010, doi: 10.1371/journal.pone.0013130, which is incorporated herein by reference in its entirety).

[0537] It has been reported that P22:

(1) acts as an antagonist in Golgi-dependent protein secretion (36); and

(2) acts as an antagonist of the immune response by altering the interferon and other cytokine signaling pathways after viral infection; and

(3) it promotes assembly of the viral replication complex.

VPg (NS5)

[0538] The norovirus VPg protein is also generated by cleavage of the initial polyprotein (where it is found between residues 876 and 1008 according to the canonical numbering system). VPg becomes linked to the 5’ end of the viral protein (reportedly via action of the viral ProPol protein), where it has been reported to facilitate viral replication, e.g., by helping to prime synthesis (Belliot et al. Virology 374:33, 2008, doi: 10.1016/j.virol.2007.12.028, which is incorporated herein by reference in its entirety) and/or by recruiting host elongation factor(s) (Daughenbaugh et al. EMBO J. 22:2852, 2003, doi: 10.1093/emboj/cdg251, which is incorporated herein by reference in its entirety) and/or capping machinery (Hosmillo et al. Elife. 8:e46681, 2019, doi: 10.7554/eLife.46681, which is incorporated herein by reference in its entirety).

[0539] Also, a conserved conserved KGKxKxGRG motif found in the N-terminal region of the VPg proteins of all norovirus genogroups, except for GUI has been reported to be involved in inducing cell cycle arrest (in G1/G0) in infected cells (McSweeney et al. Viruses. 11:217, 2019, doi: 10.3390/vl 1030217, which is incorporated herein by reference in its entirety).

Protease (NS 6)

[0540] The norovirus protease protein (NS6) cleaves the polyprotein encoded by ORF1 via a two-stage process in which “early” sites (p48/NTPase and NTPase/p22) are cleaved first, followed by “late” sites (p22/VPg, Vpg/Pro, and Pro/Pol); it is worth noting that the ProPol precursor protein itself also shows cleavage ability, which has been reported to be comparable to that of the Pro protein alone (May et al. Virology 444:218, 2013, doi: 10.1016/j.virol.2013.06.013, which is incorporated herein by reference in its entirety).

[0541] Some differences have been reported between Pro proteins of different genotypes. Specifically, the GII.4 protease crystal structure reveals differences in the substrate binding pocket and catalytic triad residues relative to that of the GI protein. The GII.4 protease active site also includes a conserved arginine residue that interacts with the catalytic histidine (Viskovska et al. J Virol. 93:e01479, 2019, doi: 10.1128/JVI.01479-18, which is incorporated herein by reference in its entirety). Polymerase (NS7)

[0542] The norovirus Pol protein (NS7) is also generated by cleavage of the polyprotein encoded by ORF1 (where it is found between residues 1190 and 1699, using the canonical numbering system). As noted above, the ProPol precursor protein has been reported to share the protease activity of the released Pro protein; it has also been reported to have replicase activity of the released Pol protein (Belliot et al. J Virol. 77:10957, 2003, doi: 10.1128/JVI.77.20.10957- 10974.2003; Belliot et al. J Virol. 79:2393, 2005, each of which is incorporated herein by reference in its entirety).

[0543] Phylogenetic comparisons of Pol protein sequences have been used to classify human noroviruses into sixty (60) different P types and P groups: fourteen (14) GI P types, thirty-seven (37) GII P types, two (2) GUI P types, two (2) GIV P types, two (2) GVI P types, one (1) GVII P types, one (1) GX P type, two tentative P groups, and fourteen (14) tentative P types (Chhabra et al. J Gen Virol. 100: 1393, 2019, doi: 10.1099/jgv.0.001318, which is incorporated herein by reference in its entirety).

Antigen Formats

[0544] In some embodiments, an antigen utilized as described herein is or comprises a full-length viral protein (e.g., a full-length VP1 or VP2, etc.). In some embodiments, an antigen utilized as described herein is or comprises a fragment or domain of a viral protein (e.g., an S domain of a VP1 protein), or an antigenic portion thereof. In some embodiments, an antigen utilized as described herein is a membrane-tethered antigen (e.g., a full-length protein, such asVPl or VP2, or a fragment thereof, such as a VP1, an S domain or antigenic fragment thereof, that is fused with a membrane-associating moiety, such as for example, a transmembrane moiety). In some embodiments, a provided pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) comprises or delivers antigen sequences that are or comprise one or more antibody epitopes.

[0545] In some embodiments, an antigen utilized as described herein includes one or more variant sequences relative to a relevant reference antigen. For example, in some embodiments, a protease cleavage site is removed or blocked; alternatively or additionally, in some embodiments, a terminally truncated antigen is utilized. [0546] In some embodiments, an antigen utilized as described herein includes a multimerization element (e.g., a heterologous multimerization element).

[0547] In some embodiments, an antigen utilized as described herein includes a membrane association element (e.g., a heterologous membrane association element), such as a transmembrane domain.

[0548] In some embodiments, an antigen utilized as described herein includes a secretion signal (e.g., a heterologous secretion signal).

[0549] In some embodiments, utilized sequences may be longer (and, e.g., may therefore include more epitopes) than a viral protein found in nature.

[0550] In some embodiments, utilized sequences may be from a different strain or plurality of strains (e.g., as may be circulating in and/or otherwise relevant to a population to which a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) is administered).

[0551] In some embodiments, an antigen utilized as described herein may include a plurality of epitopes (e.g., B-cell and/or T-cell epitopes) arranged in a non-natural configuration (e.g., in a string construct as described herein). In some embodiments, an antigen utilized as described herein may include a plurality of epitopes predicted or demonstrated to bind HLA alleles reflective of a population to which a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) composition is to be administered as described herein.

[0552] In some embodiments, a provided pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) may comprise or deliver a plurality of antigens, one or more antigens that includes B cell epitopes and one or more antigens that includes T cell epitopes. In some embodiments, a provided pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) may comprise or deliver one antigen that includes both B cell and CD4 epitopes and a separate CSP antigen that includes CD8 epitopes.

Herpes Simplex Virus (HSV)

[0553] Herpes simplex virus (HSV) belongs to the alpha subfamily of the human herpesvirus family and includes HSV-1 and HSV-2. The structure of HSV- 1 and HSV-2 mainly include (from inside to outside) a DNA core, capsid, tegument and envelope. Each of HSV-1 and HSV-2 have a double stranded DNA genome of about 153kb, encoding at least 80 genes. The DNA core is enclosed by an icosapentahedral capsid composed of 162 capsomeres, 150 hexons and 12 pentons, made of six different viral proteins. The DNA is surrounded by at least 20 different viral tegument proteins that have structural and regulatory roles. Some of them participating in capsid transport to the nucleus and other organelles, viral DNA entry into the nucleus, activation of early genes transcription, suppression of cellular protein biosynthesis, and mRNA degradation. The viral envelope surrounding the tegument has at least 12 different glycoproteins (B-N) on their surface. The glycoproteins may exist as heterodimers (H/L and E/I) with most existing as monomers.

[0554] HSV-1 and HSV-2 are responsible for a number of minor, moderate and severe pathologies, including oral and genital ulceration, virally induced blindness, viral encephalitis and disseminated infection of neonates. HSV-1 and HSV-2 are usually transmitted by different routes and affect different areas of the body, but the signs and symptoms that they cause can overlap. Infections caused by HS V - 1 represent one of the more widespread infections of the orofacial region and commonly causes herpes labialis, herpetic stomatitis, and keratitis. HSV-2 typically causes genital herpes and is transmitted primarily by direct sexual contact with lesions. Most genital HSV infections are caused by HSV-2, however, an increasing number of genital HSV infections have been attributed to HSV-1. Genital HSV-1 infections are typically less severe and less prone to occurrence than genital HSV-2 infections.

[0555] HSV infections are transmitted through contact with herpetic lesions, mucosal surfaces, genital secretions, or oral secretions. The average incubation period after exposure is typically 4 days, but may range between 2 and 12 days. HSV particles can infect neuronal prolongations enervating peripheral tissues and establish latency in these cells, namely in the trigeminal ganglia and dorsal root ganglia of the sacral area from where they can sporadically reactivate. Additionally, similar to other herpesviruses, HSV infections are lifelong and generally asymptomatic. Without wishing to be bound by any particular theory, it is understood that HSV particles can be shed from infected individuals independent of the occurrence of clinical manifestations. [0556] HSV infections are rarely fatal, but are characterized by blisters that can rupture and become painful. There are few clear differences in clinical presentation based on the type of infecting virus. However, as discussed above, HSV-1 infections tend to be less severe than HSV- 2 infections, and patients infected with HSV-2 generally have more outbreaks.

A. HSV Lifecycle

[0557] As described herein, to initiate infection, an HSV (HSV-1 or HSV-2) particle binds to the cell surface using viral glycoproteins and fuses its envelope with the plasma membrane. After the fusion of membranes, the viral capsid and tegument proteins are internalized in the cytoplasm. Once in the cytoplasm, the viral capsid accumulates in the nucleus and releases viral DNA into the nucleus. HSV replicates by three rounds of transcription that yield: a (immediate early) proteins that mainly regulate viral replication; 0 (early) proteins that synthesise and package DNA; and y (late) proteins, most of which are virion proteins (see, Whitley et.al., Lancet 2001 May 12;357(9267); Taylor et.al., Front Biosci. 2002 Mar l;7:d752- 64; and Ibanez et.al., Front Microbiol. 2018 Oct 11;9:2406; each of which is incorporated herein by reference in its entirety) (see, e.g., Fig. 2, Steps 4-6).

[0558] HSV capsids are assembled within the nucleus of infected cells. Once assembly of viral capsids has been completed in the nucleus, these particles will continue their maturation process in this same compartment through the acquisition of tegument proteins. After leaving the nucleus, additional tegument proteins will be added to the capsids. Meanwhile, the glycoproteins are translated and glycosylated in the endoplasmic reticulum and processed in the trans-Golgi network (TGN) and then directed to multivesicular bodies (see, e.g., Fig. 2, Step 8). Then, they are exported to the plasma membrane glycoproteins within early endosomes (see, e.g., Fig. 2, Step 9). Viral capsids in the cytoplasm will then fuse with HS V-glycoprotein-containing endosomes to form infectious virions within vesicles.

[0559] HSV (HSV-1 or HSV-2) are able to establish a latent infection. After primary infection, HSV either replicates productively in epithelial cells or enters sensory neuron axons and moves to the neuronal cell nucleus. There, the viral DNA remains as circular, extra- chromosomal DNA, and does not possess any lytic gene expression; however, latency associated transcripts are expressed and then spliced to produce mRNA. This general transcriptional silence may allow the virus to remain hidden in the cell by avoiding immune surveillance. In some aspects, provided herein are technologies (e.g., compositions and methods) for augmenting, inducing, promoting, enhancing and/or improving an immune response against HSV (e.g., HSV- 1 and/or HSV-2) or a component thereof (e.g., a protein or fragment thereof). In some embodiments, technologies provided herein are designed to augment, induce, promote, enhance and/or improve immunological memory against HSV or a component thereof (e.g., a protein or fragment thereof). In some embodiments, technologies described herein are designed to act as an immunological boost to a primary vaccine, such as a vaccine directed to an epitope and/or epitopes of HSV (e.g., HSV-1 and/or HSV-2).

[0560] The virus remains in this state for the lifetime of the host, or until the proper signals reactivate the virus and new progeny are generated. Progeny virus then travel through the neuron axis to the site of the primary infection to re-initiate a lytic replication cycle.

B. HSV Genome

[0561] The genome of HSV-1 and the genome of HSV-2 are both approximately 150 kb long of double-stranded DNA, varying slightly between subtypes and strains. The genome encodes more than 80 genes and has high GC contents: 67 and 69% for HSV-1 and HSV-2, respectively (see, Whitley et.al., Lancet 2001 May 12;357(9267); Taylor et.al., Front Biosci.

2002 Mar l;7:d752-64; and Jiao et.al., Microbiol Resour Announc. 2019 Sep; 8(39): e00993-19, which is incorporated herein by reference in its entirety).

[0562] The genome is organized as unique long region (UL) and a unique short region (US). The UL is typically bounded by terminal long (TRL) and internal long (IRL) repeats. The US is typically bounded by terminal short (IRS) and internal short (TRS) repeats. The genes found in the unique regions are present in the genome as a single copy, but genes that are encoded in the repeat regions are present in the genome in two copies (see, Whitley et.al., Lancet 2001 May 12;357(9267); Taylor et.al., Front Biosci. 2002 Mar l;7:d752-64; and Jiao et.al., Microbiol Resour Announc. 2019 Sep; 8(39): e00993-19, which is incorporated herein by reference in its entirety).

[0563] HSV contains three origins of replication within the genome that are named depending upon their location in either the Long (oriL) or Short (oriS) region of the genome. OriL is found as a single copy in the UL segment, but oriS is located in the repeat region of the Short segment; thus, it is present in the genome in two copies. Both oriL and oriS are palindromic sequences consisting of an AT-rich center region flanked by inverted repeats that contain multiple binding sites of varying affinity for the viral origin binding protein (UL9). Either oriL or one of the oriS sequences is sufficient for viral replication (see, Whitley et.al., Lancet 2001 May 12;357(9267); Taylor et.al., Front Biosci. 2002 Mar l;7:d752-64; and Jiao et.al., Microbiol Resour Announc. 2019 Sep; 8(39): e00993-19, which is incorporated herein by reference in its entirety).

[0564] The viral genome also contains signals that orchestrate proper processing of the newly synthesized genomes for packaging into pre-formed capsids. Progeny genomes are generated in long concatemers that require cleavage into unit-length monomers. For this purpose, the viral genome contains two DNA sequence elements, pacl and pac2, that ensure proper cleavage and packaging of unit-length progeny genomes. These elements are located within the direct repeats (DR) found within the inverted repeat regions at the ends of the viral genome (see, Whitley et.al., Lancet 2001 May 12;357(9267); Taylor et.al., Front Biosci. 2002 Mar l;7:d752- 64; and Jiao et.al., Microbiol Resour Announc. 2019 Sep; 8(39): e00993-19, which is incorporated herein by reference in its entirety).

C. HSV Vaccines

[0565] Several HSV vaccines, mainly targeting HSV-2 and primarily focused on the generation of neutralizing antibodies (nAbs) targeting the viral envelope glycoprotein D as the correlate of immune protection, have been developed and evaluated in human clinical trial, see Table 1C below. Despite these vaccines exhibiting protection against HSV in preclinical studies and in some cases Phase 2 studies, none of these vaccines has demonstrated sufficient efficacy for further development or commercialization.

Table 1C: Certain HSV Vaccines Under Clinical Development

Respiratory syncytial (RSV)

[0566] Respiratory syncytial virus (RSV), also called human respiratory syncytial virus (hRSV) and human orthopneumo virus, is a common, contagious virus that causes infections of the respiratory tract. It is a negative-sense, single-stranded RNA virus. Its name is derived from the large cells known as syncytia that form when infected cells fuse.

[0567] RSV is the single most common cause of respiratory hospitalization in infants, and reinfection remains common in later life: it is a notable pathogen in all age groups. Infection rates are typically higher during the cold winter months, causing bronchiolitis in infants, common colds in adults, and more serious respiratory illnesses such as pneumonia in the elderly and immunocompromised.

[0568] RSV can cause outbreaks both in the community and in hospital settings. Following initial infection via the eyes or nose, the virus infects the epithelial cells of the upper and lower airway, causing inflammation, cell damage, and airway obstruction. A variety of methods are available for viral detection and diagnosis of RSV including antigen testing, molecular testing, and viral culture. The main prevention measures include hand-washing and avoiding close contact with infected individuals; prophylactic use of palivizumab is also available to prevent RSV infection in high-risk infants. Currently, there is no vaccine against RSV.

[0569] Most people recover in a week or two, but RSV can be serious, especially for infants and older adults. RSV is the most common cause of bronchiolitis (inflammation of the small airways in the lung) and pneumonia (infection of the lungs) in children younger than 1 year of age in the United States. Symptoms

[0570] People infected with RSV usually show symptoms within 4 to 6 days after getting infected. Symptoms of RSV infection usually include

• Runny nose

• Decrease in appetite

• Coughing

• Sneezing

• Fever

• Wheezing

[0571] These symptoms usually appear in stages and not all at once. In very young infants with RSV, the only symptoms may be irritability, decreased activity, and breathing difficulties.

[0572] Almost all children will have had an RSV infection by their second birthday.

Care

[0573] RSV can cause more serious health problems

RSV can also cause more severe infections such as bronchiolitis, an inflammation of the small airways in the lung, and pneumonia, an infection of the lungs. It is the most common cause of bronchiolitis and pneumonia in children younger than 1 year of age.

[0574] Healthy adults and infants infected with RSV do not usually need to be hospitalized. But some people with RSV infection, especially older adults and infants younger than 6 months of age, may need to be hospitalized if they are having trouble breathing or are dehydrated. In the most severe cases, a person may require additional oxygen, or IV fluids (if they can’t eat or drink enough), or intubation (have a breathing tube inserted through the mouth and down to the airway) with mechanical ventilation (a machine to help a person breathe). In most of these cases, hospitalization only lasts a few days.

[0575] RSV can be dangerous for some infants and young children. Each year in the United States, an estimated 58,000-80,000 children younger than 5 years old are hospitalized due to RSV infection. Those at greatest risk for severe illness from RSV include Premature infants

• Infants, especially those 6 months and younger

• Children younger than 2 years old with chronic lung disease or congenital (present from birth) heart disease

• Children with weakened immune systems

• Children who have neuromuscular disorders, including those who have difficulty swallowing or clearing mucus secretions

Severe RSV Infection

[0576] Virtually all children get an RSV infection by the time they are 2 years old. Most of the time RSV will cause a mild, cold-like illness, but it can also cause severe illness such as

• Bronchiolitis (inflammation of the small airways in the lung)

• Pneumonia (infection of the lungs)

• One to two out of every 100 children younger than 6 months of age with RSV infection may need to be hospitalized. Those who are hospitalized may require oxygen, IV fluids (if they aren’t eating and drinking), and/or mechanical ventilation (a machine to help with breathing). Most improve with this type of supportive care and are discharged in a few days.

[0577] RSV season in most regions of the U.S. starts in the fall and peaks in the winter.

[0578] RSV is divided into two antigenic subtypes, A and B, based on the reactivity of the F and G surface proteins to monoclonal antibodies. The subtypes tend to circulate simultaneously within local epidemics, although subtype A tends to be more prevalent.

Generally, RSV subtype A (RSVA) is thought to be more virulent than RSV subtype B (RSVB), with higher viral loads and faster transmission time. To date, 16 RSVA and 22 RSVB clades have been identified. Among RSVA, the GAI, GA2, GA5, and GA7 clades predominate; GA7 is found only in the United States. Among RSVB, the BA clade predominates worldwide.

[0579] F and G proteins are the primary targets for neutralizing antibodies during natural infection.

[0580] Surface protein G (glycoprotein) is primarily responsible for viral attachment to host cells. This protein is highly variable between strains. G protein exists in both membrane- bound and secreted forms. The membrane-found form is responsible for attachment by binding to glycosaminoglycans (GAGs), such as heparan sulfate, on the surface of host cells. The secreted form acts as a decoy, interacting with antigen presenting cells to inhibit antibody- mediated neutralization. G protein also contains a CX3C fractalkine-like motif that binds to the CX3C chemokine receptor 1 (CX3CR1) on the surface of ciliated bronchial host cells. This binding may alter cellular chemotaxis and reduce the migration of immune cells into the lungs of infected individuals. G protein also alters host immune response by inhibiting signaling from several toll-like receptors, including TLR4.

[0581] The RSV G protein was first described by Seymour Levine as a heavily glycosylated 80 kDa protein in purified virions produced in HeLa cells (Levine 1977). He later showed that rabbit antibodies to G protein, but not to F protein, prevented virions from binding to HeLa cells, indicating that the G protein is the major virus attachment protein (Levine et al. 1987). The G protein backbone contains 289 to 299 amino acids (32-33 kDa), depending on the strain, and is palmitoylated (Collins and Mottet 1992). With 30-40 O-linked glycans and 4-5 N- linked glycans, the G protein is similar to mucins produced in the airways although much smaller in molecular mass (Satake et al. 1985; Wertz et al. 1985). Approximately 60% of the G protein molecular mass is carbohydrate. The size of the G protein varies depending on the cell type in which it is produced: 80-100 kDa in immortalized cell lines (Garcia-Beato et al. 1996) but 180 kDa in primary HAE cultures (Kwilas et al. 2009). This larger form is not a disulfide-linked dimer because it does not dissociate in reducing conditions, but could be a dimer held together by a different bond, or a more heavily glycosylated monomer.

[0582] The central region of the G protein contains a 13 -amino acid highly conserved domain, partially overlapping the cysteine noose domain with 4 cysteines linked 1-4 and 2-3 (Gorman et al. 1997), followed by a highly basic heparin-binding domain (HBD). The HBD is the likely attachment site for heparan sulfate (HS) found on the surface of most cells. A peptide from the G protein HBD (amino acids 184-198) binds efficiently to HEp-2 cells and inhibits RSV infection (Feldman et al. 1999). Two large mucin-like domains flank the central region (Fig. 3B) and are highly variable in sequence, making the G protein the most variable RSV protein, a useful characteristic for RSV evolution studies. The overall Ser and Thr content of these two regions is relatively stable, suggesting that they may provide substrates for O-linked glycan decoration rather than any particular sequence or specific function. The appearance of a 20 amino acid repeat in the second mucin-like domain of the G protein of a B strain virus (Trento et al. 2003) and a 24 amino acid insertion in the same region of an A strain virus (Eshaghi et al. 2012) underscore the flexibility of this region. This B strain spread throughout the world in the decade since it appeared, suggesting that the repeat provides some advantage to the virus.

[0583] The F gene encodes a type I integral membrane protein that is synthesized as a 574 amino acid inactive precursor, F0, decorated with 5 to 6 N-linked glycans, depending on the strain (Collins et al. 1984). It is also palmitoylated at a cysteine in its cytoplasmic domain (Arumugham et al. 1989). Three F0 monomers assemble into a trimer and, as the trimer passes through the Golgi, the monomers are activated by a furin-like host protease (Bolt et al. 2000; Collins and Mottet 1991). The protease cleaves twice, after amino acids 109 and 136 (Gonzalez- Reyes et al. 2001; Zimmer et al. 2001a), generating three polypeptides (Fig. 1). The N-terminal and C-terminal cleavage products are the F2 and Fl subunits (named in order of size), respectively, and are covalently linked to each other by two disulfide bonds (Gruber and Eevine 1983; Day et al. 2006). The intervening 27 amino acid peptide, pep27, contains 2 or 3 N-linked glycans, but dissociates after cleavage (Begona Ruiz-Arguello et al. 2002). The F2 subunit contains two N-linked glycans, whereas the larger Fl subunit contains a single N-linked site. Unlike the others, this Fl glycan is essential for the protein to cause membrane fusion (Ei et al. 2007; Zimmer et al. 2001b).

[0584] The functional F protein trimer in the virion membrane is in a metastable, prefusion form. It is not yet clear what causes the F protein to trigger, but the result is a major refolding into its postfusion form. At the N-terminus of each Fl subunit is the fusion peptide (FP), a stretch of hydrophobic residues that insert into the target membrane (Collins et al. 1984). The FP is mirrored by the transmembrane (TM) domain near the C-terminus of Fl, and each is connected to a heptad repeat (HR) in this order: FP-HRA-HRB-TM. Upon triggering the pre- HRA refolds into the long HRA helix and trimerizes. The F protein folds in the center as the target and viral membranes approach each other, enabling HRB to bind to the grooves in the HRA trimer, forming a hairpin 6-helix bundle (6HB) (Zhao et al. 2000). The F glycoprotein is highly conserved among RSV isolates from both A and B subgroups, with amino acid sequence identities of 90% or higher. Much of the variability in F (-25%) is found within an antigenic site at the apex of the prefusion trimer (antigenic site 0) composed of an a-helix from Fl (aa 196— 210) and a strand from F2 (aa 62-69) and may be a site that determines subtype-specific immunity (McLellan et al. 2013). This relative sequence conservation combined with its surface location on the virion and its obligatory role in viral entry and antigenic sites associated with potent neutralization make F an ideal target for neutralizing antibodies (Anderson et al. 1988; Walsh and Hruska 1983). Antigenic site 0 is the most variable portion of the F protein, suggesting it may be subject to immune pressure.

[0585] Surface protein F (fusion protein) is responsible for fusion of viral and host cell membranes, as well as syncytium formation between viral particles. Its sequence is highly conserved between strains. While viral attachment appears to involve both F and G proteins, F fusion occurs independently of G. F protein exists in multiple conformational forms. In the prefusion state (PreF), the protein exists in a trimeric form and contains the major antigenic site 0. 0 serves as a primary target of neutralizing antibodies in the body. After binding to its target on the host cell surface (its exact ligand remains unclear), PreF undergoes a conformational change during which 0 is lost. This change enables the protein to insert itself into the host cell membrane and leads to fusion of the viral and host cell membranes. A final conformational shift results in a more stable and elongated form of the protein (postfusion, PostF). Opposite of the RSV G protein, the RSV F protein also binds to and activates toll-like receptor 4 (TLR4), initiating the innate immune response and signal transduction.

[0586] Much of the variability in F (-25%) is found antigenic site 0 (at the apex of the antigenic trimer). 0 is composed of an a-helix from Fi (aa 196-210) and a strand from F2 (aa 62-69) and may be a site that determines subtype-specific immunity.

Immune Imprinting

[0587] Immune imprinting is a phenomenon whereby initial exposure to a particular antigen can limit (e.g., subsequent) development of immune responses against epitopes that are unique to new variants of the antigen. In particular, when exposed to a new, previously un- encountered infectious agent, such as a virus, immune systems respond, among other things, by generating antibodies that bind to and neutralize portions of antigen(s) of the agent, in a highly specific fashion. Subsequently, the immune system retains a ‘memory’ of the antigen(s), along with the ability to produce the particular antibodies that target it, in the form of memory B and T cells. [0588] On the one hand, following an initial exposure to a particular agent, this immune memory allows the body to rapidly recognize and defend against it when it is subsequently encountered. On the other hand, processes such as natural mutation and evolution can give rise to variants of the agent that are similar enough to the originally encountered strain to be recognized and trigger a memory response, prompting production of antibodies that were generated to defend against the original strain, rather than being expressly tailored to the new variant. If the new variant includes sufficient mutations in key regions (e.g., implicated in host cell infection, viral replication, etc.) targeted by these antibodies, the efficacy of this memory response can be reduced. Accordingly, immune imprinting can be particularly concerning for pathogens having a high concentration of mutations at neutralization sensitive epitopes.

[0589] In the context of viral infection and vaccination, this immune imprinting phenomenon can lead to increased rates or reinfection by mutated variants and limit efficacy of vaccination in individuals after their initial exposure to an earlier strain (e.g., whether due to natural infection or an earlier vaccine dose). Immune imprinting can, according, be especially problematic for vaccination against viruses that have higher mutation rates, such as RNA viruses. These include, without limitation, influenza, coronavirus (e.g., severe acute respiratory syndrome-related coronavirus), human immunodeficiency virus (HIV), Respiratory syncytial virus (RSV), and the like.

[0590] For example, in the context of the recent SARS-CoV 2 pandemic, mutation of circulating virus has given rise to tens of thousands of viral variants, several of which - such as Omicron and recently emergent XBB (e.g., XBB.1.5) - are characterized by their immune escape potential. In particular, these variants include several mutations that allow them to evade existing (e.g., memory) immune responses that individuals have developed as a result of prior exposure - either through vaccination and/or natural infection - to previous strains, such as the original Wild-Type (WT) Wuhan variant.

[0591] A schematic illustrating the immune imprinting phenomenon is shown in Fig. 1. Subjects administered a vaccine that delivers a wild-type (WT) antigen produce antibodies and form memory B cells recognizing the WT antigen. As new Variants of Concern (VOC) arise that evade the immune response induced by the first vaccine, VOC-adapted booster shots are developed and administered to subjects. VOCs often evade the immune system by acquiring mutations at neutralization sensitive epitopes (regions prone to mutation shown in different colors in Fig. 1). Subjects exposed to a VOC-adapted vaccine have a predisposition to activate memory B cells that were formed in response to the initial WT vaccine rather than activate naive B cells. As a result, administering the VOC-adapted vaccine induces production of antibodies that recognize both the WT virus and the VOC but few or no antibodies that are specific to the VOC. So long as the VOC retains some neutralization epitopes from the WT virus, a neutralization response against the VOC can still be induced. As new variants continue to lose neutralization epitopes from the WT strain, however, the immune response induced by VOC- adapted vaccines become less and less effective. Further discussion of the imprinting phenomenon in the SARS-CoV-2 context can be found in Wheatley et al., Trends Immunol, 2021 , the contents of which are incorporated by reference herein in their entirety. Among other things, the present disclosure provides the insight that immune imprinting can be an issue for vaccine updates that address virus strains comprising a number of mutations at neutralization sensitive sites, i.e. exhibit close to no conserved neutralizing epitopes.

[0592] Immune imprinting can have serious implications for vaccine development. As shown in Fig. 2 and Example 1, a single exposure to Omicron BA.l, BA.2, or BA.4/5 SARS- CoV-2 variants has not been found to induce neutralization of Omicron XBB. Without wishing to be bound by theory, this failure to cross neutralize the XBB variant may be due to the variant’s low retention of neutralizing B-cell epitopes relative to the original Wuhan strain (see Fig. 5(B)). In short, exposure to BA.l, BA.2, and BA.4/5 may be activating memory B cells that recognize epitopes in both Wuhan and these Omicron variants, and not generating immune responses that recognize features that are unique to these variants. If true, these results suggest that variant adapted vaccines may not produce effective immune responses to variants that retain few neutralization epitopes relative to a previously encountered SARS-CoV-2 variant (e.g., a variant that a subject was first infected with or vaccinated against). The present disclosure provides certain insights useful in overcoming this immune imprinting phenomenon in SARS- CoV-2. Specifically, among other things, the present disclosure provides an insight that eliminating conserved B cell epitopes, e.g., by use of a subdomain of a SARS-CoV-2 S protein (e.g., a subdomain lacking regions comprising a high number of conserved, non-neutralizing epitopes and/or a low number of neutralizing epitopes) can induce more of a de novo immune response. This approach is new and fundamentally different from strategies previously described in the art, which, e.g., attempt to overcome imprinting by identifying certain conserved, neutralizing epitopes (e.g., as described in WO2021202734A2). The present disclosure also provides specific compositions and methods that can be used to induce de novo neutralizing responses.

[0593] Among other things, the present disclosure provides technologies that are useful for increasing the breadth of immune response. In some embodiments, such an immune response is or comprises a B cell immune response. In some embodiments, a B cell immune response is or comprises an antibody response (e.g., neutralizing antibody response) to arisen epitopes in variant polypeptides.

[0594] Among other things, the present disclosure provides an insight that it may be particularly desirable, especially for circulating infectious diseases (e.g., for which variants can be expected to arise), to encourage immune responses, specifically including antibody responses (e.g., neutralizing responses) to arisen epitopes. In particular, the present disclosure, among other things, provides an insight that it may be desirable for SARS-CoV-2 infection (e.g., for which variants can be expected to arise), to encourage immune responses, specifically including antibody responses (e.g., neutralizing responses) to arisen epitopes. In some embodiments, such circulating infectious disease is a bacterial infectious disease. In some embodiments, such circulating infectious disease is a parasitic infectious disease. An exemplary parasitic infectious disease is malaria. In some embodiments, such circulating infectious disease is a viral infectious disease. In some embodiments, a viral infectious disease is associated with an RNA virus. Exemplary viral infectious diseases include, but are not limited to coronavirus, ebolavirus, influenza viruses, norovirus, rotavirus, respiratory syncytial virus, alphaherpesvirus, etc.

[0595] Among other things, and without wishing to be bound by any particular theory, the present disclosure provides an insight that, where an antigen (e.g., S protein of SARS-CoV-2) includes one or more “memory epitopes”, such presence may bias an immune response to the antigen toward activation of memory B cells, in at least some instances to the detriment of developing a sufficiently effective antibody response (e.g., a neutralizing antibody response) to arisen epitope(s).

[0596] In some embodiments, the present disclosure, among other things, provides technologies for modulating the balance of immune response toward de no priming response to arisen epitopes in variant polypeptides (e.g., unique epitopes arisen from variant polypeptides of a reference antigen, wherein the unique epitopes are not present in the reference antigen) and SARS-CoV-2 (e.g., in some embodiments XBB variant of SARS-CoV-2). In some embodiments, the present disclosure, among other things, provides technologies for increasing activation of naive B cell immune response to at least one of the arisen epitopes. In some embodiments, such arisen epitopes are neutralizing epitopes. In some embodiments, the present disclosure, among other things, provides technologies for inducing a priming-favorable cytokine milieu, for example, in lymphoid tissues. Without wishing to be bound by a particular theory, in some embodiments, induction of a priming-favorable cytokine milieu can be mediated through interferon alpha (IFNa). Without wishing to be bound by a particular theory, in some embodiments, induction of a priming-favorable cytokine milieu can be mediated through a CD4+ T cell immune response.

[0597] In some embodiments, technologies provided herein may be particularly useful to subjects who have been previously exposed (e.g., via infection and/or vaccination) to a reference antigen of an infectious agent (e.g., SARS-CoV-2) and are receiving an immunogenic composition that delivers a variant polypeptide of the reference antigen (e.g., SARS-CoV-2), or an immunogenic portion thereof. In some embodiments, such a variant polypeptide comprises arisen epitopes. In some embodiments, such arisen epitopes are or comprise neutralizing epitopes (e.g., neutralizing antibody epitopes). In some embodiments, technologies provided herein may be particularly used to induce activation of naive B cell immune response (e.g., in some embodiments antibody response, e.g., neutralizing antibody response) to at least one of the arisen epitopes (e.g., in some embodiments at least one of the neutralizing epitopes).

[0598] The present disclosure exemplifies certain aspects of provided technologies through administering a combination of a modified RNA vaccine that delivers a variant polypeptide of a reference antigen of an infectious agent, e.g., a vaccine that delivers a variant of a coronavirus S protein or an immunogenic portion thereof, and a particular interferon-alpha (IFNa)-inducing agent, e.g., a non-modified RNA. In some embodiments, such a non-modified RNA encodes at least one or more T cell epitopes. In some embodiments, such a non-modified RNA encodes at least one or more B cell epitopes. A skilled person, having read the disclosure, will appreciate that such strategies utilized in coronavirus vaccines may be also useful in other infectious diseases, e.g., circulating infectious diseases. [0599] In one aspect, the present disclosure provides a combination comprising (i) a composition that comprises or delivers at least one polypeptide comprising or consisting of a variant polypeptide of a reference antigen of an infectious agent (e.g., SARS-CoV-2), or an immunogenic portion thereof, wherein the variant polypeptide comprises neutralizing epitopes that are absent in the reference antigen; and (ii) an agent that induces a priming -favorable cytokine milieu in lymphoid tissues, wherein the agent is present at a dose that is effective to increase activation of naive B cell immune response to at least one of the neutralizing epitopes

[0600] In some embodiments, such a combination is provided in the same composition.

[0601] In some embodiments, such a combination is provided in separate compositions.

Exemplary Antigen Formats

[0602] In some embodiments, an antigen utilized as described herein is or comprises a full-length viral protein. In some embodiments, an antigen utilized as described herein is or comprises an immunogenic portion or domain of a viral polypeptide. In some embodiments, an antigen utilized as described herein is a membrane-tethered antigen (e.g., an antigenic fragment thereof fused with a membrane-associating moiety, such as for example, a transmembrane moiety). In some embodiments, a provided pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) comprises or delivers antigen sequences that are or comprise one or more antibody epitopes and/or one or more CD4 T cell and/or CD8 T cell epitopes.

[0603] In some embodiments, an antigen utilized as described herein includes one or more variant sequences relative to a relevant reference antigen. For example, in some embodiments, a protease cleavage site is removed or blocked; alternatively or additionally, in some embodiments, a terminally truncated antigen is utilized, and/or one or more mutations associated with a viral variant (e.g., a SARS-CoV-2 variant of concern) is present in the antigen.

[0604] In some embodiments, an antigen utilized as described herein includes a multimerization element (e.g., a heterologous multimerization element).

[0605] In some embodiments, an antigen utilized as described herein includes a membrane association element (e.g., a homologous membrane association element), such as a transmembrane domain. [0606] In some embodiments, an antigen utilized as described herein includes a secretion signal (e.g., a homologous secretion signal).

[0607] In some embodiments, utilized sequences may comprise one or more mutations associated with a viral variant (e.g., a variant that prevalent and/or that is predicted to be highly immune escaping). In some embodiments, utilized sequences comprise one or more mutations associated with a variant of concern (e.g., a variant of concern identified by WHO). In some embodiments, utilized sequences comprise one or more mutations associated with a viral variant that has been determined to be or has been predicted to be highly immune escaping (e.g., highly immune escaping relative to an immune response developed in subjects administered a previously approved vaccine and/or a previously prevalent viral variant).

[0608] Among other things, described herein are certain SARS-CoV-2 antigens for use in inducing an immunogenic response. In some embodiments, a SARS-CoV-2 antigen comprise immunogenic portions of a full-length SARS-CoV-2 polypeptide (e.g., an SI domain of a SARS- COV-2 S protein and/or an RBD of a SARS-CoV-2 S protein). In some embodiments, such antigens are delivered as protein antigens to induce an immunogenic response. In some embodiments, such antigens are delivered using RNA (e.g., modRNA encoding an SI domain and/or RBD of a SARS-CoV-2 S protein and formulated in LNP particles) to induce an immunogenic response.

Secretory Signals

[0609] In some embodiments, an antigen construct described herein includes a secretory signal, e.g., that is functional in mammalian cells. In some embodiments, a utilized secretory signal is a heterologous secretory signal. In some embodiments, a utilized secretory signal is a homologous secretory signal (e.g., the N-terminal 16 or 19 amino acids of a SARS-CoV-2 S protein). In some embodiments, a secretory signal comprises or consists of a non-human secretory signal. In some embodiments, a secretory signal comprises or consists of a viral secretory signal. In some embodiments, a viral secretory signal comprises or consists of an HSV secretory signal (e.g., an HSV-1 or HSV-2 secretory signal).

[0610] In some embodiments, a secretory signal comprises or consists of an Ebola virus secretory signal. In some embodiments, an Ebola virus secretory signal comprises or consists of an Ebola virus spike glycoprotein (SGP) secretory signal. [0611] In some embodiments, a secretory signal is characterized by a length of about 15 to 30 amino acids.

[0612] In many embodiments, a secretory signal is positioned at the N-terminus of an antigen construct as described herein. In some embodiments, a secretory signal preferably allows transport of the antigen construct with which it is associated into a defined cellular compartment, preferably a cell surface, endoplasmic reticulum (ER) or endosomal-lysosomal compartment.

[0613] In some embodiments, a secretory signal is selected from an S1S2 signal peptide (e.g., aa 1-16 or 1-19), an immunoglobulin secretory signal (e.g., aa 1-22), an HSV-1 gD signal peptide (MGGAAARLGAVILFVVIVGLHGVRSKY; SEQ ID NO: 7), an HSV-2 gD signal peptide (MGRLTSGVGTAALLVVAVGLRVVCA; SEQ ID NO: 8); a human SPARC signal peptide, a human insulin isoform 1 signal, a human albumin signal peptide, etc. Those skilled in the art will be aware of other secretory signal such as, for example, as disclosed in W02017/081082, which is incorporated herein by reference in its entirety (e.g., SEQ ID NOs: 1- 1115 and 1728, or fragments variants thereof).

[0614] In some embodiments, an antigen construct described herein does not comprise a secretory signal.

[0615] In certain embodiments, a signal peptide is an IgG signal peptide, such as an IgG kappa signal peptide.

[0616] In some embodiments, a secretory signal comprises or consists of an HSV glycoprotein D (gD) secretory signal.

[0617] In some embodiments, a string construct sequence encodes an antigen that may comprise or otherwise be linked to a signal sequence (e.g., secretory signal), such as those listed in Table 2 or at least a sequence having 1, 2, 3, 4, or 5 amino acid differences relative thereto. In some embodiments, a secretory signal such as MFVFLVLLPLVSSQCVNLT (SEQ ID NO: 9), or at least a sequence having 1, 2, 3, 4, or at the most 5 amino acid differences relative thereto is utilized..

[0618] In some embodiments, a secretory signal is selected from a gl signal peptide. In some embodiments, a secretory signal such as MPGRSLQGLAILGLWVCATGLVVR (SEQ ID NO: 10), or at least a sequence having 1, 2, 3, 4, or at the most 5 amino acid differences relative thereto is utilized. In some embodiments, a secretory signal such as MPGRSLQGLAILGLWVCATGL (SEQ ID NO: 11), or at least a sequence having 1, 2, 3, 4, or at the most 5 amino acid differences relative thereto is utilized.

[0619] In some embodiments, a secretory signal is one listed in Table 2 and/or Table 3, or a secretory signal having 1, 2, 3, 4, or 5 amino acid differences relative thereto. In some embodiments, a secretory signal is selected from those included in the Table 2 below and/or those encoded by the sequences in Table 3 below.

