WO2023092069A1

WO2023092069A1 - Sars-cov-2 mrna domain vaccines and methods of use

Info

Publication number: WO2023092069A1
Application number: PCT/US2022/080141
Authority: WO
Inventors: Guillaume Stewart-Jones; Robert PARIS
Original assignee: Modernatx, Inc.
Priority date: 2021-11-18
Filing date: 2022-11-18
Publication date: 2023-05-25

Abstract

The disclosure provides coronavirus mRNA vaccines, including booster vaccines, directed against one or more spike proteins of one or more variant strains of SARS-CoV-2, as well as methods of using the vaccines.

Description

SARS-COV-2 MRNA DOMAIN VACCINES AND METHODS OF USE

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 63/281,018, filed November 18, 2021, which is incorporated by reference herein in its entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (M137870207WO00-SEQ-JXV.xml; Size: 24,042 bytes; and Date of Creation: October 11, 2022) is herein incorporated by reference in its entirety.

BACKGROUND

Human coronaviruses are highly contagious enveloped, positive sense single-stranded RNA viruses of the Coronaviridae family. Two sub-families of Coronaviridae are known to cause human disease. The most important being the β- coronaviruses (betacoronaviruses). The (β- coronaviruses are common etiological agents of mild to moderate upper respiratory tract infections. Outbreaks of novel coronavirus infections such as the infections caused by a coronavirus initially identified from the Chinese city of Wuhan in December 2019; however, have been associated with a high mortality rate death toll. This recently identified coronavirus, referred to as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) (formerly referred to as a “2019 novel coronavirus,” or a “2019-nCoV”) has rapidly infected hundreds of thousands of people. The pandemic disease that the SARS-CoV-2 virus causes has been named by World Health Organization (WHO) as COVID-19 (Coronavirus Disease 2019). The first genome sequence of a SARS-CoV-2 isolate (Wuhan-Hu-1; USA-WA1/2020 isolate) was released by investigators from the Chinese CDC in Beijing on January 10, 2020 at Virological, a UK-based discussion forum for analysis and interpretation of virus molecular evolution and epidemiology. The sequence was then deposited in GenBank on January 12, 2020, having Genbank Accession number MN908947.1. Subsequently, a number of SARS-CoV-2 strain variants have been identified, some of which are more infectious than the SARS-CoV-2 isolate.

As of the time of worldwide emergency use authorization of the authorized SARS-CoV- 2 nucleic acid-based vaccines, there is not yet a strategy for combatting the recently-discovered and later-emerging SARS-CoV-2 variants of concern (VOC). The continuing health problems and mortality associated with coronavirus infections, particularly the SARS-CoV-2 pandemic, are of tremendous concern internationally. The public health crisis caused by SARS-CoV-2 and its variants reinforces the importance of rapidly developing effective and safe vaccine candidates against these viruses.

The emergence of SARS-CoV-2 variants with substitutions in the receptor binding domain (RBD) and N-terminal domain (NTD) of the viral S protein has raised concerns among scientists and health officials. The entry of coronavirus into host cells is mediated by interaction between the RBD of the viral S protein and host angiotensin-converting enzyme 2 (ACE2). Vaccine development has focused on inducing antibody responses against this region of SARS- CoV-2 S protein. More recently, a neutralization “supersite” has also been identified in the NTD. A significant decrease in vaccine efficacy has been correlated with amino acid substitutions in the RBD (eg, K417N, E484K, and N501Y) and NTD (e.g., L18F, D80A, D215G, and A242-244) of the S protein. Some of the most recently circulating isolates containing these substitutions from the United Kingdom (B.l.1.7), Republic of South Africa (B.1.351), Brazil (P.l lineage), New York (B.1.526), and California (B.1.427/B.1.429 or CAL.20C lineage), have shown a reduction in neutralization from convalescent serum in pseudovirus neutralization (PsVN) assays and resistance to certain monoclonal antibodies. In particular, mutations in the NTD subdomain, and specifically the neutralization supersite, are most extensive in the B.1.351 lineage virus. See McCallum, M. et al. N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2. Cell, doi:10.1016/j.cell.2021.03.028 (2021).

Using 2 orthogonal vesicular stomatitis virus (VSV) and lentivirus PsVN assays expressing S variants of 20E (EU1), 20A.EU2, D614G-N439K, mink cluster 5, B.l.1.7, P.l, B.1.427/B.1.429, B.1.1.7+E484K, and B.1.351, the assessment of the neutralizing capacity of sera from Phase 1 participants and non-human primates (NHPs) that received 2 doses of mRNA- 1273 was reported. See Wu, K. et al. Serum Neutralizing Activity Elicited by mRNA-1273 Vaccine. N Engl J Med, doi:10.1056/NEJMc2102179 (2021). Subsequent studies demonstrated reduced neutralization titers against the full B.1.351 variant following mRNA-1273 vaccination, although levels are still significant and expected to be protective. Despite this prediction of continued efficacy of mRNA-1273 against this key variant of concern, the duration of vaccine mediated protection is still unknown.

There remains a need for development and evaluation of further COVID-19 vaccines against SARS-CoV-2 variants encoding the prefusion stabilized S protein of SARS-CoV-2 that incorporates key mutations present in the B.1.351 variant, including L18F, D80A, D215G, L242-244del, R₂46I, K417N, E484K, N501Y, D614G, A701V. Additional vaccines are necessary to expand the breadth of coverage to multiple circulating variants as well as the ancestral wild-type virus that is still circulating globally.

SUMMARY

A SARS-CoV-2 vaccine, mRNA-1273 (developed by Modema Therapeutics), has been shown to elicit high viral neutralizing titers in Phase 1 trial human participants (Jackson et al, 2020; Anderson et al, 2020) and is highly efficacious in prevention of symptomatic COVID- 19 disease and severe disease (Baden et al., 2020). However, the recent emergence of SARS-CoV-2 variants have raised concerns due to their increased rates of transmission as well as their potential to circumvent immunity elicited by natural infection or vaccination (Volz et al., 2021; Tegally et al., 2020; Wibmer et al., 2021; Wang et al., 2021; Collier et al., 2021).

The B.1.351 variant emerged in South Africa, and increased rates of transmission and higher viral burden after infection have been reported (Tegally et al., 2020). The mutations located in the S protein are more extensive than the B.l.1.7 variant with changes of L18F, D80A, D215G, L242-244del, R₂46I, K417N, E484K, N501Y, D614G, and A701V, with three of these mutations located in the RBD (K417N, E484K, N501Y). B.1.351 shares key mutations in the RBD with a reported variant in Brazil (Tegally et al., 2020; Naveca et al., 2021). As the RBD is the predominant target for neutralizing antibodies, these mutations could impact the effectiveness of monoclonal antibodies already approved and in advanced development as well as of polyclonal antibody elicited by infection or vaccination in neutralizing the virus (Greaney et al., 2021, Wibmer et al, 2021).

Recent data have suggested that the key mutation present in the B.1.351 variant, E484K, confers resistance to SARS-CoV-2 neutralizing antibodies, potentially limiting the therapeutic effectiveness of monoclonal antibody therapies (Wang et al., 2021; Greaney et al., 2020; Weisblum et al., 2020; Liu et al., 2020; Wibmer et al., 2021). Moreover, the E484K mutation was shown to reduce neutralization against a panel of convalescent sera (Weisblum et al., 2020; Liu et al., 2020; Wibmer et al., 2021). In terms of vaccination, it is clear that mRNA-1273 induces significantly higher neutralizing titers than convalescent sera against the USA- WA 1/2020 isolate (Jackson et al, 2020). A recent study using a recombinant VSV PsVN assay showed that sera of mRNA-1273 vaccinated participants had reduced neutralizing titers against E484K or K417N/E484K/N50Y combination (Wang et al, 2021), however there has been no assessment of sera from mRNA-1273 clinical trial participants against the full constellation of S mutations found in the B.l.1.7 or B.1.351 variants.

The invention pertains, inter alia, to booster vaccines comprising a single polynucleotide encoding a SARS-CoV-2 antigen comprising an RBD and NTD and transmembrane domain joined by linkers (either from the USA-WA1/2020 isolate or the beta variant (B.1.351)), or two polynucleotides, each encoding a different SARS-CoV-2 antigen (e.g., from the USA- WA1/2020 isolate and the beta variant (B.1.351)). Such a vaccine can be administered to seropositive or seronegative subjects. For example, a subject may be naive and not have antibodies that react with SARS-CoV-2 or may have preexisting antibodies to SARS-CoV-2 because they have previously had an infection with SARS-CoV-2 or may have previously been administered a dose of a vaccine (e.g., an mRNA vaccine) that induces antibodies against SARS-CoV-2.

Thus, the disclosure, in some aspects, provides a method comprising administering to a human subject a therapeutic dose of a composition comprising a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) that encodes a fusion protein of a receptor binding domain (RBD) of a SARS-CoV-2 Spike protein and an amino (N)-terminal domain (NTD) of a SARS-CoV-2 Spike protein, and less than the full length spike protein, wherein the mRNA is in a lipid nanoparticle, and wherein the composition is administered to the human subject following administration of a prophylactic composition against SARS-CoV-2.

In some embodiments, the composition further comprises a second mRNA comprising a second ORF that encodes a second fusion protein comprising an RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein, wherein the mRNA and the second mRNA are not identical.

In some embodiments, the mRNA and the second mRNA are present in the composition at a ratio of 1 : 1.

In some embodiments, the composition is administered to the human subject at least three months and within one year after administration of the prophylactic composition. In some embodiments, the composition is administered to the human subject at least six months after administration of the prophylactic composition.

In some embodiments, the prophylactic composition comprises an mRNA vaccine or a viral vector vaccine. In some embodiments, the subject the prophylactic composition is an mRNA vaccine and the human subject has received two doses of the mRNA vaccine.

In some embodiments, the composition comprises 1 pg of mRNA in total. In some embodiments, the composition comprises 2.5 pg of mRNA in total. In some embodiments, the composition comprises 5 pg of mRNA in total. In some embodiments, the composition comprises 10 pg of mRNA in total.

In some embodiments, the composition further comprises Tris buffer. In some embodiments, the composition with Tris buffer further comprises sucrose and sodium acetate. In some embodiments, the composition comprises 10 mM - 30 mM Tris buffer comprising 75 mg/mL - 95 mg/mL sucrose, and 5 mM - 15 mM sodium acetate, optionally wherein the composition has a pH of 6-8. In some embodiments, the composition comprises about 20 mM Tris buffer comprising 87 mg/mL sucrose, and 10.7 mM sodium acetate, optionally wherein the composition has a pH of 7.5. In some embodiments, the composition comprises about 0.25 mg/mL of the mRNA total.

In some embodiments, the composition is administered intramuscularly, optionally into a deltoid muscle of the subject’s arm.

In some embodiments, the fusion protein comprises a sequence having at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 11. In some embodiments, the fusion protein comprises SEQ ID NO: 11. In some embodiments, the ORF comprises a sequence having at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 10. In some embodiments, the ORF comprises SEQ ID NO: 10. In some embodiments, the mRNA comprises a sequence having at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 9. In some embodiments, the mRNA comprises SEQ ID NO: 9.

In some embodiments, the second fusion protein comprises a sequence having at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 23. In some embodiments, the fusion protein comprises SEQ ID NO: 23. In some embodiments, the second ORF comprises a sequence having at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 22. In some embodiments, the second ORF comprises SEQ ID NO: 22. In some embodiments, the second mRNA comprises a sequence having at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 21. In some embodiments, the second mRNA comprises SEQ ID NO: 21.

In some embodiments, the lipid nanoparticle comprises: ionizable amino lipid; neutral lipid; sterol; and PEG-modified lipid. In some embodiments, the lipid nanoparticle comprises: 40-55 mol% ionizable amino lipid; 5-15 mol% neutral lipid; 35-45 mol% sterol; and 1-5 mol% PEG-modified lipid. In some embodiments, the lipid nanoparticle comprises: 47 mol% ionizable amino lipid; 11.5 mol% neutral lipid; 38.5 mol% sterol; and 3.0 mol% PEG-modified lipid; 48 mol% ionizable amino lipid; 11 mol% neutral lipid; 38.5 mol% sterol; and 2.5 mol% PEG-modified lipid; 49 mol% ionizable amino lipid; 10.5 mol% neutral lipid; 38.5 mol% sterol; and 2.0 mol% PEG-modified lipid; 50 mol% ionizable amino lipid; 10 mol% neutral lipid; 38.5 mol% sterol; and 1.5 mol% PEG-modified lipid; or 51 mol% ionizable amino lipid; 9.5 mol% neutral lipid; 38.5 mol% sterol; and 1.0 mol% PEG-modified lipid. In some embodiments, the ionizable amino lipid is heptadecan-9-yl 8 ((2 hydroxy ethyl) (6 oxo 6-(undecyloxy)hexyl)amino)octanoate (Compound 1). In some embodiments, the neutral lipid is 1,2 distearoyl sn glycero-3 phosphocholine (DSPC). In some embodiments, the sterol is cholesterol. In some embodiments, the PEG-modified lipid is 1- monomethoxypolyethyleneglycol-2,3-dimyristylglycerol with polyethylene glycol of average molecular weight 2000 (PEG-DMG).

In some embodiments, the prophylactic composition against SARS-CoV-2 comprises an mRNA vaccine or a viral vector vaccine. In some embodiments, the prophylactic composition comprises an mRNA vaccine comprising an ORF encoding a SARS-CoV-2 prefusion stabilized S protein.

In some embodiments, the human subject has previously been administered at least one dose of the prophylactic composition. In some embodiments, the human subject has previously been administered two doses of the prophylactic composition. In some embodiments, the method comprises administering the composition to the human subject at least six months after the most recent administration of the prophylactic composition.

The disclosure, in another aspect, provides a composition comprising: 1 |ig - 50 pg of a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) that encodes a fusion protein comprising at least two domains of a SARS-CoV-2 Spike (S) protein, and less than the full length spike protein; and a lipid nanoparticle comprising a mixture of lipids that comprises an ionizable amino lipid, a non-cationic lipid, a sterol, and a PEG-modified lipid. In some embodiments, the composition comprises 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 pg of the mRNA. In some embodiments, the composition comprises 0.1-1, 0.1-2, 0.1-3, 0.1-4, 0.1-5, 0.5-1, 0.5-2, 0.5-3, 0.5-4, 0.5-5, 1-1.5, 1-2, 1-3, 1-4, 1-5, 5.0, 2.0-2.5, 2.0-3, 2.0-4, 2.0-5 or 5.0, 2.5-3, 2.5-4, 2.5-5, 3.5-4, 3.5-5, or 4.5-5 pg of the mRNA. In some embodiments the mRNA is present in the composition in any one of the foregoing doses as the only mRNA in the composition. In other embodiments the mRNA is present in the composition in any one of the foregoing doses in combination with one or more other mRNAs.

In some embodiments, the composition further comprises a second mRNA comprising a second ORF that encodes a second fusion protein comprising at least two domains of a SARS- CoV-2 Spike (S) protein and less than the full length S protein. In some embodiments, the mRNA and the second mRNA are present in the composition at a ratio of 1:1. In some embodiments, the composition comprises 2.5 pg of mRNA in total. In some embodiments, the composition comprises 5 pg of mRNA in total. In some embodiments, the composition comprises 10 pg of mRNA in total.

In some embodiments, the mRNA and/or the second mRNA comprises a chemical modification. In some embodiments, the mRNA and/or the second mRNA is fully modified. In some embodiments, the chemical modification is 1 -methylpseudouridine. In some embodiments, the mRNA and/or the second mRNA further comprises a 5’ cap analog, optionally a 7mG(5’)ppp(5’)NlmpNp cap. In some embodiments, the mRNA and/or the second mRNA further comprises a poly(A) tail, optionally having a length of 50 to 150 nucleotides.

In some embodiments, the composition further comprises Tris buffer, sucrose, and sodium acetate, or any combination thereof. In some embodiments, the composition further comprises 30-40 mM Tris buffer, 80-95 mg/mL sucrose, and 5-15 mM sodium acetate. In some embodiments, the composition has a pH value of 6-8, optionally 7.5. In some embodiments, the human subject is seropositive for SARS-CoV-2 or a variant thereof.

In some embodiments, the human subject is seronegative for SARS-CoV-2 or a variant thereof.

In some embodiments, the composition induces neutralizing antibodies against SARS- CoV-2. In some embodiments, the composition induces neutralizing antibodies against a SARS- CoV-2 variant, ancestral SARS-CoV-2, or both the SARS-CoV-2 variant and ancestral SARS- CoV-2. In some embodiments, the composition induces neutralizing antibodies against two or more SARS-CoV-2 variants.

In some embodiments, the geometric mean fold rise (GMFR) induced against ancestral SARS-CoV-2 in the subject at Day 29 post administration of a 2.5 pg dose relative to a neutralizing antibody titer prior to administration of the first dose is at least 28. In some embodiments, the GMFR induced against ancestral SARS-CoV-2 in the subject at Day 29 post administration of a 5 pg dose relative to a neutralizing antibody titer prior to administration of the first dose is at least 34. In some embodiments, the GMFR induced against ancestral SARS- CoV-2 in the subject at Day 29 post administration of a 10 pg dose relative to a neutralizing antibody titer prior to administration of the first dose is at least 42.

In some embodiments, the GMFR induced against ancestral SARS-CoV-2 in the subject at Day 29 post administration of a 5 pg dose relative to a neutralizing antibody titer prior to administration of the first dose is at least 25. In some embodiments, the GMFR induced against ancestral SARS-CoV-2 in the subject at Day 29 post administration of a 10 pg dose relative to a neutralizing antibody titer prior to administration of the first dose is at least 28.

In some embodiments, the GMFR induced against the beta SARS-CoV-2 strain in the subject at Day 29 post administration of a 2.5 pg dose relative to a neutralizing antibody titer prior to administration of the first dose is at least 27. In some embodiments, the GMFR induced against the beta SARS-CoV-2 strain in the subject at Day 29 post administration of a 5 pg dose relative to a neutralizing antibody titer prior to administration of the first dose is at least 33. In some embodiments, the GMFR induced against the beta SARS-CoV-2 strain in the subject at Day 29 post administration of a 10 pg dose relative to a neutralizing antibody titer prior to administration of the first dose is at least 44.

In some embodiments, the GMFR induced against the beta SARS-CoV-2 strain in the subject at Day 29 post administration of a 5 pg dose relative to a neutralizing antibody titer prior to administration of the first dose is at least 38. In some embodiments, the GMFR induced against ancestral SARS-CoV-2 in the subject at Day 29 post administration of a 10 pg dose relative to a neutralizing antibody titer prior to administration of the first dose is at least 41.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of graphs showing the change in binding antibody titers between prebooster and 28 days after administration of the booster for the ancestral (D614G) SARS-CoV-2, alpha, and beta strains (see Example 1).

FIG. 2 is a series of graphs showing the change in binding antibody titers between prebooster and 28 days after administration of the booster for the gamma, delta, and omicron strains (see Example 1).

