SARS-COV-2 immunogenic compositions

Abstract

Disclosed herein are compositions comprising protein antigens and RNA encoding the same (eg., compositions comprising protein antigens and RNA encoding antigens) that can be used to induce an immune response against SARS-CoV-2. Also disclosed herein are immunogenic compositions and medical preparations comprising the same, and methods of making and using the same. In some embodiments, the technologies provided herein can be used to address and / or overcome immune imprinting in SARS-CoV-2.

Description

Attorney Docket No.: 2013237-1485SARS-COV-2 IMMUNOGENIC COMPOSITIONSCross-Reference to Related Applications

[0001] The present application claims priority to United States Provisional Application No. 63 / 688,279, filed August 28, 2024, United States Provisional Application No. 63 / 700,480, filed September 27, 2024, and United States Provisional Application No. 63 / 754,514, filed February 5, 2025, the entireties of which are incorporated herein by reference.Background

[0002] Since the initial discovery of SARS-CoV-2, a number of variants have arisen around the world. The emergence of these novel circulating variants of SARS-CoV-2 has raised significant concerns about the temporal efficacy of vaccine interventions. The emergence of Omicron (B.l.1.529) variants, which comprise a number of mutations in the S protein, has been of particular concern.Summary

[0003] In some embodiments, the present disclosure provides technologies (e.g., compositions and methods) that can be used to induce an immune response against SARS-CoV-2. Technologies provided herein include, among others, immunogenic compositions (e.g., RNA compositions), methods of inducing an immune response, and methods of manufacturing immunogenic compositions. In some embodiments, an immunogenic composition delivers a SARS-CoV-2 antigen (e.g., comprises a SARS-CoV-2 antigen or a nucleic acid encoding a SARS-CoV-2 antigen). In some embodiments, an immunogenic composition delivers a SARS-CoV-2 S protein, or an immunogenic portion thereof. In some embodiments, technologies described herein can produce an immune response characterized by an increased naive immune response, a de novo immune response, and / or a decreased memory B cell response. In some embodiments, an immunogenic composition delivers an immunogenic portion of a SARS-CoV-2 S protein, including, e.g., an RBD, NTD, or a truncated SI subdomain of a SARS-CoV-2 virus, or a variant of any of the foregoing. In some embodiments, technologies provided herein can provide an improved immune response (e.g., higher neutralization antibody titers, increased naive B cell activation, and / or higher titers of antibodies recognizing an epitope unique to a variant of concern (relative to a reference antigen, including, e.g., a full-length SARS-CoV-2 S protein)) against one or more SARS-CoV-2 variants of concern (e.g., variants of concern against which current vaccine technologies produce a reduced neutralization response as compared to a Wuhan strain). In some embodiments, technologies provided herein can partially or fully address and / or overcome an immune imprinting effect.

[0004] Among other things, the present disclosure provides certain insights into the design of SARS-CoV-2 vaccines that provide significant advantages as compared to currently existing vaccines (e.g., vaccines that deliver a full-length SARS-CoV-2 S protein). These advantages include, e.g., increased antigen expression, increased antibody titers (in some embodiments, including increased neutralization titers), increased efficacy, increased stability (e.g., increased thermostability), increased cross-neutralization, and / or enhanced durability of antibody responses as compared to current SARS-CoV-2 vaccines (e.g., as compared to the first generation of SARS-CoV-2 RNA vaccines, encoding full-length Spike proteins (e.g., BNT162b2 (Comirnaty) and mRNA1273 (SpikeVax)). Among other things, the insights provided by the present disclosure include the identification of certain fragments of the S protein that provide an improved immune response as compared to the full-length S protein (e.g., increased neutralization titers and / or more durable immune responses). Among other things, the present disclosure provides the insight that a12947033vl Page 1 of 526Attorney Docket No.: 2013237-1485 truncated SI subdomain that comprises an endogenous sequence connecting the NTD and RBD regions, or polyribonucleotide encoding the same, can induce an improved immune response as compared to a full-length S protein or a polypeptide comprising an NTD and RBD connected via a heterologous flexible linker.

[0005] Also provided herein are improved antigen designs, e.g., for delivery via RNA, including the identification of preferred domains (e.g., secretory signal peptides and transmembrane regions) to link to SARS-CoV- 2 antigens and configurations thereof that result in significant improvement in immune responses. These include, e.g., improved secretory signal peptides, GS linkers (specifically, when used to attach fragments of the SARS-CoV-2 S protein to preferred domains), transmembrane domains, multimerization domains, and nucleotide sequences, and combinations and configurations thereof, each of which individually provides significant improvements as compared to existing SARS-CoV-2 vaccines, and which in combination provide even greater improvements.

[0006] In some embodiments, the present disclosure provides a polyribonucleotide comprising a nucleotide sequence that encodes a SARS-CoV-2 Spike (S) protein variant, or an immunogenic portion thereof, wherein the variant or the immunogenic portion thereof comprises from N-terminus to C-terminus: (i) an endogenous SARS-CoV- 2 secretory signal; (ii) an N-terminal domain (NTD) of a SARS-CoV-2 S protein; (iii) a receptor-binding domain (RBD) of a SARS-CoV-2 S protein; (iv) a GS linker; and (v) an endogenous SARS-CoV-2 transmembrane domain (TMD). In some embodiments, all the uridines in the polyribonucleotide are replaced with Nl-methyl pseudouridines (ml4J).

[0007] In some embodiments, a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises one or more mutations relative to the NTD and RBD of the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1). In some embodiments, one or more mutations comprise one or more mutations originating in one or more of an XBB.1.5 variant, a KP.2 variant, a JN.l variant, an XEC variant, an LP.8.1 variant, and / or a BA.3.2 variant relative to the NTD and RBD of the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1). In some embodiments, one or more mutations comprise one or more mutations originating in an XBB.1.5 variant relative to the NTD and RBD of the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1). In some embodiments, one or more mutations comprise one or more mutations originating in an KP.2 variant relative to the NTD and RBD of the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1). In some embodiments, one or more mutations comprise one or more mutations originating in an JN.l variant relative to the NTD and RBD of the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1). In some embodiments, one or more mutations comprise one or more mutations originating in an LP.8.1 variant relative to the NTD and RBD of the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1). In some embodiments, one or more mutations comprise one or more mutations originating in an BA.3.2 variant relative to the NTD and RBD of the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1).

[0008] In some embodiments, one or more mutations comprise: (i) T19I, A24-26, A27S, V83A, G142D, A145, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, or any combination thereof; (ii) insl6MPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A201A211, L202IL212I, V203GV213G, L206FL216F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, or any combination thereof; (iii) insl6MPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, or any combination thereof; (iv) insl6MPLF, T19I,12947033vl Page 2 of 526Attorney Docket No.: 2013237-1485R21T, T22N, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, F456L, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, or any combination thereof; (v) Insl6MPLF, T19I, R21T, L24del, P25del, P26del, A27S, S31del, S50L, H69del, V70del, V127F, G142D, Y144del, F157S, R158G, F186L, R190S, N211del, L212I, V213G, L216F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445R, G446S, N450D, L452W, L455S, F456L, N460K, S477N, T478K, N481K, V483del, E484K, F486P, Q493E, Q498R, N501Y, Y505H, or any combination thereof; or (vi) P9L, R21T, P26L, A67V, H69del, V70del, T95I, I101T, C136del, N137del, D138del, P139del, F140del, L141del, G142del, V143del, Y144del, Y145del, H146del, K147del, F157S, N164K, S172F, K187T, N211del, L212I, L242del, A243del, P251S, G339Y, A348P, S371F, S373P, S375F, R403K, D405N, T408S, K417N, A435S, N440R, V445A, G446D, L452W, N460K, S477N, T478N, E484K, G496S, Q498R, N501Y, K529N, C538del, V539del, N540del, F541del, or any combination thereof, wherein the one or more mutations are relative to the NTD and RBD of the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1).

[0009] In some embodiments, one or more mutations comprise: T19I, A24-26, A27S, V83A, G142D, A145, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, or any combination thereof, wherein the one or more mutations are relative to the NTD and RBD of the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1). In some embodiments, one or more mutations comprise: insl6MPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A201A211, L202IL212I, V203GV213G, L206FL216F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, or any combination thereof, or any combination thereof, wherein the one or more mutations are relative to the NTD and RBD of the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1). In some embodiments, one or more mutations comprise: insl6MPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, or any combination thereof, wherein the one or more mutations are relative to the NTD and RBD of the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1). In some embodiments, one or more mutations comprise: insl6MPLF, T19I, R21T, T22N, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, F456L, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, or any combination thereof, wherein the one or more mutations are relative to the NTD and RBD of the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1). In some embodiments, one or more mutations comprise: Insl6MPLF, T19I, R21T, L24del, P25del, P26del, A27S, S31del, S50L, H69del, V70del, V127F, G142D, Y144del, F157S, R158G, F186L, R190S, N211del, L212I, V213G, L216F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445R, G446S, N450D, L452W, L455S, F456L, N460K, S477N, T478K, N481K, V483del, E484K, F486P, Q493E, Q498R, N501Y, Y505H, or any combination thereof, wherein the one or more mutations are relative to the NTD and RBD of the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1). In some embodiments, one or more mutations comprise: P9L, R21T, P26L, A67V, H69del, V70del, T95I, I101T, C136del, N137del, D138del, P139del, F140del, L141del, G142del, V143del, Y144del, Y145del, H146del, K147del,12947033vl Page 3 of 526Attorney Docket No.: 2013237-1485F157S, N164K, S172F, K187T, N211del, L212I, L242del, A243del, P251S, G339Y, A348P, S371F, S373P, S375F, R403K, D405N, T408S, K417N, A435S, N440R, V445A, G446D, L452W, N460K, S477N, T478N, E484K, G496S, Q498R, N501Y, K529N, C538del, V539del, N540del, F541del, or any combination thereof, wherein the one or more mutations are relative to the NTD and RBD of the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1).

[0010] In some embodiments, a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of the NTD and / or RBD of any one or more of SEQ ID NO: 125, SEQ ID NO: 1501, SEQ ID NO: 853, SEQ ID NO: 878, SEQ ID NO: 893, and / or SEQ ID NO: 903. In some embodiments, a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of the NTD and / or RBD of SEQ ID NO: 125. In some embodiments, a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of the NTD and / or RBD of SEQ ID NO: 1501. In some embodiments, a SARS- CoV-2 S protein variant, or an immunogenic portion thereof, comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of the NTD and / or RBD of SEQ ID NO: 853. In some embodiments, a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of the NTD and / or RBD of SEQ ID NO: 878. In some embodiments, a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of the NTD and / or RBD of SEQ ID NO: 893. In some embodiments, a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of the NTD and / or RBD of SEQ ID NO: 903.

[0011] In some embodiments, a secretory signal comprises SEQ ID NO: 821, SEQ ID NO: 1750, SEQ ID NO: 44, SEQ ID NO: 15, SEQ ID NO: 822, SEQ ID NO: 9, SEQ ID NO: 16, or SEQ ID NO: 823. In some embodiments, a secretory signal comprises SEQ ID NO: 821. In some embodiments, a secretory signal comprises SEQ ID NO: 1750. In some embodiments, a secretory signal comprises SEQ ID NO: 44. In some embodiments, a secretory signal comprises SEQ ID NO: 15. In some embodiments, a secretory signal comprises SEQ ID NO: 822. In some embodiments, a secretory signal comprises SEQ ID NO: 9. In some embodiments, a secretory signal comprises SEQ ID NO: 16. In some embodiments, a secretory signal comprises SEQ ID NO: 823.

