Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/53—DNA (RNA) vaccination
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/70—Multivalent vaccine
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/20011—Coronaviridae
  • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• Infectious diseases represent a major threat to human health and well-being.
• pathogenic microorganisms such as bacteria, viruses, parasites or fungi
• infectious diseases also known as communicable diseases
• Vaccines which are pharmaceutical preparations that provide or improve immunity to a particular disease, are useful to protect human subjects from certain infectious diseases.
• 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).
• 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.
• an immunogenic composition delivers an infectious agent antigen (e.g., comprises an infectious agent antigen or a nucleic acid encoding an infectious agent antigen).
• 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).
• a SARS-CoV-2 antigen e.g., comprises a SARS-CoV-2 antigen or a nucleic acid encoding a SARS-CoV-2 antigen.
• an immunogenic composition delivers an infectious agent antigen, or an immunogenic portion thereof.
• an immunogenic composition delivers a SARS-CoV-2 S protein, or an immunogenic portion thereof.
• 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.
• 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).
• technologies provided herein can partially or fully address and/or overcome an immune imprinting effect.
• 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).
• FIG. 1 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.
• 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.
• 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).
• 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).
• 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).
• 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.
• 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.
• 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.
• 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).
• 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).
• an immune response is or comprises a B cell immune response.
• a B cell immune response is or comprises an antibody response (e.g., neutralizing antibody response) to arisen epitopes in variant polypeptides.
• 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.
• circulating infectious disease is a bacterial infectious disease.
• such circulating infectious disease is a parasitic infectious disease.
• An exemplary parasitic infectious disease is malaria.
• such circulating infectious disease is a viral infectious disease.
• 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.
• the present disclosure 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.
• an antigen e.g., S protein of SARS-CoV-2
• 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).
• the present disclosure 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).
• the present disclosure provides technologies for increasing activation of naive B cell immune response to at least one of the arisen epitopes.
• arisen epitopes are neutralizing epitopes.
• the present disclosure provides technologies for inducing a priming-favorable cytokine milieu, for example, in lymphoid tissues.
• the present disclosure provides technologies for inducing a priming-favorable cytokine milieu, for example, in lymphoid tissues.
• induction of a priming-favorable cytokine milieu can be mediated through interferon alpha (IFNa).
• IFNa interferon alpha
• induction of a priming-favorable cytokine milieu can be mediated through a CD4+ T cell immune response.
• 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.
• a variant polypeptide comprises arisen epitopes.
• arisen epitopes are or comprise neutralizing epitopes (e.g., neutralizing antibody epitopes).
• 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).
• naive B cell immune response e.g., in some embodiments antibody response, e.g., neutralizing antibody response
• the arisen epitopes e.g., in some embodiments at least one of the neutralizing epitopes.
• 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.
• a non-modified RNA encodes at least one or more T cell epitopes.
• such a non-modified RNA encodes at least one or more B cell epitopes.
• 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).
• an infectious agent e.g., a SARS-CoV-2 Spike (S) protein
• an immunogenic portion thereof wherein a B cell memory immune response has been established to the reference anti
• an antigen of an infectious agent 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.
• a reference antigen of the infectious agent e.g., a SARS-CoV-2 S protein
• 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).
• 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).
• an amino acid sequence encoded by an RNA comprises no more than 50% of the B cell memory epitopes present in a reference antigen.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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).
• 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.
• conserved B cell epitopes are non-neutralizing epitopes.
• one or more memory B cell epitopes comprise or consist of non-neutralizing epitopes and neutralizing epitopes.
• 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.
• an infectious agent has a high mutation rate.
• an infectious agent has a high number of variants or species.
• an infectious agent has a large number of variants or species, many of which are immune escaping.
• an infectious agent is a virus, bacteria, or Plasmodium.
• an infectious agent is a virus.
• a virus is a respiratory virus.
• a virus is an influenza virus, RSV, a norovirus, or HIV.
• an infectious agent is a coronavirus.
• a coronavirus is a betacoronavirus.
• a coronavirus is a MERS, SARS, or SARS-CoV-2 virus.
• a plasmodium is P. falciparum, P. vivax, P. ovale, or P. malariae.
• a variant polypeptide lacks regions that are not mutated frequently in immune-escaping variants of the infectious agent.
• 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).
• 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.
• conserved memory B cell epitopes are non-neutralizing epitopes.
• 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.
• an immunogenic portion of a coronavirus S protein does not comprise an S2 domain.
• an immunogenic portion of a SARS-CoV-2 S protein variant comprises or consists of the SI domain or the receptor binding domain (RBD).
• an immunogenic portion of a coronavirus S protein does not comprise an N-terminal domain (NTD).
• an immunogenic portion of a SARS-CoV-2 S protein variant comprises or consists of the RBD.
• 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.
• a reference SARS-CoV-2 S protein was previously delivered by a vaccine.
• the vaccine is a commercially approved vaccine, a protein-based vaccine, an RNA vaccine, or any combination thereof.
• a reference SARS-CoV-2 S protein is a Wuhan S protein.
• a reference SARS-CoV-2 S protein is an Omicron BA.4/5
• 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).
• 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.
• a SARS-CoV-2 variant with a high immune escape potential is an Omicron variant.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• a variant polypeptide comprises a secretion signal.
• a secretion signal is a homologous secretion signal.
• a secretion signal is a heterologous secretion signal.
• an antigen of an infectious agent or immunogenic portion thereof encoded comprises a hypervariable domain.
• a hypervariable domain has a high density of neutralization epitopes.
• a hypervariable domain is a region that is frequently mutated in variants of the infectious agent that have a high immune escape potential.
• a hypervariable domain is a receptor binding domain (RBD).
• RBD receptor binding domain
• a hypervariable domain comprises or consists of an RBD or S 1 domain of a coronavirus S protein.
• 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).
• a surface protein or surface glycoprotein is a coronavirus S protein.
• a variant polypeptide has been engineered to eliminate one or more memory B cell epitopes of a reference antigen.
• 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).
• one or more memory B cell epitopes comprise or consist of non-neutralizing epitopes.
• one or more memory B cell epitopes comprise or consist of non-neutralizing epitopes and neutralizing epitopes.
• a variant polypeptide comprises few intact memory B cell epitopes of the reference antigen.
• 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.
• an in vitro assay e.g., a viral neutralization assay
• in silico analysis e.g., sequence analysis and/or molecular dynamic simulations
• 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).
• a vaccine e.g., a commercially available vaccine and/or a vaccine previously administered to a subject.
• 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.
• a variant polypeptide comprises a secretion signal.
• a secretion signal is a homologous secretion signal. In some embodiments, a secretion signal is a heterologous secretion signal.
• a secretion signal is present in the N-terminal portion of the polypeptide (e.g., at the N-terminus of the polypeptide).
• 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.
• a SARS-CoV-2 S protein secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 15.
• a SARS-CoV-2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 9 or 16.
• a gDl secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 12.
• a gBl secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 37.
• 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.
• a gE2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 38.
• an EboZ secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 39.
• an HLA-DR secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 40.
• a variant polypeptide e.g., a SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises a multimerization domain.
• 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).
• a polypeptide comprises a multimerization domain that is C-terminal to the variant polypeptide.
• a multimerization domain is a fibritin domain.
• a fibritin domain comprises a sequence that is at least
• a SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises a transmembrane (TM) domain.
• a variant polypeptide comprises a transmembrane (TM) domain.
• TM domain is a homologous TM domain.
• TM domain is a heterologous TM domain.
• a TM domain is present in the C-terminal portion of the polypeptide (e.g., at the C-terminus).
• 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)).
• the TM domain is C-terminal to the multimerization domain
• 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)).
• an RNA comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 120.
• 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.
• an RNA comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 150.
• a nucleotide sequence that encodes a SARS-CoV-2 S protein variant (or immunogenic portion thereof) is codon-optimized for expression in mammalian subjects.
• a nucleotide sequence that encodes a SARS-CoV-2 S protein variant is codon-optimized for expression in human subjects.
• 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.
• a nucleotide sequence that encodes a variant polypeptide or the polypeptide is codon-optimized for expression in mammalian subjects.