DETAILED DESCRIPTION

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly emerging respiratory virus with high morbidity and mortality. SARS-CoV-2 has rapidly spread around the world compared with SARS-CoV, which appeared in 2002, and Middle East respiratory syndrome coronavirus (MERS-CoV), which emerged in 2012. The World Health Organization (WHO) reports that, as of March 2021, the current outbreak of COVID-19 has had over 120 million confirmed cases worldwide with more than 2.65 million deaths. New cases of COVID- 19 infection are on the rise and are still increasing rapidly. It is thus crucial that a variety of safe and effective vaccines and drugs be developed to prevent and treat COVID-19 and reduce the serious impact that COVID- 19 is having across the world. Vaccines and drugs made using a variety of modalities, and vaccines having improved safety and efficacy, are imperative. There remains a need to accelerate the advanced design and development of vaccines and therapeutic drugs against coronavirus disease and in particular coronavirus 2019 (COVID- 19).

On January 7, 2020, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the etiological agent of a novel pneumonia that emerged in December 2019, in Wuhan City, Hubei province in China (Lu H. et al. (2020) J Med Virol. Apr; 92(4):401-402.). Soon after, the virus caused an outbreak in China and has spread to the world. According to the analysis of genomic structure of SARS-CoV-2, it belongs to P-coronaviruses (CoVs) (Chan et al. 2020 Emerg Microbes Infect.', 9(l):221-236).

Subsequently, a number of SARS-CoV-2 variant strains have emerged and have predominated in particular initial geographic areas. However, some variants that quickly predominate in one geographic area can spread rapidly around the globe. These variants are known as variants of concern (VOC). Two main variants have been found since the fall of 2020, including one in the United Kingdom (20B/501Y.V1, VOC 202012/01, or B.1.1.7 lineage) and one in South Africa (20C/501Y.V2 or B.1.351 lineage). The two variants emerged separately from one another, but appear to have improved transmissibility relative to the USA-WA1/2020 isolate. Further, there are concerns that these variants as well as other circulating strains and any future variants may further mutate to avoid neutralization by existing vaccines and therapeutic modalities such as antibodies. In this way, the SARS-CoV-2 variants, and any other emerging mutant SARS-CoV-2 strains, are an international health concern.

The threat of emerging mutant strains of viruses presents a significant challenge to vaccine development. The compositions disclosed herein provide a significant advance in combatting the emerging viral strains that pose a global health concern. Disclosed herein are vaccines and vaccine protocols with broad viral neutralization capabilities that reduce the threat of infection from more than one strain of virus, through single or multiple administrations of combinations of antigens from different strains.

The present disclosure provides compositions (e.g., immunizing compositions such as RNA vaccines) that elicit potent neutralizing antibodies against coronavirus antigens (e.g., SARS-CoV-2 variants) at doses as low as 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 |lg of mRNA or 0.1-1, 0.1-2, 0.1-3, 0.1-4, 0.1-5, 0.5-1, 0.5-2, 0.5-3, 0.5-4, 0.5-5, 1-1.5, 1-2, 1-3, 1-4, 1-5, 2.0-2.5, 2.0-3, 2.0-4, 2.0-5 or 5.0, 2.5-3, 2.5-4, 2.5-5, 3.5-4, 3.5-5, or 4.5-5 pg of mRNA. In some embodiments, an immunizing composition includes a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) that encodes a fusion protein comprising at least two domains of a SARS- CoV-2 Spike (S) protein, and less than the full length spike protein; and a lipid nanoparticle. In some embodiments an immunizing composition includes a first messenger ribonucleic acid (mRNA) comprising a first open reading frame (ORF) that encodes a first fusion protein comprising at least two domains of a SARS-CoV-2 Spike (S) protein and less than the full length S protein, and a second mRNA comprising a second ORF that encodes a second fusion protein comprising at least two domains of a SARS-CoV-2 Spike (S) protein and less than the full length S protein; and a lipid nanoparticle. In some embodiments, the two RNAs are present in the composition in a 1:1 ratio. The composition, in some embodiments, is administered as a booster dose (e.g., subsequent to administration of an initial course of vaccination).

SARS-CoV-2

The genome of SARS-CoV-2 is a single- stranded positive-sense RNA (+ssRNA) with the size of 29.8-30 kb encoding about 9860 amino acids (Chan et a/.2000, supra.-, Kim et al. 2020 Cell, May 14; 181(4):914-921.el0.). SARS-CoV-2 is a polycistronic mRNA with 5'-cap and 3'-poly-A tail. The SARS-CoV-2 genome is organized into specific genes encoding structural proteins and nonstructural proteins (Nsps). The order of the structural proteins in the genome is 5'-replicase (open reading frame (ORF)l/ab)-structural proteins [Spike (S)-Envelope (E)-Membrane (M)-Nucleocapsid (N)]-3 The genome of coronaviruses includes a variable number of open reading frames that encode accessory proteins, nonstructural proteins, and structural proteins (Song et al. 2019 Vzrnses; l l(l):p. 59). Most of the antigenic peptides are located in the structural proteins (Cui et al. 2019 Nat. Rev. Microbiol.il (3') 181-192'). Spike surface glycoprotein (S), a small envelope protein (E), matrix protein (M), and nucleocapsid protein (N) are four main structural proteins. Since S-protein contributes to cell tropism it is capable of inducing neutralizing antibodies (NAb) and protective immunity, it can be considered one of the most important targets in coronavirus vaccine development among all other structural proteins. Moreover, amino acid sequence analysis has shown that S-protein contains conserved regions among the coronaviruses, which may be the basis for universal vaccine development.

Antigens

The compositions of the invention, e.g., vaccine compositions, feature nucleic acids, in particular, mRNAs, designed to encode an antigen of interest, e.g., an antigen derived from a betacoronavirus structural protein, in particular, antigens derived from SARS-CoV-2 Spike protein. The compositions of the invention, e.g., vaccine compositions, do not comprise antigens per se, but rather comprise nucleic acids, in particular, mRNA(s) that encode antigens or antigenic sequences once delivered to a cell, tissue or subject. Delivery of nucleic acid molecules, in particular mRNA(s) is achieved by formulating said nucleic acid molecules in appropriate carriers or delivery vehicles (e.g., lipid nanoparticles) such that upon administration to cells, tissues or subjects, nucleic acid is taken up by cells which, in turn, express protein(s) encoded by the nucleic acids, e.g., mRNAs. The term "antigen" as used herein refers to a substance such as a protein (e.g., glycoprotein), polypeptide, peptide, or the like, which elicits an immune response, e.g., elicits an immune response when present in a subject (for example, when present in a human or mammalian subject). The instant invention is based at least in part on the understanding that mRNA-encoded antigens, when expressed from mRNA administered to a cell or subject, can cause the immune system to produce an immune response to the expressed antigen, for example can trigger the production of antibodies against the expresses antigen, e.g., binding and/or neutralizing antibodies, can trigger B and or T cell responses specific to the expressed antigen, and ultimately can cause protective or prophylactic response against subsequent encounter with the antigen or with a pathogen with which the antigen is associated. Preferred mRNA-encoded antigens are “viral antigens”. As used herein, the term “viral antigen” refers to an antigen derived from a virus, for example from a pathogenic virus. The term antigen as used herein can refer to a full-length protein, for example, a full-length viral protein, or can refer to a fragment (e.g., a polypeptide or peptide fragment), subunit or domain of a protein, e.g., a viral protein subunit or domain.

Many proteins have a quaternary or three-dimensional structure, which consists of more than one polypeptide or several polypeptide chains that associate into an oligomeric molecule. As used herein the term “subunit” refers to a single protein molecule, for example, a polypeptide or polypeptide chain resulting from processing of a nascent protein molecule, which subunit assembles (or “coassembles”) with other protein molecules (e.g., subunits or chains) to form a protein complex. Proteins can have a relatively small number of subunits and therefore be described as “oligomeric” or can consist of a large number of subunits and therefore be described as “multimeric”. The subunits of an oligomeric or multimeric protein may be identical, homologous or totally dissimilar and dedicated to disparate tasks.

Proteins or protein subunits can further comprise domains. As used herein, the term “domain” refers to a distinct functional and/or structural unit within a protein. Typically, a “domain” is responsible for a particular function or interaction, contributing to the overall role of a protein. Domains can exist in a variety of biological contexts. Similar domains (i.e., domains sharing structural, functional and/or sequence homology) can exist within a single protein or can exist within distinct proteins having similar or different functions. A protein domain is often a conserved part of a given protein tertiary structure or sequence that can function and exist independently of the rest of the protein or subunit thereof.

In structural and molecular biology, identical, homologous or similar subunits or domains can help to classify newly identified or novel proteins, as was done immediately upon publication of the SARS-CoV-2 viral genomic sequence. As used herein, the term antigen is distinct from the term “epitope” which is a substructure of an antigen, e.g., a polypeptide or carbohydrate structure, which may be recognized by an antigen binding site but is insufficient to induce an immune response. The art describes protein antigens that are delivered to subjects or immune cells in isolated form, e.g., isolated protein, polypeptide or peptide antigens, however, the design, testing, validation, and production of protein antigens can be costly and time-consuming, especially when producing proteins at large scale. By contrast, mRNA technology is amenable to rapid design and testing of mRNA constructs encoding a variety of antigens. Moreover, rapid production of mRNA coupled with formulation in appropriate delivery vehicles (e.g., lipid nanoparticles), can proceed quickly and can rapidly produce mRNA vaccines at large scale. Potential benefit also arises from the fact that antigens encoded by the mRNAs of the invention are expressed by the cells of the subject, e.g., are expressed by the human body, and thus the subject, e.g., the human body, serves as the “factory” to produce the antigens which, in turn, elicits the desired immune response.

In preferred aspects, antigens are proteins capable of inducing an immune response (e.g., causing an immune system to produce antibodies against the antigens). Herein, use of the term “antigen” encompasses immunogenic proteins, as well as polypeptides or peptides derived from immunogenic proteins, for example immunogenic fragments (an immunogenic fragment that induces (or is capable of inducing) an immune response to an antigen, unless otherwise stated. It should be understood that the term “protein” encompasses polypeptides and peptides and the term “antigen” encompasses antigenic fragments. Other molecules may be antigenic such as bacterial polysaccharides or combinations of protein and polysaccharide structures, but for the viral vaccines included herein, viral proteins, fragments of viral proteins and designed and or mutated proteins derived from the betacoronavirus SARS-CoV-2 are the antigens featured herein.

Nucleic Acids

The compositions of the present disclosure comprise two or more RNAs, each having an open reading frame (ORF) encoding a coronavirus antigen. In some embodiments, each RNA is a messenger RNA (mRNA). In some embodiments, each RNA (e.g., mRNA) further comprises a 5' UTR, 3' UTR, a poly(A) tail and/or a 5' cap analog.

It should also be understood that the coronavirus vaccine of the present disclosure may include any 5' untranslated region (UTR) and/or any 3' UTR. UTR₈ may also be omitted from the RNA polynucleotides provided herein. Nucleic acids comprise a polymer of nucleotides (nucleotide monomers). Thus, nucleic acids are also referred to as polynucleotides. Nucleic acids may be or may include, for example, deoxyribonucleic acids (DNAs), ribonucleic acids (RNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a P-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino- a-LNA having a 2'- amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) and/or chimeras and/or combinations thereof.

Messenger RNA (mRNA) is any RNA that encodes a (at least one) protein (a naturally- occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded protein in vitro, in vivo, in situ, or ex vivo. The skilled artisan will appreciate that, except where otherwise noted, nucleic acid sequences set forth in the instant application may recite “T”s in a representative DNA sequence but where the sequence represents RNA (e.g., mRNA), the “T”s would be substituted for “U”s. Thus, any of the DNAs disclosed and identified by a particular sequence identification number herein also disclose the corresponding RNA (e.g. , mRNA) sequence complementary to the DNA, where each “T” of the DNA sequence is substituted with “U.”

An open reading frame (ORF) is a continuous stretch of DNA or RNA beginning with a start codon (e.g., methionine (ATG or AUG)) and ending with a stop codon (e.g., TAA, TAG or TGA, or UAA, UAG or UGA). An ORF typically encodes a protein. It will be understood that the sequences disclosed herein may further comprise additional elements, e.g., 5' and 3' UTR₈, but that those elements, unlike the ORF, need not necessarily be present in an RNA polynucleotide of the present disclosure.

In some embodiments, the first ORF comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the sequence of SEQ ID NO: 19. In some embodiments, the ORF comprises a nucleotide sequence having at least 80% identity to the sequence of SEQ ID NO: 19. In some embodiments, the ORF comprises a nucleotide sequence having at least 85% identity to the sequence of SEQ ID NO: 19. In some embodiments, the ORF comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 19. In some embodiments, the ORF comprises a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO: 19. In some embodiments, the ORF comprises the nucleotide sequence of SEQ ID NO: 19.

In some embodiments, the first mRNA comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the sequence of SEQ ID NO: 18. In some embodiments, the mRNA comprises a nucleotide sequence having at least 80% identity to the sequence of SEQ ID NO: 18. In some embodiments, the mRNA comprises a nucleotide sequence having at least 85% identity to the sequence of SEQ ID NO: 18. In some embodiments, the mRNA comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 18. In some embodiments, the mRNA comprises a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO: 18. In some embodiments, the mRNA comprises the nucleotide sequence of SEQ ID NO: 18.

In some embodiments, the second ORF comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the sequence of SEQ ID NO: 10. In some embodiments, the ORF comprises a nucleotide sequence having at least 80% identity to the sequence of SEQ ID NO: 10. In some embodiments, the ORF comprises a nucleotide sequence having at least 85% identity to the sequence of SEQ ID NO: 10. In some embodiments, the ORF comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 10. In some embodiments, the ORF comprises a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO: 10. In some embodiments, the ORF comprises the nucleotide sequence of SEQ ID NO: 10.

In some embodiments, the second mRNA comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the sequence of SEQ ID NO: 9. In some embodiments, the mRNA comprises a nucleotide sequence having at least 80% identity to the sequence of SEQ ID NO: 9. In some embodiments, the mRNA comprises a nucleotide sequence having at least 85% identity to the sequence of SEQ ID NO: 9. In some embodiments, the mRNA comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 9. In some embodiments, the mRNA comprises a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO: 9. In some embodiments, the mRNA comprises the nucleotide sequence of SEQ ID NO: 9.

Variants

In some embodiments, the compositions of the present disclosure include RNA that encodes a coronavirus antigen variant. Antigen variants or other polypeptide variants refers to molecules that differ in their amino acid sequence from a wild-type, native, or reference sequence. The antigen/polypeptide variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence. Ordinarily, variants possess at least 50% identity to a wild-type, native or reference sequence. In some embodiments, variants share at least 80%, or at least 90% identity with a wild-type, native, or reference sequence. Variant antigens/polypeptides encoded by nucleic acids of the disclosure may contain amino acid changes that confer any of a number of desirable properties, e.g., that enhance their immunogenicity, enhance their expression, and/or improve their stability or PK/PD properties in a subject. Variant antigens/polypeptides can be made using routine mutagenesis techniques and assayed as appropriate to determine whether they possess the desired property. Assays to determine expression levels and immunogenicity are well known in the art. Similarly, PK/PD properties of a protein variant can be measured using art recognized techniques, e.g., by determining expression of antigens in a vaccinated subject over time and/or by looking at the durability of the induced immune response. The stability of protein(s) encoded by a variant nucleic acid may be measured by assaying thermal stability or stability upon urea denaturation or may be measured using in silico prediction. Methods for such experiments and in silico determinations are known in the art.

In some embodiments, a composition comprises an RNA or an RNA ORF that comprises a nucleotide sequence of any one of the sequences provided herein (see, e.g., Table 1), or comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence of any one of the sequences provided herein.

The term “identity” refers to a relationship between the sequences of two or more polypeptides (e.g. antigens) or polynucleotides (nucleic acids), as determined by comparing the sequences. Identity also refers to the degree of sequence relatedness between or among sequences as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., “algorithms”). Identity of related antigens or nucleic acids can be readily calculated by known methods. “Percent (%) identity” as it applies to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical with the residues in the amino acid sequence or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for the alignment are well known in the art. It is understood that identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation. Generally, variants of a particular polynucleotide or polypeptide (e.g., antigen) have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, et al (1997), "Gapped BLAST and PSLBLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402). Another popular local alignment technique is based on the Smith- Waterman algorithm (Smith, T.F. & Waterman, M.S. (1981) “Identification of common molecular subsequences.” J. Mol. Biol. 147:195-197). A general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, S.B. & Wunsch, C.D. (1970) “A general method applicable to the search for similarities in the amino acid sequences of two proteins.” J. Mol. Biol. 48:443-453). More recently a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed that purportedly produces global alignment of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm.

As such, polynucleotides encoding peptides or polypeptides containing substitutions, insertions and/or additions, deletions and covalent modifications with respect to reference sequences, in particular the polypeptide (e.g., antigen) sequences disclosed herein, are included within the scope of this disclosure. For example, sequence tags or amino acids, such as one or more lysines, can be added to peptide sequences (e.g., at the N-terminal or C-terminal ends). Sequence tags can be used for peptide detection, purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation. Alternatively, amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminal or N-terminal residues) may alternatively be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence which is soluble or linked to a solid support. In some embodiments, sequences for (or encoding) signal sequences, termination sequences, transmembrane domains, linkers, multimerization domains (such as, e.g., foldon regions) and the like may be substituted with alternative sequences that achieve the same or a similar function. In some embodiments, cavities in the core of proteins can be filled to improve stability, e.g., by introducing larger amino acids. In other embodiments, buried hydrogen bond networks may be replaced with hydrophobic resides to improve stability. In yet other embodiments, glycosylation sites may be removed and replaced with appropriate residues. Such sequences are readily identifiable to one of skill in the art. It should also be understood that some of the sequences provided herein contain sequence tags or terminal peptide sequences (e.g., at the N-terminal or C-terminal ends) that may be deleted, for example, prior to use in the preparation of an RNA (e.g., mRNA) vaccine.

As recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of coronavirus antigens of interest. For example, provided herein is any protein fragment (meaning a polypeptide sequence at least one amino acid residue shorter than a reference antigen sequence but otherwise identical) of a reference protein, provided that the fragment is immunogenic and confers a protective immune response to the coronavirus. In addition to variants that are identical to the reference protein but are truncated, in some embodiments, an antigen includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations, as shown in any of the sequences provided or referenced herein. Antigens/antigenic polypeptides can range in length from about 4, 6, or 8 amino acids to full length proteins.

Signal Peptides

In some embodiments, a composition comprises an RNA (e.g., mRNA) having an ORF that encodes a signal peptide fused to the coronavirus antigen. Signal peptides, comprising the N-terminal 15-60 amino acids of proteins, are typically needed for the translocation across the membrane on the secretory pathway and, thus, universally control the entry of most proteins both in eukaryotes and prokaryotes to the secretory pathway. In eukaryotes, the signal peptide of a nascent precursor protein (pre-protein) directs the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates the transport of the growing peptide chain across it for processing. ER processing produces mature proteins, wherein the signal peptide is cleaved from precursor proteins, typically by a ER-resident signal peptidase of the host cell, or they remain uncleaved and function as a membrane anchor. A signal peptide may also facilitate the targeting of the protein to the cell membrane.

A signal peptide may have a length of 15-60 amino acids. For example, a signal peptide may have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids. In some embodiments, a signal peptide has a length of 20-60, 25-60, 30-60, 35- 60, 40-60, 45- 60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40, 20- 40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30, 25-30, 15-25, 20-25, or 15- 20 amino acids. Signal peptides from heterologous genes (which regulate expression of genes other than coronavirus antigens in nature) are known in the art and can be tested for desired properties and then incorporated into a nucleic acid of the disclosure. In some embodiments, the signal peptide may comprise one of the following sequences: MDSKGSSQKGSRLLLLLVVSNLLLPQGVVG (SEQ ID NO: 8), MDWTWILFLVAAATRVHS (SEQ ID NO: 7); METPAQLLFLLLLWLPDTTG (SEQ ID NO: 6); MLGSNSGQRVVFTILLLLVAPAYS (SEQ ID NO: 12); MKCLLYLAFLFIGVNCA (SEQ ID NO: 13); MWLVSLAIVTACAGA (SEQ ID NO: 14); or MFVFLVLLPLVSSQC (SEQ ID NO: 15).