[0012] In some embodiments, a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, does not comprise a heterologous linker between the NTD and RBD.12947033vl Page 4 of 526Attorney Docket No.: 2013237-1485

[0013] In some embodiments, a GS linker comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of a GS linker listed in Table 8.

[0014] In some embodiments, a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of any of SEQ ID NO: 2015; SEQ ID NO: 2000; SEQ ID NO: 2003; SEQ ID NO: 2006;SEQ ID NO: 2009; or SEQ ID NO: 2012. In some embodiments, a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2015. In some embodiments, a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO: 2000. In some embodiments, a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2003. In some embodiments, a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2006. In some embodiments, a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2009. In some embodiments, a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2012.

[0015] In some embodiments, a polyribonucleotide comprises a nucleotide sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence according to any one of SEQ ID NO: 2016; SEQ ID NO: 2001; SEQ ID NO: 2002; SEQ ID NO: 2004; SEQ ID NO: 2005; SEQ ID NO: 2007; SEQ ID NO: 2008; SEQ ID NO: 2010; SEQ ID NO: 2011; SEQ ID NO: 2013; or SEQ ID NO: 2014. In some embodiments, a polyribonucleotide comprises a nucleotide sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence according to SEQ ID NO: 2016. In some embodiments, a polyribonucleotide comprises a nucleotide sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence according to SEQ ID NO: 2001. In some12947033vl Page 5 of 526Attorney Docket No.: 2013237-1485 embodiments, a polyribonucleotide comprises a nucleotide sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence according to SEQ ID NO: 2002. In some embodiments, a polyribonucleotide comprises a nucleotide sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence according to SEQ ID NO: 2004. In some embodiments, a polyribonucleotide comprises a nucleotide sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence according to SEQ ID NO: 2005. In some embodiments, a polyribonucleotide comprises a nucleotide sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence according to SEQ ID NO: 2007. In some embodiments, a polyribonucleotide comprises a nucleotide sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence according to SEQ ID NO: 2008. In some embodiments, a polyribonucleotide comprises a nucleotide sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence according to SEQ ID NO: 2010. In some embodiments, a polyribonucleotide comprises a nucleotide sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence according to SEQ ID NO: 2011. In some embodiments, a polyribonucleotide comprises a nucleotide sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence according to SEQ ID NO: 2013. In some embodiments, a polyribonucleotide comprises a nucleotide sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence according to SEQ ID NO: 2014.

[0016] In some embodiments, a GS linker comprises SEQ ID NO: 848.

[0017] In some embodiments, an NTD of a SARS-CoV-2 S protein comprises amino acids located at positions1-209, 14-209, 15-209, 19-209, 20-209, 21-209, 1-305, 14-305, 15-305, 19-305, 20-305, 21-305, 1-311, 14-311, 15- 311, 19-311, 20-311, 21-311, 1-318, 14-318, 15-318, 19-318, 20-318, 21-318, 1-302, 14-302, 15-302, 19-302, 20- 302, or 21-302 of the SARS-CoV-2 S protein, wherein positions are relative to the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1).

[0018] In some embodiments, an RBD of a SARS-CoV-2 S protein comprises amino acids located at positions 319-528, 327-528, 328-528, 330-528, 319-530, 327-530, 328-530, 330-530, 319-531, 327-531, 328-531, 330-531, 319-537, 327-537, 328-537, 330-537, 330-537, 319-541, 327-541, 328-541, or 330-541 of the SARS-CoV-2 S protein, wherein positions are relative to the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1).

[0019] In some embodiments, the present disclosure provides a composition comprising a polyribonucleotide disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, a composition comprises lipid nanoparticles (LNPs), liposomes, lipoplexes (LPXs), or polyplexes (PLXs). In some embodiments, a polyribonucleotide12947033vl Page 6 of 526Attorney Docket No.: 2013237-1485 is fully or partially encapsulated within the LNPs, liposomes, LPXs, or PLXs. In some embodiments, a polyribonucleotide described herein is encapsulated in a lipid nanopartide (LNP).

[0020] The present disclosure also provides a method comprising administering to a subject a polyribonucleotide or composition comprising a polyribonucleotide described herein

[0021] In some embodiments, the present disclosure provides a method of preventing or treating a SARS-CoV- 2 infection in a subject comprising administering to the subject a polyribonucleotide or composition comprising a polyribonucleotide described herein.

[0022] In some embodiments, the present disclosure provides a method of manufacturing a polyribonucleotide described herein. In some embodiments, a method comprises in vitro transcription of a DNA template.

[0023] In some embodiments, compositions described herein can be used to induce an improved immune response as compared to a reference composition. In some embodiments, the reference composition comprises an RNA encoding a full-length SARS-CoV-2 S protein, optionally comprising one or more mutations that stabilize the prefusion confirmation.

[0024] In some embodiments, the improved immune response includes an improved B cell immune response. In some embodiments, an improved B cell immune response comprises inducing an increased number of B cells that can recognize a SARS-CoV-2 S protein. In some embodiments, an improved B cell immune response comprises an increased number of B cells that can recognize the RBD region of a SARS-CoV-2 S protein. In some embodiments, an improved B cell immune response comprises an increased number of B cells that can recognize the NTD region of a SARS-CoV-2 S protein. In some embodiments, the number of B cells that can recognize the S protein, NTD, or RBD is increased by at least 10%, 20%, 20%, 40%, 50%, 60%, 70%, 80%, ,90%, 100%, 150%, 200%, 250%, 300%, 350%, or 400% as compared to the number of B cells induced by an RNA composition delivering a full-length S protein. In some embodiments, the number of B cells that can recognize the S protein, NTD, or RBD is increased by an amount within a range having a lower bound of 10%, 20%, 20%, 40%, 50%, 60%, 70%, 80%, ,90%, or 100%, and an upper bound of 150%, 200%, 250%, 300%, 350%, or 400% as compared to the number of B cells induced by an RNA composition delivering a full-length S protein. In some embodiments, the number of B cells that can recognize the S protein, NTD, or RBD is increased by about 10% to about 1000%, about 10% to about 600%, about 10% to about 500%, about 100% to about 500%, at least about 100%, at least about 200%, at least about 400%, or at least about 500%. In some embodiments, the number of B cells that can recognize the S protein, NTD, or RBD is increased by at least about 200%.

[0025] In some embodiments, an improved immune response includes increased titers of antibodies that can neutralize a SARS-CoV-2 virus. In some embodiments, titers of neutralizing antibodies are increased by at least 10%, 20%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, or 400% as compared to the number of B cells induced by an RNA composition delivering a full-length S protein. In some embodiments, titers of neutralizing antibodies are increased by an amount within a range having a lower bound of 10%, 20%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, and an upper bound of 150%, 200%, 250%, 300%, 350%, or 400% as compared to the number of B cells induced by an RNA composition delivering a full-length S protein. In some embodiments, titers of neutralizing antibodies are increased by about 10% to about 1000%, about 10% to about 600%, about 10% to about 500%, about 100% to about 500%, at least about 100%, at least about 200%, at least about 400%, or at least about 500%. In some embodiments, titers of neutralizing antibodies are12947033vl Page 7 of 526Attorney Docket No.: 2013237-1485 increased by at least about 200%. In some embodiments, titers of neutralizing antibodies are increased by at least about 400%.

[0026] In some embodiments, technologies provided herein can provide a dose sparing effect (i.e., require a lower amount of RNA required to produce a given immune response). For example, as demonstrated in the Examples of the present disclosure, in some embodiments, a composition described herein can provide a dose sparing effect of at least about 2-fold. In some embodiments, a composition described herein can provide a dose sparing effect of at least about 3-fold. In some embodiments, a composition described herein can provide a dose sparing effect of at least about 4-fold. In some embodiments, a composition described herein can provide a dose sparing effect of at least about 5-fold.

[0027] In some embodiments, a SARS-CoV-2 S protein variant (or immunogenic portion thereof) has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of a reference SARS- CoV-2 S protein (or an amino acid sequence of the corresponding portion of a reference SARS-CoV-2 S protein).

[0028] In some embodiments, provided herein, is an RNA comprising a nucleotide sequence that encodes a polypeptide comprising a SARS-CoV-2 Spike (S) protein variant, or an immunogenic portion thereof, wherein a memory B cell immune response has been established to a reference SARS-CoV-2 S protein, and wherein the SARS- CoV-2 S protein variant or immunogenic portion thereof has an amino acid sequence that differs from that of the reference SARS-CoV-2 S protein variant in that it has been engineered to reduce activation of the memory B cell immune response relative to the SARS-CoV-2 S protein.

[0029] In some embodiments, an RNA comprises a nucleotide sequence that encodes a SARS-CoV-2 Spike (S) protein variant (or an immunogenic portion thereof) whose amino acid sequence is engineered so that at least one memory B cell epitope present in a reference SARS-CoV-2 S protein has been modified so that memory B cell activation potency of the SARS-CoV-2 S protein variant (or immunogenic portion thereof) has been reduced relative to the reference SARS-CoV-2 S protein.

[0030] In some embodiments, a SARS-CoV-2 S protein variant (or immunogenic portion thereof) has an amino acid sequence that is at least 80% identical to that of a reference SARS-CoV-2 S protein (or an amino acid sequence of the corresponding portion of a reference SARS-CoV-2 S protein).

[0031] In some embodiments, a SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises no more than 50% of the memory B cell epitopes present in a reference SARS-CoV-2 S protein.

[0032] In some embodiments, an RNA comprises a nucleotide sequence that encodes a SARS-CoV-2 Spike (S) protein variant (or an immunogenic portion thereof), wherein the amino acid sequence of the S protein variant or immunogenic portion thereof was engineered by a process comprising a step of removing memory B cell epitopes present in a reference SARS-CoV-2 S protein.

[0033] In some embodiments, a variant SARS-CoV-2 S protein (or immunogenic portion thereof) comprises few memory B cell epitopes of a reference SARS-CoV-2 S protein.

[0034] In some embodiments, one or more memory B cell epitopes in a reference SARS-CoV-2 S protein have been identified by antibody-binding studies (e.g., studies characterizing antibodies produced by subjects administered a vaccine that delivers the reference SARS-CoV-2 S protein and / or infected with a virus comprises the reference SARS-CoV-2 S protein).12947033vl Page 8 of 526Attorney Docket No.: 2013237-1485

[0035] In some embodiments, one or more memory B cell epitopes comprise or consist of non-neutralizing epitopes.

[0036] In some embodiments, one or more memory B cell epitopes comprise or consist of non-neutralizing epitopes and neutralizing epitopes.

[0037] In some embodiments, a variant polypeptide comprises 10 or fewer (e.g., 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, one or less, or no) conserved memory B cell epitopes.

[0038] In some embodiments, a SARS-CoV-2 S protein variant or immunogenic portion thereof is engineered so as to lack regions of a reference SARS-CoV-2 S protein that comprise a high number or density of conserved memory B cell epitopes.