Table 2: Exemplary secretory signals

Table 3: Exemplary polynucleotide sequences encoding secretory signals

C. Transmembrane Regions

[0620] In some embodiments, an antigen described herein is or comprises an antigen that is associated with or anchored to cell membrane of a cell (e.g., an antigen-presenting cell). In some embodiments, such a membrane-associated/anchored antigen is or comprises an immunogenic fragment, portion, or domain of a polypeptide antigen of an infectious agent coupled to with a membrane-associating moiety, such as for example, a transmembrane moiety. In some embodiments, such a membrane-associated/anchored antigen is or comprises a fusion protein that comprises an immunogenic fragment, portion, or domain of a polypeptide antigen of an infectious agent and a membrane-associating moiety. In some embodiments, an antigen utilized as described herein includes a membrane association element (e.g., a homologous membrane association element), such as a transmembrane domain or region.

[0621] In some embodiments, an antigen construct (e.g., SARS-CoV-2) as described herein includes a transmembrane region. In some embodiments, a transmembrane region is located at the N-terminus of a construct (e.g., SARS-CoV-2) . In some embodiments, a transmembrane region is located at the C-terminus of a construct (e.g., SARS-CoV-2) . In some embodiments, a transmembrane region is not located at the N-terminus or C-terminus of a construct (e.g., SARS-CoV-2) .

[0622] Transmembrane regions are known in the art, any of which can be utilized in a construct (e.g., SARS-CoV-2) described herein. In some embodiments, a transmembrane region comprises or is a transmembrane domain of a SARS-CoV-2 S protein, a transmembrane domain of a SARS-CoV-2 S protein with a C-terminal truncation (e.g., a 19 amino acid C-terminal truncation), Hemagglutinin (HA) of Influenza virus, Env of HIV- 1, equine infectious anaemia virus (EIAV), murine leukaemia virus (MLV), mouse mammary tumor virus, G protein of vesicular stomatitis virus (VSV), Rabies virus, or a seven transmembrane domain receptor.

[0623] In some embodiments, a heterologous transmembrane region does not comprise a hemagglutinin transmembrane region. In some embodiments, a heterologous transmembrane region comprises or consists of a non-human transmembrane region. In some embodiments, a heterologous transmembrane region comprises or consists of a viral transmembrane region. In some embodiments, a heterologous transmembrane region comprises or consists of an HSV transmembrane region, e.g., an HSV-1 or HSV-2 transmembrane region. In some embodiments, an HSV transmembrane region comprises or consists of an HSV gD transmembrane region, e.g., comprising or consisting of an amino acid sequence of

GLIAGAVGGSLLAALVICGIVYWMRRHTQKAPKRIRLPHIR (SEQ ID NO: 91). [0624] In some embodiments, a heterologous transmembrane region comprises or consists of a human transmembrane region. In some embodiments, a human transmembrane region comprises or consists of a human decay accelerating factor glycosylphosphatidylinositol (hDAF-GPI) anchor region. In some embodiments, an hDAF-GPI anchor region comprises or consists of an amino acid sequence of

PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID NO: 92).

[0625] In some embodiments, a utilized transmembrane region is a heterologous transmembrane region.

[0626] In some embodiments, a construct described herein does not comprise a transmembrane region.

[0627] Exemplary transmembrane are provided in the following Tables 4 and 5A:

Table 4: Exemplary transmembrane regions (amino acid sequences)

Table 5A: Exemplary nucleotide sequences encoding transmembrane regions

D. Multimerization Regions

[0628] In some embodiments, a construct (e.g., SARS-CoV-2 construct) as described herein includes one or more multimerization regions (e.g., a heterologous multimerization region).

[0629] In some embodiments, a heterologous multimerization region comprises a dimerization, trimerization or tetramerization region.

[0630] In some embodiments, a multimerization region is one described in W02017/081082, which is incorporated herein by reference in its entirety (e.g., SEQ ID NOs: 1116-1167, or fragments or variants thereof). Exemplary trimerization and tetramerization regions include, but are not limited to, engineered leucine zippers, fibritin foldon domain from enterobacteria phage T4, GCN4pll, GCN4-pll, and p53.

[0631] In some embodiments, a construct described herein is able to form a trimeric complex. For example, a provided construct may comprise a multimerization region allowing formation of a multimeric complex, such as for example a trimeric complex of a construct described herein. In some embodiments, a multimerization region allowing formation of a multimeric complex comprises a trimerization region, for example, a trimerization region described herein. In some embodiments, a construct includes a T4-fibritin-derived “foldon” trimerization region, for example, to increase its immunogenicity. In some embodiments, a construct includes a multimerization region comprising or consisting of the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 95). In some embodiments, a construct includes a multimerization region comprising or consisting of the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPG (SEQ ID NO: 96). An exemplary nucleotide sequences encoding SEQ ID NO: 96 is GGCUAUAUCCCUGAGGCUCCUAGAGAUGGCCAGGCCUACGUCAGAAAGGAUGGCG AGUGGGUCCUGCUGAGCACCUUUCUGGGCAGAUCCCUGGAAGUGCUGUUUCAAG GCCCUGGC (SEQ ID NO: 97). An exemplary nucleotide sequence encoding SEQ ID NO: 95 is GGCTATATCCCTGAGGCTCCTAGAGATGGCCAGGCCTACGTCAGAAAGGATGGCGA

GTGGGTCCTGCTGAGCACCTTTCTG (SEQ ID NO: 98).

E. Linkers

[0632] In some embodiments, a construct (e.g., SARS-CoV-2 construct) described herein includes one or more linkers. In some embodiments, a linker is or comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. In some embodiments, a linker is or comprises no more than about 30, 25, 20, 15, 10 or fewer amino acids. A linker can include any amino acid sequence and is not limited to any particular amino acids. In some embodiments, a linker comprises one or more glycine (G) amino acids. In some embodiments, a linker comprises one or more serine (S) amino acids. In some embodiments, a linker includes amino acids selected based on a cleavage predictor to generate highly-cleavable linkers.

[0633] In some embodiments, a linker is or comprises S-G4-S-G4-S (SEQ ID NO: 99). In some embodiments, a linker is or comprises GSPGSGSGS (SEQ ID NO: 100). In some embodiments, a linker is or comprises GGSGGGGSGG (SEQ ID NO: 101). In some embodiments, a linker is or comprises GSGSGS (SEQ ID NO: 102). In some embodiments, a linker is one presented in Table 5B. In some embodiments, a linker is or comprises a sequence as set forth in W02017/081082, which is incorporated herein by reference in its entirety (see SEQ ID NOs: 1509-1565, or a fragment or variant thereof).

[0634] In some embodiments, a construct (e.g., SARS-CoV-2 construct) described herein comprises a linker between a C-terminal region or fragment thereof and a transmembrane region.

[0635] Exemplary linkers are provided in the following Table 5B:

Table 5B: Exemplary linkers

Exemplary SARS-CoV-2 antigen polypeptide sequences and RNA sequences encoding the same

[0636] In some embodiments, a SARS-CoV-2 antigen included in or delivered by compositions and/or combinations described herein is an exemplary SARS-CoV-2 antigen polypeptide or a RNA construct encoding the same as described in Tables A-G, or having an amino acid or nucleotide sequence that is is at least 80% (including, e.g., at least 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher) identical to the sequence as described in Table A-G. In some embodiments where a SARS-CoV-2 antigen is delivered by a RNA molecule, the RNA molecule is a modified RNA as described herein. For example, in some embodiments, certain Us are replaced by a modified U (e.g., in some embodiments 1- methylpseudouridine). In some embodiments, all Us are replaced by a modified U (e.g., in some embodiments 1 -methylpseudouridine) . Table A: Sequences of a Soluble, Trimerized RBD (SP19-XBB.1.5_RBD-GS_Linker-Fibritin_long) and an exemplary RNA sequence encoding the same

Table B: Sequences of a Full-Length, Prefusion-Stabilized SARS-CoV-2 S Protein (XBB.1.5_P2) and an exemplary RNA sequence encoding the same

Table C: Sequences of a Soluble, Trimerized RBD (SP16-XBB.1.5_RBD-GS_Linker-Fibritin_long) and an exemplary RNA sequence encoding the same

Table D: Sequences of a Soluble Trimerized SI Domain (XBB.1.5_Sl-GS_Linker-Fibritin_long) and an exemplary RNA sequence encoding the same

Table E: Sequences of a Membrane-Tethered Spike Protein Comprising a C-terminal Truncation (Spike_deltal9) and an exemplary RNA sequence encoding the same

Table F: Sequences of a Trimerized, Membrane-Anchored RBD (SP19-XBB.1.5_RBD-GS_Einker-Fibritin_Short-GS_Einker-

TM(deltal9)) and an exemplary RNA sequence encoding the same

Table G: Sequences of a Membrane-Anchored SI Domain (SP19-XBB.1.5_Sl-GS_Linker-Fibritin_Short-GS_Linker-TM(deltal9))

Agents that induces a priming-favorable cytokine milieu

[0637] In some embodiments, one or more agents that induce a priming-favorable cytokine milieu, for example, in some embodiments in lymphoid tissues, is or comprises an agent that increases activation of naive B cell immune response to an antigen. In some embodiments, such an increase is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or higher. In some embodiments, such an increase is at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 2- fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, relative to activation of naive B cell immune response to an antigen in the absence of such an agent. Without wishing to be bound by a particular theory, in some embodiments, increase in activation of naive B cell immune response can be mediated through, for example, but not limited to enhancement in antigen presentation and/or promotion of expansion, survival, and/or effector function of T cells (e.g., in some embodiments CD4+ and/or CD 8+ T cell responses).

Exemplary IFN-inducing agents

[0638] In some embodiments, an agent that is useful for inducing a priming-favorable cytokine milieu is or comprises an agent that induces interferon (IFN) or activates signaling mediated by IFN. In some embodiments, such an agent specifically induces Type I IFN such as interferon alpha (IFNa) (“IFN-inducing agents”). In some embodiments, such an agent can induce a CD4+ T cell response.

[0639] In some embodiments, an IFN-inducing agent is or comprises IFNa.

[0640] In some embodiments, an IFNa-inducing agent is or comprises an unmodified

RNA molecule. As described herein, an “unmodified RNA” is an RNA molecule that contains substantially no artificial or synthetic modifications to the components of the nucleic acid, namely, sugars, bases and/or phosphate moieties, and/or cap portion and/or other non-coding elements (e.g., 3’ UTR, 5’ UTR, polyA tail). In some embodiments, an unmodified RNA is an RNA molecule that contains no modified ribonucleotides. In some embodiments, an unmodified RNA is an RNA molecule is an RNA molecule that contains no more than a certain level of modified ribonucleotides such that the immunogenicity of the resulting RNA is comparable to (e.g., within 10%, or within 5%, or within 3%) that of an unmodified RNA with no modified ribonucleotides (e.g., as described herein). In some embodiments, an unmodified RNA is an RNA molecule that contains no more than a certain level of modified ribonucleotides such that the resulting RNA is immunostimulatory (e.g., capable of activating naive immune response). In certain embodiments, an unmodified RNA is an RNA molecule that contains no more than a certain level of modified ribonucleotides such that the resulting RNA is capable of activating at least one pattern recognition receptor, including toll-like receptors (TLR3, TLR7, TLR8), RIG-I, and RNA-dependent protein kinase (PKR). Such a certain threshold level of modified ribonucleotides that can be present in an unmodified RNA is no more than 10% or lower, including, e.g., no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1%, or lower. In some embodiments, the threshold level of modified ribonucleotides that can be present in an unmodified RNA may vary with the structure of a selected modified ribonucleotide (e.g., 1- methyl pseudo uridine vs. 5-methylcytidine). In many embodiments described herein, an unmodified RNA is a synthetic RNA. In certain embodiments, a synthetic RNA is an in vitro transcribed (IVT) RNA.

[0641] Without wishing to be bound by theory, IVT RNA (e.g., IVT mRNA) is reported to activate various pattern recognition receptors, including, e.g., in some embodiments, toll-like receptors (TLR3, TLR7, TLR8), RIG-I, and/or RNA-dependent protein kinase (PKR), leading to undesirable mRNA immunogenicity and/or low expression of the IVT RNA. In some embodiments, the present disclosure, among other things, provides an insight that administration of unmodified RNA in a certain manner (e.g., at a certain dose and/or at a certain timing, relative to administration of a composition that delivers an antigen described herein, for example, in some embodiments a modified RNA that encodes an antigen) can provide certain beneficial therapeutic effects. Without wishing to be bound by any particular theory, in some embodiments, such beneficial therapeutic effect can include but are not limited to induction of IFN (e.g., IFNa) to a level that is priming-favorable. In some embodiments, such induction of IFN (e.g., IFNa) can be beneficial for activation of naive immune response to “new” epitopes present in a delivered antigen (e.g., in some embodiments, arisen epitopes present in a variant polypeptide as described herein).

[0642] In some embodiments, an unmodified RNA molecule that is useful in accordance with the present disclosure encodes one or more T cell epitopes. In some embodiments, an unmodified RNA molecule that is useful in accordance with the present disclosure encodes a plurality of (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, or more) T cell epitopes. In some embodiments, such T cell epitopes(s) is/are from a polypeptide of an infectious agent. In some embodiments, such T cell epitope(s) is/are from a highly conserved region of a polypeptide of an infectious agent (e.g., a region of polypeptide that is highly conserved among variants of an infectious agent) described herein. For example, in some embodiments, an unmodified RNA molecule that is useful in accordance with the present disclosure is a T string construct as described in the International Patent Application No. WO2021188969 or in the International Patent Application No. PCT/US22/44400, the relevant content of which is incorporated herein by reference for the purposes described herein. In some embodiments, an unmodified RNA molecule that is useful in accordance with the present disclosure is a T string construct as described in the International Patent Application No. PCT7US22/46799, the relevant content of which is incorporated herein by reference for the purposes described herein.

[0643] In some embodiments, an unmodified RNA molecule that is useful in accordance with the present disclosure does not encode a priming-favorable cytokine (e.g., in some embodiments Type I IFN, e.g., IFNa).

[0644] In some embodiments, the amount ratio (by mass or by moles) of a modified RNA molecule to an unmodified RNA is determined such that a combination of the modified RNA and the unmodified RNA provides a balance between activation of naive immune response and expression of the RNAs. In some embodiments, the amount ratio (by mass or moles) of a modified RNA molecule to an unmodified RNA molecule in a combination described herein is within a range of about 1:5 to about 10:1. In some embodiments, the amount ratio (by mass or moles) of the modified RNA molecule to the unmodified RNA molecule in a described combination is within a range of about 1: 1 to about 10: 1. In some embodiments, the amount ratio (by mass or moles) of a modified RNA molecule to an unmodified RNA molecule is about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2: 1, about 3:1, about 4: 1, about 5:1, about 6: 1, about 7: 1, about 8:1, about 9: 1, about 10: 1. In some such embodiments, the amount ratio (by mass or moles) of a modified RNA molecule to unmodified RNA molecule is about 1 : 1 [0645] In some embodiments, an IFNa-inducing agent described herein is or comprises a RNA replicon. In some embodiments an RNA replicon is an unmodified RNA molecule described herein. In some embodiments, an RNA replicon is a “self-replicating RNA” or “self- amplifying RNA.” In some embodiments, a self-replicating RNA is derived from or comprises elements derived from a single-stranded (ss) RNA virus, in particular a positive-stranded ssRNA virus, such as an alphavirus. Alphaviruses are typical representatives of positive-stranded RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856, which is incorporated herein by reference in its entirety). The total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5’-cap, and a 3’ poly(A) tail. The genome of alphaviruses encodes non-structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome. The four non-structural proteins (nsPl-nsP4) are typically encoded together by a first ORF beginning near the 5' terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3 ’ terminus of the genome. Typically, the first ORF is larger than the second ORF, the ratio being roughly 2: 1. In cells infected by an alpha virus, only the nucleic acid sequence encoding non-structural proteins is translated from the genomic RNA, while the genetic information encoding structural proteins is translatable from a subgenomic transcript, which is an RNA molecule that resembles eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111-124, which is incorporated herein by reference in its entirety). Following infection, i.e. at early stages of the viral life cycle, the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non-structural poly-protein (nsP1234). In some embodiments, an RNA replicon that is useful in accordance with the present disclosure is or comprises an RNA replicon as described in the International Patent Application No. WO2017/162266, the relevant contents of which are incorporated herein by reference for the purposes described herein.

[0646] In some embodiments, an RNA replicon is a “trans-replicating” or “trans- amplifying” RNA. Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms. In simple approaches, a first ORF encodes an alphavirus-derived RNA-dependent RNA polymerase (replicase), which upon translation mediates self-amplification of the RNA. A second ORF encoding alphaviral structural proteins is replaced by an open reading frame encoding an antigen or epitope described herein. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans-replication system). Trans-replication requires the presence of both these nucleic acid molecules in a given host cell. The nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow recognition and RNA synthesis by the alphaviral replicase. In some embodiments, an RNA replicon that is useful in accordance with the present disclosure is or comprises a trans-replicating RNA as described in the International Patent Application Nos. WO2017162265 and/or WO2017162461, and/or Beissert et al. “A trans-amplifying RNA vaccine strategy for inductive of potent protective immunity” Molecular Therapy (2020) 28: 119-128, the relevant contents of each of which are incorporated herein by reference for the purposes described herein.

[0647] In some embodiments, the amount ratio (by mass or by moles) of a modified RNA molecule to an RNA replicon (e.g., as described herein) is determined such that a combination of the modified RNA and the unmodified RNA provides a balance between activation of naive immune response and expression of the RNAs. In some embodiments, the amount ratio (by mass or moles) of a modified RNA molecule to an RNA replicon (e.g., as described herein) in a combination described herein is within a range of about 5: 1 to about 30: 1. In some embodiments, the amount ratio (by mass or moles) of the modified RNA molecule to an RNA replicon (e.g., as described herein) in a described combination is within a range of about 5: 1 to about 20: 1. In some embodiments, the amount ratio (by mass or moles) of a modified RNA molecule to an RNA replicon (e.g., as described herein) is about 1:1, about 2:1, about 3: 1, about 4: 1, about 5:1, about 6: 1, about 7:1, about 8: 1, about 9:1, about 10:1, about 11: 1, about 12: 1, about 13: 1, about 14: 1, about 15: 1, about 16:1, about 17:1, about 18:1, about 19:1, about 20: 1. In some such embodiments, the amount ratio (by mass or moles) of a modified RNA molecule to an RNA replicon is about 10: 1 to about 30:1.

Exemplary CD4+ T cell response-inducing agents [0648] In some embodiments, one or more agents that induce a priming-favorable cytokine milieu is or comprises an agent that induces one or more CD4+ T cell responses (a “CD4+ T cell response-inducing agent”).

[0649] In some embodiments, a CD4+ T cell response-inducing agent described herein is a composition that comprises or delivers one or more CD4+ T cell epitopes. In some embodiments, such a composition comprise one or more CD4+ T cell epitope peptides or polypeptides. In some embodiments, such a composition comprises a polynucleotide (e.g., in some embodiments RNA such as, e.g., mRNA) encoding one or more CD4+ T cell epitopes.

[0650] In some embodiments, a CD4+ T cell response-inducing agent described herein is or comprises an RNA molecule that encodes one or more CD4+ T cell epitopes. In some embodiments, a CD4+ T cell response-inducing agent described herein is or comprises an RNA molecule that encodes a plurality of (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, or more) CD4+ T cell epitopes. In some embodiments, such CD4+ T cell epitopes(s) is/are from a polypeptide of an infectious agent. In some embodiments, such CD4+ T cell epitope(s) is/are from a highly conserved region of a polypeptide of an infectious agent (e.g., a region of polypeptide that is highly conserved among variants of an infectious agent) described herein. For example, in some embodiments, a CD4+ T cell response-inducing agent described herein is a T string construct as described in the International Patent Application No. WO2021188969 or in the International Patent Application No. PCT7US22/44400, the relevant content of which is incorporated herein by reference for the purposes described herein. In some embodiments, a CD4+ T cell response-inducing agent described herein is a T string construct as described in the International Patent Application No. PCT/US22/46799, the relevant content of which is incorporated herein by reference for the purposes described herein.

[0651] In some embodiments, the amount ratio (by mass or moles) of a modified RNA molecule to a CD4+ T cell response-inducing agent (e.g., as described herein) in a combination described herein is within a range of about 1:5 to about 10: 1. In some embodiments, the amount ratio (by mass or moles) of a modified RNA molecule to a CD4+ T cell response-inducing agent (e.g., as described herein) in a described combination is within a range of about 1: 1 to about 10: 1. In some embodiments, the amount ratio (by mass or moles) of a modified RNA molecule to a CD4+ T cell response-inducing agent (e.g., as described herein) is about 1:5, about 1:4, about 1:3, about 1:2, about 1: 1, about 2:1, about 3: 1, about 4: 1, about 5:1, about 6: 1, about 7:1, about 8: 1, about 9:1, about 10:1. In some such embodiments, the amount ratio (by mass or moles) of a modified RNA molecule a CD4+ T cell response-inducing agent (e.g., as described herein) is about 1: 1.

Polyribonucleotides

[0652] In many embodiments, provided pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) deliver antigens as described herein by delivering a nucleic acid construct, e.g., in many embodiments, an RNA that encodes one or more antigens as described herein and is expressed in the subject upon administration of the pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine).

[0653] Among other things, the present disclosure encompasses the recognition that administration of nucleic acid, and particularly of RNA to achieve delivery (e.g., by expression) of encoded antigen can provide a variety of benefits relative to other strategies for immunizing against an infection (e.g., SARS-CoV-2) .

[0654] Among other things, the present disclosure provides an insight that RNA may be particularly useful and/or effective as an active agent in pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines (e.g., SARS-CoV-2)) for a variety of reasons including specifically that RNA can have intrinsic adjuvanticity. As noted herein, ability to induce very high antibody titers to proteins (e.g., SARS-CoV-2) , e.g., particularly antigens (e.g., SARS-CoV-2) associated with a variant of concern with high immune escape potential.

[0655] Still further, experience with SARS-CoV-2 vaccines has demonstrated that RNA actives, can also elicit significant and diverse T cell responses which, particularly when combined with strong antibody response, represents a combination of immune characteristics thought to potentially maximize the probability of protection.

A. Exemplary Polyribonucleotides Features

[0656] Polyribonucleotides described herein encode one or more constructs (e.g., SARS- CoV-2) described herein. In some embodiments, polyribonucleotides described herein can comprise a nucleotide sequence that encodes a 5’UTR of interest and/or a 3’ UTR of interest. In some embodiments, polynucleotides described herein can comprise a nucleotide sequence that encodes a polyA tail. In some embodiments, polyribonucleotides described herein may comprise a 5’ cap, which may be incorporated during transcription, or joined to a polyribonucleotide post- transcription.

1. 5’ Cap

[0657] A structural feature of mRNAs is cap structure at five-prime end (5’). Natural eukaryotic mRNA comprises a 7-methylguanosine cap linked to the mRNA via a 5' to 5'- triphosphate bridge resulting in capO structure (m7GpppN). In most eukaryotic mRNA and some viral mRNA, further modifications can occur at the 2' -hydroxy-group (2’ -OH) (e.g., the 2'- hydroxyl group may be methylated to form 2'-0-Me) of the first and subsequent nucleotides producing “capl” and “cap2” five-prime ends, respectively). Diamond, et al., (2014) Cytokine & growth Factor Reviews, 25:543-550, which is incorporated herein by reference in its entirety, reported that capO-mRNA cannot be translated as efficiently as capl -mRNA in which the role of 2'-0-Me in the penultimate position at the mRNA 5’ end is determinant. Lack of the 2'-O-met has been shown to trigger innate immunity and activate IFN response. Daffis, et al. (2010) Nature, 468:452-456; and Ziist et al. (2011) Nature Immunology, 12: 137-143, each of which is incorporated herein by reference in its entirety.

[0658] RNA capping is well researched and is described, e.g., in Decroly E et al. (2012) Nature Reviews 10: 51-65; and in Ramanathan A. et al., (2016) Nucleic Acids Res; 44(16): 7511-7526, the entire contents of each of which is hereby incorporated by reference. For example, in some embodiments, a 5 ’-cap structure which may be suitable in the context of the present invention is a capO (methylation of the first nucleobase, e.g. m7GpppN), capl (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), cap2 (additional methylation of the ribose of the 2nd nucleotide downstream of the m7GpppN), cap3 (additional methylation of the ribose of the 3rd nucleotide downstream of the m7GpppN), cap4 (additional methylation of the ribose of the 4th nucleotide downstream of the m7GpppN), ARCA (“anti -reverse cap analogue”), modified ARCA (e.g. phosphothioate modified ARCA), inosine, N1 -methyl- guanosine, 2’ -fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA- guanosine, and 2-azido-guanosine. [0659] The term “5'-cap” as used herein refers to a structure found on the 5'-end of an RNA, e.g., mRNA, and generally includes a guanosine nucleotide connected to an RNA, e.g., mRNA, via a 5'- to 5'-triphosphate linkage (also referred to as Gppp or G(5')ppp(5')). In some embodiments, a guanosine nucleoside included in a 5’ cap may be modified, for example, by methylation at one or more positions (e.g., at the 7-position) on a base (guanine), and/or by methylation at one or more positions of a ribose. In some embodiments, a guanosine nucleoside included in a 5’ cap comprises a 3’0 methylation at a ribose (3’0MeG). In some embodiments, a guanosine nucleoside included in a 5’ cap comprises methylation at the 7-position of guanine (m7G). In some embodiments, a guanosine nucleoside included in a 5’ cap comprises methylation at the 7-position of guanine and a 3’ O methylation at a ribose (m7(3’OMeG)). It will be understood that the notation used in the above paragraph, e.g., “(m2^{7 3} °)G” or “m7(3’OMeG)”, applies to other structures described herein.

[0660] In some embodiments, providing an RNA with a 5'-cap disclosed herein may be achieved by in vitro transcription, in which a 5'-cap is co-transcriptionally expressed into an RNA strand, or may be attached to an RNA post-transcriptionally using capping enzymes. In some embodiments, co-transcriptional capping with a cap disclosed improves the capping efficiency of an RNA compared to co-transcriptional capping with an appropriate reference comparator. In some embodiments, improving capping efficiency can increase a translation efficiency and/or translation rate of an RNA, and/or increase expression of an encoded polypeptide. In some embodiments, alterations to polynucleotides generates a non-hydrolyzable cap structure which can, for example, prevent decapping and increase RNA half-life.

[0661] In some embodiments, a utilized 5’ caps is a capO, a capl, or cap2 structure. See, e.g., Fig. 1 of Ramanathan A et al., and Fig. 1 of Decroly E et al., each of which is incorporated herein by reference in its entirety. See, e.g., Fig. 1 of Ramanathan A et al., and Fig. 1 of Decroly E et al., each of which is incorporated herein by reference in its entirety. In some embodiments, an RNA described herein comprises a capl structure. In some embodiments, an RNA described herein comprises a cap2.

[0662] In some embodiments, an RNA described herein comprises a capO structure. In some embodiments, a capO structure comprises a guanosine nucleoside methylated at the 7- position of guanine ((m⁷)G). In some embodiments, such a capO structure is connected to an RNA via a 5'- to 5 '-triphosphate linkage and is also referred to herein as (m⁷)Gppp. In some embodiments, a capO structure comprises a guanosine nucleoside methylated at the 2 ’-position of the ribose of guanosine. In some embodiments, a capO structure comprises a guanosine nucleoside methylated at the 3 ’-position of the ribose of guanosine. In some embodiments, a guanosine nucleoside included in a 5’ cap comprises methylation at the 7-position of guanine and at the 2’-position of the ribose ((m2⁷² °)G). In some embodiments, a guanosine nucleoside included in a 5’ cap comprises methylation at the 7-position of guanine and at the 2’-position of the ribose ((m2^{7 3} °)G).

[0663] In some embodiments, a capl structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m⁷)G) and optionally methylated at the 2’ or 3’ position of the ribose, and a 2’0 methylated first nucleotide in an RNA ((m² °)Ni). In some embodiments, a capl structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m⁷)G) and the 3’ position of the ribose, and a 2’0 methylated first nucleotide in an RNA ((m² °)Ni). In some embodiments, a capl structure is connected to an RNA via a 5'- to 5'- triphosphate linkage and is also referred to herein as, e.g., ((m⁷)Gppp(^{2 O})Ni) or (m2⁷’^{3 o})Gppp(^{2 -} °)Ni), wherein Ni is as defined and described herein. In some embodiments, a capl structure comprises a second nucleotide, N2, which is at position 2 and is chosen from A, G, C, or U, e.g., (m⁷)Gppp(^{2 O})NipN2 or (m2^{7 3} °)Gppp(^{2 O})NipN2 , wherein each of Ni and N2 is as defined and described herein.

[0664] In some embodiments, a cap2 structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m⁷)G) and optionally methylated at the 2’ or 3’ position pf the ribose, and a 2’0 methylated first and second nucleotides in an RNA ((m² °)Nip(m² ’ °)N2). In some embodiments, a cap2 structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m⁷)G) and the 3’ position of the ribose, and a 2’0 methylated first and second nucleotide in an RNA. In some embodiments, a cap2 structure is connected to an RNA via a 5'- to 5 '-triphosphate linkage and is also referred to herein as, e.g., ((m⁷)Gppp(^{2 -} °)Nip(^{2 O})N2) or (m2⁷’³ °)Gppp(^{2 O})Nip(^{2 O})N2), wherein each of Ni and N2 is as defined and described herein.

[0665] In some embodiments, the 5’ cap is a dinucleotide cap structure. In some embodiments, the 5’ cap is a dinucleotide cap structure comprising Ni, wherein Ni is as defined and described herein. In some embodiments, the 5’ cap is a dinucleotide cap G*Ni, wherein Ni is as defined above and herein, and:

G* comprises a structure of formula (I):

or a salt thereof, wherein each R² and R³ is -OH or -OCH3; and

X is O or S.

[0666] In some embodiments, R² is -OH. In some embodiments, R² is -OCH3. In some embodiments, R³ is -OH. In some embodiments, R³ is -OCH3. In some embodiments, R² is -OH and R³ is -OH. In some embodiments, R² is -OH and R³ is -CH3. In some embodiments, R² is - CH3 and R³ is -OH. In some embodiments, R² is -CH3 and R³ is -CH3.

[0667] In some embodiments, X is O. In some embodiments, X is S.

[0668] In some embodiments, the 5’ cap is a dinucleotide capO structure (e.g.,

(m⁷)GpppNi, (m2⁷² °)GpppNi, (m2^{7 3} °)GpppNi, (m⁷)GppSpNi, (m2^{7 2} °)GppSpNi, or (m2^{7 3} ’ °)GppSpNi), wherein Ni is as defined and described herein. In some embodiments, the 5’ cap is a dinucleotide capO structure (e.g., (m⁷)GpppNi, (m2⁷’² °)GpppNi, (m2⁷’³ °)GpppNi, (m⁷)GppSpNi, (m2⁷’² °)GppSpNi, or (m2⁷’³ °)GppSpNi), wherein Ni is G. In some embodiments, the 5’ cap is a dinucleotide capO structure (e.g., (m⁷)GpppNi, (m2⁷² °)GpppNi, (m2⁷’³ °)GpppNi, (m⁷)GppSpNi, (m2⁷’² °)GppSpNi, or (m2⁷’³ °)GppSpNi), wherein Ni is A, U, or C. In some embodiments, the 5’ cap is a dinucleotide capl structure (e.g., (m⁷)Gppp(m² °)Ni, (m2⁷’² °)Gppp(m² ’-°)Ni, (m₂ ⁷’³’ ^o)Gppp(m²’ ^o)Ni, (m⁷)GppSp(m²’ °)Ni, (m₂ ⁷’²’ °)GppSp(m²’- °)Ni, or (m2^{7 3} °)GppSp(m² °)Ni), wherein Ni is as defined and described herein. In some embodiments, the 5’ cap is selected from the group consisting of (m⁷)GpppG (“EcapO”), (m⁷)Gppp(m² °)G (“Ecapl”), (m2^{7 3} °)GpppG (“ARC A” or “DI”), and (m2^{7 2} °)GppSpG (“beta- S-ARCA”). In some embodiments, the 5’ cap is (m⁷)GpppG (“EcapO”), having a structure:

or a salt thereof.

[0669] In some embodiments, the 5’ cap is (m⁷)Gppp(m² °)G (“Ecapl”), having a structure:

or a salt thereof.

[0670] In some embodiments, the 5’ cap is (m2⁷’³ °)GpppG (“ARC A” or “DI”), having a structure:

or a salt thereof.

[0671] In some embodiments, the 5’ cap is (m2⁷² °)GppSpG (“beta-S-ARCA”), having a structure:

or a salt thereof.

[0672] In some embodiments, the 5’ cap is a trinucleotide cap structure. In some embodiments, the 5’ cap is a trinucleotide cap structure comprising NipN2, wherein Ni and N2 are as defined and described herein. In some embodiments, the 5’ cap is a dinucleotide cap G*NipN2, wherein Ni and N2 are as defined above and herein, and:

G* comprises a structure of formula (I):

or a salt thereof, wherein R², R³, and X are as defined and described herein.

[0673] In some embodiments, the 5’ cap is a trinucleotide capO structure (e.g. (m⁷)GpppNipN2, (m2⁷’² °)GpppNipN2, or (m2⁷’³ °)GpppNipN2), wherein Ni and N2 are as defined and described herein). In some embodiments, the 5’ cap is a trinucleotide capl structure (e.g., (m⁷)Gppp(m²’-°)NipN2, (m₂ ⁷’²’’^o)Gppp(m²’-°)NipN2, (m₂ ⁷’³’’^o)Gppp(m²’-°)NipN2), wherein Ni and N2 are as defined and described herein. In some embodiments, the 5’ cap is a trinucleotide cap2 structure (e.g., (m⁷)Gppp(m² °)Nip(m² °)N2, (m2^{7 2} °)Gppp(m² °)Nip(m² ’ °)N2, (m2^{7 3} °)Gppp(m² °)Nip(m² °)N2), wherein Ni and N2 are as defined and described herein. In some embodiments, the 5’ cap is selected from the group consisting of (m2⁷’³ ’ °)Gppp(m² °)ApG (“CleanCap AG”, “CC413”), (m2⁷’³’ ^o)Gppp(m²’ ^o)GpG (“CleanCap GG”), (m⁷)Gppp(m² °)ApG, (m⁷)Gppp(m²’ °)GpG, (m₂ ⁷’³’ ^o)Gppp(m₂ ⁶’²’ ^o)ApG, and (m⁷)Gppp(m²’’ °)ApU.

[0674] In some embodiments, the 5’ cap is (m2⁷’³ °)Gppp(m² °)ApG (“CleanCap AG”,

“CC413”), having a structure:

or a salt thereof.

[0675] In some embodiments, the 5’ cap is (m2⁷’³ °)Gppp(m² °)GpG (“CleanCap GG”), having a structure:

or a salt thereof.

[0676] In some embodiments, the 5’ cap is (m⁷)Gppp(m² °)ApG, having a structure:

or a salt thereof.

[0677] In some embodiments, the 5’ cap is (m⁷)Gppp(m² °)GpG, having a structure:

or a salt thereof.

[0678] In some embodiments, the 5’ cap is (m2⁷’³ °)Gppp(m2⁶’² °)ApG, having a structure:

or a salt thereof.

[0679] In some embodiments, the 5’ cap is (m⁷)Gppp(m² °)ApU, having a structure:

or a salt thereof.

[0680] In some embodiments, the 5’ cap is a tetranucleotide cap structure. In some embodiments, the 5’ cap is a tetranucleotide cap structure comprising NipN2pNs, wherein Ni, N2, and N3 are as defined and described herein. In some embodiments, the 5’ cap is a tetranucleotide cap G*NipN2pN3, wherein Ni, N2, and N3 are as defined above and herein, and:

G* comprises a structure of formula (I):

or a salt thereof, wherein R², R³, and X are as defined and described herein.

[0681] In some embodiments, the 5’ cap is a tetranucleotide capO structure (e.g. (m⁷)GpppNipN2pN3, (m2⁷’^{2 -O})GpppNipN2pN3, or (m2⁷’^{3 -O})GpppNiN2pN3), wherein Ni, N2, and N3 are as defined and described herein). In some embodiments, the 5’ cap is a tetranucleotide Capl structure (e.g., (m⁷)Gppp(m²’^-O)NipN2pN3, (m2^7,2 ■°)Gppp(m²’’°)NipN2pN3, (m2⁷’³’- °)Gppp(m² °)NipN2N3), wherein Ni, N2, and N3 are as defined and described herein. In some embodiments, the 5’ cap is a tetranucleotide Cap2 structure (e.g., (m⁷)Gppp(m² °)Nip(m² ’ °)N₂pN₃, (m₂ ⁷’²’-^o)Gppp(m²’-^o)Nip(m²’-°)N2pN3, (m₂ ⁷’³’-^o)Gppp(m²’-^o)Nip(m²’-°)N2pN3), wherein Ni, N2, and N3 are as defined and described herein. In some embodiments, the 5’ cap is selected from the group consisting of (m2⁷’³ °)Gppp(m² °)Ap(m² °)GpG, (m2⁷’³ °)Gppp(m² ’ °)Gp(m² °)GpC, (m⁷)Gppp(m² °)Ap(m² °)UpA, and (m⁷)Gppp(m²’ ^o)Ap(m²’ °)GpG.

[0682] In some embodiments, the 5’ cap is (m2^{7 3} °)Gppp(m² °)Ap(m² °)GpG, having a structure:

or a salt thereof.

[0683] In some embodiments, the 5’ cap is (m2⁷’³ °)Gppp(m² °)Gp(m² °)GpC, having a structure:

or a salt thereof. [0684] In some embodiments, the 5’ cap is (m⁷)Gppp(m² °)Ap(m² °)UpA, having a structure:

or a salt thereof.

[0685] In some embodiments, the 5’ cap is (m⁷)Gppp(m² °)Ap(m² °)GpG, having a structure:

or a salt thereof.

2. Cap Proximal Sequences

[0686] In some embodiments, a 5’ UTR utilized in accordance with the present disclosure comprises a cap proximal sequence, e.g., as disclosed herein. In some embodiments, a cap proximal sequence comprises a sequence adjacent to a 5’ cap. In some embodiments, a cap proximal sequence comprises nucleotides in positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide.

[0687] In some embodiments, a cap structure comprises one or more polynucleotides of a cap proximal sequence. In some embodiments, a cap structure comprises an m⁷ Guanosine cap and nucleotide +1 (Ni) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m⁷ Guanosine cap and nucleotide +2 (N2) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m⁷ Guanosine cap and nucleotides +1 and +2 (Ni and N2) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m⁷ Guanosine cap and nucleotides +1, +2, and +3 (Ni, N2, and N3) of an RNA polynucleotide.

[0688] Those skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, one or more residues of a cap proximal sequence (e.g., one or more of residues +1, +2, +3, +4, and/or +5) may be included in an RNA by virtue of having been included in a cap entity (e.g., a capl or cap2 structure, etc.); alternatively, in some embodiments, at least some of the residues in a cap proximal sequence may be enzymatically added (e.g., by a polymerase such as a T7 polymerase). For example, in certain exemplified embodiments where a m2⁷’³ °Gppp(mi² °)ApG cap is utilized, +1 (i.e., Ni) and +2 (i.e. N2) are the (mi² °)A and G residues of the cap, and +3, +4, and +5 are added by polymerase (e.g., T7 polymerase).

[0689] In some embodiments, the 5’ cap is a dinucleotide cap structure, wherein the cap proximal sequence comprises Ni of the 5’ cap, where Ni is any nucleotide, e.g., A, C, G or U. In some embodiments, the 5’ cap is a trinucleotide cap structure (e.g., the trinucleotide cap structures described above and herein), wherein the cap proximal sequence comprises Ni and N2 of the 5’ cap, wherein Ni and N2 are independently any nucleotide, e.g., A, C, G or U. In some embodiments, the 5’ cap is a tetranucleotide cap structure (e.g., the trinucleotide cap structures described above and herein), wherein the cap proximal sequence comprises Ni, N2, and N3 of the 5’ cap, wherein Ni, N2, and N3 are any nucleotide, e.g., A, C, G or U.

[0690] In some embodiments, e.g., where the 5’ cap is a dinucleotide cap structure, a cap proximal sequence comprises Ni of a the 5’ cap, and N2, N3, N4 and N5, wherein Ni to N5 correspond to positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide. In some embodiments, e.g., where the 5’ cap is a trinucleotide cap structure, a cap proximal sequence comprises Ni and N2 of a the 5’ cap, and N3, N4 and N5, wherein Ni to N5 correspond to positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide. In some embodiments, e.g., where the 5’ cap is a tetranucleotide cap structure, a cap proximal sequence comprises Ni, N2, and N3 of a the 5’ cap, and N4 and N5, wherein Ni to N5 correspond to positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide.