• a nucleotide sequence that encodes a variant polypeptide or an immunogenic portion thereof has been codon-optimized for expression in human subjects.
• 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%.
• an RNA comprises a poly(A) sequence.
• a poly(A) sequence is a disrupted poly(A) sequence.
• an RNA comprises a 5' cap.
• an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 136 or 138.
• an RNA is unmodified RNA.
• an RNA comprises one or more modified nucleotides.
• an RNA comprises a modified nucleotide in place of each uridine.
• an RNA is an mRNA, a self-amplifying RNA or a trans- amplifying RNA.
• an RNA is fully or partially encapsulated within LNP.
• an LNP comprises a cationically ionizable lipid, a neutral lipid, a sterol and a lipid conjugate.
• 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.
• RNA described herein or a composition described herein comprising administering an RNA described herein or a composition described herein to a subject.
• a method of inducing an immune response in a subject who has previously been exposed to a reference antigen of an infectious agent 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.
• a variant polypeptide of the reference antigen e.g., SARS-CoV-2
• an infectious agent e.g., SARS-CoV-2
• an infectious agent is an influenza virus, RSV, norovirus, HIV, coronavirus, or a plasmodium.
• 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) .
• the reference antigen e.g., SARS-CoV-2
• a reference SARS-CoV-2 S protein is a Wuhan SARS- CoV-2 S protein.
• an immune response comprises a naive B cell immune response.
• an immune response comprises a reduced memory B cell immune response or an immune response does not comprise a memory B cell immune response.
• 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) .
• an infectious agent e.g., SARS-CoV-2
• 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-Co
• a variant polypeptide comprises a sequence that corresponds to an immunogenic portion of a reference antigen.
• 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) .
• a reference antigen e.g, SARS-CoV-2
• 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.
• 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 (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).
• a reference antigen of the infectious agent e.g, SARS-CoV- 2
• a variant polypeptide of the reference antigen e.g, SARS-CoV-2 variant
• an immunogenic composition that delivers the variant polypeptide (e.g, SARS-CoV-2 variant).
• 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.
• 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.
• 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.
• infectious agent e.g, SARS-CoV-2
• 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%
• a vaccine previously administered to a subject or a large portion of a population was a first generation vaccine.
• 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.
• a reference antigen is from an infectious agent variant (e.g, SARS-CoV-2) that first became prevalent in a relevant jurisdiction.
• 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.
• 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).
• 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).
• 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).
• 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.
• 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
• an agent that induces a priming-favorable cytokine milieu in lymphoid tissues is or comprises interferon alpha (IFN ⁇ ) or an IFN ⁇ -inducing agent.
• an agent that induces a priming-favorable cytokine milieu in lymphoid tissues is or comprises a CD4+ T cell response inducing agent.
• 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).
• a reference antigen is a SARS-CoV-2 S protein of a Wuhan strain or an Omicron BA.4/5 strain.
• a reference antigen is a SARS-CoV-2 S protein of a XBB strain (e.g., XBB1, XBB1.5).
• a modified RNA molecule and an agent are co-delivered.
• an IFN ⁇ -inducing agent is or comprises an unmodified RNA molecule.
• the amount ratio of the modified RNA molecule to the unmodified RNA molecule is at least or greater than 1:1.
• the ratio of modified ribonucleotides to unmodified ribonucleotides in the immunogenic composition is about 1:2 to about 1:10.
• a modified ribonucleotide is 1- methylpseudouridine and an unmodified ribonucleotide is uridine.
• an unmodified RNA molecule encodes a polypeptide comprising an antigen of an infectious agent (e.g., SARS-CoV-2).
• an antigen is a B-cell antigen.
• an antigen is a T-cell antigen.
• 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.
• 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.
• 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.
• an IFNoc-inducing agent is or comprises a self-amplifying RNA molecule or a trans-amplifying RNA molecule.
• 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.
• PLX polyplexes
• LPLX lipidated polyplexes
• oligo- or poly-saccharide particles or liposomes.
• a modified RNA molecule comprises modified uridines.
• a modified RNA molecule comprises a modified uridine in lieu of each uridine.
• modified uridines are or comprise 1 -methyl pseudouridine.
• a modified RNA molecule encodes a polypeptide comprising an antigen of the infectious agent.
• an antigen is a B-cell antigen.
• an antigen is a T-cell antigen.
• 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
• an agent encodes a polypeptide comprising an antigen of the infectious agent (e.g., SARS-CoV-2) .
• an antigen is a B-cell antigen.
• an antigen is a T-cell antigen.
• 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.
• 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).
• an infectious agent e.g., SARS-CoV-2
• 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.
• modified ribonucleotides are 1 -methylpseudouridine and the corresponding unmodified ribonucleotides are uridine.
• 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.
• a composition that comprises or delivers a polypeptide antigen (e.g., SARS-CoV-2)
• 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.
• 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.
• a subject has previously been exposed to a variant of the antigen (e.g., SARS-CoV-2).
• a variant of the antigen e.g., SARS-CoV-2
• a step of exposing comprises administering a composition that comprises or delivers the antigen.
• an antigen is a polypeptide antigen.
• a step of exposing comprises administering a “priming adjuvant” to a subject who is or will soon be exposed to the antigen.
• 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.
• a first antigen e.g., SARS-CoV-2 antigen
• a second RNA molecule encoding a second antigen
• 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.
• first antigens e.g.
• a first RNA molecule comprises modified uridines.
• modified uridines are in place of all uridines.
• a second RNA molecule does not comprise a modified ribonucleotide.
• 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.
• a B cell antigen is a CoV-2 S antigen or immunogenic portion thereof.
• a T cell antigen is from the same infectious agent.
• a T cell antigen is a T string epitope.
• a T cell antigen is from SARS-CoV-2.
• a B cell antigen of SARS-CoV-2 is SARS-CoV-2 S antigen or immunogenic portion thereof.
• a first RNA molecule and a second RNA molecule are co-administered.
• 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.
• RNA molecule and a second RNA molecule are separately administered.
• 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.
• a reference antigen is a SARS-CoV-2 S protein of a Wuhan strain or an Omicron BA.4/5 strain.
• a reference antigen is a SARS-CoV-2 S protein of a XBB strain.
• a XBB strain is a XBBl or XBB 1.5.
• 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.
• S SARS-CoV-2 Spike
• 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.
• 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.
• 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.
• 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.
• 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.
• 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
• the SARS-CoV-2 variant is an Omicron variant.
• 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.
• 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.
• 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.
• the fragment of the second SARS-CoV-2 S protein comprises or consists of an S 1 domain
• 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.
• 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.
• 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.
• the polypeptide comprises a secretion signal.
• the secretion signal is a homologous secretion signal.
• the secretion signal is a heterologous secretion signal.
• the secretion signal is present at or near the N-terminus of the polypeptide.
• 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.
• the SARS-CoV-2 S protein secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 15.
• the SARS-CoV-2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 9.
• 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.
• the gC2 polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 35.
• the gI2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 11.
• the gE2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 38.
• the EboZ secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 39.
• the HLA-DR secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 40.
• the polypeptide further a multimerization domain.
• the multimerization domain in the C-terminal region e.g., at the C- terminus.
• the multimerization domain is a fibritin domain.
• the fibritin domain comprises a sequence that is at least 80% identical to SEQ ID NO: 95.
• the fibritin domain comprises a sequence that is at least 80% identical to SEQ ID NO: 96.
• the polypeptide comprises a transmembrane (TM) domain.
• the TM domain is a homologous TM domain.
• the TM domain is a heterologous TM domain.
• 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).
• the polypeptide comprises a multimerization domain and a TM domain at or near the C-terminus.
• the TM domain is C-terminal to the multimerization domain.
• 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.
• the TM domain is a SARS-CoV-2 S protein TM domain or an influenza TM domain.
• the SARS-CoV-2 TM domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 89.
• the SARS-CoV-2 TM domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 90.
• 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.
• 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.
• 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.
• 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.
• 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%.
• the nucleotide sequence encoding the fragment of the second SARS-CoV-2 S protein comprises a heterologous 3’ UTR or 5’UTR.