[0039] In some embodiments, conserved memory B cell epitopes are non-neutralizing epitopes.

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

[0041] In some embodiments, an immunogenic portion of a SARS-CoV-2 S protein variant does not comprise an S2 domain.

[0042] In some embodiments, an immunogenic portion of a SARS-CoV-2 S protein variant comprises or consists of the SI domain, a truncated SI domain, or a receptor binding domain (RBD), or a variant of any of the foregoing.

[0043] In some embodiments, an immunogenic portion of a SARS-CoV-2 S protein variant does not comprise an N-terminal domain (NTD).

[0044] In some embodiments, an immunogenic portion of a SARS-CoV-2 S protein variant comprises or consists of an RBD.

[0045] In some embodiments, an RNA encodes a SARS-CoV-2 antigen (e.g., one or more fragments of an S protein, including, e.g., one or more NTDs, RBDs, and / or fragments of an S2 subdomain) comprising one or more mutations of a SARS-CoV-2 variant. In some embodiments, a SARS-CoV-2 variant is a variant of concern or is predicted to become a variant of concern (e.g., by a health organization, including, e.g., WHO or the CDC). In some embodiments, a SARS-CoV-2 variant is rapidly growing in a region and / or exhibits increased immune escape potential as compared to currently prevalent SARS-CoV-2 viruses. In some embodiments, an RNA encodes a SARS-CoV-2 antigen that comprises one or more mutations of a variant that a health organization has recommended providing seasonally updated vaccines against.

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

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

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

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

[0050] In some embodiments, a SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises one or more mutations associated with a SARS-CoV-2 variant that has a high immune escape potential (e.g., a variant of concern).12947033vl Page 9 of 526Attorney Docket No.: 2013237-1485

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

[0052] In some embodiments, a SARS-CoV-2 variant with a high immune escape potential is an Omicron variant.

[0053] In some embodiments, a SARS-CoV-2 variant with a high immune escape potential is an XBB variant (e.g., an XBB.l or XBB.1.5 variant), a BQ.l variant, or a BA.3.2 variant.

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

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

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

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

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

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

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

[0061] In some embodiments, an RNA comprises a nucleotide sequence that encodes an immunogenic portion of the SARS-CoV-2 S protein variant comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 125, SEQ ID NO: 1501, SEQ ID NO: 853, SEQ ID NO: 878, SEQ ID NO: 893, or SEQ ID NO: 903.12947033vl Page 10 of 526Attorney Docket No.: 2013237-1485

[0062] In some embodiments, a variant polypeptide comprises a secretion signal. In some embodiments, a secretion signal is a homologous secretion signal. In some embodiments, a secretion signal is a heterologous secretion signal.

[0063] In some embodiments, a secretion signal is present in the N-terminal portion of a polypeptide (e.g., at the N-terminus).

[0064] In some embodiments, a secretion signal is a SARS-CoV-2 S protein secretion signal, a gD2 secretion signal, a gDl secretion signal, a gBl secretion signal, a gI2 secretion signal, a gE2 secretion signal, an Eboz secretion signal, or an HLA-DR secretion signal.

[0065] In some embodiments, a SARS-CoV-2 S protein secretion signal comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15.

[0066] In some embodiments, a SARS-CoV-2 secretion signal comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9.

[0067] In some embodiments, a SARS-CoV-2 secretion signal comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 16.

[0068] In some embodiments, a gD2 secretion signal comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 8.

[0069] In some embodiments, a gD2 secretion signal comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13.

[0070] In some embodiments, a gDl secretion signal comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12.

[0071] In some embodiments, a gBl secretion signal comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 37.

[0072] In some embodiments, a gC2 polypeptide comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 35.

[0073] In some embodiments, a gI2 secretion signal comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 11.

[0074] In some embodiments, a gE2 secretion signal comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 38.12947033vl Page 11 of 526Attorney Docket No.: 2013237-1485

[0075] In some embodiments, an EboZ secretion signal comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 39.

[0076] In some embodiments, an HLA-DR secretion signal comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 40.

[0077] In some embodiments, a SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises a multimerization domain.

[0078] In some embodiments a multimerization domain is in the C-terminal region of a SARS-COV-2 variant protein or an immunogenic portion thereof (e.g., at the C-terminus).

[0079] In some embodiments, a multimerization domain is a fibritin domain.

[0080] In some embodiments, a fibritin domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 95.

[0081] In some embodiments, a fibritin domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 96.

[0082] In some embodiments, a SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises a transmembrane (TM) domain.

[0083] In some embodiments, a TM domain is a homologous TM domain.

[0084] In some embodiments, a TM domain is a heterologous TM domain.

[0085] In some embodiments, a TM domain is present in the C-terminal portion of a polypeptide (e.g., at theC-terminus).

[0086] In some embodiments, a SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises a multimerization domain and a TM domain in the C-terminal portion of the polypeptide, wherein the TM domain is C- terminal to the multimerization domain (e.g., the TM domain is at the C-terminus of the variant polypeptide and the multimerization domain is adjacent to the TM domain (e.g., directly adjacent to the TM domain and / or connected to the TM domain via a GS linker)).

[0087] In some embodiments, a TM domain is a SARS-CoV-2 S protein TM domain, or an influenza TM domain.

[0088] In some embodiments, a SARS-CoV-2 TM domain comprises an amino acid sequence that is at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 89.

[0089] In some embodiments, a SARS-CoV-2 TM domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 90.

[0090] In some embodiments, an RNA comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 120.12947033vl Page 12 of 526Attorney Docket No.: 2013237-1485

[0091] In some embodiments, an immunogenic portion of a SARS-CoV-2 S protein variant comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 130.

[0092] In some embodiments, an RNA comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 135.

[0093] In some embodiments, an RNA comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 145.

[0094] In some embodiments, an RNA comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 150.

[0095] Among other things, the present disclosure describes an RNA comprising a nucleotide sequence that encodes a SARS-CoV-2 Spike (S) protein variant or an immunogenic portion thereof.

[0096] In some embodiments, a nucleotide sequence that encodes a SARS-CoV-2 S protein variant (or immunogenic portion thereof) is codon-optimized for expression in mammalian subjects.

[0097] In some embodiments, a nucleotide sequence that encodes a SARS-CoV-2 S protein variant (or immunogenic portion thereof) is codon-optimized for expression in human subjects.

[0098] In some embodiments, a nucleotide sequence encoding a SARS-CoV-2 S protein variant (or immunogenic portion thereof) has an enriched G / C content relative to wild-type sequence.

[0099] In some embodiments, G / C content is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%.

[0100] In some embodiments, an RNA comprises a heterologous 3' UTR or 5'UTR.

[0101] In some embodiments, a heterologous 5' UTR comprises or consists of a modified human alpha-globin5'-UTR.

[0102] In some embodiments, a heterologous 3' UTR comprises or consists of a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA.

[0103] In some embodiments, an RNA comprises a poly(A) sequence.

[0104] In some embodiments, a poly(A) sequence has a length of about 100-150 nucleotides.

[0105] In some embodiments, a poly(A) sequence is a disrupted poly(A) sequence.

[0106] In some embodiments, an RNA comprises a 5' cap.

[0107] In some embodiments, an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 122 or 124. In some embodiments, an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 131 or 133. In some embodiments, an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 136 or 138. In some embodiments, an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 146 or 148.

[0108] In some embodiments, an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 151 or 153. In some embodiments, an RNA is unmodified RNA.

[0109] In some embodiments, an RNA comprises one or more modified nucleotides.12947033vl Page 13 of 526Attorney Docket No.: 2013237-1485

[0110] In some embodiments, a modified nucleotide is pseudouridine (e.g., Nl-methyl-pseudouridine).

[0111] In some embodiments, an RNA comprises a modified nucleotide in place of each uridine.

[0112] In some embodiments an RNA is a self-amplifying RNA or trans-amplifying RNA.

[0113] In some embodiments, a composition an RNA described herein, wherein the RNA is fully or partially encapsulated within lipid nanoparticles (LNP), polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes. In some embodiments, an RNA is fully or partially encapsulated within an LNP. In some embodiments, an LNP comprises a cation ically ionizable lipid, a neutral lipid, a sterol and a lipid conjugate.

[0114] In some embodiments, an LNP comprises from about 40 to about 50 mol percent of the cation ica lly ionizable lipid; from about 5 to about 15 mol percent of the neutral lipid; from about 35 to about 45 mol percent of the sterol; and from about 1 to about 10 mol percent of the PEG-lipid.

[0115] In some embodiments, the present disclosure provides a method of inducing an immune response, comprising administering an RNA described herein, or a composition described herein.

[0116] In some embodiments, the present disclosure provides a method of inducing an immune response in a subject who has previously been exposed to a reference SARS-CoV-2 S protein, the method comprising: delivering a SARS-CoV-2 S protein variant or an immunogenic portion thereof to the subject, wherein a memory B cell immune response has been established to a reference SARS-CoV-2 S protein, and wherein the SARS-CoV-2 S protein variant or immunogenic portion thereof has an amino acid sequence that differs from that of the reference SARS-CoV-2 S protein variant in that it has been engineered to reduce activation of the memory B cell immune response relative to the SARS-CoV-2 S protein.

[0117] Among other things, the present disclosure describes an RNA comprising a nucleotide sequence that encodes a polypeptide, wherein the polypeptide comprises one or more immunogenic fragments of a SARS-CoV-2 S protein or a variant of one or more immunogenic fragments of a SARS-CoV-2 S protein.

[0118] In some embodiments, one or more immunogenic fragments of a SARS-CoV-2 S protein comprise an N- Terminal Domain (NTD) and / or a Receptor Binding Domain (RBD).

[0119] In some embodiments, an immune response is induced in a subject who has previously been administered one or more doses of one or more vaccines that deliver a reference SARS-CoV-2 S protein.

[0120] In some embodiments, a reference SARS-CoV-2 S protein is a Wuhan SARS-CoV-2 S protein.

[0121] In some embodiments, an immune response comprises a naive B cell immune response.

[0122] In some embodiments, an immune response comprises a reduced memory B cell immune response or an immune response that does not comprise a memory B cell immune response.

[0123] In some embodiments, disclosed herein is a method of manufacturing an immunogenic composition comprising:(a) providing a reference SARS-CoV-2 S protein, wherein the reference SARS-CoV-2 S protein is from a strain or variant (e.g., a strain or variant that has previously been prevalent and / or that has previously been delivered as a vaccine) of SARS-CoV-2,(b) determining a variant polypeptide of the reference SARS-CoV-2 S protein or an immunogenic portion thereof that comprises a reduced number of memory B cell epitopes relative to the reference SARS-CoV-2 S protein; and12947033vl Page 14 of 526Attorney Docket No.: 2013237-1485(c) producing an immunogenic composition that delivers the SARS-CoV-2 S protein variant or immunogenic portion thereof.