Fusion Proteins

In some embodiments, a composition of the present disclosure includes an RNA (e.g., mRNA) encoding an antigenic fusion protein. Thus, the encoded antigen or antigens may include two or more proteins (e.g., protein and/or protein fragment) joined together. Alternatively, the protein to which a protein antigen is fused does not promote a strong immune response to itself, but rather to the coronavirus antigen. Antigenic fusion proteins, in some embodiments, retain the functional property from each original protein.

Scaffold Moieties

The RNA (e.g., mRNA) vaccines as provided herein, in some embodiments, encode fusion proteins that comprise coronavirus antigens linked to scaffold moieties. In some embodiments, such scaffold moieties impart desired properties to an antigen encoded by a nucleic acid of the disclosure. For example scaffold proteins may improve the immunogenicity of an antigen, e.g., by altering the structure of the antigen, altering the uptake and processing of the antigen, and/or causing the antigen to bind to a binding partner.

In some embodiments, the scaffold moiety is protein that can self-assemble into protein nanoparticles that are highly symmetric, stable, and structurally organized, with diameters of 10-150 nm, a highly suitable size range for optimal interactions with various cells of the immune system. In some embodiments, viral proteins or virus-like particles can be used to form stable nanoparticle structures. Examples of such viral proteins are known in the art. For example, in some embodiments, the scaffold moiety is a hepatitis B surface antigen (HBsAg). HBsAg forms spherical particles with an average diameter of ~22 nm and which lacked nucleic acid and hence are non-infectious (Lopez-Sagaseta, J. et al. Computational and Structural Biotechnology Journal 14 (2016) 58-68). In some embodiments, the scaffold moiety is a hepatitis B core antigen (HBcAg) self-assembles into particles of 24-31 nm diameter, which resembled the viral cores obtained from HBV-infected human liver. HBcAg produced in self- assembles into two classes of differently sized nanoparticles of 300 A and 360 A diameter, corresponding to 180 or 240 protomers. In some embodiments, the coronavirus antigen is fused to HBsAG or HBcAG to facilitate self-assembly of nanoparticles displaying the coronavirus antigen.

In some embodiments, bacterial protein platforms may be used. Non-limiting examples of these self-assembling proteins include ferritin, lumazine and encapsulin.

Ferritin is a protein whose main function is intracellular iron storage. Ferritin is made of 24 subunits, each composed of a four- alpha-helix bundle, that self-assemble in a quaternary structure with octahedral symmetry (Cho K.J. et al. J Mol Biol. 2009;390:83-98). Several high- resolution structures of ferritin have been determined, confirming that Helicobacter pylori ferritin is made of 24 identical protomers, whereas in animals, there are ferritin light and heavy chains that can assemble alone or combine with different ratios into particles of 24 subunits (Granier T. et al. J Biol Inorg Chem. 2003;8:105-111; Lawson D.M. et al. Nature. 1991;349:541-544). Ferritin self-assembles into nanoparticles with robust thermal and chemical stability. Thus, the ferritin nanoparticle is well-suited to carry and expose antigens.

Lumazine synthase (LS) is also well- suited as a nanoparticle platform for antigen display. LS, which is responsible for the penultimate catalytic step in the biosynthesis of riboflavin, is an enzyme present in a broad variety of organisms, including archaea, bacteria, fungi, plants, and eubacteria (Weber S.E. Flavins and Flavoproteins. Methods and Protocols, Series: Methods in Molecular Biology. 2014). The LS monomer is 150 amino acids long, and consists of beta- sheets along with tandem alpha-helices flanking its sides. A number of different quaternary structures have been reported for LS, illustrating its morphological versatility: from homopentamers up to symmetrical assemblies of 12 pentamers forming capsids of 150 A diameter. Even LS cages of more than 100 subunits have been described (Zhang X. et al. J Mol Biol. 2006;362:753-770).

Encapsulin, a novel protein cage nanoparticle isolated from thermophile Thermotoga maritima, may also be used as a platform to present antigens on the surface of self-assembling nanoparticles. Encapsulin is assembled from 60 copies of identical 31 kDa monomers having a thin and icosahedral T = 1 symmetric cage structure with interior and exterior diameters of 20 and 24 nm, respectively (Sutter M. et al. Nat Struct Mol Biol. 2008, 15: 939-947). Although the exact function of encapsulin in T. maritima is not clearly understood yet, its crystal structure has been recently solved and its function was postulated as a cellular compartment that encapsulates proteins such as DyP (Dye decolorizing peroxidase) and Flp (Ferritin like protein), which are involved in oxidative stress responses (Rahmanpour R. et al. FEBS J. 2013, 280: 2097-2104). In some embodiments, an RNA of the present disclosure encodes a coronavirus antigen (e.g., SARS-CoV-2 S protein) fused to a foldon domain. The foldon domain may be, for example, obtained from bacteriophage T4 fibritin (see, e.g., Tao Y, et al. Structure. 1997 Jun 15; 5(6):789-98).

Linkers and Cleavable Peptides

In some embodiments, the mRNAs of the disclosure encode more than one polypeptide, referred to herein as fusion proteins. In some embodiments, the mRNA further encodes a linker located between at least one or each domain of the fusion protein. The linker can be, for example, a cleavable linker or protease- sensitive linker. In some embodiments, the linker is selected from the group consisting of F2A linker, P2A linker, T2A linker, E2A linker, and combinations thereof. This family of self-cleaving peptide linkers, referred to as 2A peptides, has been described in the art (see for example, Kim, J.H. et al. (2011) PLoS ONE 6:el8556). In some embodiments, the linker is an F2A linker. In some embodiments, the linker is a GGGS (SEQ ID NO: 1) linker. In some embodiments, the fusion protein contains three domains with intervening linkers, having the structure: domain-linker-domain-linker-domain.

Cleavable linkers known in the art may be used in connection with the disclosure. Exemplary such linkers include: F2A linkers, T2A linkers, P2A linkers, E2A linkers (See, e.g., WO2017127750). The skilled artisan will appreciate that other art-recognized linkers may be suitable for use in the constructs of the disclosure (e.g., encoded by the nucleic acids of the disclosure). The skilled artisan will likewise appreciate that other polycistronic constructs (mRNA encoding more than one antigen/polypeptide separately within the same molecule) may be suitable for use as provided herein.

Sequence Optimization

In some embodiments, an ORF encoding an antigen of the disclosure is codon optimized. Codon optimization methods are known in the art. For example, an ORF of any one or more of the sequences provided herein may be codon optimized. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add post translation modification sites in encoded protein (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art - non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods. In some embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms.

In some embodiments, a codon optimized sequence shares less than 95% sequence identity to a naturally-occurring or wild-type sequence ORF (e.g., a naturally-occurring or wildtype mRNA sequence encoding a coronavirus antigen). In some embodiments, a codon optimized sequence shares less than 90% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a coronavirus antigen). In some embodiments, a codon optimized sequence shares less than 85% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a coronavirus antigen). In some embodiments, a codon optimized sequence shares less than 80% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a coronavirus antigen). In some embodiments, a codon optimized sequence shares less than 75% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a coronavirus antigen).

In some embodiments, a codon optimized sequence shares between 65% and 85% (e.g., between about 67% and about 85% or between about 67% and about 80%) sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a coronavirus antigen). In some embodiments, a codon optimized sequence shares between 65% and 75% or about 80% sequence identity to a naturally-occurring or wildtype sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a coronavirus antigen).

In some embodiments, a codon-optimized sequence encodes an antigen that is as immunogenic as, or more immunogenic than (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 200% more), than a coronavirus antigen encoded by a non-codon-optimized sequence.

When transfected into mammalian host cells, the modified mRNAs have a stability of between 12-18 hours, or greater than 18 hours, e.g., 24, 36, 48, 60, 72, or greater than 72 hours and are capable of being expressed by the mammalian host cells.

In some embodiments, a codon optimized RNA may be one in which the levels of G/C are enhanced. The G/C-content of nucleic acid molecules (e.g., mRNA) may influence the stability of the RNA. RNA having an increased amount of guanine (G) and/or cytosine (C) residues may be functionally more stable than RNA containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. As an example, WO02/098443 discloses a pharmaceutical composition containing an mRNA stabilized by sequence modifications in the translated region. Due to the degeneracy of the genetic code, the modifications work by substituting existing codons for those that promote greater RNA stability without changing the resulting amino acid. The approach is limited to coding regions of the RNA.

Chemically Unmodified Nucleotides

In some embodiments, an RNA (e.g., mRNA) is not chemically modified and comprises the standard ribonucleotides consisting of adenosine, guanosine, cytosine and uridine. In some embodiments, nucleotides and nucleosides of the present disclosure comprise standard nucleoside residues such as those present in transcribed RNA (e.g. A, G, C, or U). In some embodiments, nucleotides and nucleosides of the present disclosure comprise standard deoxyribonucleosides such as those present in DNA (e.g. dA, dG, dC, or dT).

Chemical Modifications

The compositions of the present disclosure comprise, in some embodiments, an RNA having an open reading frame encoding a coronavirus antigen, wherein the nucleic acid comprises nucleotides and/or nucleosides that can be standard (unmodified) or modified as is known in the art. In some embodiments, nucleotides and nucleosides of the present disclosure comprise modified nucleotides or nucleosides. Such modified nucleotides and nucleosides can be naturally-occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications can include those at the sugar, backbone, or nucleobase portion of the nucleotide and/or nucleoside as are recognized in the art.

In some embodiments, a naturally-occurring modified nucleotide or nucleotide of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such naturally occurring modified nucleotides and nucleotides can be found, inter alia, in the widely recognized MODOMICS database.

In some embodiments, a non-naturally occurring modified nucleotide or nucleoside of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be found, inter alia, in published US application Nos. PCT/US2012/058519; PCT/US2013/075177;

PCT/US2014/058897; PCT/US2014/058891; PCT/US2014/070413; PCT/US2015/036773;

PCT/US2015/036759; PCT/US2015/036771; or PCT/IB 2017/051367 all of which are incorporated by reference herein. Hence, nucleic acids of the disclosure (e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids) can comprise standard nucleotides and nucleosides, naturally- occurring nucleotides and nucleosides, non-naturally-occurring nucleotides and nucleosides, or any combination thereof.

Nucleic acids of the disclosure (e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids), in some embodiments, comprise various (more than one) different types of standard and/or modified nucleotides and nucleosides. In some embodiments, a particular region of a nucleic acid contains one, two or more (optionally different) types of standard and/or modified nucleotides and nucleosides.

In some embodiments, a modified RNA nucleic acid (e.g., a modified mRNA nucleic acid), introduced to a cell or organism, exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.

In some embodiments, a modified RNA nucleic acid (e.g., a modified mRNA nucleic acid), introduced into a cell or organism, may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response) relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.

Nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids), in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the nucleic acids to achieve desired functions or properties. The modifications may be present on internucleotide linkages, purine or pyrimidine bases, or sugars. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a nucleic acid may be chemically modified.

The present disclosure provides for modified nucleosides and nucleotides of a nucleic acid (e.g., RNA nucleic acids, such as mRNA nucleic acids). A “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). A “nucleotide” refers to a nucleoside, including a phosphate group. Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. Nucleic acids can comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the nucleic acids would comprise regions of nucleotides. Modified nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures, such as, for example, in those nucleic acids having at least one chemical modification. One example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil. Any combination of base/sugar or linker may be incorporated into nucleic acids of the present disclosure.

In some embodiments, modified nucleobases in nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) comprise 1-methyl-pseudouridine (mly), 1-ethyl-pseudouridine (ely), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and/or pseudouridine (y). In some embodiments, modified nucleobases in nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) comprise 5-methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, and/or 5-methoxy cytidine. In some embodiments, the polyribonucleotide includes a combination of at least two (e.g., 2, 3, 4 or more) of any of the aforementioned modified nucleobases, including but not limited to chemical modifications.

In some embodiments, a mRNA of the disclosure comprises 1-methyl-pseudouridine (in h|/ substitutions at one or more or all uridine positions of the nucleic acid.

In some embodiments, a mRNA of the disclosure comprises 1-methyl-pseudouridine (in h|/) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid.

In some embodiments, a mRNA of the disclosure comprises pseudouridine (y) substitutions at one or more or all uridine positions of the nucleic acid.

In some embodiments, a mRNA of the disclosure comprises pseudouridine (y) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid.

In some embodiments, a mRNA of the disclosure comprises uridine at one or more or all uridine positions of the nucleic acid.

In some embodiments, mRNAs are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification. For example, a nucleic acid can be uniformly modified with 1-methyl-pseudouridine, meaning that all uridine residues in the mRNA sequence are replaced with 1-methyl-pseudouridine. Similarly, a nucleic acid can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.

The nucleic acids of the present disclosure may be partially or fully modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may be uniformly modified in a nucleic acid of the disclosure, or in a predetermined sequence region thereof (e.g., in the mRNA including or excluding the poly (A) tail). In some embodiments, all nucleotides X in a nucleic acid of the present disclosure (or in a sequence region thereof) are modified nucleotides, wherein X may be any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

The nucleic acid may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). It will be understood that any remaining percentage is accounted for by the presence of unmodified A, G, U, or C.

The mRNAs may contain at a minimum 1% and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides. For example, the nucleic acids may contain a modified pyrimidine such as a modified uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the nucleic acid is replaced with a modified uracil (e.g., a 5-substituted uracil). The modified uracil can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine in the nucleic acid is replaced with a modified cytosine (e.g., a 5-substituted cytosine). The modified cytosine can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).

Untranslated Regions (UTR₈)

The mRNAs of the present disclosure may comprise one or more regions or parts which act or function as an untranslated region. Where mRNAs are designed to encode at least one antigen of interest, the nucleic may comprise one or more of these untranslated regions (UTR₈). Wild-type untranslated regions of a nucleic acid are transcribed but not translated. In mRNA, the 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal. There is growing body of evidence about the regulatory roles played by the UTR₈ in terms of stability of the nucleic acid molecule and translation. The regulatory features of a UTR can be incorporated into the polynucleotides of the present disclosure to, among other things, enhance the stability of the molecule. The specific features can also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organs sites. A variety of 5 ’UTR and 3 ’UTR sequences are known and available in the art.

A 5' UTR is region of an mRNA that is directly upstream (5') from the start codon (the first codon of an mRNA transcript translated by a ribosome). A 5' UTR does not encode a protein (is non-coding). Natural 5'UTR₈ have features that play roles in translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G'.5'UTR also have been known to form secondary structures which are involved in elongation factor binding.

In some embodiments of the disclosure, a 5’ UTR is a heterologous UTR, i.e., is a UTR found in nature associated with a different ORF. In another embodiment, a 5’ UTR is a synthetic UTR, i.e., does not occur in nature. Synthetic UTR₈ include UTR₈ that have been mutated to improve their properties, e.g., which increase gene expression as well as those which are completely synthetic. Exemplary 5’ UTR₈ include Xenopus or human derived a-globin or b- globin (US8278063; US9012219), human cytochrome b-245 a polypeptide, and hydroxysteroid (17b) dehydrogenase, and Tobacco etch virus (US8278063, US9012219). CMV immediate-early 1 (IE1) gene (US2014/0206753, WO2013/185069), the sequence GGGAUCCUACC (SEQ ID NO: 17) (WO2014/144196) may also be used. In another embodiment, 5' UTR of a TOP gene is a 5' UTR of a TOP gene lacking the 5' TOP motif (the oligopyrimidine tract) (e.g., W02015/101414, W02015/101415, WO2015/062738, WO2015/024667, WO2015/024667; 5' UTR element derived from ribosomal protein Large 32 (L32) gene (W02015/101414, W02015/101415, WO2015/062738), 5' UTR element derived from the 5'UTR of an hydroxysteroid (17-|3) dehydrogenase 4 gene (HSD17B4) (WO2015/024667), or a 5' UTR element derived from the 5' UTR of ATP5A1 (WO2015/024667) can be used. In some embodiments, an internal ribosome entry site (IRES) is used instead of a 5' UTR.

In some embodiments, a 5' UTR of the present disclosure comprises the sequence of SEQ ID NO: 2.

A 3' UTR is region of an mRNA that is directly downstream (3') from the stop codon (the codon of an mRNA transcript that signals a termination of translation). A 3' UTR does not encode a protein (is non-coding). Natural or wild type 3' UTR₈ are known to have stretches of adenosines and uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo.

Introduction, removal or modification of 3' UTR AU rich elements (AREs) can be used to modulate the stability of nucleic acids (e.g., RNA) of the disclosure. When engineering specific nucleic acids, one or more copies of an ARE can be introduced to make nucleic acids of the disclosure less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein. Transfection experiments can be conducted in relevant cell lines, using nucleic acids of the disclosure and protein production can be assayed at various time points post-transfection. For example, cells can be transfected with different ARE-engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hour, 12 hour, 24 hour, 48 hour, and 7 days post-transfection.

3' UTR₈ may be heterologous or synthetic. With respect to 3’ UTR₈, globin UTR₈, including Xenopus P-globin UTR₈ and human P-globin UTR₈ are known in the art (US8278063, US9012219, US2011/0086907). A modified P-globin construct with enhanced stability in some cell types by cloning two sequential human P-globin 3 ’UTR₈ head to tail has been developed and is well known in the art (US2012/0195936, WO2014/071963). In addition a2-globin, al- globin, UTR₈ and mutants thereof are also known in the art (W02015/101415, WO2015/024667). Other 3' UTR₈ described in the mRNA constructs in the non-patent literature include CYBA (Ferizi et al., 2015) and albumin (Thess et al., 2015). Other exemplary 3' UTR₈ include that of bovine or human growth hormone (wild type or modified) (WO2013/185069, US2014/0206753, WO2014/152774), rabbit P globin and hepatitis B virus (HBV), a-globin 3' UTR and Viral VEEV 3’ UTR sequences are also known in the art. In some embodiments, the sequence UUUGAAUU (WO2014/144196) is used. In some embodiments, 3' UTR₈ of human and mouse ribosomal protein are used. Other examples include rps9 3’UTR (W02015/101414), FIG4 (W02015/101415), and human albumin 7 (W02015/101415).

In some embodiments, a 3' UTR of the present disclosure comprises the sequence of SEQ ID NO: 4.

Those of ordinary skill in the art will understand that 5 ’UTR₈ that are heterologous or synthetic may be used with any desired 3’ UTR sequence. For example, a heterologous 5’UTR may be used with a synthetic 3’UTR with a heterologous 3” UTR.

Non-UTR sequences may also be used as regions or subregions within a nucleic acid. For example, introns or portions of introns sequences may be incorporated into regions of nucleic acid of the disclosure. Incorporation of intronic sequences may increase protein production as well as nucleic acid levels.