• the heterologous 5' UTR comprises or consists of a modified human alpha-globin 5'-UTR.
• 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.
• 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.
• 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.
• the RNA is unmodified RNA.
• the RNA comprises one or more modified nucleotides.
• the modified nucleotide is pseudouridine (e.g., Nl-methyl-pseudouridine).
• the RNA comprises a modified nucleotide in place of each uridine.
• the RNA is an self-amplifying RNA or trans-amplifying RNA.
• the RNA is fully or partially encapsulated within lipid nanoparticles (LNP), polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes.
• LNP lipid nanoparticles
• PLX polyplexes
• LPLX lipidated polyplexes
• oligo- or poly-saccharide particles or liposomes.
• the RNA is fully or partially encapsulated within LNP.
• the LNP comprise a cationically ionizable lipid, a neutral lipid, a sterol and a lipid conjugate.
• 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.
• 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.
• 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.
• 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%
• the vaccine previously administered to the subject or a significant proportion of the population was a first generation vaccine.
• 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.
• the immune response comprises a B cell immune response. In some embodiments, the immune response comprises a naive B cell immune response.
• 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
• 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
• the ratio of the naive B cell immune response to the memory B cell immune response is increased.
• 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;
• 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.
• 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.
• WT wild-type
• 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.
• FIG. 2 Exemplary characterization tools for assessing memory B cell response, which can be useful to evaluate immune imprinting phenomenon.
• A Schematic of one- dimensial flow-cytometry analysis of memory B cell (BMEM) phenotyping using fluorochrome- labeled antigens
• BMEM memory B cell
• Figure 2 Schematic of one- dimensial flow-cytometry analysis of memory B cell (BMEM) phenotyping using fluorochrome- labeled antigens
• 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).
• an antigen or subdomain thereof e.g., RBD or SI domain in the case of a SARS-CoV-2 S protein
• 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).
• FIG. 3 Experimental design for assessing impact of immune imprinting.
• 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.
• 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.
• Fig. 4 Variant-induced broad neutralization can be mediated by expansion of responses against conserved epitopes (i.e. recall responses).
• conserved epitopes i.e. recall responses.
• a new variant of a infectious agent Omicron BA.l in the Figure
• 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.
• 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.
• BNT162b2 2 + 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.
• BNT162b2 3 + 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.
• 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 (“BNT162b2 3 ”) and showing no evidence of prior SARS-CoV-2 infection, (ii) administered four doses of BNT162b2 (“BNT162b2 4 ”) 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-Vax 3 + BA.2”), or (v) administered three doses of an RNA vaccine and who experienced a subsequent Omicron BA.4/5 breakthrough infection (“mRNA-Vax 3 + BA.4/5”).
• a pseudovirus neutralization assay comprising a Wuhan Spike protein (Wuhan-pVNT) or an Omicron BA.1 Spike protein (Omicron BA.l-pVNT).
• 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).
• BMEM Memory B cells
• 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
• XBB new SARS-CoV-2 variants of concern
• an RBD of an VOC attached to a trimerization domain e.g., an RBD of XBB.1.5 attached to a T4 foldon domain
• 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
• 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
• TM transmembrane
• 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).
• 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.
• BNT162b2 encoding an S protein of a Wuhan variant
• 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)
• 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)).
• BNT162b2 RNA encoding
• 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.
• 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.
• 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").
• 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.
• 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. "BNT162b2 2 " 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.
• 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.
• 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).
• 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.
• shorter constructs delivering only the RBD were more efficient in generating higher neutralization titers as compared to other constructs.
• FIG. 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).
• 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.
• b3 membrane anchored RBD
• FIG. 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).
• CD95 + indicates B cell activation, serving as a proxy to show that they recently undergo Germinal Center (GM) reaction.
• FIG. 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.
• 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.
• FIG. 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
• B shows the number of clonotypes of CD19 + B cells that bind Wuhan Spike, XBB.1.5 Spike, or both.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• an amino acid has the general structure H2N-C(H)(R)-COOH.
• an amino acid is a naturally- occurring amino acid.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• an antibody agent refers to an agent that specifically binds to a particular antigen.
• the term encompasses a polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding.
• 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.
• CDR complementarity determining region
• 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.
• 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.
• 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.
• 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.
• 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.
• an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain.
• 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).
• 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 (“SMIPsTM ); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®;
• SMIPsTM Small Modular ImmunoPharmaceuticals
• an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally.
• 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.)).
• 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.)).
• 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.
• an antigen-specific immune response may be or comprise generation of antibodies and/or antigen-specific T cells.
• an antigen is a peptide or polypeptide that comprises at least one epitope against which an immune response can be generated.
• an antigen is presented by cells of the immune system such as antigen presenting cells like dendritic cells or macrophages.
• 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).
• an antigen is a parasitic antigen.
• an antigen may be delivered by RNA molecules as described herein.
• 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.
• 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.
• 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.
• an antigen is presented by a diseased cell such as a virus-infected cell.
• an antigen receptor is a TCR which binds to an epitope of an antigen presented in the context of MHC.
• 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.
• 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.
• 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.
• a particular entity e.g., polypeptide, genetic signature, metabolite, microbe, etc
• a particular entity e.g., polypeptide, genetic signature, metabolite, microbe, etc
• 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.
• 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.
• binding- typically refers to a non-covalent association between or among entities or moieties.
• binding data are expressed in terms of “IC50”.
• 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.
• assays are run under conditions in which (e.g., limiting binding target and reference concentrations), IC50 values approximate KD values.
• 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.
• 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).
• a cap is or comprises a guanine nucleotide.
• 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.”
• 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).
• ARC As anti -reverse cap analogs
• 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).
• 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.
• 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
• 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.
• Co-administration refers to use of a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) described herein and an additional therapeutic agent.
• a pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
• an additional therapeutic agent may be performed concurrently or separately (e.g., sequentially in any order).
• a pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
• 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.
• compositions e.g., immunogenic composition, e.g., vaccine
• 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.
• 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.
• coding regions are codon-optimized for optimal expression in a subject to be treated using the RNA molecules described herein.
• codon-optimization may be performed such that codons for which frequently occurring tRNAs are available are inserted in place of “rare codons.”
• 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.
• G/C guanosine/cytosine
• conserveed 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.
• 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).
• 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.
• “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.
• 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.
• 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.
• 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.
• the term “corresponding to” refers to a relationship between two or more entities.
• 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).
• a monomeric residue in a polymer e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide
• a residue in an appropriate reference polymer may be identified as “corresponding to” a residue in an appropriate reference polymer.
• 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.
• 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.
• 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, ScalaBL
• 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).
• 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.
• derived from refers to a structural analogue of a designated amino acid sequence.
• 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.
• 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.
• Dosing regimen 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.
• 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.
• 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.
• 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.
• a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (z.e., is a therapeutic dosing regimen).
• Encode 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.
• 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).
• a gene, a cDNA, or an RNA molecule encodes a polypeptide if transcription and translation of mRNA corresponding to that gene produces the polypeptide in a cell or other biological system.
• 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.
• 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.
• Engineered refers to the aspect of having been manipulated by the hand of man.
• 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.
• Epitope refers to a moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component.
• an epitope may be recognized by a T cell, a B cell, or an antibody.
• an epitope is comprised of a plurality of chemical atoms or groups on an antigen.
• such chemical atoms or groups are surface-exposed when the antigen adopts a relevant three-dimensional conformation.
• such chemical atoms or groups are physically near to each other in space when the antigen adopts such a conformation.
• an epitope of an antigen may include a continuous or discontinuous fragment of the antigen.
• an epitope is or comprises a T cell epitope.
• 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.
• a gene product can be a transcript.
• a gene product can be a polypeptide.
• 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.
• Five prime untranslated region refers to a sequence of an mRNA molecule between a transcription start site and a start codon of a coding region of an RNA.
• “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.
• 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.
• a reference sequence is a full-length sequence of e.g. a nucleic acid sequence or an amino acid sequence.
• a fragment typically, refers to a sequence that is identical to a corresponding stretch within a reference sequence.
• 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.
• 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.
• 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.