[0124] In some embodiments, an RNA encodes a polypeptide comprising:(a) an NTD and an RBD, wherein the N- to C-terminal orientation of the NTD and the RBD is NTD-RBD or RBD-NTD, and wherein the NTD and the RBD are from the same or different SARS-CoV-2 strains or variants;(b) two RBDs, wherein the two RBDs are from the same or different SARS-CoV-2 strains or variants;(c) two NTDs and an RBD, wherein the N- to C-terminal orientation of the two NTDs and the RBD is NTD-NTD-RBD, NTD-RBD-NTD, or RBD-NTD-NTD, and wherein one, two, or three of the two NTDs and the RBD are from the same SARS-CoV-2 strain or variant (optionally wherein, if the C-terminal to N-terminal orientation is NTD- RBD-NTD, the first NTD and the RBD are from a first SARS-CoV-2 strain or variant and the second NTD is of a different SARS-CoV-2 strain or variant than the first SARS-CoV-2 strain or variant);(d) two RBDs and one NTD, wherein the N- to C-terminal orientation of the two RBDs and one NTD is RBD-RBD-NTD, RBD-NTD-RBD, or NTD-RBB-RBD, and wherein one, two, or all three of the two RBDs and the NTD are from the same SARS-CoV-2 strain or variant (optionally wherein, if the C-terminal to N-terminal orientation is NTD-RBD-RBD, the NTD and the first RBD are from a first SARS-CoV-2 strain or variant, and the second RBD is from a second SARS-CoV-2 strain or variant that is different from the first SARS-CoV-2 strain or variant); or(e) two NTDs and two RBDs, wherein the N-terminal to C-terminal orientation of the two NTDs and two RBDs is NTD-NTD-RBD-RBD, NTD-RBD-NTD-RBD, NTD-RBD-RBD-NTD, RBD-NTD-RBD-NTD, or RBD-RBD-NTD- NTD, and wherein one, two, three, or all four of the NTDs and the two RBDs are from the same SARS-CoV-2 strains or variants (and optionally, if the N-terminal to C-terminal orientation of the polypeptide is NTD-RBD-NTD-RBD, the first NTD and the first RBD are of a different SARS-CoV-2 strain or variant than the second NTD and the second RBD).

[0125] In some embodiments, an RNA encodes a polypeptide comprising:(a) an RBD and an NTD, wherein the RBD and NTD are connected via a flexible linker (e.g., a flexible linker described herein).(b) two RBDs, wherein the two RBDs are connected via a flexible linker or are immediately adjacent to one another;(c) an NTD and two RBDs, wherein the NTDs and the RBDs are each directedly adjacent to one another or are each connected to one another by a sequence that comprises a flexible linker.(d) two NTDs and two RBDs, wherein the NTDs and the RBDs are each connected to one another by a sequence that comprises a flexible linker, optionally wherein one or more of the sequences that connect the NTDs and RBDs further comprises a multimerization domain (e.g., a fibritin trimerization domain).

[0126] In some embodiments, a SARS-CoV-2 S protein variant or immunogenic portion thereof comprises a sequence that corresponds to an immunogenic portion of a reference SARS-CoV-2 S protein.

[0127] In some embodiments, a variant polypeptide or an immunogenic portion thereof comprises one or mutations at one or more B cell epitopes of a reference antigen.

[0128] In some embodiments, disclosed herein is a method of assessing, predicting, or characterizing the ability of an immunogenic composition that delivers a SARS-CoV-2 S protein (or immunogenic portion thereof) to induce activation of memory B cells in a subject or a population of subjects, the method comprising determining the12947033vl Page 15 of 526Attorney Docket No.: 2013237-1485 number of memory B cell epitopes present in the SARS-CoV-2 S protein (or immunogenic portion thereof) relative to a reference antigen.

[0129] In some embodiments, a reference SARS-CoV-2 S protein is from a strain or variant that a subject was exposed to and / or that a large portion of the population was exposed to.

[0130] In some embodiments, a method of producing a personalized SARS-CoV-2 vaccine for a subject comprises steps of:(a) determining a reference SARS-CoV-2 S protein that a subject has previously been exposed to;(b) determining a variant SARS-CoV-2 S protein that comprises fewer memory B cell epitopes relative to the reference SARS-CoV-2 S protein; and(c) producing an immunogenic composition that delivers the variant SARS-CoV-2 S protein.

[0131] In some embodiments, a reference SARS-CoV-2 S protein is from a strain or variant that a subject was first exposed to and / or that was first prevalent in a population of subjects.

[0132] In some embodiments, a reference SARS-CoV-2 S protein is a Wuhan SARS-CoV-2 S protein or an Omicron BA.4 / 5 SARS-CoV-2 S protein.

[0133] In some embodiments, a reference SARS-CoV-2 S protein is from a strain or variant that a subject has previously been vaccinated against or is delivered by one or more vaccines that a significant proportion of a population (e.g., at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least about 60%) has previously been administered.

[0134] In some embodiments, a vaccine previously administered to a subject, or a significant proportion of a population was a first generation vaccine.

[0135] In some embodiments, a reference SARS-CoV-2 S protein is from a SARS-CoV-2 strain or variant that was previously prevalent or is currently prevalent in a relevant jurisdiction.

[0136] In some embodiments, a reference SARS-CoV-2 S protein is from a variant that first became prevalent in a relevant jurisdiction.

[0137] In certain embodiments, antigens described herein can be engineered to incorporate sequences and / or mutations from two or more SARS-COV-2 variants (e.g., epitopes from RBDs, S proteins, and / or SI domains from two or more SARS-CoV-2 variants). For example, in some such embodiments, mutations of a one or more SARS- CoV-2 variants can be introduced in conserved epitopes of a variant SARS-CoV-2 S protein, or an immunogenic portion thereof (e.g., an SI domain or an RBD). Such engineering can be useful, e.g., to eliminate additional B cell epitopes (e.g., conserved B cell epitopes). Exemplary approaches for introducing mutations or sequences from two or more SARS-CoV-2 variants are described, e.g., in the US provisional application titled "Systems and Methods for Engineering Antigens to Promote Tailored Immune Responses", filed February 24, 2023, and having U.S. Provisional Application No. 63 / 448,215. Said application describes, among other things, technologies directed to in-si / ico design of custom, engineered, antigens (e.g., including engineered versions of SARS-CoV-2 variant proteins and portions thereof) for reducing an extent to which a memory immune response is triggered.

[0138] In some embodiments, the present disclosure provides an RNA encoding an immunogenic fragment of a SARS-CoV-2 S protein. In some embodiments, an immunogenic fragment of an S protein comprises a Receptor Binding Domain (RBD). In some embodiments, an RBD comprises an amino acid sequence from the region corresponding to amino acids 327 to 528 of SEQ ID NO: 1, a region corresponding to SEQ ID NO: 3, a region12947033vl Page 16 of 526Attorney Docket No.: 2013237-1485 corresponding to SEQ ID NO: 4, a region corresponding to SQ ID NO: 325, or a region corresponding to SEQ ID NO: 326. In some embodiments, an RBD comprises an amino acid sequence from the region corresponding to amino acids 327 to 528 of SEQ ID NO: 1. In some embodiments, an immunogenic fragment of the S protein comprises an SI subdomain of a SARS-CoV-2 S protein, or an immunogenic fragment thereof.

[0139] In some embodiments, an RNA encodes a polypeptide comprising an RBD and an NTD of an S protein.

[0140] In some embodiments, an RNA encodes a polypeptide comprises a truncated SI subdomain. In some embodiments, a truncated SI subdomain comprises amino acids 1-528 of SEQ ID NO: 1 or a corresponding region from the S protein of a SARS-CoV-2 variant. In some embodiments, a truncated SI subdomain comprises amino acids 14-528 of SEQ ID NO: 1 or a corresponding region from the S protein of a SARS-CoV-2 variant. In some embodiments, a truncated SI subdomain comprises amino acids 1-537 of SEQ ID NO: 1 or a corresponding region from the S protein of a SARS-CoV-2 variant. In some embodiments, a truncated SI subdomain comprises amino acids 14-537 of SEQ ID NO: 1 or a corresponding region from the S protein of a SARS-CoV-2 variant.

[0141] In some embodiments, an RNA encodes a polypeptide comprising a truncated SI subdomain.

[0142] In some embodiments, an RNA encodes a polypeptide comprising a truncated SI subdomain comprising an NTD and an RBD.

[0143] In some embodiments, an RBD is at the C-terminus of a truncated SI subdomain.

[0144] In some embodiments, an RNA encodes a polypeptide comprising one or more fragments of an S2 domain of a SARS-CoV-2 S protein.

[0145] In some embodiments, one or more fragments of an S2 domain comprise:(a) a fusion peptide (FP) region,(b) a stem helix (SH) region,(c) an HR1 region or a fragment thereof,(d) an HR2 region or a fragment thereof, or(e) any combination of (a)-(d).

[0146] In some embodiments, one or more fragments of an S2 domain comprise:(a) an SH region and a fragment of an HR2 region;(b) an FP region, an SH region, and a fragment of an HR2 region; wherein the FP region, the SH region, the fragment of the HR2 region are directed adjacent to one another or have one or more intervening sequences;(c) an FP region, an SH region, wherein the FP region and the SH region are directed adjacent to one another or are connected to one another via one or more intervening sequences;(d) an FP region, an SH region, and a fragment of an HR2 region, wherein the FP region, the SH region, and the fragment of the HR2 region are directly adjacent to one another or are connected via an intervening sequence that comprises one or more additional antigenic regions.

[0147] In some embodiments, one or more fragments of the S2 domain in a polypeptide are each independently (a) immediately adjacent to one another, (b) connected to one another via a sequence that comprises a flexible linker, and / or (c) connected to one another via a sequence that comprises one or more NTDs and / or one or more RBDs of a SARS-CoV-2 S protein.

[0148] In some embodiments, an RNA encodes a polypeptide comprising:(a) an RBD and an SH region;12947033vl Page 17 of 526Attorney Docket No.: 2013237-1485(b) two RBDs and an SH region;(c) an NTD, an RBD, and an SH region;(d) an NTD, two RBDs, and an SH region;(e) an NTD, two RBDs, and an FP region;(f) an NTD, an RBD, an FP region, and an SH region;(g) three RBDs, an FP region, an SH region, and an HR2 region;(h) three RBDs, an FP region, and an SH region; or(i) three RBDs, and NTD, an FP region, and an SH region.