Combinations of features may be included in flanking regions and may be contained within other features. For example, the ORF may be flanked by a 5' UTR which may contain a strong Kozak translational initiation signal and/or a 3' UTR which may include an oligo(dT) sequence for templated addition of a poly-A tail. 5' UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and/or different genes such as the 5' UTR₈ described in US Patent Application Publication No. 2010/0293625 and PCT/US2014/069155, herein incorporated by reference in its entirety.

It should be understood that any UTR from any gene may be incorporated into the regions of a nucleic acid. Furthermore, multiple wild-type UTR₈ of any known gene may be utilized. It is also within the scope of the present disclosure to provide artificial UTR₈ which are not variants of wild type regions. These UTR₈ or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5' or 3' UTR may be inverted, shortened, lengthened, made with one or more other 5' UTR₈ or 3' UTR₈. As used herein, the term “altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3' UTR or 5' UTR may be altered relative to a wild-type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an “altered” UTR (whether 3' or 5') comprise a variant UTR.

In some embodiments, a double, triple or quadruple UTR such as a 5' UTR or 3' UTR may be used. As used herein, a “double” UTR is one in which two copies of the same UTR are encoded either in series or substantially in series. For example, a double beta-globin 3' UTR may be used as described in US Patent publication 2010/0129877, the contents of which are incorporated herein by reference in its entirety.

It is also within the scope of the present disclosure to have patterned UTR₈. As used herein “patterned UTR₈” are those UTR₈ which reflect a repeating or alternating pattern, such as AB AB AB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level.

In some embodiments, flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature or property. For example, polypeptides of interest may belong to a family of proteins which are expressed in a particular cell, tissue or at some time during development. The UTR₈ from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide. As used herein, a “family of proteins” is used in the broadest sense to refer to a group of two or more polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern.

The untranslated region may also include translation enhancer elements (TEE). As a nonlimiting example, the TEE may include those described in US Publication No. 2009/0226470, herein incorporated by reference in its entirety, and those known in the art.

In vitro Transcription of RNA cDNA encoding the polynucleotides described herein may be transcribed using an in vitro transcription (IVT) system. In vitro transcription of RNA is known in the art and is described in International Publication WO 2014/152027, which is incorporated by reference herein in its entirety. In some embodiments, the RNA of the present disclosure is prepared in accordance with any one or more of the methods described in WO 2018/053209 and WO 2019/036682, each of which is incorporated by reference herein.

In some embodiments, the RNA transcript is generated using a non-amplified, linearized DNA template in an in vitro transcription reaction to generate the RNA transcript. In some embodiments, the template DNA is isolated DNA. In some embodiments, the template DNA is cDNA. In some embodiments, the cDNA is formed by reverse transcription of a RNA polynucleotide, for example, but not limited to coronavirus mRNA. In some embodiments, cells, e.g., bacterial cells, e.g., E. coli, e.g., DH-1 cells are transfected with the plasmid DNA template. In some embodiments, the transfected cells are cultured to replicate the plasmid DNA which is then isolated and purified. In some embodiments, the DNA template includes a RNA polymerase promoter, e.g., a T7 promoter located 5 ' to and operably linked to the gene of interest.

In some embodiments, an in vitro transcription template encodes a 5' untranslated (UTR) region, contains an open reading frame, and encodes a 3' UTR and a poly(A) tail. The particular nucleic acid sequence composition and length of an in vitro transcription template will depend on the mRNA encoded by the template.

A “5' untranslated region” (UTR) refers to a region of an mRNA that is directly upstream (i.e., 5') from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a polypeptide. When RNA transcripts are being generated, the 5’ UTR may comprise a promoter sequence. Such promoter sequences are known in the art. It should be understood that such promoter sequences will not be present in a vaccine of the disclosure.

A “3' untranslated region” (UTR) refers to a region of an mRNA that is directly downstream (i.e., 3') from the stop codon (i.e., the codon of an mRNA transcript that signals a termination of translation) that does not encode a polypeptide.

An “open reading frame” is a continuous stretch of DNA beginning with a start codon (e.g., methionine (ATG)), and ending with a stop codon (e.g., TAA, TAG or TGA) and encodes a polypeptide.

A “poly(A) tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3'), from the 3' UTR that contains multiple, consecutive adenosine monophosphates. A poly(A) tail may contain 10 to 300 adenosine monophosphates. For example, a poly(A) tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some embodiments, a poly(A) tail contains 50 to 250 adenosine monophosphates. In a relevant biological setting (e.g., in cells, in vivo) the poly(A) tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, and/or export of the mRNA from the nucleus and translation.

In some embodiments, a nucleic acid includes 200 to 3,000 nucleotides. For example, a nucleic acid may include 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides).

An in vitro transcription system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase.

The NTPs may be manufactured in house, may be selected from a supplier, or may be synthesized as described herein. The NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs.

Any number of RNA polymerases or variants may be used in the method of the present disclosure. The polymerase may be selected from, but is not limited to, a phage RNA polymerase, e.g., a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, and/or mutant polymerases such as, but not limited to, polymerases able to incorporate modified nucleic acids and/or modified nucleotides, including chemically modified nucleic acids and/or nucleotides. Some embodiments exclude the use of DNase.

In some embodiments, the RNA transcript is capped via enzymatic capping. In some embodiments, the RNA comprises 5' terminal cap, for example, 7mG(5’)ppp(5’)NlmpNp.

Chemical Synthesis

Solid-phase chemical synthesis. Nucleic acids the present disclosure may be manufactured in whole or in part using solid phase techniques. Solid-phase chemical synthesis of nucleic acids is an automated method wherein molecules are immobilized on a solid support and synthesized step by step in a reactant solution. Solid-phase synthesis is useful in site-specific introduction of chemical modifications in the nucleic acid sequences.

Liquid Phase Chemical Synthesis. The synthesis of nucleic acids of the present disclosure by the sequential addition of monomer building blocks may be carried out in a liquid phase.

Combination of Synthetic Methods. The synthetic methods discussed above each has its own advantages and limitations. Attempts have been conducted to combine these methods to overcome the limitations. Such combinations of methods are within the scope of the present disclosure. The use of solid-phase or liquid-phase chemical synthesis in combination with enzymatic ligation provides an efficient way to generate long chain nucleic acids that cannot be obtained by chemical synthesis alone.

Ligation of Nucleic Acid Regions or Subregions

Assembling nucleic acids by a ligase may also be used. DNA or RNA ligases promote intermolecular ligation of the 5’ and 3’ ends of polynucleotide chains through the formation of a phosphodiester bond. Nucleic acids such as chimeric polynucleotides and/or circular nucleic acids may be prepared by ligation of one or more regions or subregions. DNA fragments can be joined by a ligase catalyzed reaction to create recombinant DNA with different functions. Two oligodeoxynucleotides, one with a 5’ phosphoryl group and another with a free 3’ hydroxyl group, serve as substrates for a DNA ligase.

Purification

Purification of the nucleic acids described herein may include, but is not limited to, nucleic acid clean-up, quality assurance and quality control. Clean-up may be performed by methods known in the arts such as, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNATM oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark) or HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term “purified” when used in relation to a nucleic acid such as a “purified nucleic acid” refers to one that is separated from at least one contaminant. A “contaminant” is any substance that makes another unfit, impure or inferior. Thus, a purified nucleic acid (e.g., DNA and RNA) is present in a form or setting different from that in which it is found in nature, or a form or setting different from that which existed prior to subjecting it to a treatment or purification method.

A quality assurance and/or quality control check may be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC.

In some embodiments, the nucleic acids may be sequenced by methods including, but not limited to reverse-transcriptase-PCR.

Quantification

In some embodiments, the nucleic acids of the present disclosure may be quantified in exosomes or when derived from one or more bodily fluid. Bodily fluids include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood. Alternatively, exosomes may be retrieved from an organ selected from the group consisting of lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus, liver, and placenta.

Assays may be performed using construct specific probes, cytometry, qRT-PCR, real- time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof while the exosomes may be isolated using immunohistochemical methods such as enzyme linked immunosorbent assay (ELISA) methods. Exosomes may also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof.

These methods afford the investigator the ability to monitor, in real time, the level of nucleic acids remaining or delivered. This is possible because the nucleic acids of the present disclosure, in some embodiments, differ from the endogenous forms due to the structural or chemical modifications.

In some embodiments, the nucleic acid may be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UV/Vis). A non-limiting example of a UV/Vis spectrometer is a NANODROP® spectrometer (ThermoFisher, Waltham, MA). The quantified nucleic acid may be analyzed in order to determine if the nucleic acid may be of proper size, check that no degradation of the nucleic acid has occurred. Degradation of the nucleic acid may be checked by methods such as, but not limited to, agarose gel electrophoresis, HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC- HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE).

Lipid Nanoparticles (LNPs)

In some embodiments, the RNA (e.g., mRNA) of the disclosure is formulated in a lipid nanoparticle (LNP). Lipid nanoparticles typically comprise ionizable amino (cationic) lipid, non-cationic lipid, sterol and PEG lipid components along with the nucleic acid cargo of interest. The lipid nanoparticles of the disclosure can be generated using components, compositions, and methods as are generally known in the art, see for example PCT/US2016/052352; PCT/US2016/068300; PCT/US2017/037551; PCT/US2015/027400; PCT/US2016/047406; PCT/US2016/000129; PCT/US2016/014280; PCT/US2016/014280; PCT/US2017/038426; PCT/US2014/027077; PCT/US2014/055394; PCT/US2016/052117; PCT/US2012/069610; PCT/US2017/027492; PCT/US2016/059575 and PCT/US2016/069491 all of which are incorporated by reference herein in their entirety.

Vaccines of the present disclosure are typically formulated in lipid nanoparticles. The vaccines can be made, for example, using mixing processes such as microfluidics and T- junction mixing of two fluid streams, one of which contains the mRNA and the other has the lipid components. In some embodiments, the vaccines are prepared by combining an ionizable amino lipid, a phospholipid (such as DOPE or DSPC), a PEG lipid (such as 1,2-dimyristoyl-OT- glycerol methoxypoly ethylene glycol, also known as PEG-DMG), and a structural lipid (such as cholesterol) in an alcohol (e.g., ethanol). The lipids may be combined to yield desired molar ratios and diluted with water and alcohol (e.g., ethanol) to a final lipid concentration of between about 5.5 mM and about 25 mM, for example.

Vaccines including mRNA and a lipid component may be prepared, for example, by combining a lipid solution with an mRNA solution at lipid component to mRNA wt:wt ratios of between about 5:1 and about 50:1. The lipid solution may be rapidly injected using a microfluidic based system (e.g., NanoAssemblr) at flow rates between about 10 ml/min and about 18 ml/min, for example, into the mRNA solution to produce a suspension (e.g., with a water to alcohol ratio between about 1:1 and about 4:1).

Vaccines can be processed by dialysis to remove the alcohol (e.g., ethanol) and achieve buffer exchange. Formulations may be dialyzed against phosphate buffered saline (PBS), pH 7.4, for example, at volumes greater than that of the primary product (e.g., using Slide-A-Lyzer cassettes (Thermo Fisher Scientific Inc., Rockford, IL)) with a molecular weight cutoff of 10 kD, for example. The forgoing exemplary method induces nanoprecipitation and particle formation. Alternative processes including, but not limited to, T-junction and direct injection, may be used to achieve the same nanoprecipitation.

In some embodiments, the lipid nanoparticle comprises at least one ionizable amino lipid, at least one non-cationic lipid, at least one sterol, and/or at least one polyethylene glycol (PEG)-modified lipid.

In some embodiments, the lipid nanoparticle comprises 20-60 mol% ionizable amino lipid. For example, the lipid nanoparticle may comprise 20-50 mol%, 20-40 mol%, 20-30 mol%, 30-60 mol%, 30-50 mol%, 30-40 mol%, 40-60 mol%, 40-50 mol%, or 50-60 mol% ionizable amino lipid. In some embodiments, the lipid nanoparticle comprises 20 mol%, 30 mol%, 40 mol%, 50 mol%, or 60 mol% ionizable amino lipid.

In some embodiments, the lipid nanoparticle comprises 5-25 mol% non-cationic lipid. For example, the lipid nanoparticle may comprise 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% non-cationic lipid. In some embodiments, the lipid nanoparticle comprises 5 mol%, 10 mol%, 15 mol%, 20 mol%, or 25 mol% non-cationic lipid.

In some embodiments, the lipid nanoparticle comprises 25-55 mol% sterol. For example, the lipid nanoparticle may comprise 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25- 30 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35- 50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45- 50 mol%, or 50-55 mol% sterol. In some embodiments, the lipid nanoparticle comprises 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% sterol.

In some embodiments, the lipid nanoparticle comprises 0.5-15 mol% PEG-modified lipid. For example, the lipid nanoparticle may comprise 0.5-10 mol%, 0.5-5 mol%, 1-15 mol%, 1-10 mol%, 1-5 mol%, 2-15 mol%, 2-10 mol%, 2-5 mol%, 5-15 mol%, 5-10 mol%, or 10-15 mol%. In some embodiments, the lipid nanoparticle comprises 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% PEG-modified lipid.

In some embodiments, the lipid nanoparticle comprises 20-60 mol% ionizable amino lipid, 5-25 mol% non-cationic lipid, 25-55 mol% sterol, and 0.5-15 mol% PEG-modified lipid.

In some embodiments, an ionizable amino lipid of the disclosure comprises a compound of Formula (I):

or a salt or isomer thereof, wherein:

Ri is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;

R₂ and R3 are independently selected from the group consisting of H, Ci-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R₂ and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R₄ is selected from the group consisting of a C_3-6 carbocycle, -(CH₂)_nQ, -(CH₂)_nCHQR, -CHQR, -CQ(R)₂, and unsubstituted Ci-6 alkyl, where Q is selected from a carbocycle, heterocycle, -OR, -O(CH₂)_nN(R)₂, -C(O)OR, -OC(O)R, -CX₃, -CX₂H, -CXH₂, -CN, -N(R)₂, -C(O)N(R)₂, -N(R)C(O)R, -N(R)S(O)₂R, -N(R)C(O)N(R)₂, -N(R)C(S)N(R)₂, -N(R)R₈, -O(CH₂)_nOR, -N(R)C(=NR₉)N(R)₂, -N(R)C(=CHR₉)N(R)₂, -OC(O)N(R)₂, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)₂R, -N(OR)C(O)OR, -N(OR)C(O)N(R)₂, -N(OR)C(S)N(R)₂, -N(OR)C(=NR₉)N(R)₂, -N(OR)C(=CHR₉)N(R)₂, -C(=NR₉)N(R)₂, -C(=NR₉)R, -C(O)N(R)OR, and -C(R)N(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4, and 5; each R₈ is independently selected from the group consisting of C_1-3 alkyl, C₂-3 alkenyl, and H; each R₆ is independently selected from the group consisting of C_1-3 alkyl, C₂-3 alkenyl, and H;

M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)₂-, -S-S-, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C_1-3 alkyl, C₂-3 alkenyl, and H;

R₈ is selected from the group consisting of C_3-6 carbocycle and heterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C1-6 alkyl, -OR, -S(O)₂R, -S(O)₂N(R)₂, C₂-6 alkenyl, C_3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C_1-3 alkyl, C₂-3 alkenyl, and H; each R’ is independently selected from the group consisting of Ci-18 alkyl, C2-I8 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C_3-14 alkyl and C_3-14 alkenyl; each R* is independently selected from the group consisting of Ci-12 alkyl and C2-12 alkenyl; each Y is independently a C_3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

In some embodiments, a subset of compounds of Formula (I) includes those in which when R₄ is -(CH₂)_nQ, -(CH₂)_nCHQR, -CHQR, or -CQ(R)₂, then (i) Q is not -N(R)₂ when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is 1 or 2.

In some embodiments, another subset of compounds of Formula (I) includes those in which Ri is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;

R₂ and R3 are independently selected from the group consisting of H, Ci-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R₂ and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R₄ is selected from the group consisting of a C_3-6 carbocycle, -(CH2)_nQ, -(CH₂)_nCHQR, -CHQR, -CQ(R)₂, and unsubstituted C1-6 alkyl, where Q is selected from a C_3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -O(CH₂)_nN(R)₂, -C(O)OR, -OC(O)R, -CX₃, -CX₂H, -CXH₂, -CN, -C(O)N(R)₂, -N(R)C(O)R, -N(R)S(O)₂R, -N(R)C(O)N(R)₂, -N(R)C(S)N(R)₂, -CRN(R)₂C(O)OR, -N(R)R₈, -O(CH₂)_nOR, -N(R)C(=NR₉)N(R)₂, -N(R)C(=CHR₉)N(R)₂, -OC(O)N(R)₂, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)₂R, -N(OR)C(O)OR, -N(OR)C(O)N(R)₂, -N(OR)C(S)N(R)₂,-N(OR)C(=NR₉)N(R)₂, -N(OR)C(=CHR₉)N(R)₂, -C(=NR₉)N(R)₂, -C(=NR₉)R, -C(O)N(R)OR, and a 5- to 14-membered heterocycloalkyl having one or more heteroatoms selected from N, O, and S which is substituted with one or more substituents selected from oxo (=0), OH, amino, mono- or di- alkylamino, and C_1-3 alkyl, and each n is independently selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R₆ is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H;

R₇ is selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H;

R₈ is selected from the group consisting of C_3-6 carbocycle and heterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C1-6 alkyl, -OR, -S(O)2R, -S(O)₂N(R)₂, C_2-6 alkenyl, C_3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R’ is independently selected from the group consisting of C_1-18 alkyl, C_2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C_3-14 alkyl and C_3-14 alkenyl; each R* is independently selected from the group consisting of C_1-12 alkyl and C_2-12 alkenyl; each Y is independently a C_3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.

In some embodiments, another subset of compounds of Formula (I) includes those in which

R₁ is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;

R₂ and R3 are independently selected from the group consisting of H, C_1-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R₂ and R₃, together with the atom to which they are attached, form a heterocycle or carbocycle; R₄ is selected from the group consisting of a C_3-6 carbocycle, -(CH₂)_nQ, -(CH₂)_nCHQR, -CHQR, -CQ(R)₂, and unsubstituted C1-6 alkyl, where Q is selected from a C_3-6 carbocycle, a 5- to 14-membered heterocycle having one or more heteroatoms selected from N, O, and S, -OR, -O(CH₂)_nN(R)₂, -C(O)OR, -OC(O)R, -CX₃, -CX₂H, -CXH₂, -CN, -C(O)N(R)₂, -N(R)C(O)R, -N(R)S(O)₂R, -N(R)C(O)N(R)₂, -N(R)C(S)N(R)₂, -CRN(R)₂C(O)OR, -N(R)R₈, -O(CH₂)_nOR, -N(R)C(=NR₉)N(R)₂, -N(R)C(=CHR₉)N(R)₂, -OC(O)N(R)₂, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)₂R, -N(OR)C(O)OR, -N(OR)C(O)N(R)₂, -N(OR)C(S)N(R)₂, -N(OR)C(=NR₉)N(R)₂ ,-N(OR)C(=CHR₉)N(R)₂, -C(=NR₉)R, -C(O)N(R)OR, and -C(=NR₉)N(R)₂, and each n is independently selected from 1, 2, 3, 4, and 5; and when Q is a 5- to 14-membered heterocycle and (i) R₄ is -(CH₂)_nQ in which n is 1 or 2, or (ii) R₄ is -(CH₂)_nCHQR in which n is 1, or (iii) R₄ is -CHQR, and -CQ(R)₂, then Q is either a 5- to 14-membered heteroaryl or 8- to 14-membered heterocycloalkyl; each R₅ is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R₆ is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H;

R₈ is selected from the group consisting of C_3-6 carbocycle and heterocycle; R9 is selected from the group consisting of H, CN, NO₂, C1-6 alkyl, -OR, -S(O)2R, -S(O)₂N(R)₂, C2-6 alkenyl, C_3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R’ is independently selected from the group consisting of Ci-18 alkyl, C2-I8 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C_3-14 alkyl and C_3-14 alkenyl; each R* is independently selected from the group consisting of Ci-12 alkyl and C2-12 alkenyl; each Y is independently a C_3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.