• homolog refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
• polynucleotide molecules e.g., DNA molecules and/or RNA molecules
• 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.
• polynucleotide molecules e.g., DNA molecules and/or RNA molecules
• 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).
• 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.
• 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.
• Identity refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
• polynucleotide molecules e.g., DNA molecules and/or RNA molecules
• 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 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).
• 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.
• 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).
• 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.
• 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.
• the term “immunologically equivalent” is used with respect to the immunological effects or properties of antigens or antigen variants used for immunization.
• 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.
• 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.
• 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).
• immune escape potential refers to a likelihood of a given variant being able to evade previously developed immune responses.
• 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).
• an immune escape potential can be determined using one or more in vitro assay(s) (e.g., neutralization assays as described herein).
• 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.).
• Increased, Induced, or Reduced indicate values that are relative to a comparable reference measurement.
• an assessed value achieved with a provided pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
• a comparable reference pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
• 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.).
• a pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
• 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.
• 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.
• the term “reduced” or equivalent terms refers to a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero.
• 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.
• Ionizable refers to a compound or group or atom that is charged at a certain pH.
• an ionizable amino lipid such a lipid or a function group or atom thereof bears a positive charge at a certain pH.
• an ionizable amino lipid is positively charged at an acidic pH.
• 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.
• an ionizable amino lipid may have a pKa within a range of about 5 to about 7.
• Isolated means altered or removed from the natural state.
• 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.
• 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.
• a modified amino acid or nucleotide sequence refers to a change relative to a reference sequence.
• 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).
• 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).
• a modified amino acid or nucleotide sequence comprises a substitution.
• RNA lipid nanoparticle refers to a nanoparticle comprising at least one lipid and RNA molecule(s).
• an RNA lipid nanoparticle comprises at least one ionizable amino lipid.
• 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).
• 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.
• Z-average average size
• 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.
• an average size of lipid nanoparticles is determined by measuring the particle diameter.
• RNA lipid nanoparticles may be prepared by mixing lipids with RNA molecules described herein.
• lipidoid refers to a lipid-like molecule.
• a lipoid is an amphiphilic molecule with one or more lipid-like physical properties.
• the term lipid is considered to encompass lipidoids.
• Nanoparticle refers to a particle having an average size suitable for parenteral administration.
• a nanoparticle has a longest dimension (e.g., a diameter) of less than 1,000 nanometers (nm).
• a nanoparticle may be characterized by a longest dimension (e.g., a diameter) of less than 300 nm.
• a nanoparticle may be characterized by a longest dimension (e.g., a diameter) of less than 100 nm.
• 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.
• 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.
• a nanoparticle may be substantially spherical so that its longest dimension may be its diameter.
• a nanoparticle has a diameter of less than 100 nm as defined by the National Institutes of Health.
• Naturally occurring 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.
• 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.
• 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.
• 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.
• a nucleic acid particle is a lipid nanoparticle.
• a nucleic acid particle is a lipoplex particle.
• nucleic acid refers to a polymer of at least 10 nucleotides or more.
• a nucleic acid is or comprises DNA.
• a nucleic acid is or comprises RNA.
• a nucleic acid is or comprises peptide nucleic acid (PNA).
• PNA peptide nucleic acid
• a nucleic acid is or comprises a single stranded nucleic acid.
• a nucleic acid is or comprises a double-stranded nucleic acid.
• a nucleic acid comprises both single and double-stranded fragments.
• 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”.
• 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.
• natural residues e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxy thymidine, guanine, thymine, uracil.
• 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).
• 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.
• a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide.
• a nucleic acid has a nucleotide sequence that comprises one or more introns.
• 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.
• 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.
• 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.
• 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.
• a 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.
• animals e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans.
• a patient is
• PEG-conjugated lipid refers to a molecule comprising a lipid portion and a polyethylene glycol portion.
• Pharmaceutical composition refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers.
• 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.
• 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.
• compositions comprising: 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.
• 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.
• 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.
• compositions e.g., immunogenic compositions, e.g., vaccines
• an effective amount of pharmaceutical compositions 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.
• 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.
• Polypeptide refers to a polymeric chain of amino acids.
• a polypeptide has an amino acid sequence that occurs in nature.
• a polypeptide has an amino acid sequence that does not occur in nature.
• 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.
• a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both.
• a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids.
• 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.
• 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.
• 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.
• 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).
• 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%.
• a conserved region that may in some embodiments be or comprise a characteristic sequence element
• 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.
• a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide.
• 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.
• Receptor Binding Domain 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.
• 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).
• 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.
• reference describes a standard or control relative to which a comparison is performed.
• 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.
• a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest.
• a reference or control is a historical reference or control, optionally embodied in a tangible medium.
• a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment.
• a reference antigen is an antigen that a subject has previously encountered or has a high likelihood of having previously encountered.
• a reference antigen is an antigen delivered by a vaccine that was previously administered to a subject.
• 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.
• a reference antigen is an antigen of the first strain or variant of an infectious agent that a subject encountered.
• a reference antigen is an antigen of a strain or variant of an infectious agent that first became prevalent.
• 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).
• a reference antigen is a Wuhan Spike protein, or an immunogenic portion thereof.
• a reference antigen is a Spike protein of an Omicron variant (e.g., a BA.4/5 Omicron variant), or an immunogenic portion thereof.
• RNA Ribonucleic acid
• an RNA refers to a polymer of ribonucleotides.
• an RNA is single stranded.
• an RNA is double stranded.
• an RNA comprises both single and double stranded fragments.
• 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.
• RNA typically comprises at its 3’ end a poly(A) region.
• 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.
• 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).
• Ribonucleotide encompasses unmodified ribonucleotides and modified ribonucleotides.
• 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.
• end modifications e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.)
• base modifications
• 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.
• relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
• 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.
• risk may reflect one or more epigenetic events or attributes and/or one or more lifestyle or environmental events or attributes.
• 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.
• positively charged liposomes may comprise a cationic lipid, such as in some embodiments DOTMA, and additional lipids, such as in some embodiments DOPE.
• a RNA lipoplex particle is a nanoparticle.
• 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.
• 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.
• 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.
• a stable pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
• 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.
• a stable pharmaceutical composition 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.
• a stable pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
• a pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
• a pharmaceutical composition remains stable for a specified period of time under certain conditions.
• 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).
• a disease, disorder, or condition e.g., a HSV infection
• 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.
• 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.
• 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.
• Synthetic refers to an entity that is artificial, or that is made with human intervention, or that results from synthesis rather than naturally occurring.
• 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.
• the term “synthetic” refers to an entity that is made outside of biological cells.
• 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.
• 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.
• 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.
• a medical intervention e.g., surgery, radiation, phototherapy
• Three prime untranslated region refers 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.
• 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.
• 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).
• 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.
• a threshold level can be a range of values.
• Treat 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.
• 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.
• treatment may be administered to a subject at a later-stage of disease, disorder, and/or condition.
• 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.
• 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.
• vaccination includes multiple administrations, appropriately spaced in time, of a vaccine composition.
• vaccination generates an immune response to an infectious agent.
• 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.
• 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.
• 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).
• moieties e.g., carbohydrates, lipids, phosphate groups
• 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%.
• a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid.
• a reference polypeptide or nucleic acid has one or more biological activities.
• a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid.
• 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.
• 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.
• 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.
• 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.
• 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.
• a reference polypeptide or nucleic acid is one found in nature.
• Vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
• plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
• viral vector 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).
• vectors e.g., non-episomal mammalian vectors
• 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.
• 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.”
• 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 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.
• 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).
• 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.
• an immune response is or comprises a B cell immune response.
• a B cell immune response is or comprises an antibody response (e.g., neutralizing antibody response) to arisen epitopes in variant polypeptides.
• variant polypeptides are from various infectious agents.
• RNA viruses are available that can provide protection from infection.
• 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.
• RNA viruses 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.
• 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.
• HIV human immunodeficiency virus
• Infectious agent surface proteins and/or surface glycoproteins 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 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.
• 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.
• nAbs neutralizing antibodies
• vulnerable epitopes e.g., virus epitopes
• the nAb titer often correlating with the degree of protection against future infections.