[0691] In some embodiments, Ni is A. In some embodiments, Ni is C. In some embodiments, Ni is G. In some embodiments, Ni is U. In some embodiments, N2 is A. In some embodiments, N2 is C. In some embodiments, N2 is G. In some embodiments, N2 is U. In some embodiments, N3 is A. In some embodiments, N3 is C. In some embodiments, N3 is G. In some embodiments, N3 is U. In some embodiments, N4 is A. In some embodiments, N4 is C. In some embodiments, N4 is G. In some embodiments, N4 is U. In some embodiments, N5 is A. In some embodiments, N5 is C. In some embodiments, N5 is G. In some embodiments, N5 is U. It will be understood that, each of the embodiments described above and herein (e.g., for Ni through N5) may be taken singly or in combination and/or may be combined with other embodiments of variables described above and herein (e.g., 5’ caps).

[0692] In some embodiments, a cap proximal sequence comprises Ai and G2 of the Capl structure, and a sequence comprising: A3A4U5 (SEQ ID NO: 111) at positions +3, +4 and +5 respectively of the polyribonucleotide.

3. 5’ UTR

[0693] In some embodiments, a nucleic acid (e.g., DNA, RNA) utilized in accordance with the present disclosure comprises a 5'-UTR. In some embodiments, 5’-UTR may comprise a plurality of distinct sequence elements; in some embodiments, such plurality may be or comprise multiple copies of one or more particular sequence elements (e.g., as may be from a particular source or otherwise known as a functional or characteristic sequence element). In some embodiments a 5’ UTR comprises multiple different sequence elements.

[0694] The term “untranslated region” or “UTR” is commonly used in the art to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA polynucleotide, such as an mRNA molecule. An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5'-UTR) and/or 3' (downstream) of an open reading frame (3'-UTR). As used herein, the terms “five prime untranslated region” or “5' UTR” refer to a sequence of a polyribonucleotide between the 5' end of the polyribonucleotide (e.g., a transcription start site) and a start codon of a coding region of the polyribonucleotide. In some embodiments, “5' UTR” refers to a sequence of a polyribonucleotide that begins at the 5' end of the polyribonucleotide (e.g., a transcription start site) and ends one nucleotide (nt) before a start codon (usually AUG) of a coding region of the polyribonucleotide, e.g., in its natural context. In some embodiments, a 5' UTR comprises a Kozak sequence. A 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap. In some embodiments, a 5’ UTR disclosed herein comprises a cap proximal sequence, e.g., as defined and described herein. In some embodiments, a cap proximal sequence comprises a sequence adjacent to a 5’ cap.

[0695] Exemplary 5’ UTRs include a human alpha globin (hAg) 5’UTR or a fragment thereof, a TEV 5’ UTR or a fragment thereof, a HSP70 5’ UTR or a fragment thereof, or a c-Jun 5’ UTR or a fragment thereof. In some embodiments, an RNA disclosed herein comprises a hAg 5’ UTR or a fragment thereof.

[0696] In some embodiments, an RNA disclosed herein comprises a 5’ UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a 5’ UTR with the sequence AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC (SEQ ID NO: 112). In some embodiments, an RNA disclosed herein comprises a 5’ UTR having the sequence AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC (SEQ ID NO: 112).

[0697] In some embodiments, an RNA disclosed herein comprises a 5’ UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a 5’ UTR with the sequence AACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 113)(hAg-Kozak/5'UTR). In some embodiments, an RNA disclosed herein comprises a 5’ UTR having the sequence AACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 113)(hAg-Kozak/5'UTR).

4. Poly A Tail

[0698] In some embodiments, a polynucleotide (e.g., DNA, RNA) disclosed herein comprises a poly adenylate (poly A) sequence, e.g., as described herein. In some embodiments, a polyA sequence is situated downstream of a 3'-UTR, e.g., adjacent to a 3'-UTR.

[0699] As used herein, the term "poly(A) sequence" or "poly-A tail" refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the d'- end of an RNA polynucleotide. Poly(A) sequences are known to those of skill in the art and may follow the 3 ’-UTR in the RNAs described herein. An uninterrupted poly(A) sequence is characterized by consecutive adenylate residues. In nature, an uninterrupted poly(A) sequence is typical. In some embodiments, polynucleotides disclosed herein comprise an uninterrupted Poly(A) sequence. In some embodiments, polynucleotides disclosed herein comprise interrupted Poly(A) sequence. In some embodiments, RNAs disclosed herein can have a poly(A) sequence attached to the free 3'-end of the RNA by a template-independent RNA polymerase after transcription or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0700] It has been demonstrated that a poly(A) sequence of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5’) of the poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017, which is herein incorporated by reference).

[0701] In some embodiments, a poly(A) sequence in accordance with the present disclosure is not limited to a particular length; in some embodiments, a poly(A) sequence is any length. In some embodiments, a poly(A) sequence comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides. In this context, "essentially consists of" means that most nucleotides in the poly(A) sequence, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly(A) sequence are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, "consists of" means that all nucleotides in the poly(A) sequence, i.e., 100% by number of nucleotides in the poly(A) sequence, are A nucleotides. The term "A nucleotide" or "A" refers to adenylate.

[0702] In some embodiments, a poly(A) sequence is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding a poly(A) sequence (coding strand) is referred to as poly(A) cassette.

[0703] In some embodiments, the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such a cassette is disclosed in WO 2016/005324 Al, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016/005324 Al, which is herein incorporated by reference may be used in accordance with the present disclosure. A poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed. In some embodiments, the poly(A) sequence contained in an RNA polynucleotide described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.

[0704] In some embodiments, no nucleotides other than A nucleotides flank a poly(A) sequence at its 3'-end, i.e., the poly(A) sequence is not masked or followed at its 3'-end by a nucleotide other than A.

[0705] In some embodiments, the poly(A) sequence may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises about 150 nucleotides. In some embodiments, the poly(A) sequence comprises about 120 nucleotides. In some embodiments, the poly(A) sequence comprises about 100 nucleotides.

[0706] In some embodiments, a poly A tail comprises a specific number of adenosines, such as about 50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more, about 100 or more, about 120, or about 150 or about 200. In some embodiments a poly A tail of a string construct may comprise 200 A residues or less. In some embodiments, a poly A tail of a string construct may comprise about 200 A residues. In some embodiments, a poly A tail of a string construct may comprise 180 A residues or less. In some embodiments, a poly A tail of a string construct may comprise about 180 A residues. In some embodiments, a poly A tail may comprise 150 residues or less.

[0707] In some embodiments, RNA comprises a poly(A) sequence comprising the nucleotide sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAA

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AA (SEQ ID NO: 114), or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

AA (SEQ ID NO: 114). In some embodiments, a poly(A) tail comprises a plurality of A residues interrupted by a linker. In some embodiments, a linker comprises the nucleotide sequence GCATATGAC (SEQ ID NO: 115).

[0708] In some embodiments, RNA comprises a poly(A) sequence comprising the nucleotide sequence of

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AA (SEQ ID NO: 116), or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AA (SEQ ID NO: 116). In some embodiments, a poly(A) tail comprises a plurality of A residues interrupted by a linker. In some embodiments, a linker comprises the nucleotide sequence GCAUAUGAC (SEQ ID NO: 117).

5. 3’ UTR

[0709] In some embodiments, an RNA utilized in accordance with the present disclosure comprises a 3'-UTR.

[0710] As used herein, the terms “three prime untranslated region,” “3' untranslated region,” or “3' UTR” refer to a sequence of an mRNA molecule that begins following a stop codon of a coding region of an open reading frame sequence. In some embodiments, the 3' UTR begins immediately after a stop codon of a coding region of an open reading frame sequence, e.g., in its natural context. In other embodiments, the 3' UTR does not begin immediately after stop codon of the coding region of an open reading frame sequence, e.g., in its natural context. The term “3'-UTR” does preferably not include the poly(A) sequence. Thus, the 3'-UTR is upstream of the poly(A) sequence (if present), e.g. directly adjacent to the poly(A) sequence.

[0711] In some embodiments, an RNA disclosed herein comprises a 3’ UTR comprising an F element and/or an I element. In some embodiments, a 3’ UTR or a proximal sequence thereto comprises a restriction site. In some embodiments, a restriction site is a BamHI site. In some embodiments, a restriction site is a Xhol site.

[0712] In some embodiments, an RNA construct comprises an F element. In some embodiments, an F element sequence is a 3 ’-UTR of amino-terminal enhancer of split (AES).

[0713] In some embodiments, an RNA disclosed herein comprises a 3’ UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a 3’ UTR with the sequence of CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGT CTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACC TCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCC TAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTT TAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACC (SEQ ID NO: 118). In some embodiments, an RNA disclosed herein comprises a 3’ UTR with the sequence of CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGT CTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACC TCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCC TAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTT TAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACC (SEQ ID NO: 118).

[0714] In some embodiments, an RNA disclosed herein comprises a 3’ UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a 3’ UTR with the sequence of CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGA GUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACC ACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCU

UAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACG AAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACA CC (SEQ ID NO: 119). In some embodiments, an RNA disclosed herein comprises a 3’ UTR with the sequence of

CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGA GUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACC ACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCU

UAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACG AAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACA CC (SEQ ID NO: 119).

[0715] In some embodiments, an RNA disclosed herein comprises a 3’ UTR provided in

SEQ ID NO: 119.

[0716] In some embodiments, a 3 ’UTR is an FI element as described in

W02017/060314, which is herein incorporated by reference in its entirety.

Table 6: Sequences of an Exemplary RNA Construct Encoding a Soluble, Trimerized RBD (SP19-XBB.1.5_RBD-GS_Linker- Fibritin_long)

Table 7: Sequences of an Exemplary RNA Construct Encoding a Full-Length, Prefusion-Stabilized SARS-CoV-2 S Protein (XBB.1.5_P2)

Table 8: Sequences of an Exemplary RNA Construct Encoding a Soluble, Trimerized RBD (SP16-XBB.1.5_RBD-GS_Einker- Fibritin_long)

Table 9: Sequences of an Exemplary RNA Construct Encoding a Soluble Trimerized SI Domain (XBB.1.5_Sl-GS_Einker- Fibritin_long)

Table 10: Sequences of an Exemplary RNA Construct Encoding a Membrane-Tethered Spike Protein Comprising a C-terminal Truncation (Spike_deltal9)

Table 11: Sequences of an Exemplary RNA Construct Encoding a Trimerized, Membrane-Anchored RBD (SP19-XBB.1.5_RBD- GS_Linker-Fibritin_Short-GS_Linker-TM(delta 19))

Table 12: Sequences of an Exemplary RNA Construct Encoding a Membrane-Anchored SI Domain (XBB.1.5_Sl-GS_Einker- Fibritin_Short-GS_Linker-TM(delta 19))

B. RNA Formats

[0717] At least three distinct formats useful for RNA compositions (e.g., pharmaceutical compositions) have been developed, namely non-modified uridine containing mRNA (uRNA), nucleoside-modified mRNA (modRNA), and self- amplifying mRNA (saRNA). Each of these platforms displays unique features. Each of these platforms displays unique features. In general, in all three formats, RNA is capped, contains open reading frames (ORFs) flanked by untranslated regions (UTR), and have a polyA-tail at the 3' end. An ORF of an uRNA and modRNA vectors encode an antibody agent or fragment thereof. An saRNA has multiple ORFs.

[0718] In some embodiments, an RNA comprises only unmodified nucleotides. In some embodiments, an RNA comprises only unmodified nucleotides and comprise any 5’ cap described herein (including a 5’ cap comprising chemical modifications that do not occur in nature).

Modified RNA

[0719] In some embodiments, technologies described herein are particularly useful with compositions and/or methods described herein involving delivery of modified RNAs that encodes a polypeptide that comprises or consists of a variant polypeptide of a reference antigen (e.g., exemplary antigen as described herein) of an infectious agent, or an immunogenic portion thereof, wherein the variant polypeptide comprises neutralizing epitopes that are absent in the reference antigen. As described herein, a “modified RNA” is an RNA molecule that contains certain artificial or synthetic modifications to at least one or more components of the nucleic acid, namely, sugars, bases and/or phosphate moieties, and/or cap portion and/or other non- coding elements (e.g., 3’ UTR, 5’ UTR, polyA tail). In some embodiments, a modified RNA is an RNA molecule that contains at least a certain level of modified ribonucleotides such that the resulting RNA is weakly immunogenic. In some embodiments, a modified RNA is an RNA molecule that contains at least a certain level of modified ribonucleotides such that the immunogenicity of the resulting RNA is lower than an unmodified RNA with no modified ribonucleotides (e.g., as described herein). In some embodiments, a modified RNA is an RNA molecule that contains at least a certain level of modified ribonucleotides such that activation of at least one pattern recognition receptor, including toll-like receptors (TER3, TER7, TER8), RIG-I, and RNA-dependent protein kinase (PKR), is reduced. In some embodiments, such reduction in activation of at least one pattern recognition receptor is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more, as compared to such activation by an unmodified RNA with no modified ribonucleotides (e.g., as described herein). In some embodiments, a modified RNA comprises more than 10% modified ribonucleotides, including, e.g., more than 15%, more than 20%, more than 25%, more than 30%, more than 40%, or more modified ribonucleotides. In some embodiments, a modified RNA comprises modified ribonucleotides at a level of about 15% to about 50%, or about 15% to about 40% or about 20% to about 40%, or about 20% to about 30%. In certain embodiments, a modified RNA is an in vitro transcribed (IVT) RNA.

[0720] In one embodiment, RNA encoding a vaccine antigen to be administered according to the present disclosure is non-immunogenic. RNA encoding immunostimulant may be administered according to the present disclosure to provide an adjuvant effect. RNA encoding immunostimulant may be standard RNA or non-immunogenic RNA.

[0721] The term "non-immunogenic RNA" as used herein refers to RNA that does not induce a response by the immune system upon administration, e.g., to a mammal, or induces a weaker response than would have been induced by the same RNA that differs only in that it has not been subjected to the modifications and treatments that render the non-immunogenic RNA non-immunogenic, i.e., than would have been induced by standard RNA (stdRNA). In one preferred embodiment, non-immunogenic RNA, which is also termed modified RNA (modRNA) herein, is rendered non-immunogenic by incorporating modified nucleosides suppressing RNA- mediated activation of innate immune receptors into the RNA and removing double-stranded RNA (dsRNA).

[0722] For rendering the non-immunogenic RNA non-immunogenic by the incorporation of modified nucleosides, any modified nucleoside may be used as long as it lowers or suppresses immunogenicity of the RNA. Particularly preferred are modified nucleosides that suppress RNA- mediated activation of innate immune receptors. In one embodiment, the modified nucleosides comprises a replacement of one or more uridines with a nucleoside comprising a modified nucleobase. In one embodiment, the modified nucleobase is a modified uracil. In one embodiment, the nucleoside comprising a modified nucleobase is selected from the group consisting of 3-methyl-uridine (m³U), 5 -methoxy-uridine (mo⁵U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s²U), 4-thio-uridine (s⁴U), 4-thio-pseudouridine, 2-thio- pseudouridine, 5 -hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo- uridine or 5 -bromo-uridine), uridine 5-oxyacetic acid (cmo⁵U), uridine 5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U), 1 -carboxymethyl -pseudouridine, 5- carboxyhydroxymethyl-uridine (chm⁵U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U), 5-methoxycarbonylmethyl-uridine (mcm⁵U), 5-methoxycarbonylmethyl-2-thio- uridine (mcm⁵s²U), 5-aminomethyl-2-thio-uridine (nm⁵s²U), 5-methylaminomethyl-uridine (mnm⁵U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm⁵s²U), 5- methylaminomethyl-2-seleno-uridine (mnm⁵se²U), 5-carbamoylmethyl-uridine (ncm⁵U), 5- carboxymethylaminomethyl-uridine (cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (rm⁵U), 1- taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(rm5s2U), 1 -taurinomethyl-4-thio- pseudouridine), 5-methyl-2-thio-uridine (m⁵s²U), l-methyl-4-thio-pseudouridine (m ¹ s⁴vp), 4-thio- 1-methyl-pseudouridine, 3 -methyl -pseudouridine

2-thio- 1 -methyl -pseudouridine, 1- methyl-l-deaza-pseudouridine, 2-thio- 1 -methyl- 1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D), 2-thio- dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4- methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, Nl-methyl-pseudouridine, 3-(3- amino-3 -carboxypropyl )uridine (acp³U), 1 -methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ \p), 5-(isopentenylaminomethyl)uridine (inm⁵U), 5-(isopentenylaminomethyl)-2-thio- uridine (inm⁵s²U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m⁵Um), 2'-O-methyl-pseudouridine (\|/m), 2-thio-2'-O-methyl-uridine (s²Um), 5- methoxycarbonylmethyl-2'-O-methyl-uridine (mcm⁵Um), 5-carbamoylmethyl-2'-O-methyl- uridine (ncm⁵Um), 5-carboxymethylaminomethyl-2'-O-methyl -uridine (cmnm⁵Um), 3,2'-O- dimethyl-uridine (m³Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm⁵Um), 1 -thio- uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-0H-ara-uridine, 5-(2- carbomethoxyvinyl) uridine, and 5-[3-(l-E-propenylamino)uridine. In one particularly preferred embodiment, the nucleoside comprising a modified nucleobase is pseudouridine (y), Nl-methyl- pseudouridine (m h|/) or 5 -methyl -uridine (m5U), in particular N 1 -methyl-pseudouridine.

[0723] In one embodiment, the replacement of one or more uridines with a nucleoside comprising a modified nucleobase comprises a replacement of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the uridines.

[0724] In some embodiments of the present disclosure, RNA is “replicon RNA” or simply a “replicon,” in particular “self-replicating RNA” or “self-amplifying RNA.” In one particularly preferred embodiment, replicon or self-replicating RNA is derived from or comprises elements derived from a single-stranded (ss) RNA virus, in particular a positive- stranded ssRNA virus, such as an alphavirus. Alphaviruses are typical representatives of positive-stranded RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856, which is incorporated herein by reference in its entirety). The total genome length of many alpha viruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5 ’-cap, and a 3’ poly(A) tail. The genome of alpha viruses encodes non- structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome. The four non-structural proteins (nsPl-nsP4) are typically encoded together by a first ORF beginning near the 5' terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3 ’ terminus of the genome. Typically, the first ORF is larger than the second ORF, the ratio being roughly 2: 1. In cells infected by an alphavirus, only the nucleic acid sequence encoding non-structural proteins is translated from the genomic RNA, while the genetic information encoding structural proteins is translatable from a subgenomic transcript, which is an RNA molecule that resembles eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111-124, which is incorporated herein by reference in its entirety). Following infection, i.e. at early stages of the viral life cycle, the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non-structural poly-protein (nsP1234).

[0725] Alphavirus -derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms. In simple approaches, a first ORF encodes an alphavirus-derived RNA-dependent RNA polymerase (replicase), which upon translation mediates self-amplification of the RNA. A second ORF encoding alphaviral structural proteins is replaced by an open reading frame encoding a HSV (HSV-1 and/or HSV-2) construct described herein. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans-replication system). Trans-replication requires the presence of both these nucleic acid molecules in a given host cell. The nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow recognition and RNA synthesis by the alphaviral replicase.

[0726] Features of a non-modified uridine platform may include, for example, one or more of intrinsic adjuvant effect, as well as good tolerability and safety. Features of modified uridine (e.g., pseudouridine) platform may include reduced adjuvant effect, blunted immune innate immune sensor activating capacity and thus good tolerability and safety. Features of self- amplifying platform may include, for example, long duration of protein expression, good tolerability and safety, higher likelihood for efficacy with very low vaccine dose.

[0727] The present disclosure provides particular RNA constructs optimized, for example, for improved manufacturability, encapsulation, expression level (and/or timing), etc. Certain components are discussed below, and certain preferred embodiments are exemplified herein.

C. Codon Optimization and GC Enrichment

[0728] As used herein, the term “codon-optimized” refers to alteration of codons in a coding region of a nucleic acid molecule (e.g., a polyribonucleotide) to reflect the typical codon usage of a host organism (e.g., a subject receiving a nucleic acid molecule (e.g., a polyribonucleotide)) without preferably altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, in some embodiments, coding regions are codon-optimized for optimal expression in a subject to be treated using the RNA molecules described herein. In some embodiments, codon-optimization may be performed such that codons for which frequently occurring tRNAs are available are inserted in place of “rare codons.” In some embodiments, codon-optimization may include increasing guanosine/cytosine (G/C) content of a coding region of RNA described herein as compared to the G/C content of the corresponding coding sequence of a wild type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence. [0729] In some embodiments, a coding sequence (also referred to as a “coding region”) is codon optimized for expression in the subject to whom a composition (e.g., a pharmaceutical composition) is to be administered (e.g., a human). Thus, in some embodiments, sequences in such a polynucleotide (e.g., a polyribonucleotide) may differ from wild type sequences encoding the relevant antigen or antigenic fragment or epitope thereof, even when the amino acid sequence of the antigen or antigenic fragment or epitope thereof is wild type.

[0730] In some embodiments, strategies for codon optimization for expression in a relevant subject (e.g., a human), and even, in some cases, for expression in a particular cell or tissue.

[0731] In general, as is understood, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in a subject or its cells of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of a native sequence with codons that are more frequently or most frequently used in the genes of that subject or its cells while maintaining the native amino acid sequence.

[0732] Various species exhibit particular bias for certain codons of a particular amino acid. Without wishing to be bound by any one theory, codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell may generally be a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes may be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are available, for example, at the "Codon Usage Database" available at www.kazusa.orjp/codon/ and these tables may be adapted in a number of ways. Computer algorithms for codon optimizing a particular sequence for expression in a particular subject or its cells are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available.

[0733] In some embodiments, a polynucleotide (e.g., a polyribonucleotide) of the present disclosure is codon optimized, wherein the codons in the polynucleotide (e.g., the polyribonucleotide) are adapted to human codon usage (herein referred to as “human codon optimized polynucleotide”). Codons encoding the same amino acid occur at different frequencies in a subject, e.g., a human. Accordingly, in some embodiments, the coding sequence of a polynucleotide of the present disclosure is modified such that the frequency of the codons encoding the same amino acid corresponds to the naturally occurring frequency of that codon according to the human codon usage, e.g., as shown in Table 13. For example, in the case of the amino acid Ala, the wild type coding sequence is preferably adapted in a way that the codon “GCC” is used with a frequency of 0.40, the codon “GCT” is used with a frequency of 0.28, the codon “GCA” is used with a frequency of 0.22 and the codon “GCG” is used with 30 a frequency of 0.10 etc. (see Table 13). Accordingly, in some embodiments, such a procedure (as exemplified for Ala) is applied for each amino acid encoded by the coding sequence of a polynucleotide to obtain sequences adapted to human codon usage.

Table 13: Human codon usage with frequencies indicated for each amino acid

[0734] Certain strategies for codon optimization and/or G/C enrichment for human expression are described in W02002/098443, which is incorporated by reference herein in its entirety. In some embodiments, a coding sequence may be optimized using a multiparametric optimization strategy. In some embodiments, optimization parameters may include parameters that influence protein expression, which can be, for example, impacted on a transcription level, an mRNA level, and/or a translational level. In some embodiments, exemplary optimization parameters include, but are not limited to transcription-level parameters (including, e.g., GC content, consensus splice sites, cryptic splice sites, SD sequences, TATA boxes, termination signals, artificial recombination sites, and combinations thereof); mRNA-level parameters (including, e.g., RNA instability motifs, ribosomal entry sites, repetitive sequences, and combinations thereof); translation-level parameters (including, e.g., codon usage, premature poly(A) sites, ribosomal entry sites, secondary structures, and combinations thereof); or combinations thereof. In some embodiments, a coding sequence may be optimized by a GeneOptimizer algorithm as described in Fath et al. “Multiparameter RNA and Codon Optimization: A Standardized Tool to Assess and Enhance Autologous Mammalian Gene Expression” PLoS ONE 6(3): el7596; Rabb et al., which is incorporated herein by reference in its entirety, “The GeneOptimizer Algorithm: using a sliding window approach to cope with the vast sequence space in multiparameter DNA sequence optimization” Systems and Synthetic Biology (2010) 4:215-225; and Graft et al. “Codon-optimized genes that enable increased heterologous expression in mammalian cells and elicit efficient immune responses in mice after vaccination of naked DNA” Methods Mol Med (2004) 94: 197-210, the entire content of each of which is incorporated herein for the purposes described herein. In some embodiments, a coding sequence may be optimized by Eurofins’ adaption and optimization algorithm “GENEius” as described in Eurofins’ Application Notes: Eurofins’ adaption and optimization software “GENEius” in comparison to other optimization algorithms, the entire content of which is incorporated by reference for the purposes described herein.

[0735] In some embodiments, a coding sequence utilized in accordance with the present disclosure has G/C content that is increased compared to a coding sequence for a SARS-CoV-2 construct described herein. In some embodiments, guanosine/cytidine (G/C) content of a coding region is modified relative to a comparable coding sequence for SARS-CoV-2 construct described herein, but the amino acid sequence encoded by the polyribonucleotide is not modified.

[0736] Without wishing to be bound by any particular theory, it is proposed that GC enrichment may improve translation of a payload sequence. Typically, sequences having an increased G (guanosine )/C (cytidine) content are more stable than sequences having an increased A (adenosine )/U (uridine) content. In respect to the fact that several codons code for one and the same amino acid (so-called degeneration of the genetic code), the most favorable codons for the stability can be determined (so-called alternative codon usage). Depending on the amino acid to be encoded by a polyribonucleotide, there are various possibilities for modification of the ribonucleic acid sequence, compared to its wild type sequence. In particular, codons which contain A and/or U nucleosides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and/or U or contain a lower content of A and/or U nucleosides.

[0737] In some embodiments, G/C content of a coding region of a polyribonucleotide described herein is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, or even more compared to the G/C content of the coding region prior to codon optimization, e.g., of the wild type RNA. In some embodiments, G/C content of a coding region of a polyribonucleotide described herein is decreased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, or even more compared to the G/C content of the coding region prior to codon optimization, e.g., of the wild type RNA.

[0738] In some embodiments, stability and translation efficiency of a polyribonucleotide may incorporate one or more elements established to contribute to stability and/or translation efficiency of the polyribonucleotide; exemplary such elements are described, for example, in PCT/EP2006/009448 incorporated herein by reference. In some embodiments, to increase expression of a polyribonucleotide used according to the present disclosure, a polyribonucleotide may be modified within the coding region, i.e., the sequence encoding the expressed peptide or protein, without altering the sequence of the expressed peptide or protein, for example so as to increase the GC-content to increase mRNA stability and/or to perform a codon optimization and, thus, enhance translation in cells. RNA Delivery Technologies

[0739] Provided polyribonucleotides may be delivered for therapeutic applications described herein using any appropriate methods known in the art, including, e.g., delivery as naked RNAs, or delivery mediated by viral and/or non-viral vectors, polymer-based vectors, lipid compositions, nanoparticles (e.g., lipid nanoparticles, polymeric nanoparticles, lipid- polymer hybrid nanoparticles, etc.), and/or peptide -based vectors. See, e.g., Wadhwa et al. “Opportunities and Challenges in the Delivery of mRNA-Based Vaccines” Pharmaceutics (2020) 102 (27 pages), the content of which is incorporated herein by reference, for information on various approaches that may be useful for delivery polyribonucleotides described herein.

[0740] In some embodiments, one or more polyribonucleotides can be formulated with lipid nanoparticles for delivery (e.g., administration).

[0741] In some embodiments, lipid nanoparticles can be designed to protect polyribonucleotides from extracellular RNases and/or engineered for systemic delivery of the RNA to target cells. In some embodiments, such lipid nanoparticles may be particularly useful to deliver polyribonucleotides when polyribonucleotides are intravenously or intramuscularly administered to a subject.

A. Lipid Compositions

1. Lipids and Lipid-Like Materials

[0742] The terms "lipid" and "lipid-like material" are broadly defined herein as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also frequently denoted as amphiphiles. Lipids are usually poorly soluble in water. In an aqueous environment, the amphiphilic nature allows the molecules to self- assemble into organized structures and different phases. One of those phases consists of lipid bilayers, as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment. Hydrophobicity can be conferred by the inclusion of a polar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). The hydrophilic groups may comprise polar and/or charged groups and include carbohydrates, phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other like groups. [0743] Often, an amphiphilic compound has a polar head attached to a long hydrophobic tail. In some embodiments, the polar fragment is soluble in water, while the non-polar fragment is insoluble in water. In addition, the polar portion may have either a formal positive charge, or a formal negative charge. Alternatively, the polar portion may have both a formal positive and a negative charge, and be a zwitterion or inner salt. For purposes of the disclosure, the amphiphilic compound can be, but is not limited to, one or a plurality of natural or non-natural lipids and lipid-like compounds.

[0744] A "lipid-like material" is a substance that is structurally and/or functionally related to a lipid but may not be considered a lipid in a strict sense. For example, the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment and includes surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties. Generally speaking, the term refers to molecules, which comprise hydrophilic and hydrophobic moieties with different structural organization, which may or may not be similar to that of lipids.

[0745] Specific examples of amphiphilic compounds that may be included in an amphiphilic layer include, but are not limited to, phospholipids, aminolipids and sphingolipids.

[0746] Generally, lipids may be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from condensation of ketoacyl subunits), sterols and prenol lipids (derived from condensation of isoprene subunits). Although the term "lipid" is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as sterol-containing metabolites such as cholesterol.

[0747] Fatty acids are a diverse group of molecules made of a hydrocarbon chain that terminates with a carboxylic acid group; this arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water. The carbon chain, typically between four and 24 carbons long, may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, there is the possibility of either a cis or trans geometric isomerism, which significantly affects the molecule's configuration. Cis-double bonds cause the fatty acid chain to bend, an effect that is compounded with more double bonds in the chain. Other major lipid classes in the fatty acid category are the fatty esters and fatty amides.

[0748] Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the best- known being the fatty acid triesters of glycerol, called triglycerides. The word "triacylglycerol" is sometimes used synonymously with "triglyceride". In these compounds, the three hydroxyl groups of glycerol are each esterified, typically by different fatty acids. Additional subclasses of glycerolipids are represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage.

[0749] Glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid-derived "tails" by ester linkages and to one "head" group by a phosphate ester linkage. Examples of glycerophospholipids, usually referred to as phospholipids (though sphingomyelins are also classified as phospholipids) are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer).

[0750] Sphingolipids are members of a complex family of compounds that share a common structural feature, a sphingoid base backbone. The major sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with an amide-linked fatty acid. The fatty acids are typically saturated or mono-unsaturated with chain lengths from 16 to 26 carbon atoms. The major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramide phosphoinositols and mannose-containing headgroups. The glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base. Examples of these are the simple and complex glycosphingolipids such as cerebrosides and gangliosides.

[0751] Sterols, such as cholesterol and its derivatives, or tocopherol and its derivatives, are important components of membrane lipids, along with the glycerophospholipids and sphingomyelins.

[0752] Saccharolipids are compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers. In the saccharolipids, a monosaccharide substitutes for the glycerol backbone present in glycerolipids and glycerophospholipids. The most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram-negative bacteria. Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty - acyl chains. The minimal lipopolysaccharide required for growth in E. coli is Kdo2-Lipid A, a hexa-acylated disaccharide of glucosamine that is glycosylated with two 3-deoxy-D-manno- octulosonic acid (Kdo) residues.

[0753] Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0754] Lipids and lipid-like materials may be cationic, anionic or neutral. Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH.

[0755] In some embodiments, suitable lipids or lipid-like materials for use in the present disclosure include those described in W02020/128031 and US20200163878, the entire contents of each of which are incorporated herein by reference for the purposes described herein.

2. Cationic or cationically ionizable lipids or lipid-like materials

[0756] In some embodiments cationic or cationically ionizable lipids or lipid-like materials contemplated for use herein include any cationic or cationically ionizable lipids or lipid-like materials which are able to electrostatically bind nucleic acid. In one embodiment, cationic or cationically ionizable lipids or lipid-like materials contemplated for use herein can be associated with nucleic acid, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.

[0757] Cationic lipids or lipid-like materials are characterized in that they have a net positive charge (e.g., at a relevant pH). Cationic lipids or lipid-like materials bind negatively charged nucleic acid by electrostatic interaction. Generally, cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries the positive charge. [0758] In certain embodiments, a cationic lipid or lipid-like material has a net positive charge only at certain pH, in particular acidic pH, while it has preferably no net positive charge, preferably has no charge, i.e., it is neutral, at a different, preferably higher pH such as physiological pH. This ionizable behavior is thought to enhance efficacy through helping with endosomal escape and reducing toxicity as compared with particles that remain cationic at physiological pH.

[0759] In some embodiments, a cationic or cationically ionizable lipid or lipid-like material comprises a head group which includes at least one nitrogen atom (N) which is positive charged or capable of being protonated.

[0760] Examples of cationic lipids include, but are not limited to l,2-dioleoyl-3- trimethylammonium propane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N — (N',N'- dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); l,2-dioleoyl-3-dimethylammonium-propane (DODAP); l,2-diacyloxy-3- dimethylammonium propanes; l,2-dialkyloxy-3-dimethylammonium propanes; dioctadecyldimethyl ammonium chloride (DODAC), l,2-distearyloxy-N,N-dimethyl-3- aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-dimyristoyl-3- trimethylammonium propane (DMTAP), l,2-dioleyloxypropyl-3-dimethyl -hydroxyethyl ammonium bromide (DORIE), and 2,3-dioleoyloxy- N-[2(spermine carboxamide)ethyl]-N,N- dimethyl-l-propanamium trifluoroacetate (DOSPA), l,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3- beta-oxybutan-4-oxy)-l-(cis,cis-9,12-oc-tadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5- en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-l-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), l,2-N,N'-dioleylcarbamyl- 3-dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), l,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2- Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl- [l,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin- MC3-DMA), N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-l- propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3- bis(dodecyloxy)- 1 -propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N- dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (GAP-DMRIE), N-(2-Aminoethyl)- N,N-dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (PAE-DMRIE), N-(4- carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-l-aminium (DOBAQ), 2-({8-[(3P)- cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan- 1-amine (Octyl-CLinDMA), l,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1,2- dipalmitoyl-3-dimethylammonium-propane (DPDAP), N 1 -[2-((l S)- 1 -[(3-aminopropyl)amino]- 4- [di(3-amino-propyl)amino]butylcarboxamido)ethyl] -3 ,4-di [oleyloxy ] -benzamide (MVL5 ), 1,2- dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)- N,N-dimethylpropan-l-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)propan- 1 -aminium bromide (DMORIE), di((Z)-non-2-en-l-yl) 8,8'- ((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3- bis(dodecyloxy)propan- 1 -amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-l- amine (DMDMA), Di((Z)-non-2-en- 1 -yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-Dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2- dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}- ethylamino)propionamide (lipidoid 98N12-5), l-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2- [bis(2 hydroxydodecyl)amino]ethyl]piperazin-l-yl]ethyl]amino]dodecan-2-ol (lipidoid Cl 2- 200), LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1 ,2- dioleoyl-sn-3phosphoethanolamine (DOPE), from GIBCO/BRL, Grand Island, N.Y.);

LIPOFECT AMINE® (commercially available cationic liposomes comprising N-(l - (2,3dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.) or any combination of any of the foregoing. Further suitable cationic lipids for use in the present disclosure include those described in W02020/128031 and US20200163878, the entire contents of each of which are incorporated herein by reference for the purposes described herein. Further suitable cationic lipids for use in the present disclosure include those described in W02010/053572 (including Cl 2-200 described at paragraph [00225]) and W02012/170930, both of which are incorporated herein by reference for the purposes described herein. Additional suitable cationic lipids for use in the present disclosure include HGT4003, HGT5000, HGTS001, HGT5001, HGT5002 (see US20150140070A1, which is incorporated herein by reference in its entirety).

[0761] In some embodiments, formulations that are useful for pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) compositions as described herein can comprise at least one cationic lipid. Representative cationic lipids include, but are not limited to, 1 ,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1 ,2-dilinoleyoxy- 3morpholinopropane (DLin-MA), l,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1 ,2- dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1 -linoleoyl-2-linoleyloxy- 3dimethylaminopropane (DLin-2-DMAP), 1 ,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.CI), 1 ,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.CI), 1 ,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,Ndilinoleylamino)-l ,2- propanediol (DLinAP), 3-(N,N-dioleylamino)-l ,2-propanediol (DOAP), 1 ,2-dilinoleyloxo-3-(2- N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4- dimethylaminomethyl-[l ,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l ,3]-dioxolane (DLin-KC2-DMA); dilinoleyl-methyl-4- dimethylaminobutyrate (DLin-MC3-DMA); MC3 (US20100324120, which is incorporated herein by reference in its entirety).

[0762] In some embodiments, amino or cationic lipids useful in accordance with the present disclosure have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g. pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will, of course, be understood that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of lipids have to be present in the charged or neutral form. Lipids having more than one protonatable or deprotonatable group, or which are zwitterionic, are not excluded and may likewise suitable in the context of the present invention. [0763] In some embodiments, a protonatable lipid has a pKa of the protonatable group in the range of about 4 to about 11, e.g., a pKa of about 5 to about 7.

[0764] In some embodiments, a cationic lipid may comprise from about 10 mol % to about 100 mol %, about 20 mol % to about 100 mol %, about 30 mol % to about 100 mol %, about 40 mol % to about 100 mol %, or about 50 mol % to about 100 mol % of total lipid present in a lipid composition utilized in accordance with the present disclosure.

3. Additional lipids or lipid-like materials

[0765] In some embodiments, formulations utilized in accordance with the present disclosure may comprise lipids or lipid-like materials other than cationic or cationically ionizable lipids or lipid-like materials, i.e., non-cationic lipids or lipid-like materials (including non- cationically ionizable lipids or lipid-like materials). Collectively, anionic and neutral lipids or lipid-like materials are referred to herein as non-cationic lipids or lipid-like materials. In some embodiments, optimizing a formulation of nucleic acid particles by addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to an ionizable/cationic lipid or lipid-like material may, for example, enhance particle stability and efficacy of nucleic acid delivery.

[0766] In some embodiments, a lipid or lipid-like material may be incorporated which may or may not affect the overall charge of particles. In certain embodiments, such lipid or lipid- like material is a non-cationic lipid or lipid-like material.

[0767] In some embodiments, a non-cationic lipid may comprise, e.g., one or more anionic lipids and/or neutral lipids. An "anionic lipid" is negatively charged (e.g., at a selected pH).

[0768] A "neutral lipid" exists either in an uncharged or neutral zwitterionic form (e.g., at a selected pH). In some embodiments, a formulation comprises one of the following neutral lipid components: (1) a phospholipid, (2) cholesterol or a derivative thereof; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'- hydroxyethyl ether, cholesteryl-4'- hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof. [0769] Specific exemplary phospholipids that can be used include, but are not limited to, phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin. Such phospholipids include in particular diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O- octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero- 3 -phosphocholine (Cl 6 Lyso PC) and phosphatidylethanolamines, in particular diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), and further phosphatidylethanolamine lipids with different hydrophobic chains.

[0770] In certain embodiments, a formulation utilized in accordance with the present disclosure includes DSPC or DSPC and cholesterol.

[0771] In certain embodiments, formulations utilized in accordance with the present disclosure include both a cationic lipid and an additional (non-cationic) lipid.

[0772] In some embodiments, formulations herein include a polymer conjugated lipid such as a pegylated lipid. "Pegylated lipids" comprise both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art.

[0773] Without wishing to be bound by theory, the amount of (total) cationic lipid compared to the amount of other lipid(s) in formulation may affect important characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the nucleic acid. In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3: 1 to about 1:1. [0774] In some embodiments, a non-cationic lipid, in particular a neutral lipid, (e.g., one or more phospholipids and/or cholesterol) may comprise from about 0 mol % to about 90 mol %, from about 0 mol % to about 80 mol %, from about 0 mol % to about 70 mol %, from about 0 mol % to about 60 mol %, or from about 0 mol % to about 50 mol %, of the total lipid present in a formulation.

4. Lipoplex Particles

[0775] In certain embodiments of the present disclosure, the RNA described herein may be present in RNA lipoplex particles.

[0776] An "RNA lipoplex particle" contains lipid, in particular cationic lipid, and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA results in complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes may be generally synthesized using a cationic lipid, such as DOTMA, and additional lipids, such as DOPE. In one embodiment, a RNA lipoplex particle is a nanoparticle.

[0777] In certain embodiments, RNA lipoplex particles include both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

[0778] In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4: 1 to about 1:2, or about 3:1 to about 1: 1. In specific embodiments, the molar ratio may be about 3: 1, about 2.75:1, about 2.5:1, about 2.25:1, about 2: 1, about 1.75:1, about 1.5:1, about 1.25: 1, or about 1: 1. In an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2: 1.