R₂ and R3 are independently selected from the group consisting of H, Ci-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R₂ and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R₄ is selected from the group consisting of a C_3-6 carbocycle, -(CH₂)_nQ, -(CH₂)_nCHQR, -CHQR, -CQ(R)₂, and unsubstituted C_1-6 alkyl, where Q is selected from a C_3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -O(CH₂)_nN(R)₂, -C(O)OR, -OC(O)R, -CX₃, -CX₂H, -CXH₂, -CN, -C(O)N(R)₂, -N(R)C(O)R, -N(R)S(O)₂R, -N(R)C(O)N(R)₂, -N(R)C(S)N(R)₂, -CRN(R)₂C(O)OR, -N(R)R₈, -O(CH₂)_nOR, -N(R)C(=NR₉)N(R)₂, -N(R)C(=CHR₉)N(R)₂, -OC(O)N(R)₂, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)₂R, -N(OR)C(O)OR, -N(OR)C(O)N(R)₂, -N(OR)C(S)N(R)₂,-N(OR)C(=NR₉)N(R)₂, -N(OR)C(=CHR₉)N(R)₂, -C(=NR₉)R, -C(O)N(R)OR, and -C(=NR₉)N(R)₂, and each n is independently selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each Re is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)₂-, -S-S-, an aryl group, and a heteroaryl group;

R? is selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H;

R₈ is selected from the group consisting of C_3-6 carbocycle and heterocycle;

R9 is selected from the group consisting of H, CN, NO₂, C1-6 alkyl, -OR, -S(O)₂R, -S(O)₂N(R)₂, C2-6 alkenyl, C_3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R’ is independently selected from the group consisting of Ci-18 alkyl, C2-I8 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C_3-14 alkyl and C_3-14 alkenyl; each R* is independently selected from the group consisting of Ci-12 alkyl and C2-12 alkenyl; each Y is independently a C_3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.

R₂ and R3 are independently selected from the group consisting of H, C2-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R₂ and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R₄ is -(CH₂)nQ or -(CH₂)_nCHQR, where Q is -N(R)₂, and n is selected from 3, 4, and 5; each R5 is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each Re is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H;

M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)₂-, -S-S-, an aryl group, and a heteroaryl group; R₇ is selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R’ is independently selected from the group consisting of Ci-18 alkyl, C2-I8 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C_3-14 alkyl and C_3-14 alkenyl; each R* is independently selected from the group consisting of Ci-12 alkyl and Ci-12 alkenyl; each Y is independently a C_3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.

R₂ and R3 are independently selected from the group consisting of Ci-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R₂ and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R₄ is selected from the group consisting of -(CH₂)_nQ, -(CH₂)_nCHQR, -CHQR, and -CQ(R)₂, where Q is -N(R)₂, and n is selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R₆ is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H;

R₇ is selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R’ is independently selected from the group consisting of Ci-18 alkyl, C2-I8 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C_3-14 alkyl and C_3-14 alkenyl; each R* is independently selected from the group consisting of Ci-12 alkyl and Ci-12 alkenyl; each Y is independently a C_3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.

In some embodiments, a subset of compounds of Formula (I) includes those of Formula (IA):

or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; Mi is a bond or M’; R₄ is unsubstituted C_1-3 alkyl, or -(CH₂)_nQ, in which Q is OH, -NHC(S)N(R)₂, -NHC(O)N(R)₂, -N(R)C(O)R, -N(R)S(O)₂R, -N(R)R₈, -NHC(=NR₉)N(R)₂, -NHC(=CHR₉)N(R)₂, -OC(O)N(R)₂, -N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -P(O)(OR’)O-, -S-S-, an aryl group, and a heteroaryl group; and R₂ and R3 are independently selected from the group consisting of H, Ci-14 alkyl, and C2-14 alkenyl.

In some embodiments, a subset of compounds of Formula (I) includes those of Formula (II):

(II) or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and 5; Mi is a bond or M’; R₄ is unsubstituted C_1-3 alkyl, or -(CH2)_nQ, in which n is 2, 3, or 4, and Q is OH, -NHC(S)N(R)₂, -NHC(O)N(R)₂, -N(R)C(O)R, -N(R)S(O)₂R, -N(R)R₈, -NHC(=NR₉)N(R)₂, -NHC(=CHR₉)N(R)₂, -OC(O)N(R)₂, -N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -P(O)(OR’)O-, -S-S-, an aryl group, and a heteroaryl group; and R₂ and R3 are independently selected from the group consisting of H, Ci-14 alkyl, and C2-14 alkenyl. In some embodiments, a subset of compounds of Formula (I) includes those of Formula (Ila), (lib), (lie), or (lie):

or a salt or isomer thereof, wherein R₄ is as described herein.

In some embodiments, a subset of compounds of Formula (I) includes those of Formula (lid):

or a salt or isomer thereof, wherein n is 2, 3, or 4; and m, R’, R”, and R₂ through Re are as described herein. For example, each of R₂ and R3 may be independently selected from the group consisting of C5-14 alkyl and C5-14 alkenyl.

In some embodiments, an ionizable amino lipid of the disclosure comprises heptadecan- 9-yl 8 ((2 hydroxyethyl)(6 oxo 6-(undecyloxy)hexyl)amino)octanoate. Thus, in some embodiments, an ionizable amino lipid of the disclosure comprises a compound having structure:

In some embodiments, an ionizable amino lipid of the disclosure comprises a compound having structure:

In some embodiments, a non-cationic lipid of the disclosure comprises 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),

1.2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero- phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), l-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine,l,2-diarachidonoyl-sn-glycero-3-phosphocholine,

1.2-didocosahexaenoyl-sn-glycero-3-phosphocholine, l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

In some embodiments, a PEG modified lipid of the disclosure comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the PEG-modified lipid is DMG-PEG, PEG-c- DOMG (also referred to as PEG-DOMG), PEG-DSG and/or PEG-DPG. In some embodiments, a sterol of the disclosure comprises cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alphatocopherol, and mixtures thereof.

In some embodiments, a LNP of the disclosure comprises an ionizable amino lipid of Compound 1, wherein the non-cationic lipid is DSPC, the structural lipid that is cholesterol, and the PEG lipid is DMG-PEG.

In some embodiments, the lipid nanoparticle comprises 45 - 55 mole percent (mol%) ionizable amino lipid. For example, lipid nanoparticle may comprise 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mol% ionizable amino lipid.

In some embodiments, the lipid nanoparticle comprises 5 - 15 mol% DSPC. For example, the lipid nanoparticle may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mol% DSPC.

In some embodiments, the lipid nanoparticle comprises 35 - 40 mol% cholesterol. For example, the lipid nanoparticle may comprise 35, 36, 37, 38, 39, or 40 mol% cholesterol.

In some embodiments, the lipid nanoparticle comprises 1 - 2 mol% DMG-PEG. For example, the lipid nanoparticle may comprise 1, 1.5, or 2 mol% DMG-PEG.

In some embodiments, the lipid nanoparticle comprises 50 mol% ionizable amino lipid, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% DMG-PEG.

In some embodiments, a ENP of the disclosure comprises an N:P ratio of from about 2:1 to about 30:1.

In some embodiments, a LNP of the disclosure comprises an N:P ratio of about 6:1.

In some embodiments, a LNP of the disclosure comprises an N:P ratio of about 3:1.

In some embodiments, a LNP of the disclosure comprises a wt/wt ratio of the ionizable amino lipid component to the RNA of from about 10:1 to about 100:1.

In some embodiments, a LNP of the disclosure comprises a wt/wt ratio of the ionizable amino lipid component to the RNA of about 20:1.

In some embodiments, a LNP of the disclosure comprises a wt/wt ratio of the ionizable amino lipid component to the RNA of about 10:1.

In some embodiments, a LNP of the disclosure has a mean diameter from about 50 nm to about 150 nm.

In some embodiments, a LNP of the disclosure has a mean diameter from about 70 nm to about 120 nm.

Multivalent Vaccines

The compositions, as provided herein, may include RNA or multiple RNAs encoding two or more antigens of the same or different species. In some embodiments, composition includes an RNA or multiple RNAs encoding two or more coronavirus antigens. In some embodiments, the RNA may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more coronavirus antigens.

In some embodiments, two or more different RNA (e.g., mRNA) encoding antigens may be formulated in the same lipid nanoparticle. In other embodiments, two or more different RNA encoding antigens may be formulated in separate lipid nanoparticles (each RNA formulated in a single lipid nanoparticle). The lipid nanoparticles may then be combined and administered as a single vaccine composition (e.g., comprising multiple RNA encoding multiple antigens) or may be administered separately.

Combination Vaccines

The compositions, as provided herein, may include an RNA or multiple RNAs encoding two or more antigens of the same or different virus(es) or viral strain(s). In some embodiments, a composition includes RNA encoding at least one coronavirus antigen and at least one antigen of a different virus. In some embodiments, a composition includes RNA encoding at a first coronavirus antigen and a second coronavirus antigen, wherein the first and second coronavirus antigens are different from each other. Thus, compositions (e.g., RNA vaccines) of the present disclosure may target one or more antigen(s) of the same strain/species, or one or more antigen(s) of different strains/species, e.g., antigens which induce immunity to organisms which are found in the same geographic areas where the risk of coronavirus infection is high or organisms to which an individual is likely to be exposed to when exposed to a coronavirus.

Pharmaceutical Formulations

Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits and reagents for prevention or treatment of coronavirus in humans and other mammals, for example. The compositions provided herein can be used as therapeutic or prophylactic agents. They may be used in medicine to prevent and/or treat a coronavirus infection.

In some embodiments, the coronavirus vaccine containing RNA as described herein can be administered to a subject (e.g., a mammalian subject, such as a human subject), and the RNA polynucleotides are translated in vivo to produce an antigenic polypeptide (antigen).

An “effective amount” of a composition (e.g., comprising RNA) is based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the RNA (e.g., length, nucleotide composition, and/or extent of modified nucleosides), other components of the vaccine, and other determinants, such as age, body weight, height, sex and general health of the subject. Typically, an effective amount of a composition provides an induced or boosted immune response as a function of antigen production in the cells of the subject. In some embodiments, an effective amount of the composition containing RNA polynucleotides having at least one chemical modifications are more efficient than a composition containing a corresponding unmodified polynucleotide encoding the same antigen or a peptide antigen. Increased antigen production may be demonstrated by increased cell transfection (the percentage of cells transfected with the RNA vaccine), increased protein translation and/or expression from the polynucleotide, decreased nucleic acid degradation (as demonstrated, for example, by increased duration of protein translation from a modified polynucleotide), or altered antigen specific immune response of the host cell.

A composition may further comprise a buffer, for example a Tris buffer. For example, a composition may comprise 10 mM - 30 mM, 10 mM - 20 mM, or 20 mM - 30 mM Tris buffer. In some embodiments, a composition comprises 10, 15, 20, 25, or 30 mM Tris buffer. In some embodiments, a composition comprises 20 mM Tris buffer.

In some embodiments, mRNA of a vaccine composition is formulated at a concentration of 0.1 - 1 mg/mL. In some embodiments, mRNA of a vaccine composition is formulated at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/mL. In some embodiments, mRNA of a vaccine composition is formulated at a concentration of 0.5 mg/mL.

In some embodiments, a composition comprises sucrose. For example, a composition may comprise 75 mg/mL - 95 mg/mL, 75 mg/mL - 85 mg/mL, or 85 mg/mL - 95 mg/mL sucrose. In some embodiments, a composition comprises 75, 80, 85, 86, 87, 88, 89, 90, or 95 mg/mL sucrose. In some embodiments, a composition comprises 87 mg/mL sucrose.

In some embodiments, a composition comprises sodium acetate. For example, a composition may comprise 5 mM - 15 mM, 5 mM - 10 mM, or 10 mM - 15 mM sodium acetate. In some embodiments, a composition comprises 5, 10, 11, 12, 13, 14, or 15 mM sodium acetate. In some embodiments, a composition comprises 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, or 11 mM sodium acetate. In some embodiments, a composition comprises 10.7 mM sodium acetate.

A composition may have a pH value of 6-8. In some embodiments, a composition has a pH value of 6, 6.5, 7, 7.5, or 8. In some embodiments, a composition has a pH value of 7.5.

In some embodiments, the composition further comprises a mixture of lipids. The mixture of lipids typically forms a lipid nanoparticle. The mRNA described herein, in some embodiments, is formulated with a lipid nanoparticle (e.g., for administration to a subject).

In some embodiments, the lipid mixture, and thus the lipid nanoparticle, comprises: an ionizable amino lipid; a neutral lipid; a sterol; and a PEG-modified lipid. For example, the lipid mixture/lipid nanoparticle may comprise: 20-60 mol% ionizable amino lipid; 5-25 mol% neutral lipid; 25-55 mol% sterol; and 0.5-15 mol% PEG-modified lipid. In some embodiments, the lipid nanoparticle comprises: 20-60 mol% ionizable amino lipid; 5-25 mol% neutral lipid; 25-55 mol% sterol; and 0.5-15 mol% PEG-modified lipid. In some embodiments, the lipid nanoparticle comprises: 40-55 mol% ionizable amino lipid; 5-15 mol% neutral lipid; 35-45 mol% sterol; and 1-5 mol% PEG-modified lipid. For example, the lipid nanoparticle may comprise: (a) 47 mol% ionizable amino lipid; 11.5 mol% neutral lipid; 38.5 mol% sterol; and 3.0 mol% PEG-modified lipid; (b) 48 mol% ionizable amino lipid; 11 mol% neutral lipid; 38.5 mol% sterol; and 2.5 mol% PEG-modified lipid; (c) 49 mol% ionizable amino lipid; 10.5 mol% neutral lipid; 38.5 mol% sterol; and 2.0 mol% PEG-modified lipid; (d) 50 mol% ionizable amino lipid; 10 mol% neutral lipid; 38.5 mol% sterol; and 1.5 mol% PEG-modified lipid; or (e) 51 mol% ionizable amino lipid; 9.5 mol% neutral lipid; 38.5 mol% sterol; and 1.0 mol% PEG-modified lipid.

In some embodiments, the lipid mixture, and thus the lipid nanoparticle, comprises 20-55 mol%, 20-50 mol%, 20-45 mol%, 20-40 mol%, 25-60 mol%, 25-55 mol%, 25-50 mol%, 25-45 mol%, 25-40 mol%, 30-60 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 35-60 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-60 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 50-60 mol%, 50-55 mol%, or 55-60 mol% ionizable amino lipid.

In some embodiments, the lipid mixture, and thus the lipid nanoparticle, comprises 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-15 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% neutral lipid.

In some embodiments, the lipid mixture, and thus the lipid nanoparticle, comprises 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% sterol.

In some embodiments, the lipid mixture, and thus the lipid nanoparticle, comprises 0.5- 10 mol%, 0.5-5 mol%, 0.5-1 mol%, 1-15%, 1-10 mol%, 1-5 mol%, 1.5-15%, 1.5-10 mol%, 1.5- 5 mol%, 2-15%, 2-10 mol%, 2-5 mol%, 2.5-15%, 2.5-10 mol%, 2.5-5 mol%, 3-15%, 3-10 mol%, or 3-5 mol%, PEG-modified lipid.

In some embodiments, the lipid mixture comprises: 50 mol% ionizable amino lipid; 10 mol% neutral lipid; 38.5 mol% sterol; and 1.5 mol% PEG-modified lipid.

In some embodiments, the ionizable amino lipid is heptadecan-9-yl 8 ((2 hydroxy ethyl) (6 oxo 6-(undecyloxy)hexyl)amino)octanoate (Compound 1). In some embodiments, the neutral lipid is 1,2 distearoyl sn glycero-3 phosphocholine (DSPC). In some embodiments, the sterol is cholesterol. In some embodiments, the PEG-modified lipid is 1- monomethoxypolyethyleneglycol-2,3-dimyristylglycerol with polyethylene glycol of average molecular weight 2000 (PEG2000 DMG).

A composition, may further include a pharmaceutically-acceptable excipient, inert or active. A pharmaceutically acceptable excipient, after administered to a subject, does not cause undesirable physiological effects. The excipient in the pharmaceutical composition must be “acceptable” also in the sense that it is compatible with mRNA and can be capable of stabilizing it. One or more excipients (e.g., solubilizing agents) can be utilized as pharmaceutical carriers for delivery of the mRNA. Examples of a pharmaceutically acceptable excipients include, but are not limited to, biocompatible vehicles (e.g., LNPs), carriers, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other excipients include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical excipients, as well as pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences.

The term "pharmaceutical composition" refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo. A "pharmaceutically acceptable carrier," after administered to or upon a subject, does not cause undesirable physiological effects. The carrier in the pharmaceutical composition must be "acceptable" also in the sense that it is compatible with the active ingredient and can be capable of stabilizing it. One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an active agent. Examples of a pharmaceutically acceptable carrier include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences.

In some embodiments, the compositions (comprising polynucleotides and their encoded polypeptides) in accordance with the present disclosure may be used for treatment or prevention of a coronavirus infection. A composition may be administered prophylactically or therapeutically as part of an active immunization scheme to healthy individuals or early in infection during the incubation phase or during active infection after onset of symptoms. In some embodiments, the amount of RNA provided to a cell, a tissue or a subject may be an amount effective for immune prophylaxis.

The vaccine may be administered to seropositive or seronegative subjects. For example, a subject may be naive and not have antibodies that react with a virus having an antigen, wherein the antigen is the viral antigen or fragment thereof encoded by the mRNA of the vaccine (e.g., a SARS-CoV-2 antigen disclosed herein). Such a subject is said to be seronegative with respect to that vaccine. Alternatively, the subject may have preexisting antibodies to viral antigen encoded by the mRNA of the vaccine because they have previously had an infection with virus carrying the antigen or may have previously been administered a dose of a vaccine (e.g., an mRNA vaccine) that induces antibodies against the antigen (e.g., a SARS-CoV-2 antigen disclosed herein). Such a subject is said to be seropositive with respect to that vaccine. In some instances the subject may have been previously exposed to a virus but not to a specific variant or strain of the virus or a specific vaccine associated with that variant or strain. Such a subject is considered to be seronegative with respect to the specific variant or strain.

Thus, the present disclosure provides compositions (e.g., mRNA vaccines) that elicit potent neutralizing antibodies against an antigen (e.g., a SARS-CoV-2 antigen disclosed herein) in a subject. Such a composition can be administered to seropositive or seronegative subjects in some embodiments. A seronegative subject may be naive and not have antibodies that react with the specific virus (e.g., SARS-CoV-2) which the subject is being immunized against. A seropositive subject may have preexisting antibodies to the specific virus (e.g., SARS-CoV-2) because they have previously had an infection with that virus, variant or strain or may have previously been administered a dose of a vaccine (e.g., an mRNA vaccine) that induces antibodies against that virus, variant, or strain.