• 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.
• 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.
• viruses experience continued pressure to evolve mutations in vulnerable epitopes toward acquiring the ability to escape existing antibodies and re-infect hosts.
• 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.
• Infectious agents have developed various means of evading or subverting host defenses.
• an infectious agent is a virus, a bacteria, or a eukaryotic cell (e.g., a plasmodium).
• an infectious agent is a respiratory virus.
• an infectious agent is an RNA virus.
• an infectious agent is a coronavirus (e.g., MERS, SARS, or SARS-CoV-2).
• an infectious agent is HIV.
• an infectious agent is HSV (e.g., HSV-1 or HSV-2).
• an infectious agent is RSV.
• an infectious agent is a norovirus.
• an infectious agent is an influenza virus.
• an infectious agent is P. falciparum.
• 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.
• an infectious agent is a bacterium.
• the bacterium is Mycobacterium.
• the bacterium is selected from Haemophilus influenzae, Chlamydophila pneumoniae, Mycoplasma pneumonia, Staphylococcus aureus, Moraxella catarrhalis, Legionella pneumophila, and Streptococcus pneumonia.
• the bacterium is Streptococcus pneumonia.
• 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).
• 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).
• an infectious agent is prone to immune escape.
• an infectious agent is one for which seasonal, variant- adapted booster shots are regularly provided.
• an infection agent is associated with a circulating infectious disease (e.g., for which variants can be expected to arise).
• Exemplary viral infectious diseases include, but are not limited to coronavirus, ebolavirus, influenza viruses, norovirus, rotavirus, respiratory syncytial virus, alphaherpesvirus, etc.
• an antigen described herein is or comprises a B cell antigen.
• a B cell antigen comprises one or more antibody epitopes.
• such epitopes are antibody binding epitopes.
• such epitopes are antibody neutralizing epitopes.
• an antigen described herein is or comprises an antigen of an infectious agent.
• an infection agent is associated with a circulating infectious disease (e.g., for which variants can be expected to arise).
• circulating infectious disease is a bacterial infectious disease.
• circulating infectious disease is a parasitic infectious disease.
• An exemplary parasitic infectious disease is malaria.
• such circulating infectious disease is a viral infectious disease.
• 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.
• an antigen e.g., SARS-CoV-2
• a B cell antigen comprises one or more antibody epitopes.
• such epitopes are antibody binding epitopes.
• such epitopes are antibody neutralizing epitopes.
• an antigen described herein is or comprises a T cell antigen.
• a T cell antigen comprises one or more CD4 T cell and/or one or more CD8 T cell epitopes.
• 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.
• 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).
• utilized sequences comprise one or more mutations associated with a variant of concern (e.g., a variant of concern identified by WHO).
• 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).
• an antigen described herein is or comprises a surface protein or a surface glycoprotein of an infectious agent (e.g., SARS-CoV-2) .
• 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.
• 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).
• a vaccine e.g., a commercially available vaccine, an RNA vaccine, or a protein- based vaccine.
• an infectious agent antigen is solvent exposed on the surface of the infectious agent.
• an infectious agent antigen is a glyocoprotein.
• an infectious agent antigen is involved in host cell recognition.
• an infectious agent antigen is involved in host cell entry.
• an infectious agent antigen comprises one or more B cell epitopes (e.g., one or more neutralization epitopes).
• 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.
• a composition delivers a hypervariable domain.
• a “hypervariable domain” refers to a domain or region having a high frequency of mutation.
• a hypervariable domain comprises a high number of mutations relative to other polypeptide encoded by the infectious agent.
• 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).
• 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).
• 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.
• 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.
• 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.
• S spike
• RBD receptor binding domain
• NTD N-terminal domain
• M membrane protein
• E envelope protein
• nucleocapsid protein or combinations thereof.
• 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).
• such a SARS-CoV-2 S protein or an immunogenic fragment or portion thereof is from a Wuhan strain or an Omicron BA.4/5 strain.
• such a SARS-CoV-2 S protein or an immunogenic fragment or portion thereof is from a XBB strain (e.g., XBB1, XBB1.5 or sublineages thereof).
• an antigen described herein is an antigen from an influenza virus.
• an antigen described herein is from influenza A virus, including, e.g., but not limited to A(H1N1), A(H3N2), etc.
• an antigen described herein is from influenza B virus, including, e.g., B(Victoria), B(Yamagata), etc.
• an antigen described herein is from influenza C virus.
• 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.
• an antigen described herein is an antigen from a respiratory syncytial virus (RSV), e.g., as described herein.
• RSV respiratory syncytial virus
• 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.
• 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.
• an antigen described herein is an antigen from malarial polypeptide (e.g., as described herein).
• an antigen described herein is an antigen from a virus in the genus Orthopoxvirus.
• 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.
• an antigen described herein is an antigen from Herpes simplex virus (e.g., HSV-1 and HSV-2), for example, as described herein.
• Herpes simplex virus e.g., HSV-1 and HSV-2
• an antigen described herein is useful as a reference antigen of an infectious agent.
• an antigen described herein is or comprises a variant polypeptide of a reference antigen of an infectious agent, or an immunogenic portion thereof.
• an antigen described herein is or comprises a full-length polypeptide antigen of an infectious agent.
• an antigen described herein is or comprises an immunogenic fragment, portion, or domain of a polypeptide antigen of an infectious agent.
• 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.
• 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.
• an antigen described herein is an engineered antigen.
• an engineered antigen is designed to promote tailored immune responses.
• 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.
• 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.
• SARS-CoV-2 severe acute respiratory syndrome coronavirus-2
• SARS-CoV-2 MN908947.3 belongs to betacoronavirus lineage B. It has at least 70% sequence similarity to SARS-CoV.
• coronaviruses have four structural proteins, namely, envelope (E), membrane (M), nucleocapsid (N), and spike (S).
• 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.
• 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.
• 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.
• RBD receptor-binding domain
• COVID-19 The presentation of COVID-19 is generally with cough and fever, with chest radiography showing ground-glass opacities or patchy shadowing.
• many patients present without fever or radiographic changes, and infections may be asymptomatic which is relevant to controlling transmission.
• progression of disease may lead to acute respiratory distress syndrome requiring ventilation and subsequent multi-organ failure and death.
• Common symptoms in hospitalized patients 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.
• 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.
• 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.
• 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.
• 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.
• 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).).
• 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).
• a SARS-CoV-2 antigen for use in inducing an immunogenic response.
• 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).
• such antigens are delivered as protein antigens to induce an immunogenic response.
• 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.
• 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
• 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).
• 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.
• a SARS-CoV-2 S protein may be stabilized by introducing one or more proline mutations.
• S protein comprises a proline substitution at positions corresponding to residues 986 and/or 987 of SEQ ID NO: 1.
• 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
• 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.
• 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.
• 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.
• 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:
• 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.
• 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 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.
• 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.
• 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 ( “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 ( “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.
• 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.
• 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”).
• compositions described herein deliver an immunogenic portion of a full length coronavirus S protein (e.g., SARS-CoV-2 S protein).
• a full length coronavirus S protein e.g., SARS-CoV-2 S protein.
• 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).
• 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.
• an immunogenic portion of a coronavirus (e.g., SARS-CoV-2) S protein lacks a full S2 domain.
• 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.
• an immunogenic portion of a coronavirus (e.g., SARS-CoV-2) S protein lacks the entire S2 domain.
• 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).
• 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.
• a coronavirus e.g., SARS-CoV-2
• SARS-CoV-2 coronavirus-2
• 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).
• 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.
• an immunogenic portion of a coronavirus (e.g., SARS-CoV-2) S protein comprises an N-terminal domain (NTD) of the S protein.
• an immunogenic portion of a coronavirus (e.g., SARS-CoV-2) protein comprises a receptor binding domain (RBD) of the S protein.
• an immunogenic portion of a coronavirus (e.g., SARS-CoV-2) S protein comprises an S 1 domain of the S protein.
• 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.
• Coronavirus e.g., SARS-CoV-2
• 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).
• 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.