[0779] In some embodiments, RNA lipoplex particles have an average diameter that in one embodiment ranges from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm, or from about 350 nm to about 400 nm. In specific embodiments, the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm. In an embodiment, the RNA lipoplex particles have an average diameter that ranges from about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter that ranges from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

[0780] RNA lipoplex particles and compositions comprising RNA lipoplex particles described herein are useful for delivery of RNA to a target tissue after parenteral administration, in particular after intravenous administration. The RNA lipoplex particles may be prepared using liposomes that may be obtained by injecting a solution of the lipids in ethanol into water or a suitable aqueous phase. In one embodiment, the aqueous phase has an acidic pH. In one embodiment, the aqueous phase comprises acetic acid, e.g., in an amount of about 5 mM. Liposomes may be used for preparing RNA lipoplex particles by mixing the liposomes with RNA. In one embodiment, the liposomes and RNA lipoplex particles comprise at least one cationic lipid and at least one additional lipid. In one embodiment, the at least one cationic lipid comprises l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and/or 1 ,2-dioleoyl- 3-trimethylammonium-propane (DOTAP). In one embodiment, the at least one additional lipid comprises l,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Choi) and/or l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In one embodiment, the at least one cationic lipid comprises l,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA) and the at least one additional lipid comprises l,2-di-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine (DOPE). In one embodiment, the liposomes and RNA lipoplex particles comprise l,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA) and l,2-di-(9Z- octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE).

[0781] Spleen targeting RNA lipoplex particles are described in WO 2013/143683, herein incorporated by reference. It has been found that RNA lipoplex particles having a net negative charge may be used to preferentially target spleen tissue or spleen cells such as antigen- presenting cells, in particular dendritic cells. Accordingly, following administration of the RNA lipoplex particles, RNA accumulation and/or RNA expression in the spleen occurs. Thus, RNA lipoplex particles of the disclosure may be used for expressing RNA in the spleen. In an embodiment, after administration of the RNA lipoplex particles, no or essentially no RNA accumulation and/or RNA expression in the lung and/or liver occurs. In one embodiment, after administration of the RNA lipoplex particles, RNA accumulation and/or RNA expression in antigen presenting cells, such as professional antigen presenting cells in the spleen occurs. Thus, RNA lipoplex particles of the disclosure may be used for expressing RNA in such antigen presenting cells. In one embodiment, the antigen presenting cells are dendritic cells and/or macrophages.

5. Lipid Nanoparticles (LNPs)

[0782] In some embodiments, nucleic acid such as RNA described herein is administered in the form of lipid nanoparticles (LNPs). In some embodiments, LNPs may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated.

[0783] In some embodiments, an LNP comprises one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids.

[0784] In some embodiments, an LNP comprises a cationic lipid, a neutral lipid, a sterol, a polymer conjugated lipid; and an RNA, encapsulated within or associated with the lipid nanoparticle.

[0785] In some embodiments, a neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC.

[0786] In some embodiments, a sterol is cholesterol.

[0787] In some embodiments, a polymer conjugated lipid is a pegylated lipid. In some embodiments, a pegylated lipid has the following structure:

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

[0788] R¹² and R¹³ are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60. In some embodiments, R¹² and R¹³ are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In some embodiments, w has a mean value ranging from 40 to 55. In some embodiments, the average w is about 45. In some embodiments, R¹² and R¹³ are each independently a straight, saturated alkyl chain containing about 14 carbon atoms, and w has a mean value of about 45.

[0789] In some embodiments, a pegylated lipid is DMG-PEG 2000, e.g., having the following structure:

[0790] In some embodiments, a cationic lipid component of LNPs has the structure of Formula (III):

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: one of L¹ or L² is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)_X-, -S-S-, -C(=O)S-, SC(=O)-, - NR^aC(=O)-, -C(=O)NR^a-, NR^aC(=O)NR^a-, -OC(=O)NR^a- or -NR^aC(=O)O-, and the other of L¹ or L² is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)_X-, -S-S-, -C(=O)S-, SC(=O)-, -NR^aC(=O)-, - C(=O)NR^a-, NR^aC(=O)NR^a-, -OC(=O)NR^a- or -NR^aC(=O)O- or a direct bond; G¹ and G² are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene;

G³ is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;

R^a is H or C1-C12 alkyl;

R¹ and R² are each independently C6-C24 alkyl or C6-C24 alkenyl;

R³ is H, OR⁵, CN, -C(=O)OR⁴, -OC(=O)R⁴ or -NR⁵C(=O)R⁴;

R⁴ is C1-C12 alkyl;

R⁵ is H or Ci-Ce alkyl; and x is 0, 1 or 2.

[0791] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (III A) or (IIIB):

(IIIA) (IIIB) wherein:

A is a 3 to 8-membered cycloalkyl or cycloalkylene ring;

R⁶ is, at each occurrence, independently H, OH or C1-C24 alkyl; and n is an integer ranging from 1 to 15.

[0792] In some of the foregoing embodiments of Formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0793] In other embodiments of Formula (III), the lipid has one of the following structures (IIIC) or (HID):

(IIIC) (HID) wherein y and z are each independently integers ranging from 1 to 12. [0794] In any of the foregoing embodiments of Formula (III), one of L¹ or L² is -O(C=O)-. For example, in some embodiments each of L¹ and L² are -O(C=O)-. In some different embodiments of any of the foregoing, L¹ and L² are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments each of L¹ and L² is -(C=O)O-.

[0795] In some different embodiments of Formula (III), the lipid has one of the following structures (HIE) or (IIIF):

(HIE) (IIIF)

[0796] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ):

(IIII) (IIIJ)

[0797] In some of the foregoing embodiments of Formula (III), n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3,

4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is

5. In some embodiments, n is 6. [0798] In some other of the foregoing embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6. [0799] In some of the foregoing embodiments of Formula (III), R⁶ is H. In other of the foregoing embodiments, R⁶ is C1-C24 alkyl. In other embodiments, R⁶ is OH. [0800] In some embodiments of Formula (III), G³ is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G³ is linear C₁-C₂₄ alkylene or linear C1-C24 alkenylene. [0801] In some other foregoing embodiments of Formula (III), R¹ or R², or both, is C6- C₂₄ alkenyl. For example, in some embodiments, R¹ and R² each, independently have the following structure: , wherein:

R^7a and R^7b are, at each occurrence, independently H or C1-C12 alkyl; and a is an integer from 2 to 12, and wherein R^7a, R^7b and a are each selected such that R¹ and R² each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12. [0802] In some of the foregoing embodiments of Formula (III), at least one occurrence of R^7a is H. For example, in some embodiments, R^7a is H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R^7b is C1-C8 alkyl. For example, in some embodiments, C₁-C₈ alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert- butyl, n-hexyl or n-octyl. [0803] In different embodiments of Formula (III), R¹ or R², or both, has one of the following structures: 11831138v1

[0804] In some of the foregoing embodiments of Formula (III), R³ is OH,

CN, -C(=O)OR⁴, -OC(=O)R⁴ or -NHC(=O)R⁴. In some embodiments, R⁴ is methyl or ethyl.

[0805] In various different embodiments, the cationic lipid of Formula (III) has one of the structures set forth in in Table 14 below.

Table 14: Exemplary Compounds of Formula (III).

[0806] In various different embodiments, a cationic lipid has one of the structures set forth in Table 15 below.

Table 15: Exemplary Cationic Lipid Structures

[0807] In some embodiments, an LNP comprises a cationic lipid that is an ionizable lipid-like material (lipidoid). In some embodiments, a cationic lipid has the following structure:

[0808] In some embodiments, lipid nanoparticles can have an average size (e.g., mean diameter) of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 70 to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, lipid nanoparticles in accordance with the present disclosure can have an average size (e.g., mean diameter) of about 50 nm to about 100 nm. In some embodiments, lipid nanoparticles may have an average size (e.g., mean diameter) of about 50 nm to about 150 nm. In some embodiments, lipid nanoparticles may have an average size (e.g., mean diameter) of about 60 nm to about 120 nm. In some embodiments, lipid nanoparticles in accordance with the present disclosure can have an average size (e.g., mean diameter) of about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. The term “average diameter” or “mean diameter” refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Z-average with the dimension of a length, and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321, which is herein incorporated by reference). Here “average diameter,” “mean diameter,” “diameter,” or “size” for particles is used synonymously with this value of the Z-average.

[0809] In some embodiments, lipid nanoparticles described herein may exhibit a polydispersity index less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less. By way of example, lipid nanoparticles can exhibit a polydispersity index in a range of about 0.1 to about 0.3 or about 0.2 to about 0.3. The “polydispersity index” is preferably calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the “average diameter.” Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of ribonucleic acid nanoparticles (e.g., ribonucleic acid nanoparticles). [0810] Lipid nanoparticles described herein can be characterized by an “N/P ratio,” which is the molar ratio of cationic (nitrogen) groups (the “N” in N/P) in the cationic polymer to the anionic (phosphate) groups (the “P” in N/P) in RNA. It is understood that a cationic group is one that is either in cationic form (e.g., N⁺), or one that is ionizable to become cationic. Use of a single number in an N/P ratio (e.g., an N/P ratio of about 5) is intended to refer to that number over 1, e.g., an N/P ratio of about 5 is intended to mean 5:1. In some embodiments, a lipid nanoparticle described herein has an N/P ratio greater than or equal to 5. In some embodiments, a lipid nanoparticle described herein has an N/P ratio that is about 5, 6, 7, 8, 9, or 10. In some embodiments, an N/P ratio for a lipid nanoparticle described herein is from about 10 to about 50. In some embodiments, an N/P ratio for a lipid nanoparticle described herein is from about 10 to about 70. In some embodiments, an N/P ratio for a lipid nanoparticle described herein is from about 10 to about 120.

B. Exemplary Methods of Making Lipid Nanoparticles

[0811] Lipids and lipid nanoparticles comprising nucleic acids and their method of preparation are known in the art, including, e.g., as described in U.S. Patent Nos. 8,569,256, 5,965,542 and U.S. Patent Publication Nos. 2016/0199485, 2016/0009637, 2015/0273068, 2015/0265708, 2015/0203446, 2015/0005363, 2014/0308304, 2014/0200257, 2013/086373, 2013/0338210, 2013/0323269, 2013/0245107, 2013/0195920, 2013/0123338, 2013/0022649, 2013/0017223, 2012/0295832, 2012/0183581, 2012/0172411, 2012/0027803, 2012/0058188, 2011/0311583, 2011/0311582, 2011/0262527, 2011/0216622, 2011/0117125, 2011/0091525, 2011/0076335, 2011/0060032, 2010/0130588, 2007/0042031, 2006/0240093, 2006/0083780, 2006/0008910, 2005/0175682, 2005/017054, 2005/0118253, 2005/0064595, 2004/0142025, 2007/0042031, 1999/009076 and PCT Pub. Nos. WO 99/39741, WO 2018/081480, WO 2017/004143, WO 2017/075531, WO 2015/199952, WO 2014/008334, WO 2013/086373, WO 2013/086322, WO 2013/016058, WO 2013/086373, W02011/141705, and WO 2001/07548, the full disclosures each of which are herein incorporated by reference in their entirety for the purposes described herein.

[0812] For example, in some embodiments, cationic lipids, neutral lipids (e.g., DSPC, and/or cholesterol) and polymer-conjugated lipids can be solubilized in ethanol at a pre- determined molar ratio (e.g., ones described herein). In some embodiments, lipid nanoparticles (lipid nanoparticle) are prepared at a total lipid to polyribonucleotides weight ratio of approximately 10: 1 to 30: 1. In some embodiments, such polyribonucleotides can be diluted to 0.2 mg/mL in acetate buffer.

[0813] In some embodiments, using an ethanol injection technique, a colloidal lipid dispersion comprising polyribonucleotides can be formed as follows: an ethanol solution comprising lipids, such as cationic lipids, neutral lipids, and polymer- conjugated lipids, is injected into an aqueous solution comprising polyribonucleotides (e.g., ones described herein).

[0814] In some embodiments, lipid and polyribonucleotide solutions can be mixed at room temperature by pumping each solution at controlled flow rates into a mixing unit, for example, using piston pumps. In some embodiments, the flow rates of a lipid solution and a RNA solution into a mixing unit are maintained at a ratio of 1:3. Upon mixing, nucleic acid-lipid particles are formed as the ethanolic lipid solution is diluted with aqueous polyribonucleotides. The lipid solubility is decreased, while cationic lipids bearing a positive charge interact with the negatively charged RNA.

[0815] In some embodiments, a solution comprising RNA-encapsulated lipid nanoparticles can be processed by one or more of concentration adjustment, buffer exchange, formulation, and/or filtration.

[0816] In some embodiments, RNA-encapsulated lipid nanoparticles can be processed through filtration.

[0817] In some embodiments, particle size and/or internal structure of lipid nanoparticles (with or without RNAs) may be monitored by appropriate techniques such as, e.g., small-angle X-ray scattering (SAXS) and/or transmission electron cryomicroscopy (CryoTEM).

Pharmaceutical Compositions

[0818] The present disclosure provides compositions e.g., pharmaceutical compositions comprising one or more polyribonucleotides as described herein.

[0819] In some embodiments, pharmaceutical formulations comprise an active agent and one or more excipients or carriers.

[0820] In some embodiments, an active agent may be or comprise an antigen or an antigenic portion thereof, as described herein. Thus, in some embodiments, an active agent is a polypeptide or plurality of polypeptides. In some embodiments, a polypeptide active agent includes a plurality of infectious agent antigens or immunogenic portions thereof (e.g., from a single strain or variant of an infectious agent or from a plurality of different strains or variant of an infectious agent). In some embodiments, a polypeptide active agent is or comprises at least one peptide that represents a distinct antigen. In some embodiments, a polypeptide active agent includes at least one peptide that is or comprises an antigen or antigenic portion or epitope of an antigenic protein; in some such embodiments, the polypeptide active agent does not include any full-length antigenic protein.

[0821] In some embodiments, an active agent may be or comprise a cell population - for example a population of cells that expresses (e.g., internally, on its surface, and or secreting) at least one antigen as described herein.

[0822] In some embodiments, an active agent is a polynucleotide that encodes (or is complementary to one that encodes) an antigen as described herein. In some such embodiments, a polynucleotide is single-stranded; in other embodiments, a polynucleotide is double stranded. In some embodiments, a polynucleotide active agent is DNA (e.g., a DNA viral vector, such as an adenoviral, adeno-associated viral, baculoviral, poxviral [e.g., vaccinia viral] vector); in some embodiments, a polynucleotide active agent is RNA (e.g., a lentiviral vector or, more preferably, an mRNA construct as described herein).

[0823] In many embodiments, a polynucleotide active agent is RNA and is provided and/or utilized in a lipid composition such as a lipoplex preparation or, preferably, an LNP preparation.

[0824] In some embodiments, a provided formulation is a liquid formulation. In some embodiments, a provided formulation is a solid (e.g., frozen formulation. In some embodiments, a provided formulation is a dry formulation.

[0825] Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.

[0826] In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by the United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.

[0827] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.

[0828] General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).

[0829] In some embodiments, pharmaceutical compositions provided herein may be formulated with one or more pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).

[0830] Pharmaceutical compositions described herein can be administered by appropriate methods known in the art. As will be appreciated by a skilled artisan, the route and/or mode of administration may depend on a number of factors, including, e.g., but not limited to stability and/or pharmacokinetics and/or pharmacodynamics of pharmaceutical compositions described herein.

[0831] In some embodiments, pharmaceutical compositions described herein are formulated for parenteral administration, which includes modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, subcuticular, or intraarticular injection and infusion. In preferred embodiments, pharmaceutical compositions described herein are formulated for intravenous, intramuscular, or subcutaneous administration. In particularly preferred embodiments, pharmaceutical compositions described herein are formulated for intramuscular administration.

[0832] In some embodiments, pharmaceutical compositions described herein are formulated for intravenous administration. In some embodiments, pharmaceutically acceptable excipients that may be useful for intravenous administration include sterile aqueous solutions or dispersions and sterile powders for preparation of sterile injectable solutions or dispersions.

[0833] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, lipid nanoparticles, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. In some embodiments, prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0834] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization and/or microfiltration. In some embodiments, pharmaceutical compositions can be prepared as described herein and/or methods known in the art. In some embodiments, a pharmaceutical composition includes ALC-0315; ALC-0159; DSPC; Cholesterol; Sucrose; NaCl; KC1; Na2HPC>4; KH2PO4; Water for injection. In some embodiments, normal saline (isotonic 0.9% NaCl) is used as diluent.

[0835] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into pharmaceutical compositions described herein. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0836] Formulations of pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing active ingredient(s) into association with a diluent or another excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi- dose unit.

[0837] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of at least one RNA product produced using a system and/or method described herein.

[0838] Relative amounts of polyribonucleotides encapsulated in lipid nanoparticles, a pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition can vary, depending upon the subject to be treated, target cells, diseases or disorders, and may also further depend upon the route by which the composition is to be administered.

[0839] In some embodiments, pharmaceutical compositions described herein are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Actual dosage levels of the active ingredients (e.g., polyribonucleotides encapsulated in lipid nanoparticles) in the pharmaceutical compositions described herein may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0840] A physician having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician could start doses of active ingredients (e.g., polyribonucleotides encapsulated in lipid nanoparticles) employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0841] In some embodiments, a pharmaceutical composition is formulated (e.g., but not limited to, for intravenous, intramuscular, or subcutaneous administration) to deliver a dose of about 5 mg RNA/kg.

[0842] In some embodiments, a pharmaceutical composition described herein may further comprise one or more additives, for example, in some embodiments that may enhance stability of such a composition under certain conditions. Examples of additives may include but are not limited to salts, buffer substances, preservatives, and carriers. For example, in some embodiments, a pharmaceutical composition may further comprise a cryoprotectant (e.g., sucrose) and/or an aqueous buffered solution, which may in some embodiments include one or more salts, including, e.g., alkali metal salts or alkaline earth metal salts such as, e.g., sodium salts, potassium salts, and/or calcium salts.

[0843] In some embodiments, a pharmaceutical composition provided herein is a preservative-free, sterile RNA-lipid nanoparticle dispersion in an aqueous buffer for intravenous or intramuscular administration. [0844] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions that are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation.

Characterization

[0845] Without wishing to be bound by any particular theory, it is proposed that ability to induce CD8⁺ T cells may be important to effectiveness of a composition for the treatment of a infection, (e.g., a pharmaceutical composition, an immunogenic composition, or a vaccine). Alternatively or additionally, in some embodiments, a robust antibody response may be important for effectiveness. In some embodiments, it may be that both are required or useful.

[0846] In some embodiments, provided technologies (e.g., compositions and/or dosing regimens, etc) are characterized by an ability to induce (e.g., when administered to a model system and/or to a human, for example by parenteral administration such as by intramuscular administration) an immune response characterized by CD8⁺ T cells targeting one or more antigen(s) described herein. That is, in some embodiments, provided technologies are characterized in that, when administered (e.g., by parenteral administration such as by intramuscular administration) to an organism (e.g., a model organism or an animal or human organism in need of protection), provided technologies induce CD8+ T cells targeting one or more antigens. In some embodiments, provided technologies are characterized in that they induce a greater CD8⁺ T cell response with respect to one or more antigens and/or induce a CD8⁺ T cell response with broader diversity (e.g., with detectable and/or significant binding to a larger number of different T cell antigens) than is observed for non-RNA vaccines, e.g., a non-RNA vaccine described herein.

[0847] In some embodiments, provided technologies are characterized in that they induce gammadelta T cells. As those skilled in the art are aware, gammadelta T cells typically represent only a small fraction (e.g., up to about 5%) of an overall T cell population in an organism. Gammadelta T cells express TCR chains encoded by the gamma and delta gene loci; subsets of gamma delta T cells are defined by the inclusion of invariant TCR V -(D)-J segments and are tissue- or context-specific. Gammadelta T cells secrete particular effector cytokines in a subtype-and context-specific manner. Often, gammadelta T cells express certain markers (e.g., as Fc gamma RIII/CD 16 and Toll-like receptors that are often associated with natural killer cells and/or antigen-presenting cells. Gammadelta T cells typically lack CD4 and CD8.

[0848] In some embodiments, provided technologies are characterized in that they induce polyclonal high affinity antibodies.

[0849] In some embodiments, provided technologies are characterized in that they induce antibody titers to a level that provides sufficient protective response against an infectious agent, when administered to a relevant population.

[0850] In some embodiments, provided technologies are characterized in that they induce sterile protection, e.g., when evaluated in a model system such as a mouse modes.

[0851] In some embodiments, provided technologies are characterized in that they induce a CD4+ T helper cell response and/or CD8+ T cell memory responses (e.g., promoting development and/or expansion of memory CD8+ T cells).

[0852] In some embodiments, provided compositions are assessed as described herein, for example for RNA integrity, stability, level, capping efficiency, translatability, of RNA, etc and/or for one or more properties of a composition (e.g., an LNP preparation) such as, for example ability to induce an antibody response, a T cell response, a T cell response with particular features (e.g., level of antibody to one or more antigens, persistence of such level, diversity of elicited antibodies, type and/or diversity of T cell response, etc.

[0853] In some embodiments, provided formulations are identified and/or characterized with respect to one or more activities or features, including, for example, expression level, nature of immune response, level of protection (e.g., to challenge, impact on viral load, impact on health and/or survival), immunogenicity (e.g., assessment of cytokine responses, phenotyping of immune response, T cell depletion and/or protection), serology, and/or functional antibody responses. In some embodiments, assessment of provided compositions can be performed in an animal model. In many embodiments, assessment of provided compositions in human system(s) is desirable. In some embodiments, in vitro assessments are performed in human systems. To give but a few examples, in some embodiments, presentation of provided antigen(s) or antigenic fragments or epitopes thereof by human dendritic cells to stimulate human T cells is assessed in vitro. Alternatively or additionally, in some embodiments, in vitro binding of sera from infected humans to provided antigens is assessed.

[0854] In some embodiments, in vivo assessments are performed in human systems. For example, in some embodiments, one or more human trials are performed. In such trials, healthy humans (e.g., volunteers, who have typically undergone screening and/or consenting procedures) are treated with a provided composition (e.g., an immunostimulatory composition, e.g., a vaccine composition) and subsequently are inoculated with an infectious agent, and clinically monitored to assess level of protection, e.g., from established infection and/or symptomatic or serious disease. Alternatively or additionally, in some embodiments, subjects are monitored for responsiveness (e.g., increased responsiveness) to a particular known or potential anti -infectious agent therapy.

[0855] In some embodiments, a provided composition (e.g., an immunostimulatory composition, e.g., a vaccine composition) provides significant (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90% or more protection from one or more of established infection, symptomatic disease, and/or serious disease. In some embodiments, a provided composition (e.g., an immunostimulatory composition, e.g., a vaccine composition) provides significantly increased responsiveness to therapy, for example as may be assessed by one or more of delayed onset, reduction of severity, and/or faster resolution of one or more symptoms or characteristics of infection.

Patient Populations

[0856] In some aspects, technologies of the present disclosure are used for therapeutic and/or prophylactic purposes. In some embodiments, technologies of the present disclosure are used in the treatment and/or prophylactic of an infection. Prophylactic purposes of the present disclosure comprises pre-exposure prophylaxis and/or post-exposure prophylaxis.

[0857] In some embodiments, technologies of the present disclosure are used in the treatment and/or prophylaxis of a disorder related to such an infection. A disordered related to such an infectious agent infection comprises, for example, a typical symptom and/or a complication of a an infectious agent infection. [0858] In some embodiments, provided compositions may be useful to detect and/or characterize one or more features of an immune response (e.g., by detecting binding to a provided antigen by serum from an infected subject).

[0859] In some embodiments, provided compositions are useful to raise antibodies to one or more antigens included therein; such antibodies may themselves be useful, for example for detection or treatment of an infectious agent or infection thereby.

[0860] The present disclosure provides use of encoding nucleic acids (e.g., DNA or RNA) to produce encoded antigens and/or use of DNA constructs to produce RNA.

[0861] In some embodiments, technologies of the present disclosure are utilized in a non-limited subject population; in some embodiments, technologies of the present disclosure are utilized in particular subject populations.

[0862] In some embodiments, a subject population comprises an adult population. In some embodiments, an adult population comprises subjects between the ages of about 19 years and about 60 years of age (e.g., about 20, 25, 30, 35, 40, 45, 50, 55, or 60 years of age).

[0863] In some embodiments, a subject population comprises an elderly population. In some embodiments, an elderly population comprises subjects of about 60 years of age, about 70 years of age, or older (e.g., about 65, 70, 75, 80, 85, 90, 95, or 100 years of age).

[0864] In some embodiments, a subject population comprises a pediatric population. In some embodiments, a pediatric population comprises subjects approximately 18 years old or younger. In some such embodiments, a pediatric population comprises subjects between the ages of about 1 year and about 18 years (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 years of age).

[0865] In some embodiments, a subject population comprises a newborn population. In some embodiments, a newborn population comprises subjects about 12 months or younger (e.g., 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 months or younger). In some embodiments, subject populations to be treated with technologies described herein include infants (e.g., about 12 months or younger) whose mothers did not receive such technologies described herein during pregnancy. In some embodiments, subject populations to be treated with technologies described herein may include pregnant women; in some embodiments, infants whose mothers were treated with disclosed technologies during pregnancy (e.g., who received at least one dose, or alternatively only who received both doses), are not vaccinated during the first weeks, months, or even years (e.g., 1, 2, 3, 4, 5, 6, 7, 8 weeks or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months or more, or 1, 2, 3, 4, 5 years or more) post-birth. Alternatively or additionally, in some embodiments, infants whose mothers were treated with disclosed technologies during pregnancy (e.g., who received at least one dose, or alternatively only who received both doses), receive reduced treated with disclosed technologies (e.g., lower doses and/or smaller numbers of administrations - e.g., boosters - and/or lower total exposure over a given period of time) after birth, for example during the first weeks, months, or even years (e.g., 1, 2, 3, 4, 5, 6, 7, 8 weeks or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months or more, or 1, 2, 3, 4, 5 years or more) post-birth or may need reduced vaccination (e.g., lower doses and/or smaller numbers of administrations - e.g., boosters - over a given period of time), In some embodiments, compositions as provided herein are administered to subject populations that do not include pregnant women.

[0866] In some embodiments, a subject population is or comprises children aged 6 weeks to up to 17 months of age.

[0867] In some embodiments, a provided pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) may be administered in combination with (i.e., so that subject(s) are simultaneously exposed to both) another pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) or therapeutic intervention, e.g., to treat or prevent SARS-CoV-2 infection, or another disease, disorder, or condition.

[0868] In some embodiments, a provided pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) may be administered with a protein vaccine, a DNA vaccine, an RNA vaccine, a cellular vaccine, a conjugate vaccine, etc. In some embodiments, one or more doses of a provided pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) may be administered together with (e.g., in a single visit) another vaccine or other therapy.

[0869] In some embodiments, a provided pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) may be administered to subjects who have been exposed, or expect they have been exposed, to an infectious agent (e.g., SARS-CoV-2) . In some embodiments, a provided pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) may be administered to subjects who do not have symptoms of infection (e.g., SARS- CoV-2) .

Treatment Methods

[0870] In some embodiments, technologies of the present disclosure may be administered to subjects according to a particular dosing regimen. In some embodiments, a dosing regimen may involve a single administration; in some embodiments, a dosing regimen may comprise one or more “booster” administrations after the initial administration. In some embodiments, initial and boost doses are the same amount; in some embodiments they differ. In some embodiments, two or more booster doses are administered. In some embodiments, a plurality of doses are administered at regular intervals. In some embodiments, periods of time between doses become longer. In some embodiments, one or more subsequent doses is administered if a particular clinical (e.g., reduction in neutralizing antibody levels) or situational (e.g., local development of a new strain) even arises or is detected.

[0871] In some embodiments, administered pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) comprising RNA constructs that encode an infectious agent antigen(s) are administered in RNA doses of from about 0.1 pg to about 300 pg, about 0.5 pg to about 200 pg, or about 1 pg to about 100 pg, such as about 1 pg, about 3 pg, about 10 pg, about 30 pg, about 50 pg, or about 100 pg. In some embodiments, an saRNA construct is administered at a lower dose (e.g., 2, 4, 5, 10 fold or more lower) than a modRNA or uRNA construct.

[0872] In some embodiments, a first booster dose is administered within about six months of the initial dose, and preferably within about 5, 4, 3, 2, or 1 months. In some embodiments, a first booster dose is administered in a time period that begins about 1, 2, 3, or 4 weeks after the first dose, and ends about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks of the first dose (e.g., between about 1 and about 12 weeks after the first dose, or between about 2 or 3 weeks and about 5 and 6 weeks after the first dose, or about 3 weeks or about 4 weeks after the first dose).

[0873] In some embodiments, a plurality of booster doses (e.g., 2, 3, or 4) doses are administered within 6 months of the first dose, or within 12 months of the first dose. [0874] In some embodiments, 3 doses or fewer are required to achieve effective vaccination (e.g., greater than 60%, and in some embodiments greater than about 70%, about 75%, about 80%, about 85%, about 90% or more) reduction in risk of infection, or of serious disease. In some embodiments, not more than two doses are required. In some embodiments, a single dose is sufficient. In some embodiments, an RNA dose is about 60 pg or lower, 50 pg or lower, 40 pg or lower, 30 pg or lower, 20 pg or lower, 10 pg or lower, 5 pg or lower, 2.5 pg or lower, or 1 pg or lower. In some embodiments, an RNA dose is about 0.25 pg, at least 0.5 pg, at least 1 pg, at least 2 pg, at least 3 pg, at least 4 pg, at least 5 pg, at least 10 pg, at least 20 pg, at least 30 pg, or at least 40 pg. In some embodiments, an RNA dose is about 0.25 pg to 60 pg, 0.5 pg to 55 pg, 1 pg to 50 pg, 5 pg to 40 pg, or 10 pg to 30 pg may be administered per dose. In some embodiments, an RNA dose is about 30 pg. In some embodiments, at least two such doses are administered. For example, a second dose may be administered about 21 days following administration of the first dose. In some embodiments, a first booster dose is administered about one month after an initial dose. In some such embodiments, at least one further booster is administered at one-month interval(s). In some embodiments, after 2 or 3 boosters, a longer interval is introduced and no further booster is administered for at least 6, 9, 12, 18, 24, or more months. In some embodiments, a single further booster is administered after about 18 months. In some embodiments, no further booster is required unless, for example, a material change in clinical or environmental situation is observed.

[0875] In some embodiments, combinations described herein are provided to subjects previously infected with an infection. In some embodiments, combinations described herein are provided as a booster dose to a subject previously administered one or more doses (e.g., part of a priming dosing regimen, a complete dosing regimen, or a full dosing regimen) of a vaccine against an infection, e.g., by a prior naturally circulating strain or variant.

[0876] In some embodiments, combinations described herein are provided to subjects previously infected with SARS-CoV-2. In some embodiments, combinations described herein are provided as a booster dose to a subject previously administered one or more doses (e.g., part of a priming dosing regimen, a complete dosing regimen, or a full dosing regimen) of a vaccine against SARS-CoV-2, e.g., by a prior naturally circulating strain or variant. Exemplary SARS- CoV-2 vaccines include a BNT162b2 vaccine, an mRNA-1273 vaccine, an Ad26.CoV2.S vaccine, a ChAdxOxl vaccine, an NVX-CoV2373 vaccine, a CvnCoV vaccine, a GAM- COVIDOVac vaccine, a CoronaVac vaccine, a BBIBP-CorV vaccine, an Ad5-nCoV vaccine, a zf2001 vaccine, a SCB-2019 vaccine, or other approved RNA (e.g., mRNA) or adenovector vaccines, etc. In some embodiments, one or more doses of a composition described herein is administered to subjects who have received one or more doses of (e.g., a complete regimen of) a SARS-CoV-2 vaccine and have been exposed to and/or infected with SARS-CoV-2.

[0877] In some embodiments, a combination or composition described herein is administered to a subject previously administered a vaccine that delivers a full length S protein of a Wuhan variant. In some embodiments, a combination or composition described herein is administered to a subject previously administered a vaccine that delivers a full length S protein of an Omicron variant (e.g., a BA.l or BA.4/5 Omicron variant).

[0878] In some embodiments, provided technologies (e.g., combinations, compositions, methods, and/or dosing regimens, etc.) can be used in combination with (e.g., in some embodiments co-administration) one or more vaccines or therapies against a different disease (e.g., in some embodiment a different infection). In some embodiments, a different disease is associated with an infectious agent, including, but not limited to a coronavirus, an influenza virus, a pneumoviridae virus, or a paramyxoviridae virus. In some embodiments, the pneumoviridae virus is a Respiratory Syncytial Virus (RSV) or a metapneumovirus. In some embodiments, the metapneumovirus is a human metapneumovirus (hMPV). In some embodiments, the paramyxoviridae virus is a parainfluenza virus or a henipavirus. In some embodiments the parainfluenzavirus is PIV3. In some embodiments, the coronavirus is a betacoronavirus (e.g., SARS-CoV-1). In some embodiments the coronavirus is a Merbecovirus (e.g., a MERS-CoV virus).

[0879] In some embodiments, provided technologies (e.g., combinations, compositions, methods, and/or dosing regimens, etc.) can be used in combination with (e.g., in some embodiments co-administration) with an RSV vaccine (e.g., an RSV A or RSV B vaccine). In some embodiments, the RSV vaccine comprises an RSV fusion protein (F), an RSV attachment protein (G), an RSV small hydrophobic protein (SH), an RSV matrix protein (M), an RSV nucleoprotein (N), an RSV M2-1 protein, an RSV Large polymerase (L), and/or an RSV phosphoprotein (P), or an immunogenic fragment of immunogenic variant thereof, or a nucleic acid (e.g., RNA), encoding any one of the same. In some embodiments, a composition described herein is co-administered with an RSV vaccine that delivers a prefusion-stabilized F protein (e.g., an RSV vaccine described in WO2017109629A1, WO2022023896A1, WO2022023895A1, and/or W02022070129A1, the contents of which are incorporated by reference in their entirety).

[0880] In some embodiments, provided technologies (e.g., combinations, compositions, methods, and/or dosing regimens, etc.) can be used in combination with (e.g., in some embodiments co-administration) with an influenza vaccine. In some embodiments, the influenza vaccine is an alphainfluenza virus, a betainfluenza virus, a gammainfluenza virus or a deltainfluenza virus vaccine. In some embodiments the vaccine is an Influenza A virus, an Influenza B virus, an Influenza C virus, or an Influenza D virus vaccine. In some embodiments, the influenza A virus vaccine comprises a hemagglutinin selected from Hl, H2, H3, H4, H5, H6, H7, H8, H9, H10, HU, H12, H13, H14, H15, H16, H17, and H18, or an immunogenic fragment or variant of the same, or a nucleic acid (e.g., RNA) encoding any one of the same. In some embodiments the influenza A vaccine comprises or encodes a neuraminidase (NA) selected from Nl, N2, N3, N4, N5, N6, N7, N8, N9, N10, and Ni l, or an immunogenic fragment or variant of the same, or a nucleic acid (e.g., RNA) encoding any one of the same. In some embodiments, the influenza vaccine comprises at least one Influenza virus hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (Ml), matrix protein 2 (M2), non-structural protein 1 (NS1 ), non-structural protein 2 (NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic protein PB1, PB1-F2, and/or polymerase basic protein 2 (PB2), or an immunogenic fragment or variant thereof, or a nucleic acid (e.g., RNA) encoding any of one of the same.

[0881] In some embodiments, provided technologies (e.g., combinations, compositions, methods, and/or dosing regimens, etc.) can be used in combination with (e.g., in some embodiments co-administration) with two or more vaccines against different infectious diseases (e.g., as described herein). In some embodiments, provided technologies (e.g., combinations, compositions, methods, and/or dosing regimens, etc.) can be used in combination with (e.g., in some embodiments co-administration) with an influenza vaccine and an RSV vaccine.

[0882] In some embodiments, compositions described herein are provided to subjects previously infected with an infectious agent. In some embodiments, compositions described herein are provided as a booster dose to a subject previously administered one or more doses (e.g., part of a priming dosing regimen, a complete dosing regimen, or a full dosing regimen) of an infectious agent vaccine (e.g., a vaccine delivering a full length antigen and/or a commercially approved vaccine). Exemplary SARS-CoV-2 vaccines include a BNT162b2 vaccine, an mRNA- 1273 vaccine, an Ad26.CoV2.S vaccine, a ChAdxOxl vaccine, an NVX-CoV2373 vaccine, a CvnCoV vaccine, a GAM-COVIDOVac vaccine, a CoronaVac vaccine, a BBIBP-CorV vaccine, an Ad5-nCoV vaccine, a zf2001 vaccine, a SCB-2019 vaccine, or other approved RNA (e.g., mRNA) or adenovector vaccines, etc. In some embodiments, one or more doses of a composition described herein is administered to subjects who have received one or more doses of (e.g., a complete regimen of) a vaccine and have been exposed to and/or infected with SARS- CoV-2.

[0883] In some embodiments, a composition described herein is designed so as to comprise few (e.g., no) conserved B cell epitopes relative to a previously administered vaccine (e.g., the first vaccine a subject has been administered), an infectious agent strain or variant that was previously prevalent in a relevant population (e.g., a strain or variant that a subject was previously infected with or had a high probability of being infected with). In some embodiments, a composition described herein is administered to a subject previously administered a vaccine that delivers a full length infectious agent antigen.

[0884] In some embodiments, an RNA composition described herein is co-administered with one or more vaccines against a non-COVID-19 disease. In some embodiments, an RNA composition described herein is co-administered with one or more vaccines against a non- COVID-19 viral disease. In some embodiments, an RNA composition described herein is co- administered with one or more vaccines against a non-COVID-19 respiratory disease. In some embodiments, the non-COVD-19 respiratory disease results from infection with a non-SARS- CoV-2 Coronavirus, an Influenza virus, a Pneumoviridae virus, or a Paramyxoviridae virus. In some embodiments, the Pneumoviridae virus is a Respiratory syncytial virus (RSV) or a Metapneumovirus (MPV). In some embodiments, the Metapneumovirus is a human metapneumovirus (hMPV). In some embodiments, the Paramyxoviridae virus is a Parainfluenza virus or a Henipavirus. In some embodiments the parainfluenzavirus is PIV3. In some embodiments, the non-SARS-CoV-2 coronavirus is a betacoronavirus (e.g., SARS-CoV-1). In come embodiments the non-SARS-CoV-2 coronavirus is a Merbecovirus (e.g., a MERS-CoV virus). [0885] In some embodiments, an RNA composition described herein is co-administered with an RSV vaccine (e.g., a vaccine delivering an RSV A and/or RSV B antigen). In some embodiments, the RSV vaccine comprises an RSV fusion protein (F), an RSV attachment protein (G), an RSV small hydrophobic protein (SH), an RSV matrix protein (M), an RSV nucleoprotein (N), an RSV M2-1 protein, an RSV Large polymerase (L), and/or an RSV phosphoprotein (P), or an immunogenic fragment of immunogenic variant thereof, or a nucleic acid (e.g., RNA), encoding any one of the same.

[0886] Numerous RSV vaccines are known in the art, any one of which can be co- administered with an RNA composition described herein. See, for example, the list of vaccines provided on the website of PATH, a global health organization. Examples of RSV vaccines are also provide in Mazur, Natalie I., et al, "The respiratory syncytial virus vaccine landscape: lessons from the graveyard and promising candidates," The Lancet Infectious Diseases 18.10 (2018): e295-e311, the contents of which is incorporated by reference herein in its entirety. In some embodiments, an RNA composition described herein is co-administered with an RSV vaccine that has been previously published on (e.g., an RSV vaccine described on the PATH website, or in Mazur et al.). In some embodiments, an RNA composition described herein is co- administered with a live-attenuated or chimeric vaccine (e.g., rBCG-N-hRSV (developed by Ponteificia Uinersidad Catolica de Chile), RSV D46 cp AM202 (developed by Sanofi Pasteur/LID/NIAD/NIH), RSV LID AM2-2 1030s (developed by Sanofi Pasteur/LID/NIAD/NIH), RSV ANS2 Al 313/11314L (developed by Sanofi Pasteur/LID/NIAD/NIH), RSV D46 ANS2 N AM2-2 Hindlll (developed by Sanofi Pasteur/LID/NIAD/NIH) or RSV LID AM2-2 1030s (developed by Sanofi Pasteur/LID/NIAD/NIH), MV-012-968 (developed by Meissa Vaccines), SP0125 (developed by Sanofi), blb201 (developed by Blue lake), CodaVax™-RSV (developed by Cadagenix), RSVDeltaG (developed by Intravacc), or SeVRSV (developed by SIHPL and St. Jude hospital), a particle based vaccine (e.g., RSV F nanoparticle (developed by Novavax) or SynGEM (developed by Mucosis), Icosavzx (developed by IVX-121), or V-306 (developed by Virometix)), a subunit vaccine (e.g., GSK RSV F (developed by GSK), Arexvy (developed by GSK), DPX-RSV (developed by Dalousie Univeristy, Immunovaccine, and VIB), RSV F DS- Cavl (developed by NIH/NIAID/VRC), MEDL7510 (developed by Medlmmune), RSVpreF (developed by Pfizer), ADV110 (developed by Advaccine), VN-0200 (developed by Daiichi Sankyo, Inc.)), a vector vaccine (e.g., MVA-BN RSV (developed by Banarian Nordic), VXA- RSVf oral (developed by Vaxart), Ad26.RSV.pref (developed by Janssen), ChAdl55-RSV (developed by GSK) Immunovaccine, DPX-RSV (developed by VIB), or DS-Cavl (developed by NIH/NIAID/VRC) or a nucleic acid vaccine (e.g., an mRNA vaccine being developed by CureVac (currently unnamed) or mRNA- 1345 (developed by Moderna), or SP0274 (developed by Sanofi)).