[0149] In some embodiments, an RNA encodes a polypeptide comprising:(a) an RBD and an SH region, wherein the N-terminal to C-terminal orientation of the RBD and the SH region is RBD-SH, and wherein the RBD and the SH region are either directly adjacent to one another or connected to one another via a sequence that comprises a flexible linker;(b) two RBDs and an SH region, wherein the N-terminal to C-terminal orientation of the two RBDs and the SH region is RBD-RBD-SH, and wherein the two RBDs and the SH region are each independently either directly adjacent to one another or connected to one another via a sequence that comprises a flexible linker;(c) an NTD, an RBD, and an SH region, wherein the N-terminal to C-terminal orientation of the NTD, RBD, and SH region is NTD-RBD-SH, and wherein the NTD, RBD, and SH region are each independently either directly adjacent to one another or connected to one another via a sequence that comprises a flexible linker;(d) an NTD, two RBDs, and an SH region, wherein the N-terminal to C-terminal orientation of the NTD, two RBDs, and SH region is NTD-RBD-RBD-SH, and wherein the NTD, RBDs, and SH region are each independently either directly adjacent to one another or are connected to one another via a sequence that comprises a flexible linker;(e) an NTD, an RBD, an FP region, an SH region, and a fragment of an HR2 region, wherein the N- terminal to C-terminal orientation of the NTD, RBD, FP region, SH region, and fragment of an HR2 region is NTD-FP- RBD-SH-RBD-HR2, and wherein the NTD, RBD, FP region, SH region, and fragment of an HR2 region are each independently either directly adjacent to one another or connected to one another via a sequence that comprises a flexible linker;(f) an NTD, an FP region, two RBDs, and an SH region, wherein the N-terminal to C-terminal orientation of the NTD, FP region, two RBDs, and SH region is NTD-FP-RBD-SH-RBD, and wherein the NTD, FP region, two RBDs, and SH region are each independently either directly adjacent to one another or connected to one another via a sequence that comprises a flexible linker;(g) three RBDs, an FP region, an SH region, and a fragment of an HR2 region, wherein the N-terminal to C-terminal orientation of the three RBDs, FP region, SH region, and fragment of an HR2 region is RBD-FP-RBD-SH- RBD-HR2, and wherein the three RBDs, FP region, SH region, and fragment of an HR2 region are each independently either directly adjacent to one another or are connected to one another via a sequence that comprises a flexible linker;(h) three RBDs, an FP region, and an SH region, wherein the N-terminal to C-terminal orientation of the three RBDs, the FP region, and the SH region is RBD-FP-RBD-SH-RBD, and wherein the three RBDs, FP region, and SH region are each independently either directly adjacent to one another or are connected to one another via a sequence that comprises a flexible linker;12947033vl Page 18 of 526Attorney Docket No.: 2013237-1485(i) an NTD, an FP region, two SH regions, and three RBDs, wherein the N-terminal to C-terminal orientation of the NTD, FP region, two SH regions, and three RBDs is NTD-FP-RBD-SH-RBD-SH-RBD, and wherein the NTD, FP region, three RBDs, and two SH regions are each either independently directly adjacent to one another or connected to one another via a sequence that comprises a flexible linker;(j) an NTD, an FP region, a fragment of an HR1 region, an SH region, three RBDs, and a fragment of an HR2 region, wherein the N-terminal to C-terminal orientation of the NTD, FP region, fragment of an HR1 region, SH region, fragment of an HR2 region and three RBDs is NTD-FP-RBD-HR1-RBD-SH-RBD-HR2, and wherein the NTD, FP region, three RBDs, SH region, and fragment of an HR2 region are each either independently directly adjacent to one another or connected to one another via a sequence that comprises a flexible linker;(k) an NTD, an FP region, three RBDs, a truncated HR1 region, and an SH region, wherein the N- terminal to C-terminal orientation of the NTD, FP region, three RBDs, truncated HR1 region, and SH region is NTD- FP-RBD-HR1-RBD-SH-RBD, where the NTD, FP region, three RBDs, truncated HR1 region, and SH region are each either independently directly adjacent to one another or are connected to one another via a sequence that comprises a flexible linker;(l) two RBDs, an FP region, an SH region, and a fragment of an HR2 region, wherein the N-terminal to C-terminal orientation of the two RBDs, FP region, SH region, and fragment of an HR2 region is RBD-FP-RBD-SH- HR2, and wherein the two RBDs, FP region, SH region, and fragment of an HR2 region are each directly either adjacent to one another or are connected to one another via a sequence that comprises a flexible linker;(m) two RBDs, an FP region, an SH region, and a fragment of an HR2 region, wherein the N-terminal to C-terminal orientation of the two RBDs, FP region, SH region, and the fragment of an HR2 region is RBD-FP-RBD-SH- HR2, and wherein the two RBDs, FP region, SH region, and HR2 fragment are each independently either directly adjacent to one another or are connected to one another via a sequence that comprise a flexible linker;(n) two RBDs, an FP region, and an SH region, wherein the N-terminal to C-terminal orientation of the two RBDs, FP region, and SH region is RBD-FP-RBD-SH, and wherein the two RBDs, FP region, and SH region are each independently either immediately adjacent to one another or connected to one another via a sequence that comprises a flexible linker.

[0150] In some embodiments, an RBD comprises:(a) amino acids 319-528, 327-528, 328-528, 330-528, 319-530, 327-530, 328-530, 330-530, 319-531, 327-531, 328-531, 330-531, 319-537, 327-537, 328-537, 330-537, 330-537, 319-541, 327-541, 328-541, or 330-541 of SEQ ID NO: 1;(b) a region of an S protein of a SARS-CoV-2 variant [having an amino acid sequence] that corresponds to any one of the amino acid sequences recited in (a);(c) SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 325, or SEQ ID NO: 326, or a component / element? having an amino acid sequence corresponding to any of the foregoing from an S protein of a SARS-CoV-2 variant; or(d) a component / element having an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to any one of the amino acid sequences recited in (a)-(c).

[0151] In some embodiments, an NTD comprises:12947033vl Page 19 of 526Attorney Docket No.: 2013237-1485(a) amino acids 1-209, 14-209, 15-209, 19-209, 20-209, 21-209, 1-305, 14-305, 15-305, 19-305, 20- 305, 21-305, 1-311, 14-311, 15-311, 19-311, 20-311, 21-311, 1-318, 14-318, 15-318, 19-318, 20-318, 21-318, 1- 302, 14-302, 15-302, 19-302, 20-302, or 21-302 of SEQ ID NO: 1,(b) a sequence of a region of an S protein of a SARS-CoV-2 variant that corresponds to any one of the amino acid sequences recited in (a); or(c) a sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to any one of the amino acid sequences recited in (a)-(b).

[0152] In some embodiments, an RNA encodes a polypeptide comprising:(a) amino acids 1-528, 14-528, 15-528, 19-528, 20-528, 21-528, 1-530, 14-530, 15-530, 19-530, 20- 530, 21-530, 1-531, 14-531, 15-531, 19-531, 20-531, 21-531, 1-537, 14-537, 15-537, 19-537, 20-537, 21-537, 1- 541, 14-541, 15-541, 19-541, 20-541, or 21-541 of SEQ ID NO: 1;(b)QCVMPLFNLITTTQSYTNSFTRGVYYPDKVFRSSVLHLTQDLFLPFFSNVTWFHAISGTNGTKRFDNPVLPFNDGVYFASTEKSNIIR GWIFGTTLDSKTQSLLIVNNATNVFIKVCEFQFCNDPFLDVYHKNNKSWMESESGVYSSANNCTFEYVSQPFLMDLEGKQGNFKNL REFVFKNIDGYFKIYSKHTPIIGRDFPQGFSALEPLVDLPIGINITRFQTLLALNRSYLTPGDSSSGWTAGAADYYVGYLQPRTFLLKYN ENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNVTNLCPFHEVFNATTFASVYAWNRTRISNCVADYSVL YNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIKGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKHSGNYDYWYRS LRKSKLKPFERDISTEIYQAGNKPCKGKGPNCYFPLQSYGFRPTYGVGHQPYRVWLSFELLHAPATVCGPK (SEQ ID NO: 664);(c)QCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPALPFNDGVYFASTEKSNIIRG WIFGrTTLDSKTQSLLIVNNATNWIKVCEFQFCNDPFLDVYQKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKEGNFKNLRE FVFKNIDGYFKIYSKHTPINLERDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPVDSSSGWTAGAAAYYVGYLQPRTFLLKYNE NGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYN FAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRK SKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVWLSFELLHAPATVCGPK (SEQ ID NO: 656);(d) a sequence of a region of an S protein of a SARS-CoV-2 variant that corresponds to any one of the amino acid sequences recited in any one of (a)-(c); or(e) a sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to any one of the amino acid sequences recited in (a)-(d).

[0153] In some embodiments, an FP region comprises: (i) amino acids 809-843 of SEQ ID NO: 1 (PSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGD (SEQ ID NO: 800)), a sequence of a corresponding region of an S protein of a SARS-CoV-2 variant, or a sequence that is 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 800; or (ii) amino acids 809-832 of SEQ ID NO: 1, a sequence of a corresponding region of an S protein of a SARS-CoV-2 variant, or a sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 800.

[0154] In some embodiments, an SH region comprises (i) amino acids 1141-1164 of SEQ ID NO: 1 (LQPELDSFKEELDKYFKNHTSPDV (SEQ ID NO: 801)), a sequence of a corresponding region of an S protein of a SARS-CoV-2 variant, or a sequence that is 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 801; (ii) amino acids 1140-1162 of SEQ ID NO: 1 (PLQPELDSFKEELDKYFKNHTSP (SEQ ID NO: 802), a sequence of a corresponding region of an S protein of a SARS-12947033vl Page 20 of 526Attorney Docket No.: 2013237-1485CoV-2 variant, or a sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 802; (iii) amino acids 1136-1167 of SEQ ID NO: 1 (TVYDPLQPELDSFKEELDKYFKNHTSPDVDLG), a sequence of a corresponding region of an S protein of a SARS-CoV-2 variant, or a sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to TVYDPLQPELDSFKEELDKYFKNHTSPDVDLG (SEQ ID NO: 803); or (iv) amino acids 1136-1168 of SEQ ID NO: 1 (TVYDPLQPELDSFKEELDKYFKNHTSPDVDLGD (SEQ ID NO: 804)), a sequence of a corresponding region of an S protein of a SARS-CoV-2 variant, or a sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 804.

[0155] In some embodiments, a fragment of an HR1 region comprises amino acids 975-995 of SEQ ID NO: 1 (SVLNDILSRLDKVEAEVQIDR (SEQ ID NO: 805)), a corresponding region of an S protein of a SARS-CoV-2 variant, or a sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 805.

[0156] In some embodiments, an HR2 region or the fragment thereof comprises amino acids 1162-1200, 1162- 1207, 1165-1200, 1168-1200, 1169-1200, 1165-1207, 1168-1207, or 1169-1207 of SEQ ID NO: 1, a corresponding region of an S protein of a SARS-CoV-2 variant, or a sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to any one of amino acids 1162-1200, 1162-1207, 1165- 1200, 1168-1200, 1169-1200, 1165-1207, 1168-1207, or 1169-1207 of SEQ ID NO: 1.

[0157] In some embodiments, an RNA encodes a polypeptide comprising an SH region and an HR2 region of a SARS-CoV-2 S protein or a fragment thereof, optionally wherein the polypeptide comprises:(a) amino acids 1136-1200 of SEQ ID NO: 1, or a corresponding region of an S protein of a SARS-CoV- 2 variant, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 1163-1200 of SEQ ID NO: 1;(b) amino acids 1136-1207 of SEQ ID NO: 1, or a corresponding region of an S protein of a SARS-CoV- 2 variant, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 1163-1207 of SEQ ID NO: 1;(c) amino acids 1140-1200 of SEQ ID NO: 1, or a corresponding region of an S protein of a SARS-CoV- 2 variant, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 1140-1200 of SEQ ID NO: 1;(d) amino acids 1140-1207 of SEQ ID NO: 1, or a corresponding region of an S protein of a SARS-CoV- 2 variant, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 1140-1207 of SEQ ID NO: 1;(e) amino acids 1141-1200 of SEQ ID NO: 1, or a corresponding region of an S protein of a SARS-CoV- 2 variant, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 1163-1200 of SEQ ID NO: 1; or(f) amino acids 1141-1207 of SEQ ID NO: 1, or a corresponding region of an S protein of a SARS-CoV- 2 variant, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 1163-1207 of SEQ ID NO: 1;

[0158] In some embodiments, an RNA encodes a polypeptide comprising a transmembrane domain, optionally wherein the transmembrane domain is at the N-terminus or C-terminus of the polypeptide.