• an RBD of a coronavirus (e.g., SARS-CoV-2) S protein comprises the amino acid sequence:
• an SI domain of a coronavirus 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• compositions described herein deliver an immunogenic portion of an S protein of a SARS-CoV-2 variant.
• 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)).
• CDC Center for Disease Control and Prevention
• CanCOGeN Canadian CO VID Genomics Network
• WHO World Health Organization
• 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).
• 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.
• a polypeptide comprises two or more SARS-CoV-2 subdomains (e.g., two or more SI domains or RBDs).
• 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.
• 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 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.
• 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.
• RTS,S a malaria vaccine candidate known as “RTS,S”, a milestone in malaria vaccine development.
• 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; MosquirixTM).
• 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).
• 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).
• CSP circumsporozoite protein
• CSP precipitation reaction 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).
• sporozoites 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.
• 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).
• GPDH glyceraldehyde 3- phosphate dehydrogenase
• 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).
• sporozoites 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.
• 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.
• 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.
• basic red blood cell receptors basic and complement decay-accelerating factor (also known as CD55)
• 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.
• 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.
• 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).
• 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.
• 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.
• 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.
• genes encoding P. falciparum erythrocyte membrane protein 1 (PfEMPl) have been studied most extensively.
• PfEMPl P. falciparum erythrocyte membrane protein 1
• 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.
• iRBCs infected red blood cells
• 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.
• CSA chondroitin sulfate A
• EPCR endothelial protein C receptor
• ICAM1 heparan sulfate
• IgM IgM
• IgG IgG
• PECAM1 thrombospondin
• VCAM1 thrombospondin
• 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
• pir Plasmodium interspersed repeat
• 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.
• 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.
• 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.
• 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).
• SNPs Single Nucleotide Polymorphisms
• CNVs Copy Number Variations
• 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.
• Circumsporozoite protein 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 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.
• Table 1A Exemplary Sequences for CSP P. falciparum isolates from Asia, South America and Africa
• 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).
• 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).
• 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).
• 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.
• 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.
• GPI glycosylphosphatidylinositol
• Pl 13 sequences are known (see, e.g., Uniprot accession number Q8ILP3). Exemplary Pl 13 amino acid sequence is provided in Table 2A.
• Cysteine-Rich Protective Antigen 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 Ca 2+ 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)).
• 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.
• RH5 -interacting Protein 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)).
• 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.
• 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).
• El 40 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.
• 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.
• 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.
• 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.
• SPECT1 and SPECT2 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.
• mechanisms of cell traversal of these two proteins are yet to be defined in P. falciparum. See Y ang et al.
• 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 Ca 2+ -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.
• 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.
• Exported protein 1 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).
• PVM parasitophorous vacuolar membrane
• 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).
• 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).
• GST glutathione S-transferase
• 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.
• Upregulated in infective sporozoites gene 3 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).
• L-FABP liver fatty acid-binding protein
• 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).
• 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).
• 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.
• Upregulated in infective sporozoites gene 4 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).
• UIS4 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).
• Plasmodium UIS4 sequences are known (see, e.g., UniProt accession number Q8IJM9). Exemplary UIS4 amino acid sequence is provided in Table 2A.
• LISP-1 Liver specific protein 1
• PVM parasitophorous vacuolar membrane
• 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;
• 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.
• LISP-2 Liver specific protein 2
• 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).
• 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.
• Thrombospondin-related adhesion protein 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 Mg 2+ 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).
• MIDAS metal ion-dependent adhesion site
• 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).
• TSR thrombospondin repeat
• 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).
• 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.
• LS AP- 1 Liver-stage-associated protein 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).
• 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.
• 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.
• LSA-1 Liver-Stage Antigen 1
• 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).
• 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.
• 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.
• LSA-3 Liver stage antigen 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).
• 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, Q8IFS
• Glutamic acid-rich protein 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 involvement in sequestration has been proposed to occur by way of binding with a chloride/bicarbonate anion exchanger
• 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.
• PIESP2 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.
• Shizont egress antigen- 1 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.
• SEA1 sequences are known (see, e.g., UniProt accession number A0A143ZXM2), and exemplary SEA1 amino acid sequence is provided in Table 2A.
• SEQ ID NO:A 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).
• 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).
• the junction region includes an R1 region (amino acids 93-97) and amino acids ADGNPDP (SEQ ID NO: B) at positions 98- 104.
• the central domain includes a minor repeat region (amino acids 105-128) and a major repeat region (amino acids 129-272).
• the minor repeat region includes three repeats of the amino acid sequence NANPNVDP (SEQ ID NO:C).
• 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).
• the C-terminal domain includes a C-terminal region (amino acids 273-375) and a transmembrane domain (amino acids 376-397).
• the C-terminal region includes a Th2R region (amino acids 314-327) and a Th3R region (amino acids 352-363).
• 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.
• 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.
• 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.
• H1N1 circulating influenza A
• 2009 HINT A(HlNl)pdm09 viruses
• Influenza A (H3N2) viruses also comprise many separate, genetically different clades in recent years that contine to circulate.
• 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.
• 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.
• type A, B, C, D
• host of origin although for humans, the host of origin is usually not indicated
• geographical origin e.g., a virus, a virus, a virus, or a virus.
• Seasonal flu vaccines are typically formulated to provide protection against multiple influenza viruses that are known to cause epidemics.
• vaccines have been formulated as tetravalent vaccines, to provide antigens against H1N1, H3N2, B/Victoria, and B/Yamagata viruses.
• an influenza vaccine can protect both against the viruses that the vaccine comprises or delivers antigens from, and antigenically similar viruses.
• 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.
• 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..
• 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.
• 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.
• 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.
• 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.
• VLPs virus-like particles
• HBGA histo-blood group antigens
• 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).
• 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).
• 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.
• 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.
• host translation initiation factors e.g., eIF3
• 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.
• 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.
• 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.
• UTRs short untranslated regions
• 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)
• 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 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.
• 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.
• 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.
• 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.
• P22 has also been reported to contribute to trans-Golgi disassembly (36), and also to facilitate virion release from cells.
• 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).
• 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.
• multiple genotypes e.g., GI and GII
• the present disclosure provides certain norovirus antigen constructs particularly useful in effective vaccination.
• 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 components e.g., proteins or fragments or epitopes thereof, including epitopes that may comprise non-amino acid, e.g., carbohydrate moieties
• 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.
• a pharmaceutical composition described herein induces a relevant immune response effective against norovirus (e.g., by targeting a norovirus protein).
• 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.
• 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.
• a provided pharmaceutical composition e.g., immunogenic composition, e.g., norovirus vaccine
• an antigen that is included in and/or delivered by a provided pharmaceutical composition 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.
• a provided pharmaceutical composition e.g., immunogenic composition, e.g., norovirus vaccine
• 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.
• 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.
• 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.
• 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.
• 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).
• a utilized antigen induces an immune response that targets a VP1 protein from any of genogroups and/or genotypes.
• a utilized antigen induces an immune response that targets a VP1 protein from GI or GIL
• an immune response may be or comprise a T cell immune response.
• 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.
• 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
• 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).
• 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).
• a relevant region e.g., where subjects to be vaccinated are or will be present.
• 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.
• an antigen utilized in accordance with the present disclosure 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.
• 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.
• 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 variant
• 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).
• 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).
• 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
• 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).
• 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
• a provided pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
• a norovirus VP1 protein, or fragment or epitope thereof comprises or delivers a norovirus VP1 protein, or fragment or epitope thereof;
• 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).
• a provided pharmaceutical composition comprises or delivers a full-length VP1 protein or variant thereof.
• a provided pharmaceutical composition 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.
• a provided pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
• a VP1 antigen e.g., a full length or fragment VP1, or a variant thereof
• a separate RNA and/or a separate LNP e.g., from at least one other antigen (e.g., a multi-epitope antigen) as described herein.
• a provided pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
• P domain sequences e.g., P2 domain sequences
• 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.
• 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
• 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).
• 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.
• a VP1 epitope may be from GI genogroup of norovirus.
• a VP1 epitope may be from GII genogroup of norovirus.
• Viral Protein 2 (VP2)
• 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).
• 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).
• 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).