In some embodiments, an initial dose is administered followed by a booster dose. As described herein, in some embodiments, the compositions comprise a booster dose. A booster dose is a dose that is given at a certain interval after completion of the primary dose or series of doses that is/are intended to boost immunity to, and therefore prolong protection against, the disease (e.g., COVID- 19) that is to be prevented. A booster dose may be given after an earlier administration of an immunizing composition. In some embodiments, the earlier administration of an immunizing composition comprises 1 or 2 doses of the immunizing composition. The time of administration between the initial administration of an immunizing composition and the booster may be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month (e.g., 28 days, 29 days, 30 days, or 31 days), 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years or more than 99 years. In exemplary embodiments, the time of administration between the initial administration of the immunizing composition and the booster may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months or 1 year. In one embodiment, the time between the initial immunizing composition and the booster dose (e.g., the composition described herein) is six months.

In some embodiments, a composition may be administered intramuscularly, intranasally or intradermally, similarly to the administration of inactivated vaccines known in the art.

A composition may be utilized in various settings depending on the prevalence of the infection or the degree or level of unmet medical need. As a non-limiting example, the RNA vaccines may be utilized to treat and/or prevent a variety of infectious disease. RNA vaccines have superior properties in that they produce much larger antibody titers, better neutralizing immunity, produce more durable immune responses, and/or produce responses earlier than commercially available vaccines.

Provided herein are immunizing compositions including RNA and/or complexes optionally in combination with one or more pharmaceutically acceptable excipients.

The RNA may be formulated or administered alone or in conjunction with one or more other components. For example, an immunizing composition may comprise other components including, but not limited to, adjuvants.

In some embodiments, an immunizing composition does not include an adjuvant (they are adjuvant free).

An RNA may be formulated or administered in combination with one or more pharmaceutically-acceptable excipients. In some embodiments, vaccine compositions comprise at least one additional active substance, such as, for example, a therapeutically-active substance, a prophylactically-active substance, or a combination of both. Vaccine compositions may be sterile, pyrogen-free or both sterile and pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents, such as vaccine compositions, may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

In some embodiments, an immunizing composition is administered to humans, human patients or subjects. For the purposes of the present disclosure, the phrase “active ingredient” generally refers to the RNA vaccines or the polynucleotides contained therein, for example, RNA polynucleotides (e.g., mRNA polynucleotides) encoding antigens.

Formulations of the vaccine compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient (e.g., mRNA polynucleotide) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.

Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.

In some embodiments, an RNA is formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation); (4) alter the biodistribution (e.g., target to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein (antigen) in vivo. In addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, excipients can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with the RNA (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof.

Dosing/Administration

Provided herein are immunizing compositions (e.g., RNA vaccines), methods, kits and reagents for prevention and/or treatment of coronavirus infection in humans and other mammals. Immunizing compositions can be used as therapeutic or prophylactic agents. In some embodiments, immunizing compositions are used to provide prophylactic protection from coronavirus infection. In some embodiments, immunizing compositions are used to treat a coronavirus infection. In some embodiments, embodiments, immunizing compositions are used in the priming of immune effector cells, for example, to activate peripheral blood mononuclear cells (PBMCs) ex vivo, which are then infused (re-infused) into a subject. A subject may be any mammal, including non-human primate and human subjects. Typically, a subject is a human subject.

In some embodiments, an immunizing composition (e.g., RNA a vaccine) is administered to a subject (e.g., a mammalian subject, such as a human subject) in an effective amount to induce an antigen- specific immune response. The RNA encoding the coronavirus antigen is expressed and translated in vivo to produce the antigen, which then stimulates an immune response in the subject. The immunizing compositions provided herein, in some embodiments, comprise booster vaccines.

As used herein, when referring to a prophylactic composition, such as a vaccine, the term “booster” refers to an extra administration of the prophylactic (vaccine) composition following an earlier administration of a prophylactic composition (e.g., two doses of mRNA-1273 or the complete administration of another SARS-CoV-2 vaccine). As used herein, a “complete administration” refers to the FDA-approved protocol for vaccination with the SARS-CoV-2 vaccine (included Emergency Use Authorization). In some embodiments, the subject has undergone a complete administration of the SARS-CoV-2 vaccine prior to administration of the booster. In some embodiments, the subject has not undergone a completed administration of the SARS-CoV-2 vaccine (e.g., the subject has been administered one of the two FDA-approved doses) prior to administration of the booster. The time of administration between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes,

10 minutes, 15 minutes, 20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours,

11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years or more than 99 years. In exemplary embodiments, the time of administration between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months or 1 year. In one embodiment, the time of administration between the initial administration of the prophylactic composition and the booster is at least six months. Prophylactic protection from a coronavirus can be achieved following administration of an immunizing composition (e.g., an RNA vaccine) of the present disclosure. Immunizing compositions can be administered once, twice, three times, four times or more but it is likely sufficient to administer the vaccine once (as a booster). It is possible, although less desirable, to administer an immunizing compositions to an infected individual to achieve a therapeutic response. Dosing may need to be adjusted accordingly.

A method of eliciting an immune response in a subject against a coronavirus antigen (or multiple antigens) is provided in aspects of the present disclosure. In some embodiments, a method involves administering to the subject an immunizing composition comprising at least two RNAs (e.g., mRNA), each having an open reading frame encoding a coronavirus antigen, thereby inducing in the subject an immune response specific to the coronavirus antigens, wherein anti-antigen antibody titer in the subject is increased following vaccination relative to anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the antigen. An “anti-antigen antibody” is a serum antibody the binds specifically to the antigen.

A prophylactically effective dose is an effective dose that prevents infection with the virus at a clinically acceptable level. In some embodiments, the effective dose is a dose listed in a package insert for the vaccine. A traditional vaccine, as used herein, refers to a vaccine other than the mRNA vaccines of the present disclosure. For instance, a traditional vaccine includes, but is not limited, to live microorganism vaccines, killed microorganism vaccines, subunit vaccines, protein antigen vaccines, DNA vaccines, virus like particle (VLP) vaccines, etc. In exemplary embodiments, a traditional vaccine is a vaccine that has achieved regulatory approval and/or is registered by a national drug regulatory body, for example the Food and Drug Administration (FDA) in the United States or the European Medicines Agency (EMA).

In some embodiments, the anti-antigen antibody titer in the subject is increased 1 log to 10 log following vaccination relative to anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the coronavirus or an unvaccinated subject. In some embodiments, the anti-antigen antibody titer in the subject is increased 1 log, 2 log, 3 log, 4 log, 5 log, or 10 log following vaccination relative to anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the coronavirus or an unvaccinated subject.

A method of eliciting an immune response in a subject against a coronavirus is provided in other aspects of the disclosure. The method involves administering to the subject an immunizing composition (e.g., an RNA vaccine) comprising a RNA polynucleotide comprising an open reading frame encoding a coronavirus antigen, thereby inducing in the subject an immune response specific to the coronavirus, wherein the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine against the coronavirus at 2 times to 100 times the dosage level relative to the immunizing composition.

In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at twice the dosage level relative to an immunizing composition of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at three times the dosage level relative to an immunizing composition of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 4 times, 5 times, 10 times, 50 times, or 100 times the dosage level relative to an immunizing composition of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 10 times to 1000 times the dosage level relative to an immunizing composition of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 100 times to 1000 times the dosage level relative to an immunizing composition of the present disclosure.

In other embodiments, the immune response is assessed by determining [protein] antibody titer in the subject. In other embodiments, the ability of serum or antibody from an immunized subject is tested for its ability to neutralize viral uptake or reduce coronavirus transformation of human B lymphocytes. In other embodiments, the ability to promote a robust T cell response(s) is measured using art recognized techniques.

Other aspects the disclosure provide methods of eliciting an immune response in a subject against a coronavirus by administering to the subject an immunizing composition (e.g., an RNA vaccine) comprising an RNA having an open reading frame encoding a coronavirus antigen, thereby inducing in the subject an immune response specific to the coronavirus antigen, wherein the immune response in the subject is induced 2 days to 10 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the coronavirus. In some embodiments, the immune response in the subject is induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine at 2 times to 100 times the dosage level relative to an immunizing composition of the present disclosure. In some embodiments, the immune response in the subject is induced 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 5 weeks, or 10 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactic ally effective dose of a traditional vaccine.

Also provided herein are methods of eliciting an immune response in a subject against a coronavirus by administering to the subject an RNA having an open reading frame encoding a first antigen and an RNA having an open reading frame encoding a second antigen, wherein the RNA does not include a stabilization element, and wherein an adjuvant is not co-formulated or co-administered with the vaccine.

An immunizing composition (e.g., an RNA vaccine) may be administered by any route that results in a therapeutically effective outcome. These include, but are not limited, to intradermal, intramuscular, intranasal, and/or subcutaneous administration. The present disclosure provides methods comprising administering RNA vaccines to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. The RNA is typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the RNA may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

The effective amount of the RNA (total RNA), as provided herein, may range from about 1 pg - 100 pg, administered as a single dose or as multiple doses. As noted herein, the compositions of the disclosure, in some embodiments, comprise at least two RNAs, optionally in a 1:1 ratio. Accordingly, a single dose of the vaccine composition (e.g., booster), in some embodiments, comprises about 20 pg mRNA total (e.g., 10 pg of the first mRNA and 10 pg of the second mRNA, or 20 pg of a single mRNA). In some embodiments, a single dose of a vaccine composition (e.g., administered once, twice, three times, or more) comprises about 40 pg mRNA total (e.g., 20 pg of the first mRNA and 20 pg of the second mRNA, or 40 pg of a single mRNA). In some embodiments, a single dose of a vaccine composition (e.g., administered once, twice, three times, or more) comprises about 1 pg mRNA total (e.g., 0.5 pg of the first mRNA and 0.5 pg of the second mRNA, or 1 pg of a single mRNA). In some embodiments, a single dose of a vaccine composition (e.g., administered once, twice, three times, or more) comprises about 2.5 pg mRNA total (e.g., 1.25 pg of the first mRNA and 1.25 pg of the second mRNA, or 2.5 pg of a single mRNA). In some embodiments, a single dose of a vaccine composition (e.g., administered once, twice, three times, or more) comprises about 5 pg mRNA total (e.g., 2.5 pg of the first mRNA and 2.5 pg of the second mRNA, or 5 pg of a single mRNA). In some embodiments, a single dose of a vaccine composition (e.g., administered once, twice, three times, or more) comprises about 10 pg mRNA total (e.g., 5 pg of the first mRNA and 5 pg of the second mRNA, or 10 pg of a single mRNA).

In some embodiments, a single dose of a vaccine composition (e.g., administered once, twice, three times, or more) comprises at least 1 pg mRNA. In some embodiments, a single dose of a vaccine composition (e.g., administered once, twice, three times, or more) comprises less than 50 pg mRNA.

In some embodiments, a total amount of mRNA administered to a subject is about 1 pg, about 2.5 pg, about 3 pg, about 3.5 pg, about 4 pg, about 4.5 pg, about 5 pg, about 10 pg, about 20 pg, about 40 pg, or about 50 pg mRNA. In some embodiments, a total amount of mRNA administered to a subject is about 1 pg. In some embodiments, a total amount of mRNA administered to a subject is about 2.5 pg. In some embodiments, a total amount of mRNA administered to a subject is about 5 pg. In some embodiments, a total amount of mRNA administered to a subject is about 10 pg. In some embodiments, a total amount of mRNA administered to a subject is about 20 pg. In some embodiments, a total amount of mRNA administered to a subject is about 40 pg. In some embodiments, a total amount of mRNA administered to a subject is about 50 pg.

The RNA described herein can be formulated into a dosage form described herein, such as an intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous).

Vaccine Efficacy

Some aspects of the present disclosure provide formulations of the immunizing compositions (e.g., RNA vaccines), wherein the RNA is formulated in an effective amount to produce an antigen specific immune response in a subject (e.g., production of antibodies specific to a coronavirus antigen). “An effective amount” is a dose of the RNA effective to produce an antigen- specific immune response. Also provided herein are methods of inducing an antigenspecific immune response in a subject. As used herein, an immune response to a vaccine or LNP of the present disclosure is the development in a subject of a humoral and/or a cellular immune response to a (one or more) coronavirus protein(s) present in the vaccine. For purposes of the present disclosure, a “humoral” immune response refers to an immune response mediated by antibody molecules, including, e.g., secretory (IgA) or IgG molecules, while a “cellular” immune response is one mediated by T-lymphocytes (e.g., CD4+ helper and/or CD8+ T cells (e.g., CTLs) and/or other white blood cells. One important aspect of cellular immunity involves an antigen- specific response by cytolytic T-cells (CTLs). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the destruction of intracellular microbes or the lysis of cells infected with such microbes. Another aspect of cellular immunity involves and antigen- specific response by helper T-cells. Helper T-cells act to help stimulate the function, and focus the activity nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface. A cellular immune response also leads to the production of cytokines, chemokines, and other such molecules produced by activated T-cells and/or other white blood cells including those derived from CD4+ and CD8+ T-cells.

In some embodiments, the antigen- specific immune response is characterized by measuring an anti-coronavirus antigen antibody titer produced in a subject administered an immunizing composition as provided herein. An antibody titer is a measurement of the amount of antibodies within a subject, for example, antibodies that are specific to a particular antigen or epitope of an antigen. Antibody titer is typically expressed as the inverse of the greatest dilution that provides a positive result. Enzyme-linked immunosorbent assay (ELISA) is a common assay for determining antibody titers, for example.

In some embodiments, an antibody titer is used to assess whether a subject has had an infection or to determine whether immunizations are required. In some embodiments, an antibody titer is used to determine the strength of an autoimmune response, to determine whether a booster immunization is needed, to determine whether a previous vaccine was effective, and to identify any recent or prior infections. In accordance with the present disclosure, an antibody titer may be used to determine the strength of an immune response induced in a subject by an immunizing composition (e.g., RNA vaccine).

In some embodiments, an anti-coronavirus antigen antibody titer produced in a subject is increased by at least 1 log relative to a control. For example, anti-coronavirus antigen antibody titer produced in a subject may be increased by at least 1.5, at least 2, at least 2.5, or at least 3 log relative to a control. In some embodiments, the anti-coronavirus antigen antibody titer produced in the subject is increased by 1, 1.5, 2, 2.5 or 3 log relative to a control. In some embodiments, the anti-coronavirus antigen antibody titer produced in the subject is increased by 1-3 log relative to a control. For example, the anti-coronavirus antigen antibody titer produced in a subject may be increased by 1-1.5, 1-2, 1-2.5, 1-3, 1.5-2, 1.5-2.5, 1.5-3, 2-2.5, 2-3, or 2.5-3 log relative to a control.

In some embodiments, the anti-coronavirus antigen antibody titer produced in a subject is increased at least 2 times relative to a control. For example, the anti-coronavirus antigen n antibody titer produced in a subject may be increased at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times relative to a control. In some embodiments, the anti-coronavirus antigen antibody titer produced in the subject is increased 2, 3, 4, 5, 6, 7, 8, 9, or 10 times relative to a control. In some embodiments, the anti-coronavirus antigen antibody titer produced in a subject is increased 2-10 times relative to a control. For example, the anti-coronavirus antigen antibody titer produced in a subject may be increased 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8- 9, or 9-10 times relative to a control.

In some embodiments, antibody-mediated immunogenicity in a subject is assessed at one or more time points (e.g., Day 1 - Day 196, e.g., Day 1, Day 8, Day 29, Day 57, and/or Day 196). Methods of assessing antibody-mediated immunogenicity are known and include geometric mean concentration (GMC) of antibody to antigen, geometric mean fold rise (GMFR) in serum antibody, geometric mean titer (GMT), median, minimum, maximum, 95% confidence interval (CI), geometric mean ratio (GMR) of post-baseline / baseline titers, and seroconversion rate.

In some embodiments, the GMFR, defined as the ratio of post-baseline/baseline titers) of neutralizing antibody induced against an ancestral SARS-CoV-2 (D614G) strain in a subject at Day 29, following the first dose of the composition (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein), is 20-50, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, the GMFR of neutralizing antibody induced against an ancestral SARS-CoV-2 (D614G) strain in a subject at Day 29, following a 2.5 pg first dose of the vaccine (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS- CoV-2 Spike protein, and less than the full length spike protein), is approximately 28, 28.5, or 29. In some embodiments, the GMFR of neutralizing antibody induced against an ancestral SARS-CoV-2 (D614G) strain in a subject at Day 29, following a 5 pg first dose of the vaccine (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein), is approximately 34, 34.5, or 35. In some embodiments, the GMFR of neutralizing antibody induced against an ancestral SARS-CoV-2 (D614G) strain in a subject at Day 29, following a 10 pg first dose of the vaccine (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein), is approximately 42, 42.5, or 43.

In some embodiments, the GMFR, defined as the ratio of post-baseline/baseline titers) of neutralizing antibody induced against the beta SARS-CoV-2 strain in a subject at Day 29, following the first dose of the composition (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein), is 20-50, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, the GMFR of neutralizing antibody induced against the beta SARS-CoV-2 strain in a subject at Day 29, following a 2.5 pg first dose of the vaccine (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein), is approximately 27, 27.5, or 28. In some embodiments, the GMFR of neutralizing antibody induced against the beta SARS-CoV-2 strain in a subject at Day 29, following a 5 pg first dose of the vaccine (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein), is approximately 33, 33.5, or 34. In some embodiments, the GMFR of neutralizing antibody induced against the beta SARS-CoV-2 strain in a subject at Day 29, following a 10 pg first dose of the vaccine (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein), is approximately 44, 44.5, or 45.

In some embodiments, the GMFR, defined as the ratio of post-baseline/baseline titers) of neutralizing antibody induced against an ancestral SARS-CoV-2 (D614G) strain in a subject at Day 29, following the first dose of the composition (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein at a 1:1 ratio with a second mRNA comprising a second ORF that encodes a second fusion protein comprising an RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein), is 20-50, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, the GMFR of neutralizing antibody induced against an ancestral SARS-CoV-2 (D614G) strain in a subject at Day 29, following a 5 pg first dose of the vaccine (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein at a 1:1 ratio with a second mRNA comprising a second ORF that encodes a second fusion protein comprising an RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein), is approximately 25, 25.5, or 26. In some embodiments, the GMFR of neutralizing antibody induced against an ancestral SARS-CoV-2 (D614G) strain in a subject at Day 29, following a 10 pg first dose of the vaccine (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS- CoV-2 Spike protein, and less than the full length spike protein at a 1:1 ratio with a second mRNA comprising a second ORF that encodes a second fusion protein comprising an RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein), is approximately 28, 28.5, or 29.

In some embodiments, the GMFR, defined as the ratio of post-baseline/baseline titers) of neutralizing antibody induced against the beta SARS-CoV-2 strain in a subject at Day 29, following the first dose of the composition (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein at a 1:1 ratio with a second mRNA comprising a second ORF that encodes a second fusion protein comprising an RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein), is 20-50, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, the GMFR of neutralizing antibody induced against the beta SARS-CoV-2 strain in a subject at Day 29, following a 5 pg first dose of the vaccine (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein at a 1:1 ratio with a second mRNA comprising a second ORF that encodes a second fusion protein comprising an RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein), is approximately 38, 38.5, or 39. In some embodiments, the GMFR of neutralizing antibody induced against the beta SARS-CoV-2 strain in a subject at Day 29, following a 10 pg first dose of the vaccine (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS- CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein at a 1:1 ratio with a second mRNA comprising a second ORF that encodes a second fusion protein comprising an RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein), is approximately 41, 41.5, or 42.