[0159] In some embodiments, a transmembrane domain is from a viral membrane protein.12947033vl Page 21 of 526Attorney Docket No.: 2013237-1485

[0160] In some embodiments, a transmembrane domain is a SARS-CoV-2 S protein transmembrane domain or wherein the transmembrane domain is obtained from a viral protein that is not a SARS-CoV-2 S protein.

[0161] In some embodiments, a transmembrane domain comprises a transmembrane domain listed in Table 5 (e.g., comprising an amino acid sequence provided in Table 5 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence listed in Table 4) and / or wherein the transmembrane domain is encoded by a nucleotide sequence that is listed in Table 6 or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to a nucleotide sequence listed in Table 6).

[0162] In some embodiments, a transmembrane domain is a SARS-CoV-2 S protein transmembrane region (including a SARS-CoV-2 transmembrane domain; e.g., wherein the transmembrane domain comprises an amino acid sequence of EQYIKWPWYIWLGFIAGLIAIVMVTIMLCC (SEQ ID NO: 436), or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 436.

[0163] In some embodiments, an RNA encodes a polypeptide comprising, C-terminal to the transmembrane domain, a sequence that is endogenously C-terminal to the transmembrane domain.

[0164] In some embodiments, a sequence that is endogenously C-terminal to the transmembrane domain is MTSCCSCLKGCCSCGSCC (SEQ ID NO: 654), or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 654.

[0165] In some embodiments, a transmembrane domain comprises:(a) an amino acid sequence of SEQ ID NO: 90 (EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC), or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 90;(b) an amino acid sequence of SEQ ID NO: 89 (EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT), or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 89;(c) amino acids 1200-1254 or 1200-1273 of SEQ ID NO: 1, a sequence of a corresponding region of a SARS-CoV-2 S protein of a SARS-CoV-2 variant, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 1200-1254 or 1200-1273 of SEQ ID NO: 1;(d) amino acids 1206-1254 or 1206-1273 of SEQ ID NO: 1, a sequence of a corresponding region of a SARS-CoV-2 S protein of a SARS-CoV-2 variant, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 1206-1254 or 1206-1273 of SEQ ID NO: 1; or(e) amino acids 1211-1254 or 1211-1273 of SEQ ID NO: 1, a sequence of a corresponding region of a SARS-CoV-2 S protein of a SARS-CoV-2 variant, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 1211-1254 or 1211-1273 of SEQ ID NO: 1.

[0166] In some embodiments, an RNA encodes a polypeptide comprising a region of a SARS-CoV-2 S protein that comprises (i) an SH region and an HR2 region or a fragment thereof, (ii) an SH region and a transmembrane region, and / or (iii) a fragment of an HR2 region and a transmembrane region.12947033vl Page 22 of 526Attorney Docket No.: 2013237-1485

[0167] In some embodiments, an RNA encodes a polypeptide comprising:(a) a region of a SARS-CoV-2 S protein that comprises the SH region and a HR2 region or a fragment thereof that comprises amino acids 1136-1200, 1136-1205, 1136-1206, 1140-1200, 1140-1205, 1140-1206, 1141- 1200, 1141-1205, or 1141-1206 of SEQ ID NO: 1, a sequence of a corresponding region of an S protein of a SARS- CoV-2 variant, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 1136-1200, 1136-1205, 1136-1206, 1140-1200, 1140-1205, 1140- 1206, 1141-1200, 1141-1205, or 1141-1206 of SEQ ID NO: 1, TVYDPLQPELDSFKEELDKYFKNHTSPDVSDLGDISGINASWNIQKEIDRLNEVAKNLNESLIDL (SEQ ID NO: 806) or TVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASWNIQKEIDRLNEVAKNLNESLIDL (SEQ ID NO: 807);(b) a region of a SARS-CoV-2 S protein that comprises the SH region and the transmembrane region and comprises amino acids 1140-1254, 1141-1254, 1136-1254, 1140-1273, 1141-1273, or 1136-1273 of SEQ ID NO: 1, a corresponding region of an S protein of a SARS-CoV-2 variant, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to amino acids 1140-1254, 1141-1254, 1136-1254, 1140-1273, 1141-1273, or 1136-1273 of SEQ ID NO: 1;(c) a region of a SARS-CoV2 S protein that comprises the HR2 region or the fragment thereof and comprises amino acids 1136-1254, 1136-1273, 1162-1254, 1162-1273, 1165-1254, 1165-1273, 1168-1254, 1168- 1273, 1169-1254, or 1169-1273 of SEQ ID NO: 1, a corresponding region of an S protein of a SARS-CoV-2 variant, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 1136-1254, 1136-1273, 1165-1254, 1165-1273, 1168-1254, 1168-1273, 1169-1254, or 1169- 1273 of SEQ ID NO: 1.

[0168] In some embodiments, a polypeptide comprises a secretory signal peptide (e.g., a secretory signal peptide of a viral protein).

[0169] In some embodiments, a secretory signal peptide is a secretory signal peptide of a SARS-CoV-2 S protein.

[0170] In some embodiments, a heterologous secretory signal peptide is a secretory signal peptide of a viral protein that is not a SARS-CoV-2 S protein.

[0171] In some embodiments, a secretory signal peptide is a heterologous secretory signal peptide.

[0172] In some embodiments, a polypeptide comprises a secretory signal peptide that comprises (i) an amino acid sequence that is listed in Table 3 or Table 72 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence that is listed in Table 3 or Table 72, and / or (ii) wherein the RNA comprises a nucleotide sequence that is listed in Table 4 or Table 72 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% identical to an amino acid sequence that is listed in Table 3 or Table 72 sequence that is listed in Table 4 and / or Table 72.

[0173] In some embodiments, a polypeptide sequence comprises an SP24-Q7PUJ5_ANOGA secretory signal peptide (e.g., a secretory signal peptide comprising an amino acid sequence of MCRGLSAVLILLVSLSAQLHVWG (SEQ ID NO: 22) or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 22.

[0174] In some embodiments, a polypeptide sequence comprises an SP24-SP18-HEMA_CVBM secretory signal peptide (e.g., a secretory signal peptide comprising the amino acid sequence of MFLLLRFVLVSCIIGSLG (SEQ ID NO:12947033vl Page 23 of 526Attorney Docket No.: 2013237-1485391) or an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 391.

[0175] In some embodiments, a polypeptide sequence comprises an SP25-GD_HHV1K secretory signal peptide (e.g., a secretory signal peptide comprising an amino acid sequence of MGGAAARLGAVILFWIVGLHGVRG (SEQ ID NO: 12) or an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 12.

[0176] In some embodiments, a polypeptide sequence comprises an SP32-GB_HHV1K secretory signal peptide (e.g., a secretory signal peptide comprising the amino acid sequence of MHQGAPSWGRRWFWWALLGLTLGVLVASAAP (SEQ ID NO: 38) or an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 38.

[0177] In some embodiments, a polypeptide sequence comprises an SP20-A7U881_HHV2 secretory signal peptide (e.g., a secretory signal peptide comprising the amino acid sequence of MARGAGLVFFVGVWWSCLA (SEQ ID NO: 366) or an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 366.

[0178] In some embodiments, a polypeptide sequence comprises a SARS-CoV-2 secretory signal peptide (e.g., a secretory signal peptide comprising the amino acid sequence of one of the SARS-CoV-2 secretory signal peptides provided in Table 3 or an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to one or more of the amino acid sequence of the SARS-CoV-2 secretory signal peptide provided in Table 3.

[0179] In some embodiments, a polypeptide comprises one or more domains that can induce formation of a viral-like particle (VLP) when the RNA is transfected into a cell.

[0180] In some embodiments, one or more domains that can induce formation of a VLP comprise an endosomal sorting complex required for transport (ESCRT)- and ALG-2-interacting protein X (ALIX) binding region (collectively referred to as EABR).

[0181] In some embodiments, an EABR sequence comprises an amino acid sequence of FNSSINNIHEMEIQLKDALEKNQQWLVYDQQREVYVKGLLAKIFELEKKTETAAHSLP (SEQ ID NO: 667), or sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 667

[0182] In some embodiments, an EABR sequence comprises an amino acid sequence of LQSRPEPTAPPEESFRSGVETTTPPQKQEPIDKELYPLTSLRSLFGNDPSSQ (SEQ ID NO: 669), or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 669.

[0183] In some embodiments, a polypeptide comprises a transmembrane domain.

[0184] In some embodiments, a transmembrane domain comprises a SARS-CoV-2 S protein transmembrane domain, optionally wherein the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 90, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 89.

[0185] In some embodiments, an EABR and a transmembrane domain are C-terminal to one or more fragments of a SARS-CoV-2 S protein and / or a EABR is C-terminal to a transmembrane domain.

[0186] In some embodiments, a transmembrane domain and a EABR sequence are directly adjacent to one another, or a transmembrane domain and a EABR sequence are connected to one another via a sequence that comprises a flexible linker.12947033vl Page 24 of 526Attorney Docket No.: 2013237-1485

[0187] In some embodiments, a flexible linker comprises a GS linker comprising about 5, about 10, about 15, or about 20 amino acids.

[0188] In some embodiments, a polypeptide comprises a peptide having an amino acid sequence of EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCGSGSGSFNSSINNIHEMEIQLKDALEKNQQWLVYDQQR EVYVKGLLAKIFELEKKTETAAHSLP (SEQ ID NO: 820), or sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, identical thereto,

[0189] optionally wherein the peptide having an amino acid sequence of SEQ ID NO: 441 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, identical thereto is at the C- terminus of the polypeptide.

[0190] In some embodiments, a polypeptide comprises an EPM sequence.

[0191] In some embodiments, a EPM sequence comprises:(a) an amino acid sequence of ALPGNPDHREMGETLPEEVGEYRQPSGGSVPVSPGPPSGLEPTSSSSPY (SEQ ID NO: 668), or sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, identical to SEQ ID NO: 668;(b) an amino acid sequence of ALPGNPDHREMGETLPEEVGEYRQPSGGSVPVSPGPPSGLEPTSSSPY (SEQ ID NO: 840), or sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, identical to SEQ ID NO: 840; or(c) an amino acid sequence of ALPGYPECREMGETLPEKPA (SEQ ID NO: 808), or sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, identical to SEQ ID NO: 808.

[0192] In some embodiments, a polypeptide comprises:(a) an amino acid sequence of ALPGNPDHREMGETLPEEVGEYRQPSGGSVPVSPGPPSGLEPTSSSPYGGGSFNSSINNIHEMEIQLKDALEKNQQWLVYDQQREV YVKGLLAKIFELEKKTETAAHSLP (SEQ ID NO: 809), or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, identical to SEQ ID NO: 809;(b) an amino acid sequence of ALPGYPECREMGETLPEKPAGGGSFNSSINNIHEMEIQLKDALEKNQQWLVYDQQREVYVKGLLAKIFELEKKTETAAHSLP (SEQ ID NO: 810), or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, identical to SEQ ID NO: 810.