• TLR Toll-like receptors
• 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 protein also known as NS3
• NS3 is generated by cleavage of the polyprotein, in which it is located between residues 331 and 696, according to canonical numbering systems.
• 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).
• the norovirus p22 protein is another of the polypeptides formed by cleavage of the encoded preprotein.
• P22 includes a motif (YX(
• 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).
• 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.
• the norovirus protease protein 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).
• 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) Polymerase (NS7)
• 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).
• 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).
• 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).
• 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.
• a full-length viral protein e.g., a full-length VP1 or VP2, etc.
• 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.
• 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).
• a provided pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
• 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).
• an antigen utilized as described herein includes a membrane association element (e.g., a heterologous membrane association element), such as a transmembrane domain.
• a membrane association element e.g., a heterologous membrane association element
• an antigen utilized as described herein includes a secretion signal (e.g., a heterologous secretion signal).
• utilized sequences may be longer (and, e.g., may therefore include more epitopes) than a viral protein found in nature.
• 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).
• a pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
• 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).
• 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.
• a provided pharmaceutical composition 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.
• a provided pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
• HSV Herpes Simplex Virus
• Herpes simplex virus 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• an HSV 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).
• 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.
• TGN trans-Golgi network
• 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.
• technologies 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).
• 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).
• 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).
• 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.
• 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.
• 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).
• 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).
• 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.
• 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).
• DR direct repeats
• 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.
• nAbs neutralizing antibodies
• Respiratory syncytial virus also called human respiratory syncytial virus (hRSV) and human orthopneumo virus
• hRSV human respiratory syncytial virus
• 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.
• 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.
• 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.
• 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.
• 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
• 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.
• RSV subtype A RSV subtype A
• RSVB RSV subtype B
• 16 RSVA and 22 RSVB clades have been identified.
• GAI, GA2, GA5, and GA7 clades predominate; GA7 is found only in the United States.
• BA clade predominates worldwide.
• F and G proteins are the primary targets for neutralizing antibodies during natural infection.
• G protein G 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.
• TLR4 toll-like receptors
• 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).
• 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.
• 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.
• HS heparan sulfate
• 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.
• 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).
• 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.
• FP fusion peptide
• 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.
• 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).
• 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.
• prefusion state 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
• a final conformational shift results in a more stable and elongated form of the protein (postfusion, PostF).
• the RSV F protein 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.
• TLR4 toll-like receptor 4
• 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 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.
• 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.
• this immune memory allows the body to rapidly recognize and defend against it when it is subsequently encountered.
• 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.
• 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.
• RNA viruses include, without limitation, influenza, coronavirus (e.g., severe acute respiratory syndrome-related coronavirus), human immunodeficiency virus (HIV), Respiratory syncytial virus (RSV), and the like.
• 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.
• 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.
• FIG. 1 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.
• 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.
• 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.
• 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).
• 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.
• 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.
• an immune response is or comprises a B cell immune response.
• a B cell immune response is or comprises an antibody response (e.g., neutralizing antibody response) to arisen epitopes in variant polypeptides.
• 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.
• the present disclosure 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.
• such circulating infectious disease is a bacterial infectious disease.
• such circulating infectious disease is a parasitic infectious disease.
• An exemplary parasitic infectious disease is malaria.
• such circulating infectious disease is a viral infectious disease.
• 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.
• an antigen e.g., S protein of SARS-CoV-2
• 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).
• the present disclosure 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).
• the present disclosure 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.
• the present disclosure provides technologies for inducing a priming-favorable cytokine milieu, for example, in lymphoid tissues.
• induction of a priming-favorable cytokine milieu can be mediated through interferon alpha (IFNa).
• IFNa interferon alpha
• induction of a priming-favorable cytokine milieu can be mediated through a CD4+ T cell immune response.
• 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.
• a variant polypeptide comprises arisen epitopes.
• arisen epitopes are or comprise neutralizing epitopes (e.g., neutralizing antibody epitopes).
• 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).
• naive B cell immune response e.g., in some embodiments antibody response, e.g., neutralizing antibody response
• the arisen epitopes e.g., in some embodiments at least one of the neutralizing epitopes.
• 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.
• a non-modified RNA encodes at least one or more T cell epitopes.
• such a non-modified RNA encodes at least one or more B cell epitopes.
• 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
• an infectious agent e.g., SARS-CoV-2
• 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
• such a combination is provided in the same composition.
• such a combination is provided in separate compositions.
• an antigen utilized as described herein is or comprises a full-length viral protein.
• an antigen utilized as described herein is or comprises an immunogenic portion or domain of a viral polypeptide.
• 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).
• a provided pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
• an antigen utilized as described herein includes one or more variant sequences relative to a relevant reference antigen.
• 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.
• a viral variant e.g., a SARS-CoV-2 variant of concern
• an antigen utilized as described herein includes a multimerization element (e.g., a heterologous multimerization element).
• an antigen utilized as described herein includes a membrane association element (e.g., a homologous membrane association element), such as a transmembrane domain.
• a secretion signal e.g., a homologous secretion signal.
• 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).
• utilized sequences comprise one or more mutations associated with a variant of concern (e.g., a variant of concern identified by WHO).
• 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).
• a SARS-CoV-2 antigen for use in inducing an immunogenic response.
• 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).
• such antigens are delivered as protein antigens to induce an immunogenic response.
• 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.
• an antigen construct described herein includes a secretory signal, e.g., that is functional in mammalian cells.
• a utilized secretory signal is a heterologous secretory signal.
• 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).
• a secretory signal comprises or consists of a non-human secretory signal.
• a secretory signal comprises or consists of a viral secretory signal.
• a viral secretory signal comprises or consists of an HSV secretory signal (e.g., an HSV-1 or HSV-2 secretory signal).
• a secretory signal comprises or consists of an Ebola virus secretory signal.
• an Ebola virus secretory signal comprises or consists of an Ebola virus spike glycoprotein (SGP) secretory signal.
• SGP Ebola virus spike glycoprotein
• a secretory signal is characterized by a length of about 15 to 30 amino acids.
• a secretory signal is positioned at the N-terminus of an antigen construct as described herein.
• 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.
• 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.
• 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 e.g., MGAAARLGAVILFVVIVGLHGVRSKY; SEQ ID NO: 7
• an antigen construct described herein does not comprise a secretory signal.
• a signal peptide is an IgG signal peptide, such as an IgG kappa signal peptide.
• a secretory signal comprises or consists of an HSV glycoprotein D (gD) secretory signal.
• gD HSV glycoprotein D
• 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.
• 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.
• a secretory signal is selected from a gl signal peptide.
• 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.
• 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.
• 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 3 Exemplary polynucleotide sequences encoding secretory signals
• 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).
• 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.
• 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.
• 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.
• an antigen construct (e.g., SARS-CoV-2) as described herein includes a transmembrane region.
• a transmembrane region is located at the N-terminus of a construct (e.g., SARS-CoV-2) .
• a transmembrane region is located at the C-terminus of a construct (e.g., SARS-CoV-2) .
• a transmembrane region is not located at the N-terminus or C-terminus of a construct (e.g., SARS-CoV-2) .
• Transmembrane regions are known in the art, any of which can be utilized in a construct (e.g., SARS-CoV-2) described herein.
• 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.
• a transmembrane region comprises or is a transmembrane domain of a SARS-CoV-2 S protein, a transmembrane domain of
• a heterologous transmembrane region does not comprise a hemagglutinin transmembrane region.
• a heterologous transmembrane region comprises or consists of a non-human transmembrane region.
• a heterologous transmembrane region comprises or consists of a viral transmembrane region.
• a heterologous transmembrane region comprises or consists of an HSV transmembrane region, e.g., an HSV-1 or HSV-2 transmembrane region.
• an HSV transmembrane region comprises or consists of an HSV gD transmembrane region, e.g., comprising or consisting of an amino acid sequence of
• a heterologous transmembrane region comprises or consists of a human transmembrane region.
• a human transmembrane region comprises or consists of a human decay accelerating factor glycosylphosphatidylinositol (hDAF-GPI) anchor region.
• hDAF-GPI anchor region comprises or consists of an amino acid sequence of
• PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID NO: 92).