[0887] In some embodiments, an RNA composition described herein is co-administered with an influenza vaccine. In some embodiments, the influenza vaccine is an alpha-influenza virus, a beta-influenza virus, a gamma-influenza virus or a delta-influenza virus vaccine. In some embodiments the vaccine is an Influenza A virus, an Influenza B virus, an Influenza C virus, or an Influenza D virus vaccine. In some embodiments, the influenza A virus vaccine comprises a hemagglutinin selected from Hl, H2, H3, H4, H5, H6, H7, H8, H9, H10, HU, H12, H13, H14, H15, H16, H17, and H18, or an immunogenic fragment or variant of the same, or a nucleic acid (e.g., RNA) encoding any one of the same. In some embodiments the influenza A vaccine comprises or encodes a neuraminidase (NA) selected from Nl, N2, N3, N4, N5, N6, N7, N8, N9, N10, and Nl 1, or an immunogenic fragment or variant of the same, or a nucleic acid (e.g., RNA) encoding any one of the same. In some embodiments, the influenza vaccine comprises at least one Influenza virus hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (Ml), matrix protein 2 (M2), non-structural protein 1 (NS1 ), non-structural protein 2 (NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic protein PB1, PB1-F2, and/or polymerase basic protein 2 (PB2), or an immunogenic fragment or variant thereof, or a nucleic acid (e.g., RNA) encoding any of one of the same.

[0888] In some embodiments, an RNA composition described herein can be co- administered with a commercially approved influenza vaccine. In some embodiments, an RNA composition described herein can be co-administered with an inactivated influenza virus (e.g., Fluzone®, Fluzone high-dose quadrivalent®, Fluzone quadrivalent®, Fluzone intradermal quardi valent®, Fluzone quadrivalent southern hemisphere®, Fluad®, Fluad quadrivalent®, Afluria Quardivalent®, Fluarix Quadrivalent®, FluEaval Quadrivalent®, or Flucelvax Quadrivalent®), a recombinant influenza vaccine (e.g., Flublok quadrivalent®), a live attenuated influenza vaccine (e.g., FluMist Quadrivalent®), a non-adjuvanted influenza vaccine, an adjuvanted influenza vaccine, or a subunit or split vaccine. [0889] In some embodiments, an RNA composition described herein is co-administered with an influenza vaccine and/or an RSV vaccine.

[0890] In one embodiment, a composition or medical preparation is a pharmaceutical composition.

[0891] In some embodiments, a composition provided herein and the one or more vaccines against one or more additional diseases are administered at different times. In some embodiments, a composition provided herein is administered at the same time as one or more injectable vaccine(s) against additional diseases. In some such embodiments, a composition provided herein and the one or more injectable vaccine(s) are administered at different injection sites. In some embodiments, a composition provided herein is co-formulated with one or more vaccines against one or more additional diseases (e.g., an influenza vaccine, an RSV vaccine, or an influenza and RSV vaccine). In some embodiments, such co-formulations are provided as a regularly provided booster dose against current variants of concern (e.g., as an annual booster dose).

[0892] In one embodiment, a composition (e.g., RNA) described herein is co- administered with one or more T-cell epitopes of an infectious agent (e.g., SARS-CoV-2) or RNA encoding the same. In some embodiments, a composition (e.g., RNA) described herein is co-administered one or more T-cell epitopes, or RNA encoding the same, derived from an M protein, an N protein, and/or an ORFlab protein of SARS-CoV-2, e.g., a composition disclosed in WO2021188969, the contents of which is incorporated by reference herein in its entirety. In some embodiments, RNA described herein is co-administered with a T-string construct described in WO2021188969 (e.g., an RNA encoding SEQ ID NO: RS C7p2full of WO2021/188969 and/or comprising SEQ ID NO: RS C7n2 or RS C7n2full). In some embodiments, RNA described herein and a T-string construct described in WO2021188969 are administered in a combination of up to about 100 ug RNA total. In some embodiments, subjects are administered at least 2 doses of RNA described herein (e.g., in some embodiments at 30 pg per dose) in combination with a T-string construct (e.g., an RNA encoding SEQ ID NO: RS C7p2full of WO2021/188969), e.g., each dose of a combination of RNA described herein and an RNA encoding SEQ ID NO: RS C7p2full of up to about 100 ug RNA total, wherein the two doses are administered, for example, at least 4 weeks or longer (including, e.g., at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, or at least 12 weeks, or longer) apart from one another. In some embodiments, subjects are administered at least 3 doses of RNA described herein (e.g., in some embodiments at 30 ug each) in combination with a T-string construct (e.g., an RNA encoding SEQ ID NO: RS C7p2full of WO2021/188969), e.g., each dose of a combination of RNA described herein and an RNA encoding SEQ ID NO: RS C7p2full of up to about 100 ug RNA total, wherein the first and the second doses and the second and third doses are each independently administered at least 4 weeks or longer (including, e.g., at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, or at least 12 weeks, or longer) apart from one another. In some embodiments, the RNA described herein and the T-string construct may be co-administered as separate formulations (e.g., formulations administered on the same day to separate injection sites). In some embodiments, the RNA described herein and the T-string construct may be co-administered as a co-formulation (e.g., a formulation comprising RNA described herein and the T-string construct as separate LNP formulations or as LNP formulations comprising both a T-string construct and RNA described herein).

[0893] In some embodiments, populations to be treated with compositions described herein (e.g., mRNA compositions) may include certain populations with a blood type that may have been determined to more susceptible to infection. In some embodiments, populations to be treated with compositions (e.g., mRNA compositions) described herein may include immunocompromised subjects (e.g., those with HIV/AIDS; cancer patients (e.g., receiving antitumor treatment); patients who are taking certain immunosuppressive drugs (e.g., transplant patients, cancer patients, etc.); autoimmune diseases or other physiological conditions expected to warrant immunosuppressive therapy (e.g., within 3 months, within 6 months, or more); and those with inherited diseases that affect the immune system (e.g., congenital agammaglobulinemia, congenital IgA deficiency)). In some embodiments, populations to be treated with compositions (e.g., mRNA compositions) described herein may include those with an infectious disease. For example, in some embodiments, populations to be treated with compositions (e.g., mRNA compositions) described herein may include those infected with human immunodeficiency virus (HIV) and/or a hepatitis virus (e.g., HBV, HCV). In some embodiments, populations to be treated with compositions (e.g., mRNA compositions) described herein may include those with underlying medical conditions. Examples of such underlying medical conditions may include, but are not limited to hypertension, cardiovascular disease, diabetes, chronic respiratory disease, e.g., chronic pulmonary disease, asthma, etc., cancer, and other chronic diseases such as, e.g., lupus, rheumatoid arthritis, chronic liver diseases, chronic kidney diseases (e.g., Stage 3 or worse such as in some embodiments as characterized by a glomerular filtration rate (GFR) of less than 60 mL/min/1.73m2). In some embodiments, populations to be treated with compositions (e.g., mRNA compositions) described herein may include overweight or obese subjects, e.g., specifically including those with a body mass index (BMI) above about 30 kg/m2. In some embodiments, populations to be treated with compositions (e.g., mRNA compositions) described herein may include subjects who have prior diagnosis of COVID-19 or evidence of current or prior infection, e.g., based on serology or nasal swab. In some embodiments, populations to be treated include white and/or non-Hispanic/non- Latino.

[0894] In some embodiments, a composition (e.g., mRNA composition) as provided herein is administered to and/or assessed in subject(s) who have been determined not to show evidence of prior infection, and/or of present infection, before administration; in some embodiments, evidence of prior infection and/or of present infection, may be or include evidence of intact virus, or any viral nucleic acid, protein, lipid etc. present in the subject (e.g., in a biological sample thereof, such as blood, cells, mucus, and/or tissue), and/or evidence of a subject’s immune response to the same. In some embodiments, a composition (e.g., mRNA composition) as provided herein is administered to and/or assessed in subject(s) who have been determined to show evidence of prior infection, and/or of present infection, before administration; in some embodiments, evidence of prior infection and/or of present infection, may be or include evidence of intact virus, or any viral nucleic acid, protein, lipid etc. present in the subject (e.g., in a biological sample thereof, such as blood, cells, mucus, and/or tissue), and/or evidence of a subject’s immune response to the same. In some embodiments, a subject is considered to have a prior infection based on having a positive N-binding antibody test result or positive nucleic acid amplification test (NAAT) result on the day of Dose 1.

[0895] In some embodiments, a composition (e.g., mRNA composition) described herein may be administered to a subject, or population of subjects, in and/or from a geographic region where one or more certain variants (e.g., SARS-CoV2 variants) are prevalent. For example, in some embodiments, a SARS-CoV2 variant is known or assessed to be prevalent in a geographic region, and a composition (e.g., mRNA composition) described herein, that encodes such SARS- CoV2 variant or portion thereof, is administered to a subject or population of subjects in and/or from such geographic location.

[0896] In some embodiments, a composition (e.g., mRNA composition) described herein may be delivered to a draining lymph node of a subject in need thereof, for example, for vaccine priming. In some embodiments, such delivery may be performed by intramuscular administration of a provided RNA (e.g., mRNA) composition.

[0897] In some embodiments, one or more compositions (e.g., mRNA compositions) described herein may be administered according to a regimen established to produce neutralizing antibodies directed to an antigen delivered by the composition and/or an immunogenic portion thereof (e.g., RBD) as measured in serum from a subject that achieves or exceeds a reference level (e.g., a reference level determined based on human infection by the infectious agent /disease convalescent sera) for a period of time and/or induction of cell-mediated immune response (e.g., a T cell response against a given infectious agent), including, e.g., in some embodiments induction of T cells that recognize at least one or more MHC-restricted (e.g., MHC class I-restricted) epitopes within a infectious agent polypeptide and/or an immunogenic portion thereof (e.g., RBD) for a period of time. In some such embodiments, the period of time may be at least 2 months, 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months or longer.

[0898] In some embodiments (e.g., in some embodiments of assessing efficacy), a subject is determined to have experienced infection if one or more of the following is established: detection of infectious agent nucleic acid in a sample from the subject, detection of antibodies that specifically recognize an infectious agent (e.g., an infectious agent glycoprotein), one or more symptoms of infectious agent infection, and combinations thereof. In some such embodiments, detection of infectious agent nucleic acid may involve, for example, NAAT testing on a mid-turbinatae swap sample. In some such embodiments, detection of relevant antibodies may involve serological testing of a blood sample or portion thereof. For example, in some such embodiments, symptoms of CO VID- 19 infection may be or include: fever, new or increased cough, new or increased shortness of breath, chills, new or increased muscle pain, new loss of taste or smell, sore throat, diarrhea, vomiting and combinations thereof. In some embodiments, symptoms of COVID-19 infection may be or include: fever, new or increased cough, new or increased shortness of breath, chills, new or increased muscle pain, new loss of taste or smell, sore throat, diarrhea, vomiting, fatigue, headache, nasal congestion or runny nose, nausea, and combinations thereof. In some embodiments, a subject is determined to have experienced infection if such subject both has experienced one such symptom and also has received a positive test for infectious agent nucleic acid or antibodies, or both. In some such embodiments, a subject is determined to have experienced infection if such subject both has experienced one such symptom and also has received a positive test for nucleic acid. In some such embodiments, a subject is determined to have experienced infection if such subject both has experienced one such symptom and also has received a positive test for infectious agent antibodies.

[0899] In some embodiments, a treatment effect conferred by one or more compositions (e.g., mRNA compositions) described herein may be characterized by (i) a anti-antigen binding antibody level above a pre-determined threshold; (ii) a anti-RBD binding antibody level above a pre-determined threshold; and/or (iii) a infectious agent serum neutralizing titer above a threshold level, e.g., at baseline, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, and/or 24 months after completion of vaccination. In some embodiments, antibody levels and/or serum neutralizing titers may be characterized by geometric mean concentration (GMC), geometric mean titer (GMT), or geometric mean fold-rise (GMFR).

[0900] In some embodiments, a composition (e.g., mRNA composition) as described herein may be shipped, stored, and/or utilized, in a container (such as a vial or syringe), e.g., a glass container (such as a glass vial or syringe), which, in some embodiments, may be a single- dose container or a multi-dose container (e.g., may be arranged and constructed to hold, and/or in some embodiments may hold, a single dose, or multiple doses of a product for administration). In some embodiments, a multi-dose container (such as a multi-dose vial or syringe) may be arranged and constructed to hold, and/or may hold 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses; in some particular embodiments, it may be designed to hold and/or may hold 5 doses. In some embodiments, a single-dose or multi-dose container (such as a single-dose or multi-dose vial or syringe) may be arranged and constructed to hold and/or may hold a volume or amount greater than the indicated number of doses, e.g., in order to permit some loss in transfer and/or administration. In some embodiments, a composition (e.g., mRNA composition) as described herein may be shipped, stored, and/or utilized, in a preservative-free glass container (e.g., a preservative-free glass vial or syringe, e.g., a single-dose or multi-dose preservative-free glass vial or syringe). In some embodiments, a composition (e.g., mRNA composition) as described herein may be shipped, stored, and/or utilized, in a preservative-free glass container (e.g., a preservative-free glass vial or syringe, e.g., a single-dose or multi-dose preservative-free glass vial or syringe) that contains a frozen liquid, e.g., in some embodiments 0.45 ml of frozen liquid (e.g., including 5 doses). In some embodiments, a composition (e.g., mRNA composition) as described herein and/or a container (e.g., a vial or syringe) in which it is disposed, is shipped, stored, and/or utilized may be maintained at a temperature below room temperature, at or below 4 °C, at or below 0 °C, at or below -20 °C, at or below -60 °C, at or below -70 °C, at or below - 80 °C , at or below -90 °C, etc. In some embodiments, a composition (e.g., mRNA composition) as described herein and/or a container (e.g., a viral or syringe) in which it is disposed, is shipped, stored, and/or utilized may be maintained at a temperature between -80°C and -60°C and in some embodiments protected from light. In some embodiments, a composition (e.g., mRNA composition) as described herein and/or a container (e.g., a viral or syringe) in which it is disposed, is shipped, stored, and/or utilized may be maintained at a temperature below about 25°C, and in some embodiments protected from light. In some embodiments, (e.g., mRNA composition) as described herein and/or a container (e.g., a viral or syringe) in which it is disposed, is shipped, stored, and/or utilized may be maintained at a temperature below about 5°C (e.g., below about 4°C), and in some embodiments protected from light. In some embodiments, (e.g., mRNA composition) as described herein and/or a container (e.g., a viral or syringe) in which it is disposed, is shipped, stored, and/or utilized may be maintained at a temperature below about -20°C, and in some embodiments protected from light. In some embodiments, (e.g., mRNA composition) as described herein and/or a container (e.g., a viral or syringe) in which it is disposed, is shipped, stored, and/or utilized may be maintained at a temperature above about - 60°C (e.g., in some embodiments at or above about -20°C, and in some embodiments at or above about 4-5°C, in either case optionally below about 25°C), and in some embodiments protected from light, or otherwise without affirmative steps (e.g., cooling measures) taken to achieve a storage temperature materially below about -20°C. [0901] In some embodiments, a composition (e.g., mRNA composition) as described herein and/or a container (e.g., a vial or syringe) in which it is disposed is shipped, stored, and/or utilized together with and/or in the context of a thermally protective material or container and/or of a temperature adjusting material. For example, in some embodiments, a composition (e.g., mRNA composition) as described herein and/or a container (e.g., a vial or syringe) in which it is disposed is shipped, stored, and/or utilized together with ice and/or dry ice and/or with an insulating material. In some particular embodiments, a container (e.g., a vial or syringe) in which a composition (e.g., mRNA composition) is disposed is positioned in a tray or other retaining device and is further contacted with (or otherwise in the presence of) temperature adjusting (e.g., ice and/or dry ice) material and/or insulating material. In some embodiments, multiple containers (e.g., multiple vials or syringes such as single use or multi-use vials or syringes as described herein) in which a provided composition (e.g., mRNA composition) is disposed are co-localized (e.g., in a common tray, rack, box, etc.) and packaged with (or otherwise in the presence of) temperature adjusting (e.g., ice and/or dry ice) material and/or insulating material. To give but one example, in some embodiments, multiple containers (e.g., multiple vials or syringes such as single use or multi-use vials or syringes as described herein) in which a composition (e.g., mRNA composition) is disposed are positioned in a common tray or rack, and multiple such trays or racks are stacked in a carton that is surrounded by a temperature adjusting material (e.g., dry ice) in a thermal (e.g., insulated) shipper. In some embodiments, temperature adjusting material is replenished periodically (e.g., within 24 hours of arrival at a site, and/or every 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, etc.).

Preferably, re-entry into a thermal shipper should be infrequent, and desirably should not occur more than twice a day. In some embodiments, a thermal shipper is re-closed within 5, 4, 3, 2, or 1 minute, or less, of having been opened. In some embodiments, a provided composition (e.g., mRNA composition) that has been stored within a thermal shipper for a period of time, optionally within a particular temperature range remains useful. For example, in some embodiments, if a thermal shipper as described herein containing a provided composition (e.g., mRNA composition) is or has been maintained (e.g., stored) at a temperature within a range of about 15 °C to about 25 °C, the composition (e.g., mRNA composition) may be used for up to 10 days; that is, in some embodiments, a composition (e.g., mRNA composition) that has been maintained within a thermal shipper, which thermal shipper is at a temperature within a range of about 15 °C to about 25 °C, for a period of not more than 10 days is administered to a subject. Alternatively or additionally, in some embodiments, if a provided composition (e.g., mRNA composition) is or has been maintained (e.g., stored) within a thermal shipper, which thermal shipper has been maintained (e.g., stored) at a temperature within a range of about 15 °C to about 25 °C, it may be used for up to 10 days; that is, in some embodiments, a provided composition (e.g., mRNA composition) that has been maintained within a thermal shipper, which thermal shipper has been maintained at a temperature within a range of about 15 °C to about 25 °C for a period of not more than 10 days is administered to a subject.

Exemplary Combination Methods

[0902] In one aspect, combination methods that increases activation of naive B cell immune response to “new” epitopes (e.g., “new” neutralizing epitopes) arisen from a variant of an infectious agent are described herein. In some embodiments, a method of inducing a priming immune response comprises (a) administering to a subject one or both of: (i) a composition that comprises or delivers a variant polypeptide of a reference antigen of an infectious agent (e.g., as described herein); and an agent that induces a priming-favorable cytokine milieu in lymphoid tissues, wherein the agent is present at a dose that is effective to increase activation of naive B cell immune response to at least one of the neutralizing epitopes. In some embodiments, a subject amenable to a method described herein has previously received a composition that comprises or delivers the reference antigen. In some embodiments, a subject amenable to a combination method described herein has previously received the agent that induces a priming- favorable cytokine milieu in lymphoid tissues.

[0903] In some embodiments, such an agent for inducing a priming-favorable cytokine milieu in lymphoid tissues is an IFNa-inducing agent (e.g., as described herein). For example, in some embodiments, such an IFNa-inducing agent is or comprises an unmodified RNA (e.g., as described herein). In some embodiments, such an unmodified RNA encodes one or more T cell epitopes (e.g., as described herein). In some embodiments, such an agent for inducing a priming- favorable cytokine milieu is an agent that induces CD4+ T cell immune response. [0904] In some embodiments, the dose of the agent that is effective to increase activation of naive B cell immune response to such neutralizing epitope(s) can be characterized, for example, in an animal model, by measuring the level of a priming-favorable cytokine in lymphoid tissues. For example, in some embodiments, the dose of the agent that is effective to increase activation of naive B cell immune response to such neutralizing epitope(s) can be effective to increase the level of IFN, e.g., Type I IFN (e.g., IFNa), for example, in lymphoid tissues, to a certain level that is sufficient for “priming.” In some embodiments, the dose of the agent can be effective to increase the level of IFN, e.g., Type I IFN (e.g., IFNa), for example, in lymphoid tissues, to a certain pre-determined level, for example in some embodiments a level that is sufficient to “prime” an immune response. In some embodiments, the dose of the agent can be effective to increase the level of IFN, e.g., Type I IFN (e.g., IFNa), for example, in lymphoid tissues, by at least 30% (including, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more), as compared to the level of IFN in the absence of such an agent. In some embodiments, the dose of the agent can be effective to increase the level of IFN, e.g., Type I IFN (e.g., IFNa), for example, in lymphoid tissues, by at least 1.1-fold (including, e.g., at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or more), as compared to the level of IFN in the absence of such an agent.

[0905] In some embodiments, the dose of the agent can be effective to increase T cell immune response, for example in some embodiments CD4+ T cell immune response. In some embodiments, the dose of the agent can be effective to increase CD4+ T cell immune response (e.g., increasing CD4+ T cell population) by at least 30% (including, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more), as compared to the CD4+ T cell immune response in the absence of such an agent. In some embodiments, the dose of the agent can be effective to increase CD4+ T cell immune response (e.g., increasing CD4+ T cell population) by at least 1.1 -fold (including, e.g., at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or more), as compared to the CD4+ T cell immune response in the absence of such an agent.

[0906] In some embodiments, described herein is a method of inducing or supporting a priming immune response to an antigen in a subject comprises exposing the subject to the antigen under immune priming conditions. In some embodiments, an antigen is or comprises a variant polypeptide of a reference antigen of an infectious agent. As described herein, an immune priming condition is a particular state or condition that favors an immune response toward de no immune response (e.g., in some embodiments to “new” epitopes arisen from variant polypeptides of a reference antigen of an infection agent (e.g., as described herein) and/or reduces and/or delay memory response (e.g., in some embodiments to epitopes such as in some embodiments non-neutralizing epitopes that are shared between the variant polypeptide(s) and the reference antigen). In some embodiments, such an immune priming condition is characterized by increasing the level of IFN, e.g., Type I IFN (e.g., IFNa), for example, in lymphoid tissues, to a pre-determined level. In some embodiments, such an immune priming condition is characterized by increasing CD4+ T cell immune response (e.g., increasing the population of CD4+ T cells) to a pre-determined level.

[0907] In some embodiments, a subject amenable to a method described herein has previously been exposed to a reference antigen of an infectious agent. In some embodiments, a subject amenable to a method described herein has previously been exposed to a variant polypeptide of the reference antigen. In some embodiments, such a variant polypeptide has an amino acid sequence that is at least 80% (including, e.g., at least 85%, at least 90%, at least 92%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99%) identical to the amino acid sequence of a reference antigen. In some embodiments, such a variant polypeptide can have a certain portion that has an amino acid sequence that is lower than 80% identical to the corresponding portion of the reference antigen, but has a percent identity that is higher than 80% over the entire length of the polypeptide. In some embodiments, such a variant polypeptide comprises neutralizing epitopes that are absent in the reference antigen. In some embodiments, a subject amenable to a method described herein has previously received a composition that comprises or delivers the reference antigen. In some embodiments, a subject amenable to a combination method described herein has previously received the agent that induces a priming- favorable cytokine milieu in lymphoid tissues.

[0908] In some embodiments, the step of exposing comprises administering a composition (e.g., as described herein) that comprises or delivers a variant polypeptide of the reference antigen. In some embodiments, a composition comprises a variant polypeptide of the reference antigen. In some embodiments, a composition comprises a polyribonucleotide encoding a variant polypeptide of the reference antigen. [0909] In some embodiments, the step of exposing comprises administering a “priming adjuvant” to a subject who is or will soon be exposed to an antigen, e.g., a variant polypeptide of the reference antigen. In some embodiments, a “priming adjuvant” is or comprises an aagent that induces a priming-favorable cytokine milieu in lymphoid tissues (e.g., as described herein).

[0910] In some embodiments, methods of inducing an immune response in a subject in need thereof are also provided herein. In some embodiments, such a method comprises administering to a subject a first RNA molecule encoding a first antigen and a second RNA molecule encoding a second antigen, wherein the first RNA molecule is a modified RNA molecule and the second RNA molecule (i) does not comprise a modified ribonucleotide or (ii) is an RNA replicon (e.g., in some embodiments a self-amplifying RNA molecule or a trans- amplifying RNA molecule) as described herein. In some embodiments, such a method comprises administering to a subject a composition comprising a first plurality of RNA molecules encoding first antigens and a second plurality of RNA molecules encoding second antigens, wherein at least 10% (including, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% , at least 80%, at least 90%, at least 95%, at least 98%, and up to 100%) of the first plurality of RNA molecules are modified RNA molecules, and at least 10% (including, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% , at least 80%, at least 90%, at least 95%, at least 98%, and up to 100%) of the second plurality of RNA molecules (i) do not comprise a modified ribonucleotide or (ii) are self-amplifying RNA molecules or trans- amplifying RNA molecules. In some embodiments, such a method comprises administering to a subject a first dose of a composition comprising a first RNA molecule encoding a first antigen, and a second dose of a composition comprising a second RNA molecule encoding a second antigen, wherein the first RNA molecule is a modified RNA molecule, and the second RNA molecule (i) does not comprise a modified ribonucleotide or (ii) is an RNA replicon (e.g., in some embodiments a self-amplifying RNA molecule or a trans-amplifying RNA molecule) as described herein.

[0911] In some embodiments of various methods described herein, the first RNA molecule comprises modified uridines (e.g., in some embodiments 1 -methylpseudouridine). In some embodiments, such modified uridines are in place of all uridines. [0912] In some embodiments of various methods described herein, the second RNA molecule does not comprise a modified ribonucleotide.

[0913] In some embodiments of various methods described herein, the first antigen is or comprises a B cell antigen of an infectious agent (e.g., as described herein) and the second antigen is or comprises a T cell antigen. In some embodiments, a T cell antigen is or comprises at least one or more T cell epitopes. In some embodiments of various methods described herein, a T cell antigen is from the same infectious agent from which a first antigen is derived.

[0914] In some embodiments of various methods described herein, the first RNA molecule and the second RNA molecule are co- administered. For example, in some embodiments, the first RNA molecule and the second RNA molecule are separately or co- formulated in lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes. In some embodiments of various methods described herein, the first RNA molecule and the second RNA molecule are separately administered.

[0915] In some embodiments of various methods described herein, a subject amenable to such a method described herein has previously been exposed (e.g., by prior vaccination and/or by infection) to a reference antigen of an infectious agent. In some embodiments, such a subject has previously been administered one or more doses of one or more vaccines directed to a reference antigen of an infectious agent, wherein the reference antigen is from an earlier strain or lineage of the infectious agent, and wherein a B cell memory immune response has been established to the reference antigen. In some embodiments, a subject amenable to a method described herein has previously been exposed (e.g., by prior vaccination and/or by infection) to a variant polypeptide of the reference antigen. In some embodiments, such a variant polypeptide has an amino acid sequence that is at least 80% (including, e.g., at least 85%, at least 90%, at least 92%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99%) identical to the amino acid sequence of a reference antigen. In some embodiments, such a variant polypeptide can have a certain portion that has an amino acid sequence that is lower than 80% identical to the corresponding portion of the reference antigen, but has a percent identity that is higher than 80% over the entire length of the polypeptide. In some embodiments, such a variant polypeptide comprises neutralizing epitopes that are absent in the reference antigen. In some embodiments, a subject amenable to a method described herein has previously received a composition that comprises or delivers the reference antigen. In some embodiments, a subject amenable to a combination method described herein has previously received the agent that induces a priming- favorable cytokine milieu in lymphoid tissues. [0916] In some embodiments, described herein is a combination comprising: (i) a modified RNA molecule encoding a polypeptide comprising or consisting of a variant polypeptide of a reference antigen of an infectious agent, or an immunogenic portion thereof, wherein the variant polypeptide comprises neutralizing epitopes that are absent in the reference antigen; and (ii) an agent that induces a priming-favorable cytokine milieu in lymphoid tissues, wherein the agent is present at a dose that is effective to increase activation of naïve B cell immune response to at least one of the neutralizing epitopes. In some embodiments, the reference antigen is (i) a surface protein or surface glycoprotein of an infectious agent strain or variant that was previously and/or is currently prevalent; and/or (ii) a surface protein or surface glycoprotein of an infectious agent that has been previously delivered in a vaccine (e.g., a commercially available vaccine, an RNA vaccine, or a protein-based vaccine). In some embodiments, the infectious agent is associated with a circulating infectious disease (e.g., for which variants can be expected to arise). In some embodiments, such circulating infectious disease is a bacterial infectious disease. In some embodiments, such circulating infectious disease is a parasitic infectious disease. An exemplary parasitic infectious disease is malaria. In some embodiments, such circulating infectious disease is a viral infectious disease. In some embodiments, a viral infectious disease is associated with an RNA virus. Exemplary viral infectious diseases include, but are not limited to coronavirus, ebolavirus, influenza viruses, norovirus, rotavirus, respiratory syncytial virus, alphaherpesvirus, etc. [0917] In some embodiments, a modified RNA molecule included in a described combination comprises modified ribonucleotides. In some embodiments, such modified ribonucleotides is 1-methylpseudouridine. [0918] In some embodiments, a modified RNA molecule included in a described combination encodes a polypeptide comprising or consisting of a variant polypeptide of a reference antigen of a coronavirus, or an immunogenic portion thereof, wherein the variant polypeptide comprises neutralizing epitopes that are absent in the reference antigen. In some embodiments, such a coronavirus is SARS-CoV-2. In some embodiments, such a reference 11831138v1 antigen is a SARS-CoV-2 S protein of a Wuhan strain or an Omicron BA.4/5 strain, or an immunogenic fragment or portion thereof, including, e.g., SI, RBD, and/or NTD. In some embodiments, such a reference antigen is a SARS-CoV-2 S protein of XBB strain (e.g., XBB1, XBB1.5), or an immunogenic fragment or portion thereof, including, e.g., SI, RBD and/or NTD.

[0919] In some embodiments, a modified RNA molecule comprises a nucleotide sequence that encodes the amino acid sequence of a SARS-CoV-2 antigen polypeptide as disclosed in Tables A-G or an amino acid sequence that is at least 80% (including, e.g., at least 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher) identical to the amino acid sequence of a SARS-CoV-2 antigen polypeptide as described in Table A-G.

[0920] In some embodiments, a modified RNA molecule comprises a nucleotide sequence as disclosed in Tables A-G or a nucleotide sequence that is at least 80% (including, e.g., at least 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher) identical to the polynucleotide sequence encoding a SARS-CoV-2 antigen as described in Table A-G.

[0921] In some embodiments, an agent included in a described composition that induces a priming-favorable cytokine milieu in lymphoid tissues is or comprises an IFNa-inducing agent. In some embodiments, such an IFNa-inducing agent is or comprises a RNA replicon (e.g., as described herein). In some embodiments, such an IFNa-inducing agent is or comprises an unmodified RNA molecule (e.g., as described herein). In some embodiments, the amount ratio (by mass or moles) of the modified RNA molecule to the unmodified RNA molecule in a described composition is within a range of about 1:5 to about 10: 1. In some embodiments, the amount ratio (by mass or moles) of the modified RNA molecule to the unmodified RNA molecule in a described composition is within a range of about 1: 1 to about 10: 1. In some embodiments, the amount ratio (by mass or moles) of the modified RNA molecule to the unmodified RNA molecule is about 1:5, about 1:4, about 1:3, about 1:2, about 1: 1, about 2:1, about 3: 1, about 4:1, about 5: 1, about 6:1, about 7: 1, about 8: 1, about 9:1, about 10:1. Without wishing to be bound by a particular theory, in some embodiments, such an unmodified RNA molecule is characterized in that it induces CD4+ T cell response. In some embodiments, such an unmodified RNA molecules encodes one or more T cell epitopes. In some embodiments, such an unmodified RNA molecule is a T string construct as described in the International Patent Application No. WO2021188969 or in the International Patent Application No. PCT/US22/44400, the relevant content of which is incorporated herein by reference for the purposes described herein. In some such embodiments, the amount ratio (by mass or moles) of the modified RNA molecule to the unmodified RNA molecule is about 1 : 1

[0922] In some embodiments, an unmodified RNA molecule described herein comprises a nucleotide sequence that encodes a polypeptide comprising one or more T cell epitopes, wherein the polypeptide is represented by the amino acid sequence set forth below:

GGSGGGGSGGSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSA AEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAP SASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPK KDKFKAAMVTNNTFTLKVPHVGEIPVAYRKVLLKTIQPRVEKYLFDESGEFKLSEVGPE HSLAEYYIFFASFYYKRNGFAYANRNRFLYIIKLIFLWLLWPVTLACFVLAAVYRINWIT GGIAIAMACLVGLMWLSYFIASFRLFYNSFHTTDPSFLGRYMSALFADDLNQLTGYHTD FSSEIIGYQLMCQPILLAEAELAKNVSLILGTVSWNLKKNGLGRCDIKDLPKEITVATSRT LSYYKLGASQRVAYRSYLLSAGIFGAITDVFYKENSYKVPTDNYITTYARMAAPKEIIFL EGETLFGDDTVIEVAIILASFSASTRRRGGGSGGGGSGG. In some embodiments, an exemplary nucleotide sequence that encodes such a polypeptide is set forth below:

GGCGGCUCUGGAGGAGGCGGCUCCGGAGGCUCUCCUGCCAGAAUGGCUGGAAAUG GAGGAGAUGCUGCUCUGGCUCUGCUGCUGCUGGAUAGACUGAAUCAGCUGGAAA GCAAAAUGAGCGGAAAAGGACAGCAGCAGCAGGGACAAACAGUGACAAAGAAAU CUGCUGCUGAAGCCAGCAAGAAACCAAGACAGAAAAGAACAGCCACAAAAGCCUA CAAUGUGACACAGGCCUUUGGAAGAAGGGGACCUGAACAGACACAGGGAAAUUU UGGAGAUCAGGAACUGAUCAGACAGGGAACAGAUUACAAACACUGGCCUCAGAU CGCCCAGUUUGCCCCAUCUGCCUCUGCCUUCUUUGGAAUGAGCAGAAUUGGAAUG GAAGUGACACCUUCUGGAACAUGGCUGACAUACACAGGAGCCAUCAAACUGGAU GAUAAAGAUCCAAAUUUUAAAGAUCAGGUGAUUCUGCUGAAUAAACACAUUGAU GCCUACAAAACAUUUCCACCAACAGAACCAAAGAAAGAUAAAUUCAAAGCUGCUA

UGGUGACAAACAACACAUUUACACUGAAAGUGCCUCAUGUGGGAGAAAUUCCUG UGGCUUACAGAAAAGUGCUGCUGAAAACAAUUCAACCAAGAGUGGAAAAAUACC UGUUUGAUGAAUCUGGAGAAUUUAAACUUUCUGAAGUGGGACCUGAACACUCUC UUGCUGAAUACUACAUUUUCUUUGCUUCUUUUUACUAUAAGAGGAAUGGAUUUG CAUACGCAAAUAGAAAUAGAUUUCUGUACAUUAUUAAACUGAUUUUUCUGUGGC UGCUGUGGCCUGUGACACUGGCUUGCUUUGUGCUGGCUGCUGUGUACAGAAUAA AUUGGAUAACAGGAGGAAUUGCAAUUGCAAUGGCAUGCUUGGUGGGAUUGAUGU GGUUGUCUUACUUUAUUGCUUCUUUUAGACUGUUUUACAACUCUUUUCACACAA CUGAUCCAUCUUUUCUGGGAAGAUACAUGUCUGCUCUUUUUGCUGAUGAUUUAA AUCAGUUAACAGGAUAUCACACAGAUUUUUCUUCUGAAAUCAUUGGAUACCAGU UGAUGUGUCAACCAAUUUUGUUGGCUGAAGCUGAAUUGGCAAAAAACGUGUCUU UGAUUCUUGGAACAGUUUCUUGGAAUCUUAAGAAAAACGGACUGGGAAGAUGCG AUAUUAAAGAUCUGCCAAAAGAAAUUACAGUGGCAACAAGCAGAACACUGAGCU ACUACAAACUGGGAGCAAGCCAGAGAGUGGCAUACAGAUCCUACUUAUUAUCUG CUGGAAUUUUUGGAGCUAUCACAGAUGUGUUUUACAAAGAAAACUCAUACAAAG UGCCAACAGACAAUUACAUAACAACAUACGCUAGAAUGGCUGCUCCAAAAGAAA UCAUCUUCCUUGAAGGAGAAACACUUUUUGGAGAUGAUACAGUGAUUGAAGUGG CAAUUAUUCUGGCAUCUUUUUCUGCAUCCACAAGAAGAAGGGGAGGAGGAUCCG GUGGUGGCGGCAGCGGCGGC

In some embodiments, such a polypeptide can further comprise a secretory signal (e.g., as described herein) and/or a transmembrane region (e.g., as described herein). In some such embodiments, the amount ratio (by mass or moles) of the modified RNA molecule to the unmodified RNA molecule is about 1:1.

[0923] In some embodiments, an unmodified RNA molecule described herein comprises a nucleotide sequence that encodes a polypeptide comprising one or more T cell epitopes, wherein the polypeptide is represented by the amino acid sequence set forth below:

MFVFLVLLPLVSSQCVNLTGGSGGGGSGGSPARMAGNGGDAALALLLLDRLNQLESK MSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQE LIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQ VILLNKHIDAYKTFPPTEPKKDKFKAAMVTNNTFTLKVPHVGEIPVAYRKVLLKTIQPRV EKYLFDESGEFKLSEVGPEHSLAEYYIFFASFYYKRNGFAYANRNRFLYIIKLIFLWLLWP VTLACFVLAAVYRINWITGGIAIAMACLVGLMWLSYFIASFRLFYNSFHTTDPSFLGRY MSALFADDLNQLTGYHTDFSSEIIGYQLMCQPILLAEAELAKNVSLILGTVSWNLKKNG LGRCDIKDLPKEITVATSRTLSYYKLGASQRVAYRSYLLSAGIFGAITDVFYKENSYKVP TDNYITTYARMAAPKEIIFLEGETLFGDDTVIEVAIILASFSASTRRRGGGSGGGGSGGEQ YIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKG VKLHYT. In some embodiments, an exemplary nucleotide sequence that encodes such a polypeptide is set forth below:

AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAU GUUUGUUUUUCUUGUUUUACUUCCUUUAGUCUCAUCUCAGUGUGUCAAUUUGAC AGGCGGCUCUGGAGGAGGCGGCUCCGGAGGCUCUCCUGCCAGAAUGGCUGGAAAU GGAGGAGAUGCUGCUCUGGCUCUGCUGCUGCUGGAUAGACUGAAUCAGCUGGAA

AGCAAAAUGAGCGGAAAAGGACAGCAGCAGCAGGGACAAACAGUGACAAAGAAA UCUGCUGCUGAAGCCAGCAAGAAACCAAGACAGAAAAGAACAGCCACAAAAGCCU ACAAUGUGACACAGGCCUUUGGAAGAAGGGGACCUGAACAGACACAGGGAAAUU UUGGAGAUCAGGAACUGAUCAGACAGGGAACAGAUUACAAACACUGGCCUCAGA UCGCCCAGUUUGCCCCAUCUGCCUCUGCCUUCUUUGGAAUGAGCAGAAUUGGAAU GGAAGUGACACCUUCUGGAACAUGGCUGACAUACACAGGAGCCAUCAAACUGGA

UGAUAAAGAUCCAAAUUUUAAAGAUCAGGUGAUUCUGCUGAAUAAACACAUUGA

UGCCUACAAAACAUUUCCACCAACAGAACCAAAGAAAGAUAAAUUCAAAGCUGCU

AUGGUGACAAACAACACAUUUACACUGAAAGUGCCUCAUGUGGGAGAAAUUCCU

GUGGCUUACAGAAAAGUGCUGCUGAAAACAAUUCAACCAAGAGUGGAAAAAUAC

CUGUUUGAUGAAUCUGGAGAAUUUAAACUUUCUGAAGUGGGACCUGAACACUCU

CUUGCUGAAUACUACAUUUUCUUUGCUUCUUUUUACUAUAAGAGGAAUGGAUUU

GCAUACGCAAAUAGAAAUAGAUUUCUGUACAUUAUUAAACUGAUUUUUCUGUGG

CUGCUGUGGCCUGUGACACUGGCUUGCUUUGUGCUGGCUGCUGUGUACAGAAUA

AAUUGGAUAACAGGAGGAAUUGCAAUUGCAAUGGCAUGCUUGGUGGGAUUGAUG

UGGUUGUCUUACUUUAUUGCUUCUUUUAGACUGUUUUACAACUCUUUUCACACA

ACUGAUCCAUCUUUUCUGGGAAGAUACAUGUCUGCUCUUUUUGCUGAUGAUUUA

AAUCAGUUAACAGGAUAUCACACAGAUUUUUCUUCUGAAAUCAUUGGAUACCAG

UUGAUGUGUCAACCAAUUUUGUUGGCUGAAGCUGAAUUGGCAAAAAACGUGUCU

UUGAUUCUUGGAACAGUUUCUUGGAAUCUUAAGAAAAACGGACUGGGAAGAUGC

GAUAUUAAAGAUCUGCCAAAAGAAAUUACAGUGGCAACAAGCAGAACACUGAGC

UACUACAAACUGGGAGCAAGCCAGAGAGUGGCAUACAGAUCCUACUUAUUAUCU

GCUGGAAUUUUUGGAGCUAUCACAGAUGUGUUUUACAAAGAAAACUCAUACAAA

GUGCCAACAGACAAUUACAUAACAACAUACGCUAGAAUGGCUGCUCCAAAAGAA

AUCAUCUUCCUUGAAGGAGAAACACUUUUUGGAGAUGAUACAGUGAUUGAAGUG

GCAAUUAUUCUGGCAUCUUUUUCUGCAUCCACAAGAAGAAGGGGAGGAGGAUCC

GGUGGUGGCGGCAGCGGCGGCGAACAGUACAUUAAAUGGCCUUGGUACAUUUGG

CUUGGAUUUAUUGCAGGAUUAAUUGCAAUUGUGAUGGUGACAAUUAUGUUAUGU

UGUAUGACAUCAUGUUGUUCUUGUUUAAAAGGAUGUUGUUCUUGUGGAAGCUGU

UGUAAAUUUGAUGAAGAUGAUUCUGAACCUGUGUUAAAAGGAGUGAAAUUGCAU

UACACAUGAUGACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUC

CCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACC

UCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGC

AAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUU

AACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGU

CAAUUUCGUGCCAGCCACACCCUGGAGCUAGCAAAAAAAAAAAAAAAAAAAAAA

AAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

In some such embodiments, the amount ratio (by mass or moles) of the modified RNA molecule to the unmodified RNA molecule is about 1: 1.