[0193] In some embodiments, a polypeptide comprises a multimerization domain (e.g., a trimerization domain, including, e.g., a T4 fibritin trimerization domain).

[0194] In some embodiments, a polypeptide comprises a multimerization domain comprising an amino acid sequence of:(a) GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 95), an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications thereto;(b) DKVEVELQDIRSRLDKVEVELQDIRSRLDKVEVELQDIR (SEQ ID NO: 811), an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications thereto;(c) DKVEVELQDIRSRLDKVEVELQDIRSRLDKVEVELQDIRS (SEQ ID NO: 812), an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications thereto;(d) KVEVELQDIRSRLDKVEVNLSDIRSRLDKVEVELQDIRPDVDL (SEQ ID NO: 813), an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications thereto;12947033vl Page 25 of 526Attorney Docket No.: 2013237-1485(e) DKVEANLSDIRSRLDKVEANLSDIRSRLDKVEANLSDIRS (SEQ ID NO: 814, an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications thereto; or(f) DVDLGSVLNDILSRLDKVEAEVQIDR (SEQ ID NO: 815), an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications thereto.

[0195] In some embodiments, a polypeptide does not comprise a soluble multimerization domain (e.g., a trimerization domain, e.g., a T4 fibritin trimerization domain).

[0196] In some embodiments, a polypeptide comprises:(1) a truncated an SI subdomain (e.g., amino acids 1-528, 1-537, or 1-541 of SEQ ID NO: 1 or a corresponding region of an S protein of a SARS-CoV-2 variant);(2) a secretory signal peptide (e.g., a SP24-Q7PUJ5_ANOGA, SP24-SP18-HEMA_CVBM, SP25- GD_HHV1K, SP32-GD_HHV1K, or SP20-A7U881_HHV2 secretory signal peptide described herein); and(3) a transmembrane domain (e.g., a viral transmembrane domain, a transmembrane domain of a coronavirus S protein, a transmembrane domain of an influenza virus HA protein, or a transmembrane domain of a SARS-CoV-2 S protein).

[0197] In some embodiments, a polypeptide comprises:(i) a truncated SI subdomain of a SARS-CoV-2 S protein or a variant thereof;(ii) a heterologous secretory signal peptide; and(iii) a homologous transmembrane domain,

[0198] wherein the N-terminal to C-terminal orientation of the truncated SI subdomain or variant thereof, heterologous secretory signal peptide, and homologous transmembrane domain is (secretory signal peptide)- (truncated SI subdomain)-(transmembrane domain).

[0199] In some embodiments, a secretory signal peptide comprises:(i) an amino acid sequence of SEQ ID NO: 22 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 22;(ii) an amino acid sequence of SEQ ID NO: 391 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 391;(iii) an amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 12;(iv) an amino acid sequence of SEQ ID NO: 38 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 38.(v) an amino acid sequence of SEQ ID NO: 366 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 366.

[0200] In some embodiments, a transmembrane domain comprises:(i) an amino acid sequence of SEQ ID NO: 90, or an amino acid sequence that is at least 70%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 90; or(ii) an amino acid sequence of SEQ ID NO: 89, or an amino acid sequence that is at least 70%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 89.

[0201] In some embodiments, a polypeptide does not comprise a soluble multimerization domain (e.g., a trimerization domain, e.g., a T4 fibritin trimerization domain).12947033vl Page 26 of 526Attorney Docket No.: 2013237-1485

[0202] In some embodiments, a truncated SI subdomain or variant thereof and a transmembrane domain are connected to one another by a flexible linker, wherein the flexible linker comprises about 10 to about 20 amino acids (e.g., about 15 amino acids).

[0203] In some embodiments, a flexible linker comprises a (G4S)2 (SEQ ID NO: 489), (G4S)3 (SEQ ID NO: 490), or (G4S)4 (SEQ ID NO: 491) sequence.

[0204] In some embodiments, the N-terminal to C-terminal orientation of a polypeptide is: (secretory signal peptide)-(truncated SI subdomain)-(transmembrane domain).

[0205] In some embodiments, (1) a secretory signal peptide and a truncated SI subdomain; and / or (2) a truncated of SI subdomain and a transmembrane domain are connected to one another by a linker (e.g., an artificial linker, a flexible linker, a flexible linker comprising a GS sequence).

[0206] In some embodiments, a GS sequence comprises a sequence provided in Table 8.

[0207] In some embodiments, a GS sequence comprises at least 5, at least 10, at least 15, or at least 20 amino acids (e.g., G or S amino acids).

[0208] In some embodiments, a GS sequence comprises (G4S)1(SEQ ID NO: 488), (G4S)2 (SEQ ID NO: 489), (G4S)3 (SEQ ID NO: 490), or (G4S)4 (SEQ ID NO: 491) sequence.

[0209] In some embodiments, a truncated SI subdomain and a transmembrane domain are connected via a flexible linker that comprises 10-20 amino acids (e.g., about 15 amino acids).

[0210] In some embodiments, a polypeptide comprises:(a) a truncated SI subdomain or a variant thereof and a transmembrane domain of a SARS-CoV-2 S protein;(b) a truncated SI subdomain or a variant thereof and an HSV-1 gD secretory signal peptide;(c) a truncated SI subdomain or a variant thereof, a transmembrane domain, and an HSV-1 gD secretory signal peptide;(d) a truncated SI subdomain or a variant thereof, a transmembrane domain of a SARS-CoV-2 S protein, and a secretory signal peptide; or(e) a truncated SI subdomain or a variant thereof, a transmembrane domain of a SARS-CoV-2 S protein, and an HSV-1 gD secretory signal peptide.

[0211] In some embodiments, a(a) a truncated SI subdomain or a variant thereof comprises amino acids 1-528, 14-528, 15-528, 19- 528, 20-528, 21-528, 1-530, 14-530, 15-530, 19-530, 20-530, 21-530, 1-531, 14-531, 15-531, 19-531, 20-531, 21- 531, 1-537, 14-537, 15-537, 19-537, 20-537, 21-537, 1-541, 14-541, 15-541, 19-541, 20-541, or 21-541 of SEQ ID NO: 1, or a sequence of a corresponding region of an S protein of a SARS-CoV-2 variant;(b) a secretory signal peptide comprises an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 12; and / or(c) a transmembrane domain) comprises an amino acid sequence of SEQ ID NO: 90, or an amino acid sequence that is that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 90.

[0212] In some embodiments, a polypeptide comprises a transmembrane, and the N-terminal to C-terminal orientation of the polypeptide is (secretory signal peptide)-(truncated SI subdomain)-(transmembrane domain), and12947033vl Page 27 of 526Attorney Docket No.: 2013237-1485 the truncated SI subdomain or a variant thereof and the transmembrane domain are connected via a sequence that comprises a GS linker, optionally wherein the GS linker comprises about 10-20 residues (e.g., about 15 or about 20 residues).

[0213] In some embodiments, a polypeptide comprises a secretory signal peptide (e.g., a secretory signal peptide of a viral protein). In some embodiments, a polypeptide comprises a secretory signal peptide that comprises (i) an amino acid sequence that is listed in Table 3 or Table 72 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence that is listed in Table 3 or Table 72, and / or (ii) wherein the RNA comprises a nucleotide sequence that is listed in Table 3 or Table XXXI or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence that is listed in Table 3 or Table 72 sequence that is listed in Table 4 and / or Table 72.

[0214] In some embodiments, a polypeptide sequence comprises an SP24-Q7PUJ5_ANOGA secretory signal peptide (e.g., a secretory signal peptide comprising an amino acid sequence of the SP24-Q7PUJ5_ANOGA secretory signal peptide provided in Table 72) or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of the SP24-Q7PUJ5_ANOGA secretory signal peptide provided in Table 72.

[0215] In some embodiments, a polypeptide sequence comprises an SP24-SP18-HEMA_CVBM secretory signal peptide (e.g., a secretory signal peptide comprising the amino acid sequence of the SP24-SP18-HEMA_CVBM secretory signal peptide provided in Table 72 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of the SP24- SP18-HEMA_CVBM secretory signal peptide provided in Table 72.

[0216] In some embodiments, a polypeptide sequence comprises an SP25-GD_HHV1K secretory signal peptide (e.g., a secretory signal peptide comprising an amino acid sequence of the SP25-GD_HHV1K secretory signal peptide provided in Table 72 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of the SP25-GD_HHV1K secretory signal peptide provided in Table 72.

[0217] In some embodiments, a polypeptide sequence comprises an SP32-GD_HHV1K secretory signal peptide (e.g., a secretory signal peptide comprising the amino acid sequence of the SP32-GD_HHV1K secretory signal peptide provided in Table 72 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of the SP32-GD_HHV1K secretory signal peptide provided in Table 72.

[0218] In some embodiments, a polypeptide sequence comprises an SP20-A7U881_HHV2 secretory signal peptide (e.g., a secretory signal peptide comprising the amino acid sequence of the SP20-A7U881_HHV2 secretory signal peptide provided in Table 72 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of the SP20-A7U881_HHV2 secretory signal peptide provided in Table 72.

[0219] In some embodiments, a polypeptide sequence comprises a SARS-CoV-2 secretory signal peptide (e.g., a secretory signal peptide comprising the amino acid sequence of one of the SARS-CoV-2 secretory signal peptides provided in Table 3 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,12947033vl Page 28 of 526Attorney Docket No.: 2013237-148596%, 97%, 98%, 99% or more identical to one or more of the amino acid sequence of the SARS-CoV-2 secretory signal peptide provided in Table 3).

[0220] In some embodiments, a polypeptide comprises a transmembrane domain. In some embodiments, a transmembrane domain is from a viral membrane protein. In some embodiments, a transmembrane domain is a SARS-CoV-2 S protein transmembrane domain. In some embodiments, a transmembrane domain is obtained from a viral protein that is not a SARS-CoV-2 S protein. In some embodiments, a transmembrane domain comprises a transmembrane domain that is listed in Table 5 (e.g., comprising an amino acid sequence provided in Table 4 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence listed in Table 4) and / or wherein the transmembrane domain is encoded by a nucleotide sequence that is listed in Table 4B or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to a nucleotide sequence listed in Table 6).

[0221] In some embodiments, a transmembrane domain comprises an amino acid sequence of SEQ ID NO: 90, or an amino acid sequence that is at least 70%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 90.

[0222] In some embodiments, a polypeptide comprises a soluble multimerization domain (e.g., a trimerization domain, e.g., a T4 fibritin domain). In some embodiments, a polypeptide does not comprise a soluble multimerization domain (e.g., a trimerization domain, e.g., a T4 fibritin domain).

[0223] In some embodiments, a polypeptide comprises:(1) a fragment of an SI polypeptide (e.g., amino acids 1-528 of SEQ ID NO: 1 or a corresponding region of a SARS-CoV-2 variant);(2) a secretory signal peptide (e.g., a SP24-Q7PUJ5_ANOGA, SP24-SP18-HEMA_CVBM, SP25- GD_HHV1K, SP32-GD_HHV1K, or SP20-A7U881_HHV2 secretory signal peptide described herein); and(3) a transmembrane domain (e.g., a viral transmembrane domain, a transmembrane domain of a coronavirus S protein, a transmembrane domain of an influenza virus HA protein, or a transmembrane domain of a SARS-CoV-2 S protein).