• a utilized transmembrane region is a heterologous transmembrane region.
• a construct described herein does not comprise a transmembrane region.
• Table 5A Exemplary nucleotide sequences encoding transmembrane regions
• a construct e.g., SARS-CoV-2 construct
• includes one or more multimerization regions e.g., a heterologous multimerization region.
• a heterologous multimerization region comprises a dimerization, trimerization or tetramerization region.
• 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.
• a construct described herein is able to form a trimeric complex.
• 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.
• a multimerization region allowing formation of a multimeric complex comprises a trimerization region, for example, a trimerization region described herein.
• a construct includes a T4-fibritin-derived “foldon” trimerization region, for example, to increase its immunogenicity.
• 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
• a construct e.g., SARS-CoV-2 construct
• a linker is or comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.
• 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.
• a linker comprises one or more glycine (G) amino acids.
• a linker comprises one or more serine (S) amino acids.
• a linker includes amino acids selected based on a cleavage predictor to generate highly-cleavable linkers.
• a linker is or comprises S-G4-S-G4-S (SEQ ID NO: 99). In some embodiments, a linker is or comprises GSPGSGS (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).
• a construct e.g., SARS-CoV-2 construct
• SARS-CoV-2 antigen polypeptide sequences and RNA sequences encoding the same
• 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.
• the RNA molecule is a modified RNA as described herein.
• certain Us are replaced by a modified U (e.g., in some embodiments 1- methylpseudouridine).
• 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-
• one or more agents that induce a priming-favorable cytokine milieu 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.
• 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.
• 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).
• 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.
• IFN interferon
• such an agent specifically induces Type I IFN such as interferon alpha (IFNa) (“IFN-inducing agents”).
• IFN-inducing agents such an agent can induce a CD4+ T cell response.
• an IFN-inducing agent is or comprises IFNa.
• an IFNa-inducing agent is or comprises an unmodified
• 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).
• an unmodified RNA is an RNA molecule that contains no modified ribonucleotides.
• 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).
• 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).
• 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).
• TLR3, TLR7, TLR8 toll-like receptors
• RIG-I RNA-dependent protein kinase
• IVT RNA e.g., IVT mRNA
• 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.
• TLR3, TLR7, TLR8 RNA-dependent protein kinase
• PLR RNA-dependent protein kinase
• the present disclosure 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.
• such beneficial therapeutic effect can include but are not limited to induction of IFN (e.g., IFNa) to a level that is priming-favorable.
• IFN 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).
• a delivered antigen e.g., in some embodiments, arisen epitopes present in a variant polypeptide as described herein.
• an unmodified RNA molecule that is useful in accordance with the present disclosure encodes one or more T cell epitopes.
• 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.
• T cell epitopes(s) is/are from a polypeptide of an infectious agent.
• 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.
• 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.
• 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.
• 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).
• a priming-favorable cytokine e.g., in some embodiments Type I IFN, e.g., IFNa.
• 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.
• 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.
• 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.
• 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.
• the amount ratio (by mass or moles) of a modified RNA molecule to unmodified RNA molecule is about 1 : 1 [0645]
• an IFNa-inducing agent described herein is or comprises a RNA replicon.
• an RNA replicon is an unmodified RNA molecule described herein.
• an RNA replicon is a “self-replicating RNA” or “self- amplifying RNA.”
• 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.
• ss single-stranded
• 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.
• the first ORF is larger than the second ORF, the ratio being roughly 2: 1.
• 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).
• mRNA eukaryotic messenger RNA
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• the amount ratio (by mass or moles) of a modified RNA molecule to an RNA replicon is about 10: 1 to about 30:1.
• 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”).
• a CD4+ T cell response-inducing agent described herein is a composition that comprises or delivers one or more CD4+ T cell epitopes.
• such a composition comprise one or more CD4+ T cell epitope peptides or polypeptides.
• 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.
• a CD4+ T cell response-inducing agent described herein is or comprises an RNA molecule that encodes one or more CD4+ T cell epitopes.
• 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.
• 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.
• 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.
• 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.
• 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.
• the amount ratio (by mass or moles) of a modified RNA molecule to a CD4+ T cell response-inducing agent 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.
• 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.
• provided pharmaceutical compositions e.g., immunogenic compositions, e.g., vaccines
• 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) .
• an infection e.g., SARS-CoV-2
• 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.
• pharmaceutical compositions e.g., immunogenic compositions, e.g., vaccines (e.g., SARS-CoV-2)
• SARS-CoV-2 e.g., antigens associated with a variant of concern with high immune escape potential.
• 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.
• Polyribonucleotides described herein encode one or more constructs (e.g., SARS- CoV-2) described herein.
• polyribonucleotides described herein can comprise a nucleotide sequence that encodes a 5’UTR of interest and/or a 3’ UTR of interest.
• polynucleotides described herein can comprise a nucleotide sequence that encodes a polyA tail.
• polyribonucleotides described herein may comprise a 5’ cap, which may be incorporated during transcription, or joined to a polyribonucleotide post- transcription.
• 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).
• capO structure m7GpppN
• 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).
• 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.
• 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.
• 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.
• RNA e.g., mRNA
• 5'-cap 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')).
• 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.
• a guanosine nucleoside included in a 5’ cap comprises a 3’0 methylation at a ribose (3’0MeG).
• a guanosine nucleoside included in a 5’ cap comprises methylation at the 7-position of guanine (m7G).
• 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)).
• m7(3’OMeG) methylation at the 7-position of guanine and a 3’ O methylation at a ribose
• 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.
• co-transcriptional capping with a cap disclosed improves the capping efficiency of an RNA compared to co-transcriptional capping with an appropriate reference comparator.
• improving capping efficiency can increase a translation efficiency and/or translation rate of an RNA, and/or increase expression of an encoded polypeptide.
• alterations to polynucleotides generates a non-hydrolyzable cap structure which can, for example, prevent decapping and increase RNA half-life.
• 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.
• an RNA described herein comprises a capl structure. In some embodiments, an RNA described herein comprises a cap2.
• an RNA described herein comprises a capO structure.
• a capO structure comprises a guanosine nucleoside methylated at the 7- position of guanine ((m 7 )G).
• such a capO structure is connected to an RNA via a 5'- to 5 '-triphosphate linkage and is also referred to herein as (m 7 )Gppp.
• a capO structure comprises a guanosine nucleoside methylated at the 2 ’-position of the ribose of guanosine.
• a capO structure comprises a guanosine nucleoside methylated at the 3 ’-position of the ribose of guanosine.
• 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 72 °)G).
• 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).
• a capl structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m 7 )G) and optionally methylated at the 2’ or 3’ position of the ribose, and a 2’0 methylated first nucleotide in an RNA ((m 2 °)Ni).
• a capl structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m 7 )G) and the 3’ position of the ribose, and a 2’0 methylated first nucleotide in an RNA ((m 2 °)Ni).
• a capl structure is connected to an RNA via a 5'- to 5'- triphosphate linkage and is also referred to herein as, e.g., ((m 7 )Gppp( 2 O )Ni) or (m2 7 ’ 3 o )Gppp( 2 - °)Ni), wherein Ni is as defined and described herein.
• a capl structure comprises a second nucleotide, N2, which is at position 2 and is chosen from A, G, C, or U, e.g., (m 7 )Gppp( 2 O )NipN2 or (m2 7 3 °)Gppp( 2 O )NipN2 , wherein each of Ni and N2 is as defined and described herein.
• a cap2 structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m 7 )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 ((m 2 °)Nip(m 2 ’ °)N2).
• a cap2 structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m 7 )G) and the 3’ position of the ribose, and a 2’0 methylated first and second nucleotide in an RNA.
• a cap2 structure is connected to an RNA via a 5'- to 5 '-triphosphate linkage and is also referred to herein as, e.g., ((m 7 )Gppp( 2 - °)Nip( 2 O )N2) or (m2 7 ’ 3 °)Gppp( 2 O )Nip( 2 O )N2), wherein each of Ni and N2 is as defined and described herein.
• 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 R 2 and R 3 is -OH or -OCH3; and

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