[0924] In some embodiments, the modified RNA molecule and the unmodified RNA molecule included in a combination described herein are separately or co-formulated in lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes.

[0925] In some embodiments, described herein is a combination comprising: (i) a composition that comprises or delivers a polypeptide comprising or consisting of a variant polypeptide of a reference antigen of an infectious agent, or an immunogenic portion thereof, wherein the variant polypeptide comprises neutralizing epitopes that are absent in the reference antigen; and (ii) an agent that induces a priming-favorable cytokine milieu in lymphoid tissues, wherein the agent is present at a dose that is effective to increase activation of naive B cell immune response to at least one of the neutralizing epitopes, and wherein the agent is or comprises (i) an unmodified RNA molecule (e.g., as described herein) or (ii) a RNA replicon (e.g., as described herein), and wherein the RNA molecule or the RNA replicon is formulated in lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes. In some embodiments, the reference antigen is (i) a surface protein or surface glycoprotein of an infectious agent strain or variant that was previously and/or is currently prevalent; and/or (ii) a surface protein or surface glycoprotein of an infectious agent that has been previously delivered in a vaccine (e.g., a commercially available vaccine, an RNA vaccine, or a protein-based vaccine). In some embodiments, the infectious agent is associated with a circulating infectious disease (e.g., for which variants can be expected to arise). In some embodiments, such circulating infectious disease is a bacterial infectious disease. In some embodiments, such circulating infectious disease is a parasitic infectious disease. An exemplary parasitic infectious disease is malaria. In some embodiments, such circulating infectious disease is a viral infectious disease. In some embodiments, a viral infectious disease is associated with an RNA virus. Exemplary viral infectious diseases include, but are not limited to coronavirus, ebolavirus, influenza viruses, norovirus, rotavirus, respiratory syncytial virus, alphaherpesvirus, etc.

[0926] In some embodiments, a polypeptide included in a described combination comprises or consists of a variant polypeptide of a reference antigen of an infectious agent, or an immunogenic portion thereof, wherein the variant polypeptide comprises neutralizing epitopes that are absent in the reference antigen. In some embodiments, a polypeptide included in a described combination comprises or consists of a variant polypeptide of a reference antigen of a coronavirus, or an immunogenic portion thereof, wherein the variant polypeptide comprises neutralizing epitopes that are absent in the reference antigen. In some embodiments, such a coronavirus is SARS-CoV-2. In some embodiments, such a reference antigen is a SARS-CoV-2 S protein of a Wuhan strain or an Omicron BA.4/5 strain, or an immunogenic fragment or portion thereof, including, e.g., SI, RBD, and/or NTD. In some embodiments, such a reference antigen is a SARS-CoV-2 S protein of XBB strain (e.g., XBB1, XBB1.5), or an immunogenic fragment or portion thereof, including, e.g., SI, RBD and/or NTD.

[0927] In some embodiments, the amount ratio (by mass or moles) of the polypeptide to the agent (comprising an unmodified RNA molecule or a RNA replicon) in a described composition is about 1:5 to about 20:1. In some embodiments, the amount ratio (by mass or moles) of the polypeptide to the agent (comprising an unmodified RNA molecule or a RNA replicon) in a described composition is within a range of about 1:1 to about 20:1. In some embodiments, the amount ratio (by mass or moles) of the polypeptide to the agent (comprising an unmodified RNA molecule or a RNA replicon) is about 1:5, about 1:4, about 1:3, about 1:2, about 1: 1, about 2:1, about 3: 1, about 4: 1, about 5:1, about 6: 1, about 7:1, about 8: 1, about 9:1, about 10: 1, about 15: 1, or about 20: 1. Without wishing to be bound by a particular theory, in some embodiments, such an agent (comprising an unmodified RNA molecule or a RNA replicon) is characterized in that it induces CD4+ T cell response. In some embodiments, such an agent (comprising an unmodified RNA molecule or a RNA replicon) encodes one or more T cell epitopes. In some embodiments, such an agent (comprising an unmodified RNA molecule or a RNA replicon) is a T string construct as described in the International Patent Application No. WO2021188969 or in the International Patent Application No. PCT/US22/44400, the relevant content of which is incorporated herein by reference for the purposes described herein.

[0928] In some embodiments, a combination described herein are provided in the same composition.

[0929] In some embodiments, a combination described herein are provided in separate compositions.

[0930] In some embodiments, a combination described herein are provided in a single molecule. For example, in some embodiments, a combination described herein are provided in a RNA molecule. Accordingly, in some embodiments, described herein is an RNA molecule comprising a nucleotide sequence that includes modified ribonucleotides and corresponding unmodified ribonucleotides, wherein the ratio of the modified ribonucleotides to the corresponding unmodified ribonucleotides is within a range of about 1: 10 to about 1: 1; and wherein the nucleotide sequence encodes an antigen of an infectious agent (e.g., as described herein). In some embodiments, such a nucleotide sequence comprises a first domain and a second domain, wherein at least one of the first domain and the second domain comprises modified ribonucleotides and the other domain comprises no modified ribonucleotides. In some embodiments, the modified ribonucleotides are 1 -methylpseudouridine and the corresponding unmodified ribonucleotides are uridine.

Methods of Manufacturing Polyribonucleotides

[0931] Individual polyribonucleotides can be produced by methods known in the art. For example, in some embodiments, polyribonucleotides can be produced by in vitro transcription, for example, using a DNA template. A plasmid DNA used as a template for in vitro transcription to generate a polyribonucleotide described herein is also within the scope of the present disclosure.

[0932] A DNA template is used for in vitro RNA synthesis in the presence of an appropriate RNA polymerase (e.g., a recombinant RNA-polymerase such as a T7 RNA- polymerase) with ribonucleotide triphosphates (e.g., ATP, CTP, GTP, UTP). In some embodiments, polyribonucleotides (e.g., ones described herein) can be synthesized in the presence of modified ribonucleotide triphosphates. By way of example only, in some embodiments, pseudouridine (y), N1 -methyl-pseudouridine (mh|/), or 5 -methyl -uridine (m5U) can be used to replace uridine triphosphate (UTP). In some embodiments, pseudouridine (y) can be used to replace uridine triphosphate (UTP). In some embodiments, N 1 -methyl-pseudouridine (mh|/) can be used to replace uridine triphosphate (UTP). In some embodiments, 5-methyl- uridine (m5U) can be used to replace uridine triphosphate (UTP).

[0933] As will be clear to those skilled in the art, during in vitro transcription, an RNA polymerase (e.g., as described and/or utilized herein) typically traverses at least a fragment of a single-stranded DNA template in the 3'— > 5' direction to produce a single-stranded complementary RNA in the 5'— > 3' direction.

[0934] In some embodiments where a polyribonucleotide comprises a polyA tail, one of those skill in the art will appreciate that such a polyA tail may be encoded in a DNA template, e.g., by using an appropriately tailed PCR primer, or it can be added to a polyribonucleotide after in vitro transcription, e.g., by enzymatic treatment (e.g., using a poly(A) polymerase such as an E. coli Poly(A) polymerase). Suitable poly(A) tails are described herein above. For example, in some embodiments, a poly(A) tail comprises a nucleotide sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AA (SEQ ID NO: 114). In some embodiments, a poly(A) tail comprises a plurality of A residues interrupted by a linker. In some embodiments, a linker comprises the nucleotide sequence GCATATGAC (SEQ ID NO: 115).

[0935] In some embodiments, those skilled in the art will appreciate that addition of a 5' cap to an RNA (e.g., mRNA) can facilitate recognition and attachment of the RNA to a ribosome to initiate translation and enhances translation efficiency. Those skilled in the art will also appreciate that a 5' cap can also protect an RNA product from 5' exonuclease mediated degradation and thus increases half-life. Methods for capping are known in the art; one of ordinary skill in the art will appreciate that in some embodiments, capping may be performed after in vitro transcription in the presence of a capping system (e.g., an enzyme -based capping system such as, e.g., capping enzymes of vaccinia virus). In some embodiments, a cap may be introduced during in vitro transcription, along with a plurality of ribonucleotide triphosphates such that a cap is incorporated into a polyribonucleotide during transcription (also known as co- transcriptional capping). In some embodiments, a GTP fed-batch procedure with multiple additions in the course of the reaction may be used to maintain a low concentration of GTP in order to effectively cap the RNA. Suitable 5' cap are described herein above. For example, in some embodiments, a 5' cap comprises m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

[0936] Following RNA transcription, a DNA template is digested. In some embodiments, digestion can be achieved with the use of DNase I under appropriate conditions.

[0937] In some embodiments, in-vitro transcribed polyribonucleotides may be provided in a buffered solution, for example, in a buffer such as HEPES, a phosphate buffer solution, a citrate buffer solution, an acetate buffer solution; in some embodiments, such solution may be buffered to a pH within a range of, for example, about 6.5 to about 7.5; in some embodiments approximately 7.0. In some embodiments, production of polyribonucleotides may further include one or more of the following steps: purification, mixing, filtration, and/or filling.

[0938] In some embodiments, polyribonucleotides can be purified (e.g., in some embodiments after in vitro transcription reaction), for example, to remove components utilized or formed in the course of the production, like, e.g., proteins, DNA fragments, and/or or nucleotides. Various nucleic acid purifications that are known in the art can be used in accordance with the present disclosure. Certain purification steps may be or include, for example, one or more of precipitation, column chromatography (including, e.g., but not limited to anionic, cationic, hydrophobic interaction chromatography (HIC)), solid substrate -based purification (e.g., magnetic bead-based purification). In some embodiments, polyribonucleotides may be purified using magnetic bead-based purification, which in some embodiments may be or comprise magnetic bead-based chromatography. In some embodiments, polyribonucleotides may be purified using hydrophobic interaction chromatography (HIC) and/or diafiltration. In some embodiments, polyribonucleotides may be purified using HIC followed by diafiltration.

[0939] In some embodiments, dsRNA may be obtained as side product during in vitro transcription. In some such embodiments, a second purification step may be performed to remove dsRNA contamination. For example, in some embodiments, cellulose materials (e.g., microcrystalline cellulose) may be used to remove dsRNA contamination, for examples in some embodiments in a chromatographic format. In some embodiments, cellulose materials (e.g., microcrystalline cellulose) can be pretreated to inactivate potential RNase contamination, for example in some embodiments by autoclaving followed by incubation with aqueous basic solution, e.g., NaOH. In some embodiments, cellulose materials may be used to purify polyribonucleotides according to methods described in WO 2017/182524, the entire content of which is incorporated herein by reference.

[0940] In some embodiments, a batch of polyribonucleotides may be further processed by one or more steps of filtration and/or concentration. For example, in some embodiments, polyribonucleotide(s), for example, after removal of dsRNA contamination, may be further subject to diafiltration (e.g., in some embodiments by tangential flow filtration), for example, to adjust the concentration of polyribonucleotides to a desirable RNA concentration and/or to exchange buffer to a drug substance buffer.

[0941] In some embodiments, polyribonucleotides may be processed through 0.2 pm filtration before they are filled into appropriate containers.

[0942] In some embodiments, polyribonucleotides and compositions thereof may be manufactured in accordance with a process as described herein, or as otherwise known in the art. [0943] In some embodiments, polyribonucleotides and compositions thereof may be manufactured at a large scale. For example, in some embodiments, a batch of polyribonucleotides can be manufactured at a scale of greater than 1 g, greater than 2 g, greater than 3 g, greater than 4 g, greater than 5 g, greater than 6 g, greater than 7 g, greater than 8 g, greater than 9 g, greater than 10 g, greater than 15 g, greater than 20 g, or higher.

[0944] In some embodiments, RNA quality control may be performed and/or monitored at any time during production process of polyribonucleotides and/or compositions comprising the same. For example, in some embodiments, RNA quality control parameters, including one or more of RNA identity (e.g., sequence, length, and/or RNA natures), RNA integrity, RNA concentration, residual DNA template, and residual dsRNA, may be assessed and/or monitored after each or certain steps of a polyribonucleotide manufacturing process, e.g., after in vitro transcription, and/or each purification step.

[0945] In some embodiments, the stability of polyribonucleotides (e.g., produced by in vitro transcription) and/or compositions comprising polyribonucleotides can be assessed under various test storage conditions, for example, at room temperatures vs. fridge or sub-zero temperatures over a period of time (e.g., at least 3 months, at least 6 months, at least 9 months, at least 12 months, or longer). In some embodiments, polyribonucleotides (e.g., ones described herein) and/or compositions thereof may be stored stable at a fridge temperature (e.g., about 4°C to about 10°C) for at least 1 month or longer including, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months or longer. In some embodiments, polyribonucleotides (e.g., ones described herein) and/or compositions thereof may be stored stable at a sub-zero temperature (e.g., -20°C or below) for at least 1 month or longer including, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months or longer. In some embodiments, polyribonucleotides (e.g., ones described herein) and/or compositions thereof may be stored stable at room temperature (e.g., at about 25 °C) for at least 1 month or longer.

[0946] In some embodiments, one or more assessments may be utilized during manufacture, or other preparation or use of polyribonucleotides (e.g., as a release test). [0947] In some embodiments, one or more quality control parameters may be assessed to determine whether polyribonucleotides described herein meet or exceed acceptance criteria (e.g., for subsequent formulation and/or release for distribution). In some embodiments, such quality control parameters may include, but are not limited to RNA integrity, RNA concentration, residual DNA template and/or residual dsRNA. Certain methods for assessing RNA quality are known in the art; for example, one of skill in the art will recognize that in some embodiments, one or more analytical tests can be used for RNA quality assessment. Examples of such certain analytical tests may include but are not limited to gel electrophoresis, UV absorption, and/or PCR assay.

[0948] In some embodiments, a batch of polyribonucleotides may be assessed for one or more features as described herein to determine next action step(s). For example, a batch of polyribonucleotides can be designated for one or more further steps of manufacturing and/or formulation and/or distribution if RNA quality assessment indicates that such a batch of polyribonucleotides meet or exceed the relevant acceptance criteria. Otherwise, an alternative action can be taken (e.g., discarding the batch) if such a batch of polyribonucleotides does not meet or exceed the acceptance criteria.

[0949] In some embodiments, a batch of polyribonucleotides that satisfy assessment results can be utilized for one or more further steps of manufacturing and/or formulation and/or distribution.

A. RNA Production

[0950] Those skilled in the art are aware of a variety of techniques that can be used to produce RNAs as described herein, including chemical or enzymatic (e.g., by polymerization) synthesis. In many embodiments, RNA is produced by transcription, e.g., by in vivo or in vitro transcription. Indeed, one advantage of RNA as an active agent for use in pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) or other therapeutic contexts is its facile production by in vitro transcription. Particularly given that relatively modest adjustments to manufacturing processes can often optimize production of related sequences, the present disclosure teaches that RNA modalities are particularly desirable for use as active agents in pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines). Moreover, the present disclosure provides a particular insight that RNA is particularly useful as an active agent in SARS-CoV-2 vaccines, as, among other advantages, it permits facile adaptation (e.g., sequence alteration) to emerging or locally-relevant strains and/or antigens (e.g., permitting customization of antigen sequences in light of, for example, circulating strains and/or HLA allele diversity within relevant populations (e.g., in a particular geography/region). Furthermore, production of RNA requires only a single development and manufacturing platform, irrespective of encoded pathogen antigens. Thus, RNA can allow for rapid, cost-efficient, high-volume manufacturing and flexible stockpiling (long term storage of low-volume libraries of frozen plasmid and unformulated RNA, which can be rapidly formulated and distributed). Particularly for a SARS-CoV-2 infection, where timing of administration (e.g., vaccine administration) relative to season and/or incidence of outbreak may materially impact effectiveness, such ability to store and promptly reconstitute may prove an important advantage relative to alternative strategies.

[0951] Typically, RNA is transcribed in vitro from a linearized (e.g., by restriction digestion) or amplified (e.g., PCR- amplified) DNA template. Those skilled in the art are aware of a variety of promoters useful for directing RNA synthesis by a transcription of a DNA template, for example by a DNA-dependent RNA polymerase such as, for example, T7, T3, SP6, or Syn5 RNA polymerase.

[0952] A typical in vitro transcription reaction will include a DNA template, rNTPs for the four bases (i.e., adenine, cytosine, guanine and uracil), optionally a cap analog, the relevant RNA polymerase, and appropriate buffers and/or salts. In some embodiments, one or more of a ribonuclease (RNase) inhibitor and/or a pyrophosphatase may be included.

[0953] In some embodiments, rNTPs utilized in an in vitro transcription reaction include one or more nucleotide analogs. In some embodiments, a nucleotide analog is 2-amino-6- chloropurineriboside-5’ -triphosphate, 2-Aminopurine-riboside-5’ -triphosphate; 2- aminoadenosine-5 ’ -triphosphate, 2’ -Amino-2’ -deoxy cytidine-triphosphate, 2-thiocytidine-5 ’ - triphosphate, 2-thiouridine-5’ -triphosphate, 2’-Fluorothymidine-5’-triphosphate, 2’-0-Methyl- inosine-5 ’ -triphosphate 4-thiouridine-5 ’ -triphosphate, 5-aminoallylcytidine-5 ’ -triphosphate, 5- aminoallyluridine-5 ’ -triphosphate, 5 -bromocytidine- 5 ’ -triphosphate, 5-bromouridine-5 ’ - triphosphate, 5-Bromo-2’-deoxycytidine-5’ -triphosphate, 5-Bromo-2’-deoxyuridine-5’- triphosphate, 5-iodocytidine-5’ -triphosphate, 5-lodo-2’ -deoxy cytidine-5’ -triphosphate, 5- iodouridine- 5 ’ -triphosphate, 5 -lodo-2’ -deoxyuridine- 5 ’ -triphosphate, 5 -methylcytidine- 5 ’ - triphosphate, 5-methyluridine-5’ -triphosphate, 5-Propynyl-2’-deoxycytidine-5’-triphosphate, 5- Propynyl-2’ -deoxyuridine- 5 ’ -triphosphate, 6-azacytidine-5 ’ -triphosphate, 6-azauridine-5 ’ - triphosphate, 6-chloropurineriboside-5’ -triphosphate, 7-deazaadenosine-5’-triphosphate, 7- deazaguanosine-5’ -triphosphate, 8-azaadenosine-5’ -triphosphate, 8-azidoadenosine-5’- triphosphate, benzimidazole-riboside-5’ -triphosphate, N1 -methyladenosine-5’ -triphosphate, N1 -methylguanosine-5 ’ -triphosphate, N6-methyladenosine-5 ’ -triphosphate, 06-methylguanosine- 5 ’-triphosphate, pseudouridine- 5’ -triphosphate, or puromycin-5’ -triphosphate, xanthosine-5’- triphosphate. Particular preference is given to nucleotides for base modifications selected from the group of base-modified nucleotides consisting of 5-methylcytidine-5’ -triphosphate, 7- deazaguanosine-5’ -triphosphate, 5-bromocytidine-5’ -triphosphate, and pseudouridine-5’- triphosphate, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4- thio-pseudouridine, 2-thio-pseudouridine, 5 -hydroxyuridine, 3-methyluridine, 5 -carboxymethyl- uridine, 1 -carboxymethyl-pseudouridine, 5-propynyl-uridine, 1 -propynyl-pseudouridine, 5- taurinomethyluridine, 1 -taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1 - taurinomethyl-4-thio-uridine, 5 -methyl -uridine, 1 -methyl-pseudouridine, 4-thio-l -methyl- pseudouridine, 2-thio-l -methyl-pseudouridine, 1 -methyl- 1 -deaza-pseudouridine, 2-thio-l - methyl- 1 -deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2- thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy- pseudouridine, and 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3- methyl-cytidine, N4-acetylcytidine, 5 -formylcytidine, N4-methylcytidine, 5- hydroxymethylcytidine, 1 -methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo- pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l - methyl-pseudoisocytidine, 4-thio-l -methyl- 1 -deaza-pseudoisocytidine, 1 -methyl- 1 -deaza- pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2- thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy- pseudoisocytidine, and 4-methoxy- 1 -methyl-pseudoisocytidine, 2-aminopurine, 2, 6- diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza- 2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1 - methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2- methoxy-adenine, inosine, 1 -methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza- 8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine,

7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1 - methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-

8-oxo-guanosine, 1 -methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2- dimethyl-6-thio-guanosine, 5’-0-(l -thiophosphate)-adenosine, 5’-0-(l -thiophosphate)-cytidine, 5’-0-(l -thiophosphate)-guanosine, 5’-0-(l -thiophosphate)-uridine, 5’-0-(l -thiophosphate)- pseudouridine, 6-aza-cytidine, 2-thio-cytidine, alpha- thio-cytidine, Pseudo-iso-cytidine, 5- aminoallyl -uridine, 5-iodo-uridine, N1 -methyl-pseudouridine, 5,6-dihydrouridine, alpha-thio- uridine, 4-thio-uridine, 6- aza-uridine, 5 -hydroxy -uridine, deoxy-thymidine, 5 -methyl -uridine, Pyrrolo-cytidine, inosine, alpha-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo- guanosine, 7-deaza-guanosine, N1 -methyl-adenosine, 2-amino-6-Chloro-purine, N6-methyl-2- amino-purine, Pseudo-iso-cytidine, 6-Chloro-purine, N6-methyl-adenosine, alpha-thio- adenosine, 8-azido-adenosine, 7-deaza-adenosine, pseudouridine, N1 -methylpseudouridine, N1 - ethylpseudouridine, 2-thiouridine, 4 ’-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-l - methyl- 1 -deaza-pseudouridine, 2-thio-l -methyl-pseudouridine, 2-thio-5 -aza-uridine, 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-l -methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2’-0-methyl uridine, or a combination thereof.

[0954] In some embodiments, uridine analog(s) are utilized. In some embodiments, no natural uridine is utilized. Thus, in some embodiments 100% of the uracil in a sequence has a chemical modification (relative to uridine); in many embodiments at the 5-position. In particular embodiments, pseudouridine is utilized.

[0955] In particular embodiments, utilized nucleotide analogs comprise pseudouridine, N1 -methylpseudouridine, 5-methylcytosine, -methoxyuridine, and combinations thereof.

[0956] In some embodiments, four rNTPs are utilized in equimolar concentrations in in vitro transcription reactions; in some embodiments, they are not equimolar. For example, in some embodiments, the initial concentration of one or more rNTPs at the start of an in vitro reaction is at a relatively lower concentration and, in some embodiments, may be supplemented by one or more “feedings” over time during the reaction. In some particular embodiments, rGTP is fed over time (so that the IVT reaction is a “G fed batch” process). Alternatively or additionally, in some particular embodiments, rUTP is fed over time (so that the IVT reaction is a “U fed batch” or “G/U fed batch” process).

[0957] In some embodiments, one or more of rNTP concentration, salt concentration, metal concentration, pH, temperature etc, is adjusted for production of a particular RNA construct in order to optimize, for example, one or more of RNA integrity, capping efficiency, contaminant (e.g., dsRNA) level, intact transcript level (e.g., relative to template DNA concentration in the reaction), etc.

[0958] In some embodiments, exemplary reagents used in RNA in vitro transcription include: a DNA template (linearized plasmid DNA or PCR product) with a promoter sequence that has a high binding affinity for its respective RNA polymerase such as bacteriophage- encoded RNA polymerases (T7, T3, SP6, or Syn5); ribonucleotide triphosphates (NTPs) for the four bases (adenine, cytosine, guanine and uracil); optionally, a cap analogue as defined herein (e.g. m7G(5')ppp(5')G (m7G)); optionally, further modified nucleotides as defined herein; a DNA-dependent RNA polymerase capable of binding to the promoter sequence within the DNA template (e.g. T7, T3, SP6, or Syn5 RNA polymerase); optionally, a ribonuclease (RNase) inhibitor to inactivate any potentially contaminating RNase; optionally, a pyrophosphatase to degrade pyrophosphate, which may inhibit RNA in vitro transcription; MgC12, which supplies Mg2+ ions as a co-factor for the polymerase; a buffer (TRIS or HEPES) to maintain a suitable pH value, which can also contain antioxidants (e.g. OTT), and/or polyamines such as spermidine at optimal concentrations, e.g. a buffer system comprising TRIS-Citrate as disclosed in W02017/109161.

[0959] In the context of RNA production, in some embodiments, it may be desired to provide GMP-grade RNA. In some embodiments, GMP-grade RNA may be produced using a manufacturing process approved by regulatory authorities. In some embodiments, RNA production is performed under current good manufacturing practice (GMP), implementing various quality control steps on DNA and/or RNA level, for example, in some embodiments according to quality steps described in W02016/180430. In some embodiments, RNA of the present disclosure is a GMP-grade RNA.

DNA Constructs

[0960] Among other things, the present disclosure provides DNA constructs, for example that may encode one or more RNA constructs described herein, or components thereof. In some embodiments, DNA constructs provided by and/or utilized in accordance with the present disclosure are comprised in a vector.

[0961] Non-limiting examples of a vector include plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retroviral, adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC). In some embodiments, a vector is an expression vector. In some embodiments, a vector is a cloning vector. In general, a vector is a nucleic acid construct that can receive or otherwise become linked to a nucleic acid element of interest (e.g., a construct that is or encodes a pay load, or that imparts a particular functionality, etc.)

[0962] Expression vectors, which may be plasmid or viral or other vectors, typically include an expressible sequence of interest (e.g., a coding sequence) that is functionally linked with one or more control elements (e.g., promoters, enhancers, transcription terminators, etc.). Typically, such control elements are selected for expression in a system of interest. In some embodiments, a system is ex vivo (e.g., an in vitro transcription system); in some embodiments, a system is in vivo (e.g., a bacterial, yeast, plant, insect, fish, vertebrate, mammalian cell or tissue, etc.).

[0963] Cloning vectors are generally used to modify, engineer, and/or duplicate (e.g., by replication in vivo, for example in a simple system such as bacteria or yeast, or in vitro, such as by amplification such as polymerase chain reaction or other amplification process). In some embodiments, a cloning vector may lack expression signals.

[0964] In many embodiments, a vector may include replication elements such as primer binding site(s) and/or origin(s) of replication. In many embodiments, a vector may include insertion or modification sites such as restriction endonuclease recognition sites and/or guide RNA binding sites, etc.

[0965] In some embodiments, a vector is a viral vector (e.g., an AAV vector). In some embodiments, a vector is a non-viral vector. In some embodiments, a vector is a plasmid.

[0966] Those skilled in the art are aware of a variety of technologies useful for the production of recombinant polynucleotides (e.g., DNA or RNA) as described herein. For example, restriction digestion, reverse transcription, amplification (e.g., by polymerase chain reaction), Gibson assembly, etc., are well established and useful tools and technologies. Alternatively or additionally, certain nucleic acids may be prepared or assembled by chemical and/or enzymatic synthesis. In some embodiments, a combination of known methods is utilized to prepare a recombinant polynucleotide.

[0967] In some embodiments, polynucleotide(s) of the present disclosure are included in a DNA construct (e.g., a vector) amenable to transcription and/or translation.

[0968] In some embodiments, an expression vector comprises a polynucleotide that encodes proteins and/or polypeptides of the present disclosure operatively linked to a sequence or sequences that control expression (e.g., promoters, start signals, stop signals, polyadenylation signals, activators, repressors, etc.). In some embodiments, a sequence or sequences that control expression are selected to achieve a desired level of expression. In some embodiments, more than one sequence that controls expression (e.g., promoters) are utilized. In some embodiments, more than one sequence that controls expression (e.g., promoters) are utilized to achieve a desired level of expression of a plurality of polynucleotides that encode a plurality proteins and/or polypeptides. In some embodiments, a plurality of recombinant proteins and/or polypeptides are expressed from the same vector (e.g., a bi-cistronic vector, a tri-cistronic vector, multi-cistronic). In some embodiments, a plurality of polypeptides are expressed, each of which is expressed from a separate vector.

[0969] In some embodiments, an expression vector comprising a polynucleotide of the present disclosure is used to produce an RNA and/or protein and/or polypeptide in a host cell. In some embodiments, a host cell may be in vitro (e.g., a cell line) - for example a cell or cell line (e.g., Human Embryonic Kidney (HEK cells), Chinese Hamster Ovary cells, etc.) suitable for producing polynucleotides of the present disclosure and proteins and/or polypeptides encoded by said polynucleotides.

[0970] In some embodiments, an expression vector is an RNA expression vector. In some embodiments, an RNA expression vector comprises a polynucleotide template used to produce a RNA in cell-free enzymatic mix. In some embodiments, an RNA expression vector comprising a polynucleotide template is enzymatically linearized prior to in vitro transcription. In some embodiments, a polynucleotide template is generated through PCR as a linear polynucleotide template. In some embodiments, a linearized polynucleotide is mixed with enzymes suitable for RNA synthesis, RNA capping and/or purification. In some embodiments, the resulting RNA is suitable for producing proteins encoded by the RNA.

[0971] A variety of methods are known in the art to introduce an expression vector into host cells. In some embodiments, a vector may be introduced into host cells using transfection. In some embodiments, transfection is completed, for example, using calcium phosphate transfection, lipofection, or polyethylenimine-mediated transfection. In some embodiments, a vector may be introduced into a host cell using transduction.

[0972] In some embodiments, transformed host cells are cultured following introduction of a vector into a host cell to allow for expression of said recombinant polynucleotides. In some embodiments, a transformed host cells are cultured for at least 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours 40 hours, 44 hours, 48 hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours or longer. Transformed host cells are cultured in growth conditions (e.g., temperature, carbon-dioxide levels, growth medium) in accordance with the requirements of a host cell selected. A skilled artisan would recognize culture conditions for host cells selected are well known in the art.

EMBODIMENTS

1. An RNA comprising a nucleotide sequence that encodes a polypeptide comprising or consisting of a variant polypeptide of a reference antigen of an infectious agent, or an immunogenic portion thereof, wherein a B cell memory immune response has been established to the reference antigen, and wherein the variant polypeptide has an amino acid sequence that differs from that of the reference antigen in that it has been engineered to reduce the variant’s activation of the B cell memory immune response to the reference antigen.

2. An RNA encoding an antigen of an infectious agent (or portion thereof) whose amino acid sequence is engineered so that at least one B cell memory epitope present in a reference antigen of the infectious agent is modified so that the memory activation potency of the reference antigen (or portion thereof) is reduced.

3. The RNA of embodiment 2, wherein the amino acid sequence encoded by the RNA is at least 80% identical to the corresponding portion of the reference antigen.

4. The RNA of embodiment 2, wherein the amino acid sequence encoded by the RNA comprises no more than 50% of the B cell memory epitopes present in the reference antigen.

5. An RNA encoding an antigen of an infectious agent (or a portion thereof), wherein the amino acid sequence of the antigen was engineered by a process comprising a step of removing memory B cell epitopes of a reference antigen or an immunogenic portion thereof.

6. The RNA of any of the preceding embodiments, where the antigen of the infectious agent or immunogenic portion thereof encoded by the RNA was engineered so as to lack regions of the reference antigen comprising a high number (or density) of conserved B cell epitopes.

7. The RNA of embodiment 6, wherein the conserved B cell epitopes are non-neutralizing epitopes.

8. The RNA of embodiment 6, wherein the antigen of the infectious agent or immunogenic portion thereof encoded by the RNA was engineered so as to lack conserved neutralizing B cell epitopes and conserved non-neutralizing B cell epitopes.

9. The RNA of any of the preceding embodiments, wherein the infectious agent has a high mutation rate.

10. The RNA of any of the preceding embodiments, wherein the infectious agent has a large number of variants or species. 11. The RNA of any of the preceding embodiments, where the infectious agent has a large number of variants or species, many of which are immune escaping.

12. The RNA of any of the preceding embodiments, wherein the infectious agent is a virus, bacteria, or Plasmodium.

13. The RNA of embodiment 12, wherein the infectious agent is a virus.

14. The RNA of embodiment 13, wherein the virus is a respiratory virus.

15. The RNA of embodiment 13, wherein the virus is an influenza virus, RSV, a norovirus, or HIV.

16. The RNA of embodiment 13, wherein the infectious agent is a coronavirus.

17. The RNA of embodiment 16, wherein the coronavirus is a betacoronavirus.

18. The RNA of embodiment 17, wherein the coronavirus is MERS, SARS, or SARS-CoV-2.

19. The RNA of embodiment 12, wherein the plasmodium is P. falciparum, P. vivax, P. ovale, or P. malariae.

20. The RNA of any of the preceding embodiments, wherein the variant polypeptide lacks regions that are not mutated frequently in immune-escaping variants of the infectious agent.

21. The RNA of embodiment 14, wherein the variant polypeptide comprises an immunogenic portion of a coronavirus S protein that lacks sequences corresponding to regions outside of the SI domain or the receptor binding domain (RBD).

22. The RNA of embodiment 21, wherein the immunogenic portion of the coronavirus S protein does not comprise an S2 domain.

23. The RNA of embodiment 21 or 22, wherein the coronavirus S protein does not comprise an N-terminal domain (NTD).

24. The RNA of any of the preceding embodiments, wherein the antigen of the infectious agent or immunogenic portion thereof encoded by the RNA comprises a hypervariable domain.

25. The RNA of embodiment 24, wherein the hypervariable domain is a region of the antigen that has a high mutation rate.

26. The RNA of embodiment 25, wherein the hypervariable domain has a high density of neutralization epitopes.

27. The RNA of any one of embodiments 24-26, wherein the hypervariable domain is a region that is frequently mutated in variants of the infectious agent that have a high immune escape potential.

28. The RNA of any one of embodiments 24-27, wherein the hypervariable domain is a receptor binding domain (RBD).

29. The RNA of any one of embodiments 24-28, wherein the hypervariable domain comprises or consists of the RBD or SI domain of a coronavirus S protein.

30. The RNA of any one of the previous embodiments, where the reference antigen is:

(i) a surface protein or surface glycoprotein of an infectious agent strain or variant that was previously and/or is currently prevalent; and/or

(ii) a surface protein or surface glycoprotein of an infectious agent that has been previously delivered in a vaccine (e.g., a commercially available vaccine, an RNA vaccine, or a protein- based vaccine).

31. The RNA of embodiment 30, wherein the surface protein or surface glycoprotein of (i) or (ii) is a coronavirus S protein.

32. The RNA of any one of the preceding embodiments, wherein the variant polypeptide encoded by the RNA has been engineered to eliminate one or more memory B cell epitopes of the reference antigen.

33. The RNA of embodiment 32, wherein the one or more memory B cell epitopes have previously been determined to be bound by antibodies and/or B cells produced by a subject exposed to the reference antigen (e.g., via a vaccine that delivers the reference antigen and/or infection with a virus that comprises the reference antigen).

34. The RNA of embodiment 32 or 33, wherein the one or more memory B cell epitopes comprise or consist of non-neutralizing epitopes.

35. The RNA of any one of embodiments 32-34, wherein the one or more memory B cell epitopes comprise or consist of non-neutralizing epitopes and neutralizing epitopes. 36. The RNA of any one of the preceding embodiments, wherein the variant polypeptide comprises few intact memory B cell epitopes of the reference antigen.

37. The RNA of any one of the preceding embodiments, wherein the variant polypeptide comprises one or more mutations associated with an infectious agent variant that has a high immune escape potential.

38. The RNA of embodiment 37, wherein the infectious agent variant has been determined to have a high immune escape potential using an in vitro assay (e.g., a viral neutralization assay), via in silico analysis (e.g., sequence analysis and/or molecular dynamic simulations), and/or based on infection rates in subjects in a relevant population.

39. The RNA of embodiment 38, wherein the variant polypeptide comprises few conserved memory B-cell epitopes relative to:

(i) a reference antigen of a strain or variant that was previously or is currently prevalent in a relevant population, and/or

(ii) one or more reference antigens that have previously been delivered in a vaccine (e.g., a commercially available vaccine and/or a vaccine previously administered to a subject).

40. The RNA of any one of embodiments 36-39, wherein the variant polypeptide comprises 10 or fewer (e.g., 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, one or less, or no) conserved memory B cell epitopes.

41. The RNA of any one of the preceding embodiments, wherein the variant polypeptide comprises a secretion signal.

42. The RNA of embodiment 41, wherein the secretion signal is a homologous secretion signal.

43. The RNA of embodiment 41, wherein the secretion signal is a heterologous secretion signal.

44. The RNA of any one of embodiments 41-43, wherein the secretion signal is present in the N- terminal portion of the polypeptide (e.g., at the N-terminus of the polypeptide).

45. The RNA of embodiment 41 or 44, wherein the secretion signal is a SARS-CoV-2 S protein secretion signal, a gD2 secretion signal, a gDl secretion signal, a gBl secretion signal, a gI2 secretion signal, a gE2 secretion signal, an Eboz secretion signal, or an HLA-DR secretion signal. 46. The RNA of embodiment 45, wherein the SARS-CoV-2 S protein secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 15.

47. The RNA of embodiment 45, wherein the SARS-CoV-2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 9 or 16.

48. The RNA of embodiment 45, wherein the gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 8.

49. The RNA of embodiment 45, wherein the gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 13.

50. The RNA of embodiment 45, wherein the gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 33.

51. The RNA of embodiment 45, wherein the gDl secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 12.

52. The RNA of embodiment 45, wherein the gBl secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 37.

53. The RNA of embodiment 45, wherein the gC2 polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 32.

54. The RNA of embodiment 45, wherein the gI2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NOTO or 11.

55. The RNA of embodiment 45, wherein the gE2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 32.

56. The RNA of embodiment 45, wherein the EboZ secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 39.

57. The RNA of embodiment 45, wherein the HLA-DR secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 40.

58. The RNA of any one of the preceding embodiments, wherein the variant polypeptide encoded by the RNA comprises a multimerization domain.

59. The RNA of embodiment 58, wherein the polypeptide comprises a multimerization domain that is C-terminal to the variant polypeptide. 60. The RNA of embodiment 58 or 59, wherein the multimerization domain is a fibritin domain.

61. The RNA of embodiment 60, wherein the fibritin domain comprises a sequence that is at least 80% identical to SEQ ID NO: 95 or 96.

62. The RNA of any one of the preceding embodiments, wherein the variant polypeptide encoded by the RNA comprises a transmembrane (TM) domain.

63. The RNA of embodiment 62, wherein the TM domain is a homologous TM domain.

64. The RNA of embodiment 62, wherein the TM domain is a heterologous TM domain.

65. The RNA of any one of embodiments 62-64, wherein the TM domain is present in the C- terminal portion of the polypeptide (e.g., at the C-terminus).

66. The RNA of embodiment 65, wherein the variant polypeptide encoded by the RNA comprises a multimerization domain and a TM domain in the C-terminal portion of the polypeptide, wherein the TM domain is C-terminal to the multimerization domain (e.g., the TM domain is at the C-terminus of the variant polypeptide and the multimerization domain is adjacent to the TM domain (e.g., directly adjacent to the TM domain and/or connected to the TM domain via a GS linker)).

67. The RNA of any one of embodiments 62-66, wherein the TM domain is a SARS-CoV-2 S protein TM domain, or an influenza TM domain.

68. The RNA of embodiment 67, wherein the SARS-CoV-2 TM domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 89.

69. The RNA of embodiment 67, wherein the SARS-CoV-2 TM domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 90.

70. The RNA of any one of the preceding embodiments, wherein the nucleotide sequence that encodes the variant polypeptide or the polypeptide has been codon-optimized for expression in mammalian subjects.

71. The RNA of any one of the preceding embodiments, wherein the nucleotide sequence that encodes the variant polypeptide or the immunogenic portion thereof has been codon-optimized for expression in human subjects.

72. The RNA of any one of the preceding embodiments, wherein the nucleotide sequence encoding the variant polypeptide or the portion thereof has an enriched G/C content relative to wild-type sequence.