In some embodiments, the geometric mean titer (GMT) of neutralizing antibody induced against an ancestral SARS-CoV-2 (D614G) strain in a subject at Day 29, following the first dose of the composition (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein), is 2800-12,000, e.g., 2800, 2900, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 11,000, or 12,000. In some embodiments, the GMT of neutralizing antibody induced against an ancestral SARS-CoV-2 (D614G) strain in a subject at Day 29, following a 2.5 pg first dose of the vaccine (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein), is approximately 2800-7000 (e.g., approximately 4000, 4500, or 5000). In some embodiments, the GMT of neutralizing antibody induced against an ancestral SARS-CoV-2 (D614G) strain in a subject at Day 29, following a 5 pg first dose of the vaccine (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein), is approximately 4000-8000 (e.g., approximately 5000, 5500, or 6000). In some embodiments, the GMT of neutralizing antibody induced against an ancestral SARS-CoV-2 (D614G) strain in a subject at Day 29, following a 10 pg first dose of the vaccine (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein), is approximately 5000-11,000 (e.g., 7000, 7500, or 8000).

In some embodiments, the GMT of neutralizing antibody induced against the beta SARS-CoV-2 strain in a subject at Day 29, following the first dose of the composition (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein), is 400-3000, e.g., 400, 500, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, or 3000. In some embodiments, the GMT of neutralizing antibody induced against the beta SARS-CoV-2 strain in a subject at Day 29, following a 2.5 pg first dose of the vaccine (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS- CoV-2 Spike protein, and less than the full length spike protein), is approximately 500-1500 (e.g., approximately 800, 850, or 900). In some embodiments, the GMT of neutralizing antibody induced against the beta SARS-CoV-2 strain in a subject at Day 29, following a 5 pg first dose of the vaccine (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS- CoV-2 Spike protein, and less than the full length spike protein), is approximately 800-1800 (e.g., approximately 1200, 1250, or 1300). In some embodiments, the GMT of neutralizing antibody induced against the beta SARS-CoV-2 strain in a subject at Day 29, following a 10 pg first dose of the vaccine (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS- CoV-2 Spike protein, and less than the full length spike protein), is approximately 1200-2700 (e.g., approximately 1750, 1800, or 1850).

In some embodiments, the geometric mean titer (GMT) of neutralizing antibody induced against an ancestral SARS-CoV-2 (D614G) strain in a subject at Day 29, following the first dose of the composition (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein at a 1:1 ratio with a second mRNA comprising a second ORF that encodes a second fusion protein comprising an RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein), is 2000-9000, e.g., 2000, 2500, 3000, 3500, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, or 9000. In some embodiments, the GMT of neutralizing antibody induced against an ancestral SARS-CoV-2 (D614G) strain in a subject at Day 29, following a 5 pg first dose of the vaccine (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein at a 1:1 ratio with a second mRNA comprising a second ORF that encodes a second fusion protein comprising an RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein), is approximately 2900-8600 (e.g., about 4500 or about 5000). In some embodiments, the GMT of neutralizing antibody induced against an ancestral SARS-CoV-2 (D614G) strain in a subject at Day 29, following a 10 pg first dose of the vaccine (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein at a 1:1 ratio with a second mRNA comprising a second ORF that encodes a second fusion protein comprising an RBD of a SARS- CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein), is approximately 2500-8700 (e.g., approximately 4500 or 5000).

In some embodiments, the geometric mean titer (GMT) of neutralizing antibody induced against the beta SARS-CoV-2 strain in a subject at Day 29, following the first dose of the composition (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein at a 1:1 ratio with a second mRNA comprising a second ORF that encodes a second fusion protein comprising an RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein), is 800-2300, e.g., 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, or 2300. In some embodiments, the GMT of neutralizing antibody induced against the beta SARS-CoV-2 strain in a subject at Day 29, following a 5 pg first dose of the vaccine (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein at a 1:1 ratio with a second mRNA comprising a second ORF that encodes a second fusion protein comprising an RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein), is approximately 900-2750 (e.g., about 1600 or 1603). In some embodiments, the GMT of neutralizing antibody induced against the beta SARS-CoV-2 strain in a subject at Day 29, following a 10 pg first dose of the vaccine (e.g., a composition comprising an mRNA comprising an ORF that encodes a fusion protein of a RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein at a 1:1 ratio with a second mRNA comprising a second ORF that encodes a second fusion protein comprising an RBD of a SARS-CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein), is approximately 600-2300 (e.g., approximately 1200, 1250, or 1300). The GMC is the average antibody concentration for a group of subjects calculated by multiplying all values and taking the nth root of this number, where n is the number of subjects with available data. GMT is the average antibody titer for a group of subjects calculated by multiplying all values and taking the nth root of this number, where n is the number of subjects with available data.

A control, in some embodiments, is an anti-coronavirus antigen antibody titer produced in a subject who has not been administered an immunizing composition (e.g., RNA vaccine), or who has been administered a saline placebo (un unvaccinated subject). In some embodiments, a control is an anti-coronavirus antigen antibody titer produced in a subject administered a recombinant or purified protein vaccine (e.g., protein subunit vaccine). Recombinant protein vaccines typically include protein antigens that either have been produced in a heterologous expression system (e.g., bacteria or yeast) or purified from large amounts of the pathogenic organism. A control may be, for example, a subject administered a live attenuated viral vaccine or an inactivated viral vaccine.

In some embodiments, the ability of an immunizing composition (e.g., RNA vaccine) to be effective is measured in a murine model. For example, an immunizing composition may be administered to a murine model and the murine model assayed for induction of neutralizing antibody titers. Viral challenge studies may also be used to assess the efficacy of a vaccine of the present disclosure. For example, an immunizing composition may be administered to a murine model, the murine model challenged with virus, and the murine model assayed for survival and/or immune response (e.g., neutralizing antibody response, T cell response (e.g., cytokine response)).

In some embodiments, an effective amount of an immunizing composition (e.g., RNA vaccine) is a dose that is reduced compared to the standard of care dose of a recombinant protein vaccine. A “standard of care,” as provided herein, refers to a medical or psychological treatment guideline and can be general or specific. “Standard of care” specifies appropriate treatment based on scientific evidence and collaboration between medical professionals involved in the treatment of a given condition. It is the diagnostic and treatment process that a physician/ clinician should follow for a certain type of patient, illness or clinical circumstance. A “standard of care dose,” as provided herein, refers to the dose of a recombinant or purified protein vaccine, or a live attenuated or inactivated vaccine, or a VLP vaccine, that a physician/clinician or other medical professional would administer to a subject to treat or prevent coronavirus infection or a related condition, while following the standard of care guideline for treating or preventing coronavirus infection or a related condition. In some embodiments, the anti-coronavirus antigen antibody titer produced in a subject administered an effective amount of an immunizing composition is equivalent to an anticoronavirus antigen antibody titer produced in a control subject administered a standard of care dose of a recombinant or purified protein vaccine, or a live attenuated or inactivated vaccine, or a VLP vaccine.

Vaccine efficacy may be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis. 2010 Jun 1 ;201(l 1): 1607-10). For example, vaccine efficacy may be measured by double-blind, randomized, clinical controlled trials. Vaccine efficacy may be expressed as a proportionate reduction in disease attack rate (AR) between the unvaccinated (ARU) and vaccinated (ARV) study cohorts and can be calculated from the relative risk (RR) of disease among the vaccinated group with use of the following formulas:

Efficacy = (ARU - ARV)/ARU x 100; and

Efficacy = (1-RR) x 100.

Likewise, vaccine effectiveness may be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis. 2010 Jun 1 ;201(l 1): 1607-10). Vaccine effectiveness is an assessment of how a vaccine (which may have already proven to have high vaccine efficacy) reduces disease in a population. This measure can assess the net balance of benefits and adverse effects of a vaccination program, not just the vaccine itself, under natural field conditions rather than in a controlled clinical trial. Vaccine effectiveness is proportional to vaccine efficacy (potency) but is also affected by how well target groups in the population are immunized, as well as by other non- vaccine-related factors that influence the ‘real-world’ outcomes of hospitalizations, ambulatory visits, or costs. For example, a retrospective case control analysis may be used, in which the rates of vaccination among a set of infected cases and appropriate controls are compared. Vaccine effectiveness may be expressed as a rate difference, with use of the odds ratio (OR) for developing infection despite vaccination:

Effectiveness = (1 - OR) x 100.

In some embodiments, efficacy of the immunizing composition (e.g., RNA vaccine) is at least 60% relative to unvaccinated control subjects. For example, efficacy of the immunizing composition may be at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, at least 98%, or 100% relative to unvaccinated control subjects.

Sterilizing Immunity. Sterilizing immunity refers to a unique immune status that prevents effective pathogen infection into the host. In some embodiments, the effective amount of an immunizing composition of the present disclosure is sufficient to provide sterilizing immunity in the subject for at least 1 year. For example, the effective amount of an immunizing composition of the present disclosure is sufficient to provide sterilizing immunity in the subject for at least 2 years, at least 3 years, at least 4 years, or at least 5 years. In some embodiments, the effective amount of an immunizing composition of the present disclosure is sufficient to provide sterilizing immunity in the subject at an at least 5-fold lower dose relative to control. For example, the effective amount may be sufficient to provide sterilizing immunity in the subject at an at least 10-fold lower, 15-fold, or 20-fold lower dose relative to a control.

Detectable Antigen. In some embodiments, the effective amount of an immunizing composition of the present disclosure is sufficient to produce detectable levels of coronavirus antigen as measured in serum of the subject at 1-72 hours post administration.

Titer. An antibody titer is a measurement of the amount of antibodies within a subject, for example, antibodies that are specific to a particular antigen (e.g., an anti-coronavirus antigen). Antibody titer is typically expressed as the inverse of the greatest dilution that provides a positive result. Enzyme-linked immunosorbent assay (ELISA) is a common assay for determining antibody titers, for example.

A neutralizing immune response is an immune response that is a neutralizing antibody response and/or an effective neutralizing T cell response. In some embodiments a neutralizing antibody response produces a level of antibodies that meet or exceed a seroprotection threshold.

An effective T cell response is a response which produces a baseline level of viral activated or viral specific T cells including CD8+ and CD4+ T helper type 1 cells. CD8+ cytotoxic T lymphocytes typically clear the intracellular virus compartment and CD4+ T cells exert various functions in the body such as helping B and other T cells, promoting memory generation and indirect or direct cytotoxic activity. In some embodiments the effective T cells comprises a high proportion of CD8+ T cells and/or CD4+ T cells, relative to a baseline level (in a naive subject). In some embodiments these T cells are differentiated towards an early- differentiated memory phenotype with co-expression of CD27 and CD28.

In some embodiments, the effective amount of an immunizing composition of the present disclosure is sufficient to produce a 1,000-10,000 neutralizing antibody titer produced by neutralizing antibody against the coronavirus antigen as measured in serum of the subject at 1- 72 hours post administration. In some embodiments, the effective amount is sufficient to produce a 1,000-5,000 neutralizing antibody titer produced by neutralizing antibody against the coronavirus antigen as measured in serum of the subject at 1-72 hours post administration. In some embodiments, the effective amount is sufficient to produce a 5,000-10,000 neutralizing antibody titer produced by neutralizing antibody against the coronavirus antigen as measured in serum of the subject at 1-72 hours post administration. In some embodiments, the neutralizing antibody titer is at least 100 NT50. For example, the neutralizing antibody titer may be at least 200, 300, 400, 500, 600, 700, 800, 900 or 1000 NT50. In some embodiments, the neutralizing antibody titer is at least 10,000 NT50.

In some embodiments, the neutralizing antibody titer is at least 100 neutralizing units per milliliter (NU/mL). For example, the neutralizing antibody titer may be at least 200, 300, 400, 500, 600, 700, 800, 900 or 1000 NU/mL. In some embodiments, the neutralizing antibody titer is at least 10,000 NU/mL.

In some embodiments, an anti-coronavirus antigen antibody titer produced in the subject is increased by at least 1 log relative to a control. For example, an anti-coronavirus antigen antibody titer produced in the subject may be increased by at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 log relative to a control.

In some embodiments, an anti-coronavirus antigen antibody titer produced in the subject is increased at least 2 times relative to a control. For example, an anti-coronavirus antigen antibody titer produced in the subject is increased by at least 3, 4, 5, 6, 7, 8, 9 or 10 times relative to a control.

In some embodiments, a geometric mean, which is the nth root of the product of n numbers, is generally used to describe proportional growth. Geometric mean, in some embodiments, is used to characterize antibody titer produced in a subject.

EXAMPLES

Example 1: Phase 2 Clinical Trial

Study Design and Methodology

This is an observer blind, Phase 2, stratified, randomized study to evaluate the immunogenicity, safety, and reactogenicity, of mRNA-1283 (SEQ ID NO: 9) and mRNA- 1283.211 (a 1:1 mixture of SEQ ID NO: 9 and SEQ ID NO: 21) administered as a single booster dose to participants 18 years and older that were previously vaccinated with mRNA-1273 .

This study assessed whether a single dose of mRNA 1283 at three different dose levels (2.5 pg, or 5 pg, or 10 pg) or mRNA 1283.211 at two different dose levels (5 pg or 10 pg) will boost antibody responses to the B.1.351 or prototype mRNA-1273 strain viruses and will also be used to select a dose for subsequent clinical evaluation. Participants who received mRNA-1273 (100 pg) with appropriate documentation at least 6 months prior will be randomized 1:1: 1:1:1 to receive a single boost of mRNA-1283 at one of three dose levels or a single boost of mRNA- 1283.211 at one of two dose levels. Enrollment in this study was stratified by age with two age strata: 18-55 years of age and >56 years of age, with at least 20%, but not more than 50% of participants, 56 years of age or older. Those with documented prior SARS CoV-2 infection were eligible to participate if also previously vaccinated with mRNA-1273. Prior infection status was confirmed by anti-nucleocapsid antibody testing of all participants.

Treatment Groups:

Participants had up to 6 visits, 5 study visits if screening and randomization are performed on the same day. The study vaccine wase administered as a single dose on Day 1. Additional safety and immunogenicity study visits occurred on Days 8 (safety call only), 15, 29, 181, and 366 (end of study). Study visits included scheduled safety phone calls every two weeks to collect medically attended adverse events (MAAEs), adverse events of special interest (AESIs), AEs leading to withdrawal, serious adverse events (SAEs), and information about concomitant medications associated with these events, as well as to collect information about receipt of nonstudy vaccinations temporally associated with these events.

Investigational Product, Dosage, and Mode of Administration:

The term “investigational product” (IP) refers to the mRNA-1283 and mRNA- 1283.211 vaccines administered in this study.

The mRNA-1283 Drug Product was a lipid nanoparticle (LNP) dispersion containing a single mRNA sequence that encodes a protein made up of 2 segments of the SARS CoV-2 S protein: the NTD and RBD, which are linked together with a 7 amino acid flexible linker. These 3 proteins (NTD, linker, and RBD) were attached to a 23 amino acid HATM via a 5 amino acid flexible linker. This entire protein made up the NTD-RBD-HATM antigen encoded by mRNA- 1283 (SEQ ID NO: 9). The mRNA was combined in a mixture of 4 lipids: Compound 1, cholesterol, DSPC, and PEG-DMG.

The mRNA- 1283.211 formulation was a lipid nanoparticle (LNP) dispersion containing a 1:1 mixture of mRNA-1283 and mRNA- 1283.351. mRNA- 1283.351 was a single mRNA sequence that encodes a protein made up of 2 segments of the B.1.351 SARS CoV-2 S protein variant (e.g., having the following mutations relative to wild-type SARS- CoV-2: L18F, D80A, D215G, L242-244del, R₂46I, K417N, E484K, N501Y, D614G, and A701V) and includes the NTD and RBD, which were linked together with a 7 amino acid flexible linker. These 3 proteins (NTD, linker, and RBD) were attached to a 23 amino acid HATM via a 5 amino acid flexible linker. This entire protein made up the NTD-RBD-HATM antigen encoded by mRNA-1283 (SEQ ID NO: 21). The mRNA was combined in a mixture of 4 lipids: Compound 1, cholesterol, DSPC, and PEG-DMG. mRNA-1283 and mRNA-1283.211 were provided as sterile liquids for injection, as a white to off white dispersion in appearance, at a concentration of 0.4 mg/mL in 20 mM Tris buffer containing 87 mg/mL sucrose and 10.7 mM sodium acetate, at pH 7.5. mRNA-1283 was packaged in 2R USP Type I borosilicate glass vials with PLASCAP vial seal containing a 13 mm FluroTec coated plug stopper and has a 0.6 mL nominal fill volume.

Each dose of the product was prepared for each participant based on the treatment group. Each injection had a volume of 0.25 mL. The vaccines contained mRNA-1283 at the doses of 2.5 pg , 5 pg and 10 pg and mRNA 1283.211 at the doses of 5 pg and lOpg.

Investigational product was administered as an intamuscular injection into the deltoid muscle on Day 1. Preferably, the vaccine was administered into the nondominant arm. Participants were monitored for a minimum of 30 minutes after vaccination. Assessments included vital sign measurements and monitoring for local or systemic AR₈.

Primary Endpoints:

• To assess the safety and reactogenicity of mRNA-1283 and mRNA- 1283.211 o Frequency and grade of each solicited local and systemic reactogenicity adverse reaction (AR) during a 7 day follow up period after vaccination o Frequency and severity of any unsolicited adverse events (AEs) during the 28 day follow up period after vaccination o Frequency of any serious AEs (SAEs), medically attended AEs (MAAEs), AEs leading to withdrawal from study participation, and AEs of special interest (AESIs) from Day 1 to end of study (EoS)

• To assess the immunogenicity of mRNA-1283 and mRNA- 1283.211 o Immune response of mRNA-1283 and mRNA- 1283.211 against the ancestral strain of severe acute respiratory syndrome coronavirus 2 (SARS CoV 2) and against SARS CoV 2 variants, including B.1.351, at Day 29 by geometric mean titer (GMT), geometric mean fold rise (GMFR), and seroresponse rate (SRR) Secondary Endpoint:

• To evaluate the immunogenicity of the booster administration at all time points o Immune response of mRNA-1283 and mRNA- 1283.211 against ancestral SARS- CoV-2 strain and against SARS-CoV-2 variants, at all immunogenicity time points by geometric mean titer (GMT), geometric mean fold rise (GMFR), and seroresponse rate Exploratory Endpoints:

• To characterize cellular immunogenicity o Frequency, magnitude, and phenotype of antigen- specific B- and T-cells, to include B-cell and T-cell receptor repertoires

• To conduct active detection of symptomatic and asymptomatic SARS-CoV-2 infection o Laboratory confirmed asymptomatic or symptomatic SARS-CoV-2 infection will be defined in participants:

■ Negative SARS-CoV-2 anti-nucleocapsid antibody blood test at Day 1 that becomes positive at Day 29 or later, or

■ Positive SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) from nasopharyngeal swab

• To evaluate the genetic and/or phenotypic relationships of isolated SARS-CoV-2 strains to the vaccine sequence o Comparison of the SARS-CoV-2 spike genetic sequence of viral isolates with the vaccine sequence and characterization of immune responses to vaccine breakthrough isolates

Safety Assessments:

Safety assessments will include monitoring and recording of the following for each participant:

• Solicited local and systemic AR₈ that occur during the 7 days following vaccination (i.e., the day of injection and 6 subsequent days). Solicited AR₈ will be recorded daily using eDiaries.