73. The RNA of embodiment 72, wherein G/C content has been increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%.

74. The RNA of any one of the preceding embodiments, comprising a heterologous 3’ UTR or 5 ’UTR.

75. The RNA of embodiment 74, wherein the heterologous 5' UTR comprises or consists of a modified human alpha-globin 5 '-UTR.

76. The RNA of embodiment 74 or 75, wherein the heterologous 3’ UTR comprises or consists of a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA.

77. The RNA of any one of the preceding embodiments, comprising a poly(A) sequence.

78. The RNA of embodiment 77, wherein the poly(A) sequence has a length of about 100-150 nucleotides.

79. The RNA of embodiment 77 or 78, wherein the poly(A) sequence is a disrupted poly(A) sequence.

80. The RNA of any one of the preceding embodiments, further comprising a 5' cap.

81. The RNA of any one of the preceding embodiments, wherein the RNA is unmodified RNA.

82. The RNA of any one of embodiments 1-80, comprising one or more modified nucleotides.

83. The RNA of embodiment 82, wherein the modified nucleotide is pseudouridine (e.g., Nl- methyl-pseudouridine).

84. The RNA of embodiment 82 or 83, wherein the RNA comprises a modified nucleotide in place of each uridine.

85. The RNA of any one of the preceding embodiments, wherein the RNA is an self-amplifying RNA or trans-amplifying RNA. 86. A composition comprising the RNA of any one of the preceding embodiments, wherein the RNA is fully or partially encapsulated within lipid nanoparticles (LNP), polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes.

87. The composition of embodiment 86, wherein the RNA is fully or partially encapsulated within LNP.

88. The composition of embodiment 87, wherein the LNP comprise a cationically ionizable lipid, a neutral lipid, a sterol and a lipid conjugate.

89. The composition of embodiment 88, wherein the LNP comprise from about 40 to about 50 mol percent of the cationically ionizable lipid; from about 5 to about 15 mol percent of the neutral lipid; from about 35 to about 45 mol percent of the sterol; and from about 1 to about 10 mol percent of the PEG-lipid.

90. A method of inducing an immune response, comprising administering the RNA of any one of embodiments 1-85, or the composition of any one of embodiments 86-89 to a subject.

91. A method of inducing an immune response in a subject who has previously been exposed to a reference antigen of an infectious agent, the method comprising: delivering a variant polypeptide of the reference antigen or an immunogenic portion thereof to the subject, wherein a B cell memory immune response has been established to the reference antigen, and wherein the variant polypeptide has an amino acid sequence that differs from that of the reference antigen in that it has been engineered to reduce the variant polypeptide’s activation of the B cell memory immune response.

92. The method of embodiment 90 or 91, wherein the infectious agent is an influenza virus, RSV, norovirus, HIV, coronavirus, or a plasmodium.

93. The method of any one of embodiments 90-92, wherein the subject has previously been administered one or more doses of one or more vaccines that deliver the reference antigen.

94. The method of any one of embodiments 90-93, wherein the immune response comprises a naive B cell immune response. 95. The method of any one of embodiments 90-94, wherein the immune response comprises a reduced memory B cell immune response or the immune response does not comprise a memory B cell immune response.

96. A method of manufacturing an immunogenic composition comprising:

(a) providing a reference antigen of an infectious agent, wherein the reference antigen is from a strain or variant (e.g., a strain or variant that has previously been prevalent and/or that has previously been delivered as a vaccine) of the infectious agent,

(b) determining a variant polypeptide of the reference antigen that comprises fewer memory B cell epitopes relative to the reference antigen; and

(c) producing an immunogenic composition that delivers the variant polypeptide.

97. The method of embodiment 96, wherein the variant polypeptide comprises a sequence that corresponds to an immunogenic portion of the reference antigen.

98. The method of embodiment 96 or 97, wherein the variant polypeptide comprises one or mutations at one or more B cell epitopes of the reference antigen.

99. A method of assessing, predicting, or characterizing the ability of an immunogenic composition that delivers an antigen of an infectious agent to induce activation of memory B cells in a subject or a population of subjects, the method comprising determining the number of memory B cell epitopes present in the antigen relative to a reference antigen.

100. The method of embodiment 99, wherein the reference antigen is from a strain or variant of the infectious agent that the subject was exposed to and/or that a large portion of the population was exposed to.

101. A method of producing a personalized vaccine for a subject against an infectious agent, the method comprising steps of:

(a) determining a reference antigen of the infectious agent that a subject has previously been exposed to;

(c) producing an immunogenic composition that delivers the variant polypeptide.

102. The method of any one of embodiments 99-101, wherein the reference antigen is from a strain or variant of the infectious agent that the subject was first exposed to and/or that was first prevalent in the population of subjects.

103. The method of embodiment 102, where the reference antigen is from an infectious agent that a subject has previously been vaccinated against or is delivered by one or more vaccines that a significant proportion of the population (e.g., at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least abut 45%, at least about 50%, at least about 55%, or at least about 60%) has previously been administered.

104. The method of embodiment 103, wherein the vaccine previously administered to the subject or a significant proportion of the population was a first generation vaccine.

105. The method of embodiment 102, where the reference antigen is from an infectious agent that was previously prevalent or is currently prevalent in a relevant jurisdiction.

106. The method of embodiment 103, where the reference antigen is from an infectious agent variant that first became prevalent in a relevant jurisdiction.107. An RNA comprising a nucleotide sequence that encodes a SARS-CoV-2 Spike (S) protein variant, or an immunogenic portion thereof, wherein a memory B cell immune response has been established to a reference SARS- CoV-2 S protein, and wherein the SARS-CoV-2 S protein variant or immunogenic portion thereof has an amino acid sequence that differs from that of the reference SARS-CoV-2 S protein variant in that it has been engineered to reduce activation of the memory B cell immune response relative to the SARS-CoV-2 S protein.

108. An RNA comprising a nucleotide sequence that encodes a SARS-CoV-2 Spike (S) protein variant (or an immunogenic portion thereof) whose amino acid sequence is engineered so that at least one memory B cell epitope present in a reference SARS-CoV-2 S protein has been modified so that memory B cell activation potency of the SARS-CoV-2 S protein variant (or immunogenic portion thereof) has been reduced relative to the reference SARS-CoV-2 S protein.

109. The RNA of embodiment 108, wherein the SARS-CoV-2 S protein variant (or immunogenic portion thereof) has an amino acid sequence that is at least 80% identical to that of the reference SARS-CoV-2 S protein (or the amino acid sequence of the corresponding portion of the reference SARS-CoV-2 S protein).

110. The RNA of embodiment 108 or 109, wherein the SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises no more than 50% of the memory B cell epitopes present in the reference SARS-CoV-2 S protein.

111. An RNA comprising a nucleotide sequence that encodes a SARS-CoV-2 Spike (S) protein variant (or an immunogenic portion thereof), wherein the amino acid sequence of the S protein variant or immunogenic portion thereof was engineered by a process comprising a step of removing memory B cell epitopes present in a reference SARS-CoV-2 S protein.

112. The RNA of any one of the preceding embodiments, wherein the variant SARS-CoV-2 S protein (or immunogenic portion thereof) comprises few memory B cell epitopes of the reference SARS-CoV-2 S protein.

113. The RNA of embodiment 112, wherein the one or more memory B cell epitopes in the reference SARS-CoV-2 S protein have been identified by antibody-binding studies (e.g., studies characterizing antibodies produced by subjects administered a vaccine that delivers the reference SARS-CoV-2 S protein and/or infected with a virus comprises the reference SARS-CoV-2 S protein).

114. The RNA of embodiment 112 or 113, wherein the one or more memory B cell epitopes comprise or consist of non-neutralizing epitopes.

115. The RNA of any one of embodiments 112-114, wherein the one or more memory B cell epitopes comprise or consist of non-neutralizing epitopes and neutralizing epitopes.

116. The RNA of any one of embodiments 112-115, wherein the variant SARS-CoV-2 S protein comprises 10 or fewer (e.g., 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, one or less, or no) conserved memory B cell epitopes.

117. The RNA of any of the preceding embodiments, where the SARS-CoV-2 S protein variant or immunogenic portion thereof was engineered so as to lack regions of the reference SARS- CoV-2 S protein that comprise a high number or density of conserved memory B cell epitopes. 118. The RNA of embodiment 117, wherein the conserved memory B cell epitopes are non- neutralizing epitopes.

119. The RNA of any of the preceding embodiments, wherein the variant SARS-CoV-2 S protein or immunogenic portion thereof was engineered to lack regions that are not mutated frequently in immune-escaping SARS-CoV-2 variants.

120. The RNA of any one of the previous embodiments, wherein the immunogenic portion of the SARS-CoV-2 S protein variant does not comprise an S2 domain.

121. The RNA of embodiment 120, wherein the immunogenic portion of the SARS-CoV-2 S protein variant comprises or consists of the SI domain or the receptor binding domain (RBD).

122. The RNA of embodiment 121, wherein the immunogenic portion of the SARS-CoV-2 S protein variant does not comprise the N-terminal domain (NTD).

123. The RNA of any one of embodiments 119-122, wherein the immunogenic portion of the SARS-CoV-2 S protein variant comprises or consists of the RBD.

124. The RNA of any one of the preceding embodiments, wherein the reference SARS-CoV-2 S protein is from a strain or variant that was previously prevalent or is currently prevalent in a relevant population of subjects.

125. The RNA of any one of the preceding embodiments, wherein the reference SARS-CoV-2 S protein was previously delivered by a vaccine.

126. The RNA of embodiment 125, wherein the vaccine is a commercially approved vaccine, a protein-based vaccine, an RNA vaccine, or any combination thereof.

127. The RNA of any one of the previous embodiments, wherein the reference SARS-CoV-2 S protein is a Wuhan S protein.

128. The RNA of any one of the previous embodiments, wherein the reference SARS-CoV-2 S protein is an Omicron BA.4/5 S protein.

129. The RNA of any one of the preceding embodiments, wherein the SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises one or more mutations associated with a SARS-CoV-2 variant that has a high immune escape potential (e.g., a variant of concern).

130. The RNA of embodiment 129, wherein the SARS-CoV-2 variant has been determined to have a high immune escape potential using an in vitro assay (e.g., a viral neutralization assay), in silico analysis (e.g., sequence analysis and/or molecular dynamic simulations), and/or based on infection rates and/or growth rates. 131. The RNA of embodiment 129, wherein the SARS-CoV-2 variant is an Omicron variant.

132. The RNA of embodiment 131, wherein the Omicron variant is an XBB variant (e.g., an XBB.l or XBB.1.5 variant) or a BQ.l variant.

133. The RNA of embodiment 132, wherein the one or more mutations associated with an XBB.1.5 variant are T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K, where the positions of the one or more mutations are indicated relative to SEQ ID NO: 1.

134. The RNA of embodiment 132, wherein nucleotide sequence encodes a SARS-CoV-2 S protein comprising an RBD, and wherein the one or more mutations associated with an XBB.1.5 variant are G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, and Y505H, where the positions of the one or more mutations are indicated relative to SEQ ID NO: 1.

135. The RNA of embodiment 132, wherein nucleotide sequence encodes a SARS-CoV-2 S protein comprising an SI domain, and wherein the one or more mutations associated with an XBB.1.5 variant are T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H,

136. The RNA of embodiment 132, wherein nucleotide sequence encodes a SARS-CoV-2 S protein comprising an SI domain, and wherein the one or more mutations associated with an XBB.1.5 variant are T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K,

137. The RNA of embodiment 132, wherein the nucleotide sequence encodes a SARS-CoV-2 S protein comprising an SI domain, and wherein the one or more mutations associated with an XBB.1.5 variant are T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y,

138. The RNA of any one of embodiments 132-134, wherein the RNA comprises a nucleotide sequence that encodes an immunogenic portion of the SARS-CoV-2 S protein variant comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 3.

139. The RNA of embodiment 132 or 135, wherein the RNA comprises a nucleotide sequence that encodes an immunogenic portion of the SARS-CoV-2 S protein variant comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 5.

140. The RNA of any one of the preceding embodiments, wherein the variant SARS-CoV-2 S protein comprises a secretion signal.

141. The RNA of embodiment 140, wherein the secretion signal is a homologous secretion signal.

142. The RNA of embodiment 140, wherein the secretion signal is a heterologous secretion signal.

143. The RNA of any one of embodiments 140-142, wherein the secretion signal is present in the N-terminal portion of the SARS-CoV-2 S protein or immunogenic portion thereof (e.g., at the N- terminus).

144. The RNA of embodiment 141 or 143, wherein the secretion signal is a SARS-CoV-2 S protein secretion signal, a gD2 secretion signal, a gDl secretion signal, a gBl secretion signal, a gI2 secretion signal, a gE2 secretion signal, an Eboz secretion signal, or an HLA-DR secretion signal.

145. The RNA of embodiment 144, wherein the SARS-CoV-2 S protein secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 15.

146. The RNA of embodiment 144, wherein the SARS-CoV-2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 9.

147. The RNA of embodiment 144, wherein the SARS-CoV-2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 16.

148. The RNA of embodiment 144, wherein the gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 8.

149. The RNA of embodiment 144, wherein the gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 13. 150. The RNA of embodiment 144, wherein the gDl secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 12.

151. The RNA of embodiment 144, wherein the gBl secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 37.

152. The RNA of embodiment 144, wherein the gC2 polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 35.

153. The RNA of embodiment 144, wherein the gI2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 11.

154. The RNA of embodiment 144, wherein the gE2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 38.

155. The RNA of embodiment 144, wherein the EboZ secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 39.

156. The RNA of embodiment 144, wherein the HLA-DR secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 40.

157. The RNA of any one of the preceding embodiments, wherein the SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises a multimerization domain.

158. The RNA of embodiment 157, wherein the multimerization domain in the C-terminal region (e.g., at the C-terminus).

159. The RNA of embodiment 157 or 158, wherein the multimerization domain is a fibritin domain.

160. The RNA of embodiment 159, wherein the fibritin domain comprises a sequence that is at least 80% identical to SEQ ID NO: 95.

161. The RNA of embodiment 159, wherein the fibritin domain comprises a sequence that is at least 80% identical to SEQ ID NO: 96.

162. The RNA of any one of the preceding embodiments, wherein the SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises a transmembrane (TM) domain.

163. The RNA of embodiment 162, wherein the TM domain is a homologous TM domain.

164. The RNA of embodiment 162, wherein the TM domain is a heterologous TM domain.

165. The RNA of any one of embodiments 162-164, wherein the TM domain is present in the C- terminal portion of the SARS-CoV-2 S protein variant or immunogenic portion thereof (e.g., at the C-terminus). 166. The RNA of embodiment 165, wherein the SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises a multimerization domain and a TM domain in the C- terminal portion of the SARS-CoV-2 S protein variant or immunogenic portion thereof, wherein the TM domain is C-terminal to the multimerization domain (e.g., the TM domain is at the C- terminus of the variant SARS-CoV-2 S protein variant or immunogenic portion thereof and the multimerization domain is adjacent to the TM domain (e.g., directly adjacent to the TM domain and/or connected to the TM domain via a GS linker)).

167. The RNA of any one of embodiments 162-166, wherein the TM domain is a SARS-CoV-2 S protein TM domain, or an influenza TM domain.

168. The RNA of embodiment 167, wherein the SARS-CoV-2 TM domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 89.

169. The RNA of embodiment 167, wherein the SARS-CoV-2 TM domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 90.

170. The RNA of any one of embodiments 107-169, wherein the RNA comprises a nucleotide sequence that encodes an immunogenic portion of the SARS-CoV-2 S protein variant comprising a sequence that is at least 80% identical to SEQ ID NO: 120.

171. The RNA of any one of embodiments 107-170, wherein the RNA comprises a nucleotide sequence that encodes an immunogenic portion of the SARS-CoV-2 S protein variant comprising a sequence that is at least 80% identical to SEQ ID NO: 130.

172. The RNA of any one of embodiments 107-170, wherein the RNA comprises a nucleotide sequence that encodes an immunogenic portion of the SARS-CoV-2 S protein variant comprising a sequence that is at least 80% identical to SEQ ID NO: 135.

173. The RNA of any one of embodiments 107-170, wherein the RNA comprises a nucleotide sequence that encodes an immunogenic portion of the SARS-CoV-2 S protein variant comprising a sequence that is at least 80% identical to SEQ ID NO: 145.

174. The RNA of any one of embodiments 107-170, wherein the RNA comprises a nucleotide sequence that encodes an immunogenic portion of the SARS-CoV-2 S protein variant comprising a sequence that is at least 80% identical to SEQ ID NO: 150.

175. The RNA of any one of the preceding embodiments, wherein the nucleotide sequence that encodes the SARS-CoV-2 S protein variant (or immunogenic portion thereof) has been codon- optimized for expression in mammalian subjects. 176. The RNA of any one of the preceding embodiments, wherein the nucleotide sequence that encodes the SARS-CoV-2 S protein variant (or immunogenic portion thereof) has been codon- optimized for expression in human subjects.

177. The RNA of any one of the preceding embodiments, wherein the nucleotide sequence encoding the SARS-CoV-2 S protein variant (or immunogenic portion thereof) has an enriched G/C content relative to wild-type sequence.

178. The RNA of embodiment 177, wherein G/C content has been increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%.

179. The RNA of any one of the preceding embodiments, comprising a heterologous 3’ UTR or 5 ’UTR.

180. The RNA of embodiment 179, wherein the heterologous 5' UTR comprises or consists of a modified human alpha-globin 5 '-UTR.

181. The RNA of embodiment 179 or 180, wherein the heterologous 3’ UTR comprises or consists of a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA.

182. The RNA of any one of the preceding embodiments, comprising a poly(A) sequence.

183. The RNA of embodiment 182, wherein the poly(A) sequence has a length of about 100-150 nucleotides.

184. The RNA of embodiment 182 or 183, wherein the poly(A) sequence is a disrupted poly(A) sequence.

185. The RNA of any one of the preceding embodiments, comprising a 5' cap.

186. The RNA of any one of embodiment 107-185, comprising a sequence that is at least 80% identical to SEQ ID NO: 122 or 124.

187. The RNA of any one of embodiment 107-185, comprising a sequence that is at least 80% identical to SEQ ID NO: 131 or 133.

188. The RNA of any one of embodiment 107-185, comprising a sequence that is at least 80% identical to SEQ ID NO: 136 or 138.

189. The RNA of any one of embodiments 107-185, comprising a sequence that is at least 80% identical to SEQ ID NO: 146 or 148. 190. The RNA of any one of embodiments 107-185, comprising a sequence that is at least 80% identical to SEQ ID NO: 151 or 153.

191. The RNA of any one of the preceding embodiments, wherein the RNA is unmodified RNA.

192. The RNA of any one of embodiments 107-190, comprising one or more modified nucleotides.

193. The RNA of embodiment 1926, wherein the modified nucleotide is pseudouridine (e.g., N 1 -methyl-pseudouridine).

194. The RNA of embodiment 192 or 193, wherein the RNA comprises a modified nucleotide in place of each uridine.

195. The RNA of any one of the preceding embodiments, wherein the RNA is an self-amplifying RNA or trans-amplifying RNA.

196. A composition comprising the RNA of any one of the preceding embodiments, wherein the RNA is fully or partially encapsulated within lipid nanoparticles (LNP), polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes.

197. The composition of embodiment 196, wherein the RNA is fully or partially encapsulated within LNP.

198. The composition of embodiment 196, wherein the LNP comprise a cationically ionizable lipid, a neutral lipid, a sterol and a lipid conjugate.

199. The composition of embodiment 198, wherein the LNP comprise from about 40 to about 50 mol percent of the cationically ionizable lipid; from about 5 to about 15 mol percent of the neutral lipid; from about 35 to about 45 mol percent of the sterol; and from about 1 to about 10 mol percent of the PEG-lipid.

200. A method of inducing an immune response, comprising administering the RNA of any one of embodiments 107-195, or the composition of any one of embodiment 196-199 to a subject.

201. A method of inducing an immune response in a subject who has previously been exposed to a reference SARS-CoV-2 S protein, the method comprising: delivering a SARS-CoV-2 S protein variant or an immunogenic portion thereof to the subject, wherein a B cell memory immune response has been established to the reference SARS- CoV-2 S protein, and wherein the SARS-CoV-2 S protein variant or immunogenic portion thereof has an amino acid sequence that differs from that of the reference SARS-CoV-2 S protein in that it has been engineered to reduce activation of the B cell memory immune response.

202. The method of embodiment 201, wherein the subject has previously been administered one or more doses of one or more vaccines that deliver the reference SARS-CoV-2 S protein.

203. The method of embodiment 201 or 202, wherein the reference SARS-CoV-2 S protein is a Wuhan SARS-CoV-2 S protein.

204. The method of any one of embodiments 200-203, wherein the immune response comprises a naive B cell immune response.

205. The method of any one of embodiments 200-204, wherein the immune response comprises a reduced memory B cell immune response or the immune response does not comprise a memory B cell immune response.

206. A method of manufacturing an immunogenic composition comprising:

(a) providing a reference SARS-CoV-2 S protein, wherein the reference SARS-CoV-2 S protein is from a strain or variant (e.g., a strain or variant that has previously been prevalent and/or that has previously been delivered as a vaccine) of SARS-CoV-2,

(b) determining a variant SARS-CoV-2 S protein or an immunogenic portion thereof of the reference SARS-CoV-2 S protein that comprises a reduced number of memory B cell epitopes relative to the reference SARS-CoV-2 S protein; and

(c) producing an immunogenic composition that delivers the SARS-CoV-2 S protein variant or an immunogenic portion thereof.

207. The method of embodiment 206, wherein the SARS-CoV-2 S protein variant or immunogenic portion thereof comprises a sequence that corresponds to an immunogenic portion of the reference SARS-CoV-2 S protein.

208. The method of embodiment 206 or 207, wherein the variant SARS-CoV-2 S protein variant comprises one or mutations at one or more B cell epitopes of the reference antigen.

209. A method of assessing, predicting, or characterizing the ability of an immunogenic composition that delivers a SARS-CoV-2 S protein (or immunogenic portion thereof) to induce activation of memory B cells in a subject or a population of subjects, the method comprising determining the number of memory B cell epitopes present in the SARS-CoV-2 S protein (or immunogenic portion thereof) relative to a reference antigen.

210. The method of embodiment 209, wherein the reference SARS-CoV-2 S protein is from a strain or variant that the subject was exposed to and/or that a large portion of the population was exposed to.

211. A method of producing a personalized SARS-CoV-2 vaccine for a subject, the method comprising steps of:

(a) determining a reference SARS-CoV-2 S protein that a subject has previously been exposed to;

(b) determining a variant SARS-CoV-2 S protein that comprises fewer memory B cell epitopes relative to the reference SARS-CoV-2 S protein; and

(c) producing an immunogenic composition that delivers the variant SARS-CoV-2 S protein.

212. The method of any one of embodiments 209-211, wherein the reference SARS-CoV-2 S protein is from a strain or variant that the subject was first exposed to and/or that was first prevalent in the population of subjects.

213. The method of any one of embodiments 209-212, wherein the reference SARS-CoV-2 S protein is a Wuhan SARS-CoV-2 S protein or an Omicron BA.4/5 SARS-CoV-2 S protein.

214. The method of embodiment 211, where the reference SARS-CoV-2 S protein is from a strain or variant that a subject has previously been vaccinated against or is delivered by one or more vaccines that a significant proportion of the population (e.g., at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least abut 45%, at least about 50%, at least about 55%, or at least about 60%) has previously been administered.

215. The method of embodiment 214, wherein the vaccine previously administered to the subject or a significant proportion of the population was a first generation vaccine.

216. The method of embodiment 214, where the reference SARS-CoV-2 S protein is from a SARS-CoV-2 strain or variant that was previously prevalent or is currently prevalent in a relevant jurisdiction.

217. The method of embodiment 214, where the reference SARS-CoV-2 S protein is from a variant that first became prevalent in a relevant jurisdiction. 218. A combination comprising:

(i) a composition that comprises or delivers polypeptide comprising or consisting of a variant polypeptide of a reference antigen of an infectious agent, or an immunogenic portion thereof, wherein the variant polypeptide comprises neutralizing epitopes that are absent in the reference antigen; and

(ii) an agent that induces a priming-favorable cytokine milieu in lymphoid tissues, wherein the agent is present at a dose that is effective to increase activation of naive B cell immune response to at least one of the neutralizing epitopes, and wherein the agent is or comprises (i) an unmodified RNA molecule or (ii) a self-amplifying RNA molecule or a trans-amplifying RNA molecule, and wherein the RNA molecule is formulated in lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes.

219. The combination of embodiment 218, wherein the agent encodes a polypeptide comprising an antigen of the infectious agent.

220. The composition of embodiment 219, wherein the antigen is a B-cell antigen.

221. The composition of embodiment 219, wherein the antigen is a T-cell antigen.

222. The composition of embodiment 218, wherein the amount ratio of the polypeptide to the agent is within a range of about 1 : 1 to about 20: 1.

223. An RNA molecule comprising a nucleotide sequence that includes modified ribonucleotides and corresponding unmodified ribonucleotides, wherein the ratio of the modified ribonucleotides to the corresponding unmodified ribonucleotides is within a range of about 1: 10 to about 1: 1; and wherein the nucleotide sequence encodes an antigen of an infectious agent.

224. The RNA molecule of embodiment 223, wherein the nucleotide sequence comprises a first domain and a second domain, wherein at least one of the first domain and the second domain comprises modified ribonucleotides and the other domain comprises no modified ribonucleotides.

225. The RNA molecule of any one of embodiments 223-224, wherein the modified ribonucleotides are 1 -methylpseudouridine and the corresponding unmodified ribonucleotides are uridine.

226. A method of inducing a priming immune response by: administering to a subject one or both of:

(i) a composition that comprises or delivers a polypeptide antigen; and (ii) an agent that induces a priming-favorable cytokine milieu in lymphoid tissues, wherein the agent is present at a dose that is effective to increase activation of naive B cell immune response to at least one of the neutralizing epitopes.

227. A method of inducing or supporting a priming immune response to an antigen in a subject by exposing the subject to the antigen under immune priming conditions.

228. The method of embodiment 226 or 227, wherein the subject has previously been exposed to a variant of the antigen.

229. The method of embodiment 227 or 228, wherein the step of exposing comprises administering a composition that comprises or delivers the antigen.

230. The method of embodiment 229, wherein the antigen is a polypeptide antigen.

231. The method of any one of embodiments 227-230, wherein the step of exposing comprises administering a “priming adjuvant” to a subject who is or will soon be exposed to the antigen.

232. A method of inducing an immune response in a subject in need thereof, comprising administering to the subject a first RNA molecule encoding a first antigen and a second RNA molecule encoding a second antigen, wherein the first RNA molecule is a modified RNA molecule and the second RNA molecule (i) does not comprise a modified ribonucleotide or (ii) is a self-amplifying RNA molecule or a trans-amplifying RNA molecule.

233. A method of inducing an immune response in a subject in need thereof, comprising administering to the subject a composition comprising a first plurality of RNA molecules encoding first antigens, and a second plurality of RNA molecules encoding second antigens, wherein at least 10% (including, e.g., at least 20%, at least 30, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) of the first plurality of RNA molecules are modified RNA molecules, and at least 10% (including, e.g., at least 20%, at least 30, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) of the second plurality of RNA molecules (i) do not comprise a modified ribonucleotide or (ii) are self- amplifying RNA molecules or trans-amplifying RNA molecules.

234. A method of inducing an immune response in a subject in need thereof, comprising administering to the subject a first dose of a composition comprising a first RNA molecule encoding a first antigen, and a second dose of a composition comprising a second RNA molecule encoding a second antigen, wherein the first RNA molecule is a modified RNA molecule, and the second RNA molecule (i) does not comprise a modified ribonucleotide or (ii) is a self- amplifying RNA molecule or trans-amplifying RNA molecule.

235. The method of any one of embodiments 232-234, wherein the first RNA molecule comprises modified uridines.

236. The method of embodiment 235, wherein the modified uridines are in place of all uridines.

237. The method of any one of embodiments 232-236, wherein the second RNA molecule does not comprise a modified ribonucleotide.

238. The method of any one of embodiments 232-237, wherein the first antigen is or comprises a B cell antigen of an infectious agent and the second antigen is or comprises a T cell antigen.

239. The method of embodiment 238, wherein the B cell antigen is a SAR-CoV-2 S protein or immunogenic portion thereof.

240. The method of embodiment 238 or 239, wherein the T cell antigen is from the same infectious agent.

241. The method of any one of embodiments 238-240, wherein the T cell antigen comprises at least one or more T cell epitopes.

242. The method of any one of embodiments 232-241, wherein the first RNA molecule and the second RNA molecule are co-administered.

243. The method of any one of embodiments 232-242, wherein the first RNA molecule and the second RNA molecule are separately or co-formulated in lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes.

244. The method of any one of embodiments 232-243, wherein the first RNA molecule and the second RNA molecule are separately administered.

245. The method of embodiments 232-244, wherein the subject has previously been administered one or more doses of one or more vaccines directed to a reference antigen of an infectious agent, wherein the reference antigen is from an earlier strain or lineage of the infectious agent, and wherein a B cell memory immune response has been established to the reference antigen.

246. A combination comprising:

(i) a composition that comprises or delivers polypeptide comprising or consisting of a variant polypeptide of a reference antigen of SARS-CoV-2, or an immunogenic portion thereof, wherein the variant polypeptide comprises neutralizing epitopes that are absent in the reference antigen; and (ii) an agent that induces a priming-favorable cytokine milieu in lymphoid tissues, wherein the agent is present at a dose that is effective to increase activation of naive B cell immune response to at least one of the neutralizing epitopes, and wherein the agent is or comprises (i) an unmodified RNA molecule or (ii) a self-amplifying RNA molecule or a trans- amplifying RNA molecule, and wherein the RNA molecule is formulated in lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes.

247. The combination of embodiment 246, wherein the agent encodes a polypeptide comprising an antigen of the SARS-CoV-2.

248. The combination of embodiment 247 wherein the antigen is a B-cell antigen.

249. The combination of embodiment 247, wherein the antigen is a T-cell antigen.

250. The combination of any one of embodiments 246-249, wherein the amount ratio (by mass or moles) of the polypeptide to the agent is within a range of about 1:5 to about 20: 1.

251. An RNA molecule comprising a nucleotide sequence that includes modified ribonucleotides and corresponding unmodified ribonucleotides, wherein the ratio of the modified ribonucleotides to the corresponding unmodified ribonucleotides is within a range of about 1: 10 to about 1: 1; and wherein the nucleotide sequence encodes an antigen of SARS-CoV-2.

252. The RNA molecule of embodiment 251, wherein the nucleotide sequence comprises a first domain and a second domain, wherein at least one of the first domain and the second domain comprises modified ribonucleotides and the other domain comprises no modified ribonucleotides.

253. The RNA molecule of any one of embodiments 251-252, wherein the modified ribonucleotides are 1 -methylpseudouridine and the corresponding unmodified ribonucleotides are uridine.

254. A method of inducing a priming immune response by:

(a) administering to a subject one or both of:

(i) a composition that comprises or delivers a polypeptide antigen of SARS-CoV-2; and

(ii) an agent that induces a priming-favorable cytokine milieu in lymphoid tissues, wherein the agent is present at a dose that is effective to increase activation of naive B cell immune response to at least one of the neutralizing epitopes. 255. A method of inducing or supporting a priming immune response to an antigen in a subject by exposing the subject to the antigen under immune priming conditions.

256. The method of embodiments 254 or 255, wherein the subject has previously been exposed to a variant of the antigen of SARS-CoV-2.

257. The method of embodiments 255 or 256, wherein the step of exposing comprises administering a composition that comprises or delivers the antigen.

258. The method of embodiment 257, wherein the antigen is a polypeptide antigen.

259. The method of any one of embodiments 255-257, wherein the step of exposing comprises administering a “priming adjuvant” to a subject who is or will soon be exposed to the antigen.

260. A method of inducing an immune response in a subject in need thereof, comprising administering to the subject a first RNA molecule encoding SARS-CoV-2 antigen and a second RNA molecule encoding an antigen, wherein the first RNA molecule is a modified RNA molecule and the second RNA molecule (i) does not comprise a modified ribonucleotide or (ii) is a self-amplifying RNA molecule or a trans-amplifying RNA molecule.

261. A method of inducing an immune response in a subject in need thereof, comprising administering to the subject a composition comprising a first plurality of RNA molecules each encoding a SARS-CoV-2 antigen and a second plurality of RNA molecules each encoding an antigenantigens, wherein at least 10%, such as at least 50%, such as at least 80%, such as 100% of the first plurality of RNA molecules are modified RNA molecules, and at 10%, such as at least 50%, such as at least 80%, such as 100% of the second plurality of RNA molecules (i) do not comprise a modified ribonucleotide or (ii) are self-amplifying RNA molecules or trans- amplifying RNA molecules.

262. A method of inducing an immune response in a subject in need thereof, comprising administering to the subject a first dose of a composition comprising a first RNA molecule encoding a SARS-CoV-2 antigen, and a second dose of a composition comprising a second RNA molecule encoding an antigen, wherein the first RNA molecule is a modified RNA molecule, and the second RNA molecule (i) does not comprise a modified ribonucleotide or (ii) is a self- amplifying RNA molecule or trans-amplifying RNA molecule.

263. The method of any one of embodiments 260-262, wherein the first RNA molecule comprises modified uridines. 264. The method of any one of embodiments 260-263, wherein the modified uridines are in place of all uridines.

265. The method of any one of embodiments 260-264, wherein the second RNA molecule does not comprise a modified ribonucleotide.

266. The method of any one of embodiments 260-265, wherein the SARS-CoV-2 antigen encoded by the first RNA is or comprises a B cell antigen of SARS-CoV-2 and the antigen encoded by the second RNA is or comprises a T cell antigen.

267. The method of embodiment 266, wherein the T cell antigen comprises one or more T cell epitopes.

268. The method of any one of embodiments 266-267, wherein the T cell antigen is from SARS- CoV-2.

269. The method of embodiment 266, wherein the B cell antigen of SARS-CoV-2 is SARS- CoV-2 S antigen or immunogenic portion thereof.

270. The method of any one of embodiments 260-269, wherein the antigen encoded by the second RNA is or comprises one or more T cell epitopes from at least one of an M protein, an N protein, and an ORFlab protein of SARS-CoV-2.

271. The method of any one of embodiments 260-270, wherein the antigen encoded by the second RNA is or comprises one or more T cell epitopes from at least two of an M protein, an N protein, and an ORFlab protein of SARS-CoV-2.

272. The method of any one of embodiments 260-271, wherein the first RNA molecule and the second RNA molecule are co-administered.

273. The method of any one of embodiments 260-272, wherein the first RNA molecule and the second RNA molecule are separately or co-formulated in lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes.

274. The method of any one of embodiments 260-272, wherein the first RNA molecule and the second RNA molecule are separately administered.

275. The method of any one of embodiments 260-274, wherein the subject has previously been administered one or more doses of one or more vaccines directed to a reference antigen of SARS-CoV-2, wherein the reference antigen is from an earlier strain or lineage of the SARS- CoV-2, and wherein a B cell memory immune response has been established to the reference antigen. 276. The method of embodiment 275, wherein the reference antigen is a SARS-CoV-2 S protein of a Wuhan strain or an Omicron BA.4/5 strain.

277. The method of embodiment 275, wherein the reference antigen is a SARS-CoV-2 S protein of a XBB strain.

278. The method of embodiment 277, wherein the XBB strain is a XBB 1 or XBB 1.5.

279. A method of inducing an immune response in a subject who was previously exposed to a first SARS-CoV-2 Spike (S) protein, the method comprising a step of delivering a polypeptide comprising a fragment of a second SARS-CoV-2 S protein to the subject, wherein the fragment of the second SARS-CoV-2 S protein comprises or consists a Receptor Binding Domain (RBD) or an SI domain of the second SARS-CoV-2 S protein, and wherein the fragment of the second SARS-CoV-2 S protein comprises one or more mutations of one or more SARS-CoV-2 variants.

280. The method of embodiment 279, wherein the first SARS-CoV-2 S protein is from a strain or variant that was previously prevalent or is currently prevalent in a relevant jurisdiction.

281. The method of embodiment 279 or 280, wherein the subject was previously exposed to the first SARS-CoV-2 S protein by:

(a) administration of one or more doses of one or more vaccines that deliver the first SARS- CoV-2 S protein,

(b) previous infection by a SARS-CoV-2 virus comprising the first SARS-CoV-2 S protein, and/or

(c) presence in a jurisdiction where a SARS-CoV-2 strain or variant comprising the first SARS- CoV-2 S protein was prevalent.

282. The method of embodiment 279 or 280, wherein the fragment of the second SARS-CoV-2 S protein does not comprise one or more regions of a SARS-CoV-2 S protein that are infrequently mutated in SARS-CoV-2 variants.

283. The method of any one of embodiments 279-282, wherein the fragment of the second SARS-CoV-2 S protein does not comprise an S2 domain.

284. The method of any one of embodiments 279-283, wherein the fragment of the second SARS-CoV-2 S protein does not comprise an N-terminal domain (NTD).

285. The method of any one of embodiments 279-284, wherein the fragment of the second SARS-CoV-2 S protein comprises or consists of the RBD. 286. The method of any one of embodiments 279-283, wherein the fragment of the second SARS-CoV-2 S protein comprises or consists of the SI domain.

287. The method of any one of the preceding embodiments, wherein the fragment of the second SARS-CoV-2 S protein comprises one or mutations associated with a SARS-CoV-2 variant that is prevalent, predicted to be prevalent, predicted to continue to be prevalent, and/or predicted to increase in prevalence in a relevant jurisdiction.

288. The method of any one of the preceding embodiments, wherein the fragment of the second SARS-CoV-2 S protein comprises one or more mutations associated with a SARS-CoV-2 variant that has a high immune escape potential.

289. The method of embodiment 288, wherein the SARS-CoV-2 variant has been determined to have a high immune escape potential using an in vitro assay (e.g., a viral neutralization assay), in silico analysis (e.g., sequence analysis and/or molecular dynamic simulations), in vivo studies (e.g., mouse or rat studies), and/or based on an infection rate and/or growth rate in a human population.

290. The method of embodiment 288 or 289, wherein the SARS-CoV-2 variant is an Omicron variant.

291. The method of embodiment 290, wherein the Omicron variant is an XBB variant (e.g., an XBB.l or XBB.1.5 variant), a BQ.l variant, a BA.2.86 variant, or a JN variant.

292. The method of embodiment 291, wherein the one or more mutations associated with an XBB.l.5 variant are T19I, A24-26, A27S, V83A, G142D, A145, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, or N969K, or a combination thereof, where the positions of the one or more mutations are indicated relative to SEQ ID NO:1.

293. The method of any one of embodiments 279-292, wherein the fragment of the second SARS-CoV-2 S protein comprises or consists of an RBD of an XBB.1.5 SARS-CoV-2 variant, and wherein the RBD comprises one or more of the following mutations relative to SEQ ID NO:1: G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, or Y505H, or any combination thereof. 294. The method of any one of embodiments 279-293, wherein the fragment of the second SARS-CoV-2 S protein comprises or consists of an SI domain, and wherein the one or more mutations associated with an XBB.1.5 variant are selected from: T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, and P681H, or any combination thereof, wherein the positions of the one or more mutations are shown relative to SEQ ID NO: 1.

295. The method of any one of embodiments 279-294, wherein the polypeptide comprising the fragment of the second SARS-CoV-2 S protein is delivered by administering an RNA that comprises a nucleotide sequence encoding the fragment of the second SARS-CoV-2 protein.

296. The method of any one of embodiments 279-294, wherein the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 3.

297. The method of any one of embodiments 279-296, wherein the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 5.

298. The method of any one of the preceding embodiments, wherein the polypeptide comprises a secretion signal.

299. The method of embodiment 298, wherein the secretion signal is a homologous secretion signal.

300. The method of embodiment 298, wherein the secretion signal is a heterologous secretion signal.

301. The method of any one of embodiments 298-300, wherein the secretion signal is present at or near the N-terminus of the polypeptide.

302. The method of embodiment 298, wherein the secretion signal is a SARS-CoV-2 S protein secretion signal, a gD2 secretion signal, a gDl secretion signal, a gBl secretion signal, a gI2 secretion signal, a gE2 secretion signal, an Eboz secretion signal, or an HLA-DR secretion signal.

303. The method of embodiment 302, wherein the SARS-CoV-2 S protein secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 15. 304. The method of embodiment 302, wherein the SARS-CoV-2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 9.

305. The method of embodiment 302, wherein the SARS-CoV-2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 16.

306. The method of embodiment 302, wherein the gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 8.

307. The method of embodiment 302, wherein the gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 13.

308. The method of embodiment 302, wherein the gDl secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 12.

309. The method of embodiment 302, wherein the gBl secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 37.

310. The method of embodiment 302, wherein the gC2 polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 35.

311. The method of embodiment 302, wherein the gI2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 11.

312. The method of embodiment 302, wherein the gE2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 38.

313. The method of embodiment 302, wherein the EboZ secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 39.

314. The method of embodiment 302, wherein the HLA-DR secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 40.

315. The method of any one of the preceding embodiments, wherein the polypeptide further a multimerization domain.

316. The method of embodiment 315, wherein the multimerization domain in the C-terminal region (e.g., at the C-terminus).

317. The method of embodiment 315 or 316, wherein the multimerization domain is a fibritin domain.

318. The method of embodiment 317, wherein the fibritin domain comprises a sequence that is at least 80% identical to SEQ ID NO: 95. 319. The method of embodiment 317, wherein the fibritin domain comprises a sequence that is at least 80% identical to SEQ ID NO: 96.

320. The method of any one of the preceding embodiments, wherein the polypeptide comprises a transmembrane (TM) domain.

321. The method of embodiment 320, wherein the TM domain is a homologous TM domain.

322. The method of embodiment 320, wherein the TM domain is a heterologous TM domain.

323. The method of any one of embodiments 320-322, wherein the TM domain is present in the C-terminal portion of the SARS-CoV-2 S protein variant or immunogenic portion thereof (e.g., at the C-terminus).

324. The method of embodiment 323, wherein the polypeptide comprises a multimerization domain and a TM domain at or near the C-terminus.

325. The method of embodiment 324, wherein the TM domain is C-terminal to the multimerization domain.

326. The method of embodiment 325, wherein:

(a) the multimerization domain is directly adjacent to the fragment of the second SARS-CoV-2 protein or connected to the fragment of the second SARS-CoV-2 protein via a flexible linker, and/or

(b) the TM domain is directly adjacent to the multimerization domain or connected to the multimerization domain via a flexible linker.

327. The method of any one of embodiments 320-326, wherein the TM domain is a SARS-CoV- 2 S protein TM domain or an influenza TM domain.

328. The method of embodiment 327, wherein the SARS-CoV-2 TM domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 89.

329. The method of embodiment 327, wherein the SARS-CoV-2 TM domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 90.

330. The method of any one of embodiments 295-329, wherein the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 120.

331. The method of any one of embodiments 295-330, wherein the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 130. 332. The method of any one of embodiments 295-330, wherein the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 135.

333. The method of any one of embodiments 295-330, wherein the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 145.

334. The method of any one of embodiments 295-330, wherein the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 150.

335. The method of any one of embodiments 295-334, wherein the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein has been codon-optimized for expression in mammalian subjects.

336. The method of any one of embodiments 295-335, wherein the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein has been codon-optimized for expression in human subjects.

337. The method of any one of embodiments 295-336, wherein the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein has an enriched G/C content relative to wild-type sequence.

338. The method of embodiment 337, wherein G/C content has been increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%.

339. The method of any one of embodiments 295-338, wherein the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein comprises a heterologous 3’ UTR or 5 ’UTR.

340. The method of embodiment 339, wherein the heterologous 5' UTR comprises or consists of a modified human alpha-globin 5 '-UTR.

341. The method of embodiment 339 or 340, wherein the heterologous 3’ UTR comprises or consists of a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA.

342. The method of any one of embodiments 295-341, wherein the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein comprises a poly(A) sequence. 343. The method of embodiment 342, wherein the poly(A) sequence has a length of about 100- 150 nucleotides.

344. The method of embodiment 342 or 343, wherein the poly(A) sequence is a disrupted poly(A) sequence.

345. The method of any one of embodiments 295-344, wherein the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein comprises a 5' cap.

346. The method of any one of embodiments 295-345, wherein the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 122 or 124.

347. The method of any one of embodiments 295-345, wherein the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 131 or 133.

348. The method of any one of embodiments 295-345, wherein the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 136 or 138.

349. The method of any one of embodiments 295-345, wherein the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 146 or 148.

350. The method of any one of embodiments 295-345, wherein the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 151 or 153.

351. The method of any one of embodiments 295-350, wherein the RNA is unmodified RNA.

352. The method of any one of embodiments 295-351, wherein the RNA comprises one or more modified nucleotides.

353. The method of embodiment 352, wherein the modified nucleotide is pseudouridine (e.g., N 1 -methyl-pseudouridine).

354. The method of embodiment 352 or 353, wherein the RNA comprises a modified nucleotide in place of each uridine.

355. The method of any one of embodiments 295-354, wherein the RNA is an self-amplifying RNA or trans-amplifying RNA.

356. The method of any one of embodiments 295-355, wherein the RNA is fully or partially encapsulated within lipid nanoparticles (LNP), polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes.

357. The method of embodiment 356, wherein the RNA is fully or partially encapsulated within LNP. 358. The method of embodiment 357, wherein the LNP comprise a cationically ionizable lipid, a neutral lipid, a sterol and a lipid conjugate.

359. The method of any one of embodiments 279-358, wherein the first SARS-CoV-2 S protein is from a strain or variant that the subject was first exposed to and/or that was first prevalent in a population of subjects.

360. The method of any one of embodiments 279-359, wherein the first SARS-CoV-2 S protein is a Wuhan SARS-CoV-2 S protein or an Omicron BA.4/5 SARS-CoV-2 S protein.

361. The method of any one of embodiments 279-360, where the first SARS-CoV-2 S protein is from a strain or variant that the subject has previously been vaccinated against or is delivered by one or more vaccines that a significant proportion of the population (e.g., at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least abut 45%, at least about 50%, at least about 55%, or at least about 60%) has previously been administered.

362. The method of embodiment 361, wherein the vaccine previously administered to the subject or a significant proportion of the population was a first generation vaccine.

363. The method of embodiment 361, wherein the first SARS-CoV-2 S protein is from a SARS- CoV-2 strain or variant that was previously prevalent or is currently prevalent in a relevant jurisdiction.

364. The method of embodiment 361, wherein the first SARS-CoV-2 S protein is from a variant that first became prevalent in a relevant jurisdiction.

365. The method of any one of embodiments 279-364, wherein the immune response comprises a B cell immune response.

366. The method of embodiment 365, wherein the immune response comprises a naive B cell immune response.

367. The method of any one of embodiments 279-366, wherein:

(a) the immune response comprises a reduced memory B cell immune response as compared to an immune response induced by administering the full length sequence of the second SARS- CoV-2 S protein, (b) the immune response comprises an increased naive B cell immune response as compared to an immune response induced by administering the full length sequence of the second SARS- CoV-2 S protein, and/or

(c) the ratio of the naive B cell immune response to the memory B cell immune response is increased.

368. The method of embodiment 367, wherein:

(a) the memory B cell immune response is reduced by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% as compared to the immune response induced by a full length sequence of the second SARS-CoV-2 protein;

(b) the memory B cell immune response is increased by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% as compared to the immune response induced by a full length sequence of the second SARS-CoV-2 protein; and/or

(c) the ratio of the naive immune response to the memory B cell immune response is increased by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% as compared to the immune response induced by a full length sequence of the second SARS-CoV-2 protein.

Examples

Example 1: Characterization of Immune Imprinting in SARS-CoV-2

[0973] The present Example is designed to assess the extent of immune imprinting in subjects previously exposed to a SARS-CoV-2 antigen (e.g., an S protein encountered via infection or vaccination). Immune imprinting is a phenomenon in which initial exposure to a virus strain or antigen limits development of immune responses against epitopes that are unique to new variant strains. Immune imprinting can be particularly concerning for pathogens having a high concentration of mutations at neutralization sensitive epitopes (e.g., SARS-CoV-2).

[0974] A schematic illustrating the immune imprinting phenomenon is shown in Fig. 1. Subjects administered a vaccine that delivers a wild-type (WT) antigen produce antibodies and form memory B cells recognizing the WT antigen. As new Variants of Concern (VOC) arise that evade the immune response induced by the first vaccine, VOC-adapted booster shots are developed and administered to subjects. VOCs often evade the immune system by acquiring mutations at neutralization sensitive epitopes (regions prone to mutation shown in different colors in Fig. 1). Subjects exposed to a VOC-adapted vaccine have a predisposition to activate memory B cells that were formed in response to the initial WT vaccine rather than activate naive B cells. As a result, administering the VOC-adapted vaccine induces production of antibodies that recognize both the WT virus and the VOC but few or no antibodies that are specific to the VOC. So long as the VOC retains some neutralization epitopes from the WT virus, a neutralization response against the VOC can still be induced. As new variants continue to lose neutralization epitopes from the WT strain, however, the immune response induced by VOC- adapted vaccines become less and less effective. Further discussion of the imprinting phenomenon in the SARS-CoV-2 context can be found in Wheatley et al., Trends Immunol, 2021 , the contents of which are incorporated by reference herein in their entirety. Immune imprinting is expected to be particularly concerning for vaccine updates that addressing virus strains comprising a number of mutations at neutralization sensitive sites, (e.g., variants that exhibit close to no conserved neutralizing epitopes).

[0975] Immune imprinting can have serious implications for vaccine development. As shown in Fig. 2, a single exposure to Omicron BA.l, BA.2, or BA.4/5 variants has not been found to induce neutralization of Omicron XBB. Without wishing to be bound by theory, this failure to cross neutralize the XBB variant may be due to its low retention of neutralizing B-cell epitopes relative to the original Wuhan strain (see Fig. 5(B)). In short, exposure to BA.l, BA.2, and BA.4/5 may be activating memory B cells that recognize epitopes in both Wuhan and these Omicron variants, and not generating immune responses that recognize features that are unique to these variants. If true, these results suggest that variant adapted vaccines may not produce effective immune responses to variants that retain few neutralization epitopes from the original SARS-CoV-2 variant.

[0976] The present Example describes a study to characterize the extent of immune imprinting in SARS-CoV-2. Fig. 3. provides a schematic summarizing the design of the study.

Sera samples were collected from subjects in the following groups:

(i) subjects administered 2 doses of a vaccine delivering a SARS-CoV-2 S protein of a Wuhan strain who experienced a subsequent Omicron BA.l infection;

(ii) subjects administered 3 doses of a vaccine delivering a SARS-CoV-2 S protein of a Wuhan strain who experienced a subsequent Omicron BA.1 infection;

(iii) subjects administered 3 doses of a vaccine delivering a SARS-CoV-2 S protein of a Wuhan strain who experienced a subsequent Omicron BA.1 infection and who also received a booster dose of a vaccine delivering a SARS-CoV-2 S protein of an Omicron BA.l variant; and

(iv) subjects administered 3 doses of a vaccine delivering a SARS-CoV-2 S protein of a Wuhan strain and two booster doses of a vaccine delivering a SARS-CoV-2 S protein of an Omicron BA.l variant.

[0977] For group (i), subjects were infected with Omicron BA.l on average 5 months after receiving the second dose of a SARS-CoV-2 vaccine. For group (ii), subjects were infected with Omicron BA.l on average 1 month after receiving the third dose of a SARS-CoV-2 vaccine. In the present Example, for each group, the vaccine delivering a SARS-CoV-2 S protein of a Wuhan strain was BNT162b2, and the vaccine delivering a SARS-CoV-2 S protein of an Omicron BA.l variant was BA.l-adapted BNT162b2. [0978] Sera samples were subjected to memory B cell phenotyping to determine binding specificity, using the FACS and depletion assays depicted in Figs. 2(A) and (B)). Pseudovirus neutralization titers were also collected for each of the sera samples.

[0979] Pseudovirus neutralization titers (Fig. 4(A)) demonstrated that exposure to Omicron BA.l can increase neutralization of Omicron BA.l, and induce a broad cross neutralization reponse. Memory B cell phenotyping, however, found that the improved neutralization titers were driven largely by activation of memory B cells that bind epitopes common to the Wuhan S protein and the Omicron BA.l S protein. Specifically, FACS analysis found that most memory B cells bound epitopes common to Omicron BA.l and Wuhan (points along the diagonal in the representative plots shown in Fig. 4(B)). A small population of memory B cells was found to be specific to the Wuhan S protein (points at the bottom of the plot), but relatively few to no memory B cells were detected that were specific to the Omicron BA.1 Spike protein.

[0980] Depletion studies showed similar results. Sera samples were subjected to two rounds of depletion. After each round, pseudovirus neutralization titers were collected using pseudoviruses comprising an S protein of a Wuhan strain or an Omicron BA.1 variant. Results are shown in Fig. 6. Serum samples incubated with unlabeled beads in the first round of depletion were shown to have high neutralization titers (as expected). For most serum samples, depleting with a full length S protein of a Wuhan strain in the first round reduced neutralization titers against Wuhan pseudvirus, demonstrating that the assay worked. Of the 13 samples tested, only one sample was found to retain significant neutralization titers after incubation with full length Wuhan S protein and Wuhan RBD, which were then lost after incubation with an Omicron BA.l RBD (red sample in Figure 6), suggesting the presence of Omicron BA.l specific antibodies. These results indicate that a single exposure to Omicron BA.l does not induce an immune response that is specific to Omicron BA.1 in the majority of subjects.

[0981] In light of the above results, it was concluded that the improved neutralization titers observed in subjects who were exposed to Omicron BA.1 was likely driven by increased titers of of antibodies that neutralize both Omicron BA.l and Wuhan, rather than the induction of Omicron BA.l specific neutralization antibodies. [0982] Further depletion studes were performed to determine which regions in the S protein Omicron BA.l specific antibodies were binding. Subjects previously administered 2 or 3 doses of RNA encoding a SARS-CoV-2 S protein of a Wuhan strain were adminsitered (i) an Omicron BA.l-adapted booster (e.g., RNA encoding a full length S protein of an Omicron BA.l variant), (ii) a Wuhan booster (e.g., BNT162b2), or (iii) no booster. Serum samples were collected immediately prior to administering a booster, 7 days after administing a booster, and 1 month after administering a booster. Memory B cell phenotyping was then performed by FACS, using Wuhan and Omicron BA.l versions of a full length S protein, an RBD, and an NTD of a Wuhan strain or an Omicron BA.l variant. Results are shown in Fig. 7.

[0983] As shown in Fig. 7, memory B cells specific to the Omicron BA.l RBD were not detected in any group. In subjects administered an Omicron BA.l-adapted booster, a small population of BMEM cells specific to the Omicron BA.l S protein was detected 1 week and 1 month after administering a booster dose. NTD and RBD labeling showed that this binding was due to BMEM cells binding the NTD of Omicron BA.1.

Example 2: Administration of Imprinting-Resistant Constructs to Vaccine experienced mice

[0984] The present Example describes an experiment to determine whether RNA described herein can induce an immune response characterized by increased naive B cell activation and/or decreased memory B cell activation in vaccine-experienced subjects (mice in the present Example). Vaccine candidates are administered to mice previously exposed to full length SARS-CoV-2 S protein (specifically, mice previously administered two doses of RNA encoding SARS-CoV-2 S protein).

[0985] Mice are split into 12 groups, each comprising 7 or 8 members, and administered a dosing regimen described in Fig. 9. As shown in the Figure, mice in each group are first administered two doses of (i) a monovalent composition comprising RNA encoding a SARS- CoV-2 S protein of a Wuhan strain (BNT162b2), or (ii) a bivalent composition comprising an RNA encoding a SARS-CoV-2 S protein of a Wuhan strain and an RNA encoding a SARS-CoV- 2 S protein comprising mutations characteristic of an Omicron BA.4/5 variant. As discussed elsewhere in the present disclosure, the S protein in Omicron BA.4 and BAA have the same amino acid sequence. As such, in the present disclosure, “BA.4/5” is used to refer to the S protein of either variant. First and second doses of vaccine are administered 21 days apart. 5 weeks after administered a first dose, groups of mice are divided into groups based on neutralization titers against the Wuhan and/or Omicron XBB.1.5 variants (e.g., mice are allocated into groups so that neutralization titers are approximately the same in each group). In some embodiments, mice can be allocated into groups based on pseudovirus neutralization titers (e.g., as shown in Fig. 9).

[0986] Each candidate vaccine is administered as a third and a fourth dose, 18 weeks and 34 weeks, respectively, after administering a first dose of vaccine. Table 16, below, lists vaccine candidates tested in the present Example, which are also listed in Fig. 9.

Table 16: Vaccine Candidates for Administering to Vaccine-Experienced Mice

[0987] Blood samples are collected immediately before, and 3 weeks, 5 weeks, 9 weeks, 13 weeks, 17 weeks, 18 weeks, 19 weeks, 22 weeks, 26 weeks, 30 weeks, 34 weeks, 35 weeks, 37 weeks, and 39 weeks after administering a first dose of RNA. 39 weeks days after administering a first dose of RNA, mice are sacrificed, and final blood, lymph node, and spleen samples collected for analysis.

Blood Sample Analysis

[0988] Blood samples are screened for titers of antibodies that bind and neutralize various SARS-CoV-2 variants (e.g., using ELISA and pseudovirus assays that described in the previous Examples). Each spleen sample can be analyzed individually. For lymph node samples, samples from two mice can be combined for analysis.

Spleen and Lymph Node Sample Analysis

[0989] B cells are isolated from spleens and lymph nodes and phenotyped to determine B cell type (e.g., naive, memory, or plasma) and binding specificity (e.g., specificity to the S proteins of various SARS-CoV-2 variants of concern). Phenotyping can be performed using the FACS-based and depletion assays described in the previous examples. BCR repertoire analysis is also performed for B cells isolated from spleen samples.

[0990] For RBD binding assays, samples from all mice can be grouped together to generate enough sample to perform the methods. Each sample can be screened for negative, Wuhan-specific, XBB.1.5 exclusive binders, and cross reaction between Wuhan and XBB.1.5 binding.

[0991] Among other things, the protocol described in the present Example can be used to characterize the binding specificity of B cells in subjects administered a booster that delivers an antigen of a variant of concern (e.g., an XBB.1.5 S protein or an immunogenic portion thereof). Specifically, this experimental protocol can be used to determine the relative number of B cells that are specific to an infectious agent variant of concern and/or what portions of the variant of concern those B cells recognize. These results can be used to determine the impact of immune imprinting on various vaccine candidates, and the ability of certain vaccine candidates to evade the immune imprinting phenomena and induce a de novo immune response.

[0992] Vaccine candidates that are less susceptible to immune imprinting (i.e., more likely to generate a de novo response) can be characterized by one or more of: (i) an increased proportion of B cells that are specific to a variant of concern delivered by the vaccine candidate, (ii) increased neutralization titers against a variant of concern encoded by the vaccine candidate, and/or (iii) an increased number of B cell receptors that recognize epitopes that are unique to an antigen encoded by the vaccine candidate (i.e., increased B cell breadth).

Example 3: Administration of Imprinting-Resistant Constructs to Vaccine Naive Mice

[0993] The present Example describes an experiment to determine the immunogenicity of RNA described herein in vaccine naive subjects (specifically, mice). Among other things, the present Example can be used to characterize how “strong” a vaccine is (e.g., the magnitude and/or breadth of immune response induced by a vaccine) in vaccine naive subjects.

[0994] Mice are split into 9 groups, each comprising 5 members, and administered a dosing regimen described in Figure 11. As shown in the Figure, mice in each group are administered two doses of a candidate vaccine, administered 21 days apart.

[0995] Table 17, below, lists the vaccine candidates that are tested in the present Example.

Table 17: Vaccine Candidates for Testing in Vaccine-Naive Mice

[0996] Blood samples are collected immediately prior to, and 2 and 3 weeks after a first dose of vaccine candidate. 5 weeks after administering a first dose, animals are sacrificed, and a final blood sample, as well as a spleen sample and lymph node sample are collected for analysis.

[0997] 35 days after administering vaccine candidates, blood samples B-cell phenotype staining including S bite (Wuhan/XBB1.5), B cell isolation (on XBB1.5) and BCR sequencing in LN and spleen for all groups. Exemplary analysis techniques are shown in Figure 12, and described in Example 2.

[0998] As noted above, the study described in the present Example, among other things, can be used to assess the “strength” of a vaccine candidate in vaccine naive subjects (e.g., the magnitude and/or breadth of immune response induced by a vaccine and how it compares to other vaccine candidates). In some embodiments, a “strong” vaccine candidate can be characterized by one or more of (i) the activation of a number of naive B cells, (ii) the generation of a number of memory B cells and/or plasma cells, (iii) the generation of B cells that recognize a number of different SARS-CoV-2 variants, (iv) induce high neutralization titers against a particular variant of concern (e.g., a variant of concern encoded by an administered RNA), and/or (v) high cross-neutralization titers against a large number of SARS-CoV-2 variants.

Example 4: Administration of Imprint-Resistant Constructs to Vaccine-Experienced Mice

[0999] The present Example provides data demonstrating that compositions described herein can provide an improved immune response in vaccine-experienced mice as compared to previous vaccines. In particular, the present example demonstrates that compositions described herein can produce an immune response characterized by (i) an increased frequency of naive B cell activation and/or decreased activation of memory B cells, and (ii) a decreased memory B cell activation as compared to previous vaccines (e.g., vaccines delivering a full-length S protein) in vaccine-experienced subjects. In the present example, immunogenicity of vaccine candidates were compared to RNA compositions encoding a full-length S protein (either adapted to the original Wuhan strain or the XBB.1.5 variant). As in the previous examples, the experiment described in the present Example focused on SARS-CoV-2, but a person of skill in the art will recognize that a similar experimental design can be used to design and test vaccines for other infectious diseases.

[1000] The dosing protocol used in the present Example is summarized in Fig. 13. Mice were split into 12 groups, each comprising 7 or 8 members. 6 groups were administered two doses of a monovalent RNA composition encoding a SARS-CoV-2 S protein of a Wuhan strain, and the other 6 groups were administered two doses of a bivalent composition comprising (i) RNA encoding a SARS-CoV-2 S protein of a Wuhan strain and RNA encoding a SARS-CoV-2 S protein of an Omicron BA.4/5 strain. For all 12 groups of mice, the two vaccines were administered about 21 days apart. Following administration of two primary doses, neutralization titers against the Wuhan and/or Omicron BA.4/5 strains was assessed, and mice were grouped so as to provide similar average neutralization titers in each group.

[1001] 17 weeks (126 days) after the first dose, a third dose was administered to the mice, comprising a candidate vaccine. Fig. 13 indicates the candidate vaccines tested in the present experiment. 34 weeks (238 days) after the first dose (17 weeks after the third dose) a further dose of candidate vaccine was administered. For each group of mice, the same vaccine was administered in both the third dose and the fourth dose. All doses were administered at 1 pg.

[1002] Blood samples were collected immediately before administering the first dose, and at at 3 weeks, 5 weeks, 17 weeks, 19 weeks, and 21 weeks thereafter. 39 weeks after the first dose, mice were sacrificed, and the experiment ended. Blood, lymph node, and spleen samples were collected from the sacrificed mice for analysis. Analysis of samples was preformed in accordance with the protocols described in the previous examples.

[1003] Neutralizing antibody titers were collected using pseudovirus neutralization assays, using pseudoviruses adapted to express an S protein of a Wuhan strain or a SARS-CoV-2 variant. B cells from lymph nodes (EN) and splenocytes (SPE) were harvested and collected for analysis. In brief, draining EN were collected from each animal after sacrifice, with all cells from each animal suspended in 200 pF. For SPE harvesting, 15 Mio cells were collected per animal and resuspended a concentration of 10 mio/mE (1.5 mF total. 5 Mio cells per animal were used for B cell bait staining, and 10 Mio cells per animal were sorted and sequenced using a lOxGenomics protocol. Draining LN samples were sorted using full length S proteins, whereas SPLs were sorted using both RBDs and full length S proteins. 219 samples in total (including both draining LN and SPL were anlyzed).

[1004] Memory B cells were identified based on CD95 expression. Suitable alternative markers to identify memory B cells are provided in the below table:

[1005] MACS® (magnetic cell separation) separation (anti-CD90.2) was used to deplete all T cells and enrich for untouched B cells prior to FACS analysis and sequencing. Cells were labeled using Wuhan-BV421 and XBB.1.5-AF647. Monocytes were discriminated based on size. IgD binding was used to discriminate against naive cells. FACS data was used to gate on CD19⁺ cells.

[1006] Fig. 14 shows neutralization titers against various SARS-CoV-2 variants in mice administered two doses of vaccine (before administering vaccine candidates). Neutralization titers were collected using a pseudovirus neutralization assay, using pseudoviruses that had been adapted to Wuhan, BQ.1.1, BA.2.75.2, XBB, or XBB.1.5 (in particular, pseudoviruses were generated comprising the S protein of each of these variants). Fig. 14(A) shows neutralization titers 42 days after the second dose (63 days after the first dose) and Fig. 14(B) shows neutralization titers 70 days after the second dose (91 days after the first dose). As shown in Fig. 14(A), relatively little intragroup variance was observed for each variant. Mice administered two doses of Wuhan-adapted vaccine exhibited high neutralization titers against the Wuhan strain, but very low neutralization titers against each of BQ.1.1, BA.2.75.2, and XBB. Mice administered two doses of the bivalent vaccine, in contrast, exhibited high neutralization titers against the Wuhan strain (although reduced relative to Wuhan), and much higher titers against each of BQ.1.1, BA.2.75.2, and XBB as compared to mice administered two doses of vaccine delivering an S protein of a Wuhan strain. Titers against BA.2.75.2 and XBB were reduced as compared to Wuhan and BQ.1.1, but still much higher than titers produced by two doses of a Wuhan-adapted vaccine. Fig. 14(B) shows similar effects were observed 70 days post dose 2, with neutralization titers against the Wuhan strain somewhat higher than at day 42, and neutralization titers against XBB.1.5 much higher than those induced by two doses of a Wuhan- adapted vaccine, but still lower than those induced against Wuhan.

[1007] Fig. 14(c) shows results from a separate experiment, performed using protocols similar to those described in the present experiment, in which mice were administered a first dose of a Wuhan-adapted vaccine and a second dose of a bivalent vaccine (comprising (a) RNA encoding a full-length S protein of a Wuhan variant, and (b) RNA encoding a full-length S protein of an Omicron BA.4/5 variant). Shown are neutralization titers collected 13 days after the second dose. As shown in the figure, and as comparing to Fig. 14(A), neutralization titers against the Wuhan strain were still high, but neutralization titers against each of BQ.1.1, BA.2.75.2 and XBB were reduced as compared to mice administered two doses of the bivalent vaccine.

[1008] Fig. 15(A) shows pseudovirus neutralization titers against XBB.1.5 collected for groups of mice administered two doses of a Wuhan-adapted, monovalent vaccine, 126, 154, 238, 245, 259, and 273 days after the first dose of vaccine. Vaccine candidates were administered on days 126 and 238 (vaccine days indicated with a syringe symbol in the figure). Fig 15(B) plots the same data. As shown in the figure, membrane-anchored RBD provided the highest neutralization titers after a single boost, with neturalization titers increased by about 64-fold on day 238 as compared to day 126. In general, shorter constructs delivering only the RBD (either membrane anchored or soluble) were more efficient in generating higher neutralization titers as compared to contracts delivering an S 1 domain.

[1009] B cells were isolated and stained with Wuhan and XBB.1.5 S proteins, labeled with different fluorophore. Labelled cells were then separated into populations that bind (i) Wuhan S protein exclusively, (ii) XBB.1.5 S protein exclusively, and (iii) Wuhan S protein and XBB.1.5 S protein. Exemplary FACS results are shown in Fig. 16. Fig. 17 shows percentage of B cells in each group. As shown in the figure, membrane anchored RBD induced a more consistent B cell response as compared to mice administered other constructs. Fig. 18 shows cells that were separated into groups based on their ability to bind different S proteins (same groups as in Fig. 17), and stained for CD95, which is indicative to recent B cell activation (e.g., within a germinal center reaction). As shown in the figure, all treatment groups immunized with XBB.1.5 constructs showed a high frequency of XBB.1.5 single positive BMEM cells that are CD95+ positive (indicating that the cells were recently activated). This effect is not observed for Wuhan single positive cells. The effect appears to be stronger for shorter constructs (that is, the percent of recently activated BMEM cells is higher for shorter constructs).

[1010] Fig. 19 displays the experimental protocol that was used to sequence individual B cells. As shown in Figure 20, the number of clonotypes relative to the number of cells decreased in mice that were administered the constructs. As shown in Figs. 21(A) and 21(B), in splenocytes obtained from mice, constructs encoding an RBD (membrane-anchored or soluble) produced the highest number of CD19+ B cells and CD19+ clonotypes. In particular, membrane- anchored RBD produced the highest number of CD 19+ B cells and clonotypes that were specific to XBB.1.5. Constructs encoding full length S protein, in contrast, induced a higher number of cells that bind both Wuhan and XBB.1.5 S protein. As shown in Fig. 22, no difference in Ig isotypes was observed between B cells exhibiting different specificities (i.e., the relative proportion of each cell type was the same in each group of cells).

[1011] Fig. 23 shows a comparison of clonotypes across B cell phenotypes. In total, 2122 B cell clonotypes were detected: 1072 clonotypes in bait negative samples and 1031 clonotypes in combined bait positive populations. 18 shared clonotypes were detected. In the bait positive populations 176 clonotypes were detected for Wuhan specific B cells, 276 clonotypes were detected for Wuhan/XBB.1.5 double specific B cells, and 591 clonotypes were detected for XBB.1.5 specific cells. Altogether 1-3 clonotypes were shared between the three populations. No relevant overlap was detected between positive/negative and positive sorted populations, indicated an efficient sorting strategy. Next, clonotypes were analyzed to identify clonotypes that were present in two or more cohorts, to determine whether there were any clonotypes that were characteristic of a given construct type (e.g., RBD or full length Spike). NaCl cohorts contained a recurrent group of shared clones, indicating that some clonotypes were “sticky” (i.e., may be binding to beads through an interaction that is not specific to the S protein. The number of shared clonotypes detected in each cohort was correlated with the total number of clonotypes detected. Shared clonotypes related to antigen design and similarity were not detected. B cells responses were mostly private. Shared responses occur proportional to the magnitude of induce clonotypes.

EQUIVALENTS

[1012] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of technologies described herein. The scope of the present disclosure is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

1. A method of inducing an immune response in a subject who was previously exposed to a first SARS-CoV-2 Spike (S) protein, the method comprising a step of delivering a polypeptide comprising a fragment of a second SARS-CoV-2 S protein to the subject, wherein the fragment of the second SARS-CoV-2 S protein comprises or consists a Receptor Binding Domain (RBD) or an SI domain of the second SARS-CoV-2 S protein, and wherein the fragment of the second SARS-CoV-2 S protein comprises one or more mutations of one or more SARS-CoV-2 variants.

2. The method of claim 1, wherein the first SARS-CoV-2 S protein is from a strain or variant that was previously prevalent or is currently prevalent in a relevant jurisdiction.

3. The method of claim 1 or 2, wherein the subject was previously exposed to the first SARS-CoV-2 S protein by:

(a) administration of one or more doses of one or more vaccines that deliver the first SARS-CoV-2 S protein,

(c) presence in a jurisdiction where a SARS-CoV-2 strain or variant comprising the first SARS-CoV-2 S protein was prevalent.

4. The method of claim 1 or 2, wherein the fragment of the second SARS-CoV-2 S protein does not comprise one or more regions of a SARS-CoV-2 S protein that are infrequently mutated in SARS-CoV-2 variants.

5. The method of any one of claims 1-4, wherein the fragment of the second SARS-CoV- 2 S protein does not comprise an S2 domain.

6. The method of any one of claims 1-5, wherein the fragment of the second SARS-CoV- 2 S protein does not comprise an N-terminal domain (NTD).

7. The method of any one of claims 1-6, wherein the fragment of the second SARS-CoV- 2 S protein comprises or consists of the RBD.

8. The method of any one of claims 1-5, wherein the fragment of the second SARS-CoV- 2 S protein comprises or consists of the SI domain.

9. The method of any one of the preceding claims, wherein the fragment of the second SARS-CoV-2 S protein comprises one or mutations associated with a SARS-CoV-2 variant that is prevalent, predicted to be prevalent, predicted to continue to be prevalent, and/or predicted to increase in prevalence in a relevant jurisdiction.

10. The method of any one of the preceding claims, wherein the fragment of the second SARS-CoV-2 S protein comprises one or more mutations associated with a SARS-CoV-2 variant that has a high immune escape potential.

11. The method of claim 10, wherein the SARS-CoV-2 variant has been determined to have a high immune escape potential using an in vitro assay (e.g., a viral neutralization assay), in silico analysis (e.g., sequence analysis and/or molecular dynamic simulations), in vivo studies (e.g., mouse or rat studies), and/or based on an infection rate and/or growth rate in a human population.

12. The method of claim 10 or 11, wherein the SARS-CoV-2 variant is an Omicron variant.

13. The method of claim 12, wherein the Omicron variant is an XBB variant (e.g., an XBB.l or XBB.1.5 variant), a BQ.l variant, a BA.2.86 variant, or a JN variant.

14. The method of claim 13, wherein the one or more mutations associated with an XBB.1.5 variant are T19I, A24-26, A27S, V83A, G142D, A145, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, or N969K, or a combination thereof, where the positions of the one or more mutations are indicated relative to SEQ ID NO: 1.

15. The method of any one of claims 1-14, wherein the fragment of the second SARS- CoV-2 S protein comprises or consists of an RBD of an XBB.1.5 SARS-CoV-2 variant, and wherein the RBD comprises one or more of the following mutations relative to SEQ ID NO: 1: G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, or Y505H, or any combination thereof.

16. The method of any one of claims 1-15, wherein the fragment of the second SARS- CoV-2 S protein comprises or consists of an SI domain, and wherein the one or more mutations associated with an XBB.1.5 variant are selected from: T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, and P681H, or any combination thereof, wherein the positions of the one or more mutations are shown relative to SEQ ID NO: 1.

17. The method of any one of claims 1-16, wherein the polypeptide comprising the fragment of the second SARS-CoV-2 S protein is delivered by administering an RNA that comprises a nucleotide sequence encoding the fragment of the second SARS-CoV-2 protein.

18. The method of any one of claims 1-16, wherein the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 3.

19. The method of any one of claims 1-18, wherein the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 5.

20. The method of any one of the preceding claims, wherein the polypeptide comprises a secretion signal.

21. The method of claim 20, wherein the secretion signal is a homologous secretion signal.

22. The method of claim 20, wherein the secretion signal is a heterologous secretion signal.

23. The method of any one of claims 20-22, wherein the secretion signal is present at or near the N-terminus of the polypeptide.

24. The method of claim 20, wherein the secretion signal is a SARS-CoV-2 S protein secretion signal, a gD2 secretion signal, a gDl secretion signal, a gBl secretion signal, a gI2 secretion signal, a gE2 secretion signal, an Eboz secretion signal, or an HLA-DR secretion signal.

25. The method of claim 24, wherein the SARS-CoV-2 S protein secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 15.

26. The method of claim 24, wherein the SARS-CoV-2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 9.

27. The method of claim 24, wherein the SARS-CoV-2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 16.

28. The method of claim 24, wherein the gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 8.

29. The method of claim 24, wherein the gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 13.

30. The method of claim 24, wherein the gDl secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 12.

31. The method of claim 24, wherein the gBl secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 37.

32. The method of claim 24, wherein the gC2 polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 35.

33. The method of claim 24, wherein the gI2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 11.

34. The method of claim 24, wherein the gE2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 38.

35. The method of claim 24, wherein the EboZ secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 39.

36. The method of claim 24, wherein the HLA-DR secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 40.

37. The method of any one of the preceding claims, wherein the polypeptide further a multimerization domain.

38. The method of claim 37, wherein the multimerization domain in the C-terminal region (e.g., at the C-terminus).

39. The method of claim 37 or 38, wherein the multimerization domain is a fibritin domain.

40. The method of claim 39, wherein the fibritin domain comprises a sequence that is at least 80% identical to SEQ ID NO: 95.

41. The method of claim 39, wherein the fibritin domain comprises a sequence that is at least 80% identical to SEQ ID NO: 96.

42. The method of any one of the preceding claims, wherein the polypeptide comprises a transmembrane (TM) domain.

43. The method of claim 42, wherein the TM domain is a homologous TM domain.

44. The method of claim 42, wherein the TM domain is a heterologous TM domain.

45. The method of any one of claims 42-44, wherein the TM domain is present in the C- terminal portion of the SARS-CoV-2 S protein variant or immunogenic portion thereof (e.g., at the C-terminus).

46. The method of claim 45, wherein the polypeptide comprises a multimerization domain and a TM domain at or near the C-terminus.

47. The method of claim 46, wherein the TM domain is C-terminal to the multimerization domain.

48. The method of claim 47, wherein:

(a) the multimerization domain is directly adjacent to the fragment of the second SARS- CoV-2 protein or connected to the fragment of the second SARS-CoV-2 protein via a flexible linker, and/or

49. The method of any one of claims 42-48, wherein the TM domain is a SARS-CoV-2 S protein TM domain or an influenza TM domain.

50. The method of claim 49, wherein the SARS-CoV-2 TM domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 89.

51. The method of claim 49, wherein the SARS-CoV-2 TM domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 90.

52. The method of any one of claims 17-51, wherein the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 120.

53. The method of any one of claims 17-52, wherein the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 130.

54. The method of any one of claims 17-52, wherein the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 135.

55. The method of any one of claims 17-52, wherein the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 145.

56. The method of any one of claims 17-52, wherein the RNA comprises a nucleotide sequence encoding a fragment of the second SARS-CoV-2 S protein comprising a sequence that is at least 80% identical to SEQ ID NO: 150.

57. The method of any one of claims 17-56, wherein the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein has been codon-optimized for expression in mammalian subjects.

58. The method of any one of claims 17-57, wherein the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein has been codon-optimized for expression in human subjects.

59. The method of any one of claims 17-58, wherein the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein has an enriched G/C content relative to wild- type sequence.

60. The method of claim 59, wherein G/C content has been increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%.

61. The method of any one of claims 17-60, wherein the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein comprises a heterologous 3’ UTR or 5’UTR.

62. The method of claim 61, wherein the heterologous 5' UTR comprises or consists of a modified human alpha-globin 5 '-UTR.

63. The method of claim 61 or 62, wherein the heterologous 3’ UTR comprises or consists of a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA.

64. The method of any one of claims 17-63, wherein the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein comprises a poly(A) sequence.

65. The method of claim 64, wherein the poly(A) sequence has a length of about 100-150 nucleotides.

66. The method of claim 64 or 65, wherein the poly (A) sequence is a disrupted poly(A) sequence.

67. The method of any one of claims 17-66, wherein the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein comprises a 5' cap.

68. The method of any one of claims 17-67, wherein the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 122 or 124.

69. The method of any one of claims 17-67, wherein the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 131 or 133.

70. The method of any one of claims 17-67, wherein the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 136 or 138.

71. The method of any one of claims 17-67, wherein the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 146 or 148.

72. The method of any one of claims 17-67, wherein the nucleotide sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 151 or 153.

73. The method of any one of claims 17-72, wherein the RNA is unmodified RNA.

74. The method of any one of claims 17-73, wherein the RNA comprises one or more modified nucleotides.

75. The method of claim 74, wherein the modified nucleotide is pseudouridine (e.g., Nl- methyl-pseudouridine).

76. The method of claim 74 or 75, wherein the RNA comprises a modified nucleotide in place of each uridine.

77. The method of any one of claims 17-76, wherein the RNA is an self-amplifying RNA or trans-amplifying RNA.

78. The method of any one of claims 17-77, wherein the RNA is fully or partially encapsulated within lipid nanoparticles (LNP), polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes.

79. The method of claim 78, wherein the RNA is fully or partially encapsulated within LNP.

80. The method of claim 79, wherein the LNP comprise a cationically ionizable lipid, a neutral lipid, a sterol and a lipid conjugate.

81. The method of any one of claims 1-80, wherein the first SARS-CoV-2 S protein is from a strain or variant that the subject was first exposed to and/or that was first prevalent in a population of subjects.

82. The method of any one of claims 1-81, wherein the first SARS-CoV-2 S protein is a Wuhan SARS-CoV-2 S protein or an Omicron BA.4/5 SARS-CoV-2 S protein.

83. The method of any one of claims 1-82, where the first SARS-CoV-2 S protein is from a strain or variant that the subject has previously been vaccinated against or is delivered by one or more vaccines that a significant proportion of the population (e.g., at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least abut 45%, at least about 50%, at least about 55%, or at least about 60%) has previously been administered.

84. The method of claim 83, wherein the vaccine previously administered to the subject or a significant proportion of the population was a first generation vaccine.

85. The method of claim 83, wherein the first SARS-CoV-2 S protein is from a SARS- CoV-2 strain or variant that was previously prevalent or is currently prevalent in a relevant jurisdiction.

86. The method of claim 83, wherein the first SARS-CoV-2 S protein is from a variant that first became prevalent in a relevant jurisdiction.

87. The method of any one of claims 1-86, wherein the immune response comprises a B cell immune response.

88. The method of claim 87, wherein the immune response comprises a naive B cell immune response.

89. The method of any one of claims 1-88, wherein:

(a) the immune response comprises a reduced memory B cell immune response as compared to an immune response induced by administering the full length sequence of the second SARS-CoV-2 S protein,

(b) the immune response comprises an increased naive B cell immune response as compared to an immune response induced by administering the full length sequence of the second SARS-CoV-2 S protein, and/or

90. The method of claim 89, wherein:

(b) the memory B cell immune response is increased by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% as compared to the immune response induced by a full length sequence of the second SARS-CoV-2 protein; and/or (c) the ratio of the naive immune response to the memory B cell immune response is increased by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% as compared to the immune response induced by a full length sequence of the second SARS-CoV-2 protein.