• Unsolicited AEs observed or reported during the 28 days following vaccination (ie, the day of injection and 27 subsequent days).

• AEs leading to withdrawal from Day 1 through EoS or withdrawal from the study.

• MAAEs from vaccination on Day 1 through EoS or withdrawal from the study.

• AESIs from vaccination on Day 1 through EoS or withdrawal from the study.

• SAEs from vaccination on Day 1 through EoS or withdrawal from the study.

• Details of all pregnancies in female participants will be collected after the start of study treatment and until the end of their participation in the study.

Immunogenicity Assessments: Blood samples for immunogenicity assessments will be collected at the time points indicated in the Schedule of Events. The following immunogenicity assessments will be measured:

• Serum binding antibody (bAb) level against SARS CoV 2 as measured by ligand-binding assay specific to the SARS CoV 2 S protein and the S protein receptor binding domain (RBD)

• Serum neutralizing antibody (nAb) level against SARS CoV 2 as measured by pseudovirus neutralization assays

• The above assays will include SARS-CoV-2 variant antigens, pseudotyped virus expressing SARS-CoV-2 S protein, or virus isolates to assess differences in immunologic responses to different SARS-CoV-2 variant S proteins.

• Testing for serologic markers for SARS CoV 2 infection as measured by anti- nucleocapsid antibodies detected by immunoassay at enrollment (Day 1) and scheduled post-baseline timepoints.

Vaccine effectiveness will not be formally assessed in this study, but active surveillance for COVID-19 and SARS-CoV-2 infection through weekly contact and bloods draws will be performed.

Immunogenicity Analyses

Immunogenicity analyses will be performed based on the PP Set for Immunogenicity and provided by treatment group.

Analysis for the Primary Immunogenicity Objective

For the primary immunogenicity endpoints of levels of SARS-CoV-2-specific nAb and SARS-CoV-2-specific bAb against the SARS-CoV-2 prototype virus strain and against the B.1.351 variant strain, and potentially other variants, immune response of each treatment group was assessed. An analysis of covariance model was employed to compare immune response between each treatment group. Day 29 antibody titers (against prototype virus strain or the variant strain) were included in the model as a dependent variable and treatment group variable (mRNA-1283 at each dose level and mRNA-1283.211 at each dose level) were included as fixed effect. The model also was adjusted for age group (< 56, > 56). The geometric mean titer (GMT) was estimated by the geometric least square mean (GLSM) from the model for each group and corresponding 95% CI will be provided. The ratio of GMTs for each treatment group were estimated by the ratio of GLSM from the model. The 95% CI for the ratio of GLSM was provided to assess the between groups difference in immune response against the prototype strain (or B.1.351 variant strain). The primary immunogenicity endpoints of the study were also assessed by the vaccine seroresponse against the SARS-CoV-2 prototype virus strain and against the B.1.351 variant and potentially other variants. The seroresponse rate (SRR) with 95% CI at Day 29 was summarized for each treatment group. The difference of SRR₈ and 95% CI at Day 29 was calculated for mRNA 1283 at each dose level and mRNA 1283.211 at each dose level.

Analysis for the Secondary and Exploratory Immunogenicity Objectives

Immunogenicity, SARS CoV 2-specific binding antibody (bAb) and neutralizing antibody (nAb), were assessed at multiple time points in this study; however, Day 29, 28 days after booster dose, was the time point of primary interest.

For each of the antibodies of interest, e.g., levels of SARS-CoV-2-specific bAb and SARS-CoV-2-specific nAb, the GMT or level with corresponding 95% CI at each time point and geometric mean fold rise of post baseline/baseline titers or levels with corresponding 95% CI at each post baseline time point were provided by treatment group. The 95% CI was calculated based on the t distribution of the log transformed values, then back transformed to the original scale for presentation. The following descriptive statistics were also provided at each time point: number of participants (n), median, minimum, and maximum.

The SRR of each treatment group against the prototype strain and variant strain, defined as the percentage of participants achieving seroresponse against the prototype strain and variant strain, respectively, were provided for each treatment group with the 95% CI calculated using the Clopper Pearson method at each post baseline time point.

Subgroup Analyses

Subgroup analyses may include age and SARS-CoV-2 infection status at baseline depending on the sample size in a subgroup.

Efficacy Analysis

No pre-specified efficacy analysis will be performed. Exploratory analyses of symptomatic and asymptomatic SARS-CoV-2infection by treatment group were performed.

Primary Analysis

The primary analysis of safety and immunogenicity was conducted after participants have completed their Day 29 visit assessments. The primary analysis was performed by a separate team of unblinded programmers and statisticians. The analysis, including any cases of COVID-19, was presented by treatment group. With the exception of appropriately delegated unblinded study staff, vaccine administrators, and monitors, all personnel involved in the conduct of the trial remained blinded to individual treatment assignment until unblinding. Investigators were blinded until after the final database lock for final analysis.

Analysis

A final analysis of all endpoints was performed after all participants have completed all planned study procedures. The final analysis includes full analyses of all safety and immunogenicity data through Day 366 (Month 12).

Results (Day 29 Immunogenicity)

Both vaccine formulations (mRNA-1283 and mRNA-1283.211) were well-tolerated. In participants without prior SARS-CoV-2 infection, there was a potent neutralizing antibody response against ancestral SARS-CoV-2 (D614G), the omicron variant, and the beta variant 28 days after administration of the mRNA-1283 or mRNA-1283.211 booster dose. The results are shown in Tables 2-6 below.

Specifically, with respect to the ancestral SARS-CoV-2 (D614G) strain, a dose-response was observed in the mRNA-1283 groups (titers were highest in the 10 pg group). Titers were also increased in the 1283.211 groups , although no dose response was observed.

With respect to the beta SARS-CoV-2 strain, there was a dose-response in the mRNA- 1283 groups and titers were highest in the 10 pg group. Titers in the mRNA- 1283.211 groups were also increased; however, no dose-response was observed in the mRNA-1283.211 groups.

Binding antibody titers with respect to the ancestral (D614G), alpha, and beta strains are shown in FIG. 1, including the change between the pre-booster level and on Day 29 (postbooster). The binding antibody titers for the gamma, delta, and omicron strains are shown in FIG. 2, including the change between pre-booster level and the level on Day 29 (post-booster).

Table 2 - Neutralizing Antibody Titers against Ancestral SARS-CoV-2 (D614G) - 28 Days after Booster Dose (participants without pre-booster infection)

Table 3 - Neutralizing Antibody Titers against Ancestral SARS-CoV-2 (D614G) - 28 Days after Booster Dose (participants with pre-booster infection)

Table 4 - Neutralizing Antibody Titers against Beta SARS-CoV-2 Strain - 28 Days after Booster Dose (participants without pre-booster infection)

Table 5 - Neutralizing Antibody Titers against Beta SARS-CoV-2 Strain - 28 Days after Booster Dose (participants with pre-booster infection)

Table 6 - Neutralizing Antibody Titers against Omicron SARS-CoV-2 Strain - 28 Days after Booster Dose (participants without pre-booster infection and excluding participants with infection through day 29)

ADDITIONAL SEQUENCES

It should be understood that any of the mRNA sequences described herein may include a 5' UTR and/or a 3' UTR. The UTR sequences may be selected from the following sequences, or other known UTR sequences may be used. It should also be understood that any of the mRNA constructs described herein may further comprise a poly(A) tail and/or cap (e.g., 7mG(5’)ppp(5’)NlmpNp). Further, while many of the mRNAs and encoded antigen sequences described herein include a signal peptide and/or a peptide tag (e.g., C-terminal His tag), it should be understood that the indicated signal peptide and/or peptide tag may be substituted for a different signal peptide and/or peptide tag, or the signal peptide and/or peptide tag may be omitted.

5’ UTR: GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (SEQ ID NO: 20)

5’ UTR: GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGCCGCCACC (SEQ ID NO: 2) 3’ UTR: UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCA CCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 22) 3’ UTR: UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCA CCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 4)

Table 1. Sequence Listing

EQUIVALENTS

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended,

to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value.

Where a range of values is provided, each value between and including the upper and lower ends of the range are specifically contemplated and described herein.

Claims

CLAIMS What is claimed is:

1. A method comprising administering to a human subject a therapeutic dose of a composition comprising a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) that encodes a fusion protein of a receptor binding domain (RBD) of a SARS- CoV-2 Spike protein and an amino (N)-terminal domain (NTD) of a SARS-CoV-2 Spike protein, and less than the full length spike protein, wherein the mRNA is in a lipid nanoparticle, wherein the composition is administered to the human subject following administration of a prophylactic composition against SARS-CoV-2, and wherein the composition comprises 2.5 pg - 5 pg total mRNA.

2. The method of claim 1, wherein the composition further comprises a second mRNA comprising a second ORF that encodes a second fusion protein comprising an RBD of a SARS- CoV-2 Spike protein and an NTD of a SARS-CoV-2 Spike protein, and less than the full length spike protein, wherein the mRNA and the second mRNA are not identical.

3. The method of claim 2, wherein the mRNA and the second mRNA are present in the composition at a ratio of 1:1.

4. The method of any one of claims 1-3, wherein the composition is administered to the human subject at least three months and within one year after administration of the prophylactic composition.

5. The method of any one of claims 1-3, wherein the composition is administered to the human subject at least six months after administration of the prophylactic composition.

6. The method of any one of claims 1-5, wherein the prophylactic composition comprises an mRNA vaccine or a viral vector vaccine.

7. The method of claim 6, wherein the prophylactic composition is an mRNA vaccine and the human subject has received two doses of the mRNA vaccine.

8. The method of claim 6, the prophylactic composition is an mRNA vaccine and the human subject has received one dose of the mRNA vaccine.

9. The method of any one of claims 1-8, wherein the composition comprises 2.5 pg of mRNA in total.

10. The method of any one of claims 1-8, wherein the composition comprises 5 pg of mRNA in total.

11. The method of any one of the preceding claims, wherein the composition further comprises Tris buffer.

12. The method of claim 11, wherein the composition with Tris buffer further comprises sucrose and sodium acetate.

13. The method of claim 12, wherein the composition comprises 10 mM - 30 mM Tris buffer comprising 75 mg/mL - 95 mg/mL sucrose, and 5 mM - 15 mM sodium acetate, optionally wherein the composition has a pH of 6-8.

14. The method of claim 13, wherein the composition comprises about 20 mM Tris buffer comprising 87 mg/mL sucrose, and 10.7 mM sodium acetate, optionally wherein the composition has a pH of 7.5.

15. The method of any one of the preceding claims, wherein the composition comprises about 0.25 mg/mL of the mRNA total.

16. The method of any one of the preceding claims, wherein the composition is administered intramuscularly, optionally into a deltoid muscle of the subject’s arm.

17. The method of any of the preceding claims, wherein the fusion protein comprises a sequence having at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 11.

18. The method of claim 17, wherein the fusion protein comprises SEQ ID NO: 11.

19. The method of any one of claims 1-18, wherein the ORF comprises a sequence having at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 10.

20. The method of claim 19, wherein the ORF comprises SEQ ID NO: 10.

21. The method of any one of claims 1-20, wherein the mRNA comprises a sequence having at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 9.

22. The method of claim 21, wherein the mRNA comprises SEQ ID NO: 9.

23. The method of any one of claims 2-22, wherein the second fusion protein comprises a sequence having at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 23.

24. The method of claim 23, wherein the fusion protein comprises SEQ ID NO: 23.

25. The method of any one of claims 2-24, wherein the second ORF comprises a sequence having at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 22.

26. The method of claim 25, wherein the second ORF comprises SEQ ID NO: 22.

27. The method of any one of claims 2-26, wherein the second mRNA comprises a sequence having at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 21.

28. The method of claim 27, wherein the second mRNA comprises SEQ ID NO: 21.

29. The method of any one of the preceding claims, wherein the lipid nanoparticle comprises: ionizable amino lipid; neutral lipid; sterol; and PEG-modified lipid.

30. The method of claim 29, wherein the lipid nanoparticle comprises: 40-55 mol% ionizable amino lipid; 5-15 mol% neutral lipid; 35-45 mol% sterol; and 1-5 mol% PEG-modified lipid.

31. The method of claim 30, wherein the lipid nanoparticle comprises: 47 mol% ionizable amino lipid; 11.5 mol% neutral lipid; 38.5 mol% sterol; and 3.0 mol% PEG-modified lipid;

48 mol% ionizable amino lipid; 11 mol% neutral lipid; 38.5 mol% sterol; and 2.5 mol% PEG-modified lipid;

49 mol% ionizable amino lipid; 10.5 mol% neutral lipid; 38.5 mol% sterol; and 2.0 mol% PEG-modified lipid;

50 mol% ionizable amino lipid; 10 mol% neutral lipid; 38.5 mol% sterol; and 1.5 mol% PEG-modified lipid; or

51 mol% ionizable amino lipid; 9.5 mol% neutral lipid; 38.5 mol% sterol; and 1.0 mol% PEG-modified lipid.

32. The method of any one of claims 29-31, wherein the ionizable amino lipid is heptadecan- 9-yl 8 ((2 hydroxyethyl)(6 oxo 6-(undecyloxy)hexyl)amino)octanoate (Compound 1).

33. The method of any one of claims 29-32, wherein the neutral lipid is 1,2 distearoyl sn glycero-3 phosphocholine (DSPC).

34. The method of any one of claims 29-33, wherein the sterol is cholesterol.

35. The method of any one of claims 29-34, wherein the PEG-modified lipid is 1- monomethoxypolyethyleneglycol-2,3-dimyristylglycerol with polyethylene glycol of average molecular weight 2000 (PEG-DMG).

36. The method of any one of the preceding claims, wherein the prophylactic composition against SARS-CoV-2 comprises an mRNA vaccine or a viral vector vaccine.

37. The method of claim 36, wherein the prophylactic composition comprises an mRNA vaccine comprising an ORF encoding a SARS-CoV-2 prefusion stabilized S protein.

38. The method of claim 36 or claim 37, wherein the human subject has previously been administered at least one dose of the prophylactic composition.

39. The method of any one of claims 36-38, wherein the human subject has previously been administered two doses of the prophylactic composition.

40. The method of any one of claims 36-39, comprising administering the composition to the human subject at least six months after the most recent administration of the prophylactic composition.

41. A composition comprising:

0.1 |lg - 6 |lg of a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) that encodes a fusion protein comprising at least two domains of a SARS-CoV-2 Spike (S) protein, and less than the full length spike protein; and a lipid nanoparticle comprising a mixture of lipids that comprises an ionizable amino lipid, a non-cationic lipid, a sterol, and a PEG-modified lipid.

42. The composition of claim 41, further comprising a second mRNA comprising a second ORF that encodes a second fusion protein comprising at least two domains of a SARS-CoV-2 Spike (S) protein and less than the full length S protein.

43. The composition of claim 42, wherein the mRNA and the second mRNA are present in the composition at a ratio of 1 : 1.

44. The composition of any one of claims 41-43, wherein the composition comprises 2.5 pg of mRNA in total.

45. The composition of any one of claims 41-43, wherein the composition comprises 5 pg of mRNA in total.

46. The composition of any one of claims 41-45, wherein the fusion protein comprises a sequence having at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 11.

47. The composition of claim 46, wherein fusion protein comprises SEQ ID NO: 11.

48. The composition of any one of claims 41-47, wherein the ORF comprises a sequence having at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 10.

49. The composition of claim 48, wherein the ORF comprises SEQ ID NO: 10.

50. The composition of any one of claims 41-49, wherein the mRNA comprises a sequence having at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 9.

51. The composition of claim 50, wherein the mRNA comprises SEQ ID NO: 9.

52. The composition of any one of claims 42-51, wherein the second fusion protein comprises a sequence having at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 23.

53. The composition of claim 52, wherein the second fusion protein comprises SEQ ID NO: 23.

54. The composition of any one of claims 42-53, wherein the second ORF comprises a sequence having at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 22.

55. The composition of claim 54, wherein the second ORF comprises SEQ ID NO: 22.

56. The composition of any one of claims 42-55, wherein the second mRNA comprises a sequence having at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 21.

57. The composition of claim 56, wherein the second mRNA comprises SEQ ID NO: 21.

58. The composition of any one of claims 41-57, wherein the mRNA and/or the second mRNA comprises a chemical modification.

59. The composition of claim 58, wherein the mRNA and/or the second mRNA is fully modified.

60. The composition of claim 58 or 59, wherein the chemical modification is 1- methy Ip seudouridine .

61. The composition of any one of claims 41-60, wherein the mRNA and/or the second mRNA further comprises a 5’ cap analog, optionally a 7mG(5’)ppp(5’)NlmpNp cap.

62. The composition of any one of claims 41-61, wherein the mRNA and/or the second mRNA further comprises a poly(A) tail, optionally having a length of 50 to 150 nucleotides.

63. The composition of any one of claims 41-62, wherein the lipid nanoparticle comprises: ionizable amino lipid; neutral lipid; sterol; and PEG-modified lipid.

64. The composition of claim 63, wherein the lipid nanoparticle comprises: 40-55 mol% ionizable amino lipid; 5-15 mol% neutral lipid; 35-45 mol% sterol; and 1-5 mol% PEG- modified lipid.

65. The composition of claim 64, wherein the lipid nanoparticle comprises:

47 mol% ionizable amino lipid; 11.5 mol% neutral lipid; 38.5 mol% sterol; and 3.0 mol% PEG-modified lipid;

66. The composition of any one of claims 63-65, wherein the ionizable amino lipid is heptadecan-9-yl 8 ((2 hydroxy ethyl)(6 oxo 6-(undecyloxy)hexyl)amino)octanoate (Compd. 1).

67. The composition of any one of claims 63-66, wherein the neutral lipid is 1,2 distearoyl sn glycero-3 phosphocholine (DSPC).

68. The composition of any one of claims 63-67, wherein the sterol is cholesterol.

69. The composition of any one of claims 63-68, wherein the PEG-modified lipid is 1- monomethoxypolyethyleneglycol-2,3-dimyristylglycerol with polyethylene glycol of average molecular weight 2000 (PEG-DMG).

70. The composition of any one of claims 41-69, wherein the composition further comprises Tris buffer, sucrose, and sodium acetate, or any combination thereof.

71. The composition of claim 70, wherein the composition further comprises 30-40 mM Tris buffer, 80-95 mg/mL sucrose, and 5-15 mM sodium acetate.

72. The composition of any one of claims 41-71, wherein the composition has a pH value of 6-8, optionally 7.5.

73. The composition of any one of claims 41-43, wherein the composition comprises 1-5 pg of mRNA in total.

74. The composition of any one of claims 41-43, wherein the composition comprises 1-4.9 pg of mRNA in total.

75. The composition of any one of claims 41-43, wherein the composition comprises 2-4 pg of mRNA in total.

76. The method of any one of claims 1-40, wherein the human subject is seropositive for SARS-CoV-2 or a variant thereof.

77. The method of any one of claims 1-40, wherein the composition induces neutralizing antibodies against SARS-CoV-2.

78. The method of claim 76, wherein the composition induces neutralizing antibodies against a SARS-CoV-2 variant, ancestral SARS-CoV-2, or both the SARS-CoV-2 variant and ancestral SARS-CoV-2.

79. The method of claim 78, wherein the composition induces neutralizing antibodies against two or more SARS-CoV-2 variants.