[0224] In some embodiments, the N-terminal to C-terminal orientation of the polypeptide is: (secretory signal peptide)-(fragment of an SI polypeptide)-(transmembrane domain). In some embodiments, (1) the secretory signal peptide and the fragment of an SI polypeptide; and / or (2) the fragment of an SI polypeptide and the transmembrane domain are connected to one another by a linker (e.g., an artificial linker, a flexible linker, a flexible linker comprising a GS sequence). In some embodiments, a GS sequence comprises a sequence provided in Table 8. In some embodiments, a GS sequence comprises at least 5, at least 10, at least 15, or at least 20 amino acids (e.g., G or S amino acids). In some embodiments, a GS sequence comprises (G4S)1 (SEQ ID NO: 488), (GRS)z (SEQ ID NO: 489), (G4S)3(SEQ ID NO: 490), or (G4S)4(SEQ ID NO: 491) sequence.

[0225] In some embodiments, a fragment of an S protein is adapted to a SARS-CoV-2 strain or variant (e.g., an Omicron variant, XBB.1.5 variant, JN.l variant , JN.l.2 variant , JN.l.6 variant , KP.2 variant , KP.3 variant, XEC variant, JN.l.7 variant, MV.l variant, MC.l variant, LP.8.1 variant, LF.7 variant, BA.3.2 variant, and / or any variant described in the present disclosure).

[0226] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 156 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 156; (ii) the nucleotide sequence of SEQ ID NO: 158 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 158; and / or (iii)12947033vl Page 29 of 526Attorney Docket No.: 2013237-1485 comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 155, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 155.

[0227] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 161 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 161; (ii) the nucleotide sequence of SEQ ID NO: 163 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 163; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 160, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 160.

[0228] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 166 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 166; (ii) the nucleotide sequence of SEQ ID NO: 168 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 168; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 165, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 165.

[0229] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 171 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 171; (ii) the nucleotide sequence of SEQ ID NO: 173 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 173; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 170, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 170.

[0230] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 176 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 176; (ii) the nucleotide sequence of SEQ ID NO: 178 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 178; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 175, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 175.

[0231] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 181 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 181; (ii) the nucleotide sequence of SEQ ID NO: 183 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 183; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 180, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 180.

[0232] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 186 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to12947033vl Page 30 of 526Attorney Docket No.: 2013237-1485SEQ ID NO: 186; (ii) the nucleotide sequence of SEQ ID NO: 188 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 188; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 185, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 185.

[0233] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 191 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 191; (ii) the nucleotide sequence of SEQ ID NO: 193 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 193; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 190, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 190.

[0234] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 196 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 196; (ii) the nucleotide sequence of SEQ ID NO: 198 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 198; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 195, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 195.

[0235] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 201 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 201; (ii) the nucleotide sequence of SEQ ID NO: 203 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 203; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 200, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 200.

[0236] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 211 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 211; (ii) the nucleotide sequence of SEQ ID NO: 213 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 213; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 210, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 210.

[0237] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 221 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 221; (ii) the nucleotide sequence of SEQ ID NO: 223 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 223; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 220, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 220.12947033vl Page 31 of 526Attorney Docket No.: 2013237-1485

[0238] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 226 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 226; (ii) the nucleotide sequence of SEQ ID NO: 228 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 228; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 225, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 225.

[0239] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 231 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 231; (ii) the nucleotide sequence of SEQ ID NO: 233 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 233; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 230, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 230.

[0240] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 236 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 236; (ii) the nucleotide sequence of SEQ ID NO: 238 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 238; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 235, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 235.

[0241] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 241 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 241; (ii) the nucleotide sequence of SEQ ID NO: 243 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 243; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 240, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 240.

[0242] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 246 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 246; (ii) the nucleotide sequence of SEQ ID NO: 248 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 248; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 245, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 245.

[0243] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 251 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 251; (ii) the nucleotide sequence of SEQ ID NO: 253 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 253; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 250, or a sequence that is at12947033vl Page 32 of 526Attorney Docket No.: 2013237-1485 least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 250.

[0244] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 256 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 256; (ii) the nucleotide sequence of SEQ ID NO: 258 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 258; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 255, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 255.

[0245] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 261 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 261; (ii) the nucleotide sequence of SEQ ID NO: 263 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 263; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 260, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 260.

[0246] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 266 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 266; (ii) the nucleotide sequence of SEQ ID NO: 268 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 268; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 265, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 265.

[0247] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 271 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 271; (ii) the nucleotide sequence of SEQ ID NO: 273 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 273; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 270, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 270.

[0248] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 276 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 276; (ii) the nucleotide sequence of SEQ ID NO: 278 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 278; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 275, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 275.

[0249] In some embodiments, an RNA comprises comprising (i) the nucleotide sequence of SEQ ID NO: 281 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 281; (ii) the nucleotide sequence of SEQ ID NO: 283 or a sequence that is at least 70%,12947033vl Page 33 of 526Attorney Docket No.: 2013237-148580%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 283; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 280, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 280.

[0250] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 286 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 286; (ii) the nucleotide sequence of SEQ ID NO: 288 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 288; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 285, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 285.

[0251] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 291 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 291; (ii) the nucleotide sequence of SEQ ID NO: 293 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO:293; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 290, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 290.

[0252] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 296 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 296; (ii) the nucleotide sequence of SEQ ID NO: 298 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 298; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 295, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 295.

[0253] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 301 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 301; (ii) the nucleotide sequence of SEQ ID NO: 303 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 303; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 300, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 300.

[0254] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 306 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 306; (ii) the nucleotide sequence of SEQ ID NO: 308 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 308; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 305, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 305.12947033vl Page 34 of 526Attorney Docket No.: 2013237-1485

[0255] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 311 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 311; (ii) the nucleotide sequence of SEQ ID NO: 313 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 313; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 310, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 310.

[0256] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 321 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 321; (ii) the nucleotide sequence of SEQ ID NO: 323 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 323; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 320, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 320.

[0257] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 333 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 333 (ii) the nucleotide sequence of SEQ ID NO: 335 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 335; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 332; or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 332.

[0258] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 338 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 338 (ii) the nucleotide sequence of SEQ ID NO: 340 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 340; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 337; or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 337.

[0259] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 343 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 343 (ii) the nucleotide sequence of SEQ ID NO: 345 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 345; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 342; or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 342.

[0260] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 348 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 348 (ii) the nucleotide sequence of SEQ ID NO: 350 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 350; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 347; or a sequence that is at12947033vl Page 35 of 526Attorney Docket No.: 2013237-1485 least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 347.

[0261] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 353 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 353 (ii) the nucleotide sequence of SEQ ID NO: 355 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 355; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 352; or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 352.

[0262] In some embodiments, an RNA comprises RNA comprises (i) the nucleotide sequence of SEQ ID NO: 358 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 358 (ii) the nucleotide sequence of SEQ ID NO: 360 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 360; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 357; or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 357.

[0263] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 362 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 362 (ii) the nucleotide sequence of SEQ ID NO: 364 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 364; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 337; or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 337.

[0264] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an RNA described herein (e.g., in a particular formulation, a lipid formulation, a lipoplex formulation, or a lipid nanoparticle formulation).

[0265] In some embodiments, provided herein is a method comprising administering an RNA or a pharmaceutical composition provided herein. In some embodiments a method provided herein is a method of inducing an immune response to a coronavirus in a subject, and wherein the method comprises administering an RNA or a pharmaceutical composition provided herein. In some embodiments, a method described herein induces an immune response against a SARS-CoV-2 virus.

[0266] In some embodiments, provided herein is a method of manufacturing an RNA, comprising in vitro transcribing an RNA provided herein.

[0267] In some embodiments, provided herein is DNA (e.g., linear DNA or a plasmid DNA) encoding an RNA provided herein. In some embodiments, provided herein is a polypeptide encoded by an RNA provided herein.

[0268] In some embodiments, a transmembrane domain comprises an amino acid sequence of SEQ ID NO: 89, or an amino acid sequence that is at least 70%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 89. In some embodiments, RNA described herein comprises (i) the nucleotide sequence of SEQ ID NO: 328 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 328 (ii) the nucleotide sequence of SEQ ID NO: 330 or a sequence that is at least 70%, 80%, 85%, 90%, 91%,12947033vl Page 36 of 526Attorney Docket No.: 2013237-148592%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 330; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 327; or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 327.

[0269] In some embodiments, an RNA described herein comprises a nucleotide sequence encoding a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 332.

[0270] In some embodiments, one or more fragments of an S protein comprises one or more mutations of a SARS-CoV-2 strain or variant (e.g., one or more mutations in the S protein of a variant described herein, e.g., one or more mutations associated with a variant listed in Table 1). In some embodiments, a fragment of an S protein comprises one or more mutations associated with a JN.l variant, JN.l.2 variant, JN.l.6 variant, KP.2 variant, KP.3 variant, XEC variant, JN.l.7 variant, MV.l variant, MC.l variant, LP.8.1 variant, LF.7 variant, BA.3.2 variant, or a descendent thereof.

[0271] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, 60, 61, 62, 63, 64, or more) of the following mutations: insl6MPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A201, L202I, V203G, L206F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, or M1229I relative to SEQ ID NO: 1.

[0272] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, or more of the following mutations: insl6MPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A201, L202I, V203G, L206F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, or Y505H relative to SEQ ID NO: 1.

[0273] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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 or more of the following mutations: insl6MPLF, T19I, R21T, A24- 26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A201, L202I, V203G, L206F, H245N, A264D, I332V, G339H, R446T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, F456L, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, or Y505H relative to SEQ ID NO: 1.

[0274] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, 60, 61, 62, 63, or more of the following mutations: insl6MPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A201, L202I, V203G, L206F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y,12947033vl Page 37 of 526Attorney Docket No.: 2013237-1485Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, or P1143L relative to SEQ ID NO: 1.

[0275] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, or more of the following mutations: insl6MPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A201, L202I, V203G, L206F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, or Y505H relative to SEQ ID NO: 1.

[0276] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, 60, 61, 62, 63, or more of the following mutations: insl6MPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A201, L202I, V203G, L206F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, or P1143L relative to SEQ ID NO: 1.

[0277] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, or more of the following mutations: insl6MPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A201, L202I, V203G, L206F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, or Y505H relative to SEQ ID NO: 1.

[0278] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, 60, 61, 62, 63, or more of the following mutations: insl6MPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145. F157S, R158G, A201, L202I, V203G, L206F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, T572I, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, or E1150D relative to SEQ ID NO: 1.

[0279] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, or more of the following mutations: insl6MPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145. F157S, R158G, A201, L202I, V203G, L206F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, or Y505H relative to SEQ ID NO: 1.

[0280] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, 60, 61, 62, 63, or more of the following12947033vl Page 38 of 526Attorney Docket No.: 2013237-1485 mutations: insl6MPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A201, L202I, V203G, L206F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, F456L, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, V1104L, or P1143L relative to SEQ ID NO: 1.

[0281] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, or more of the following mutations: insl6MPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A201, L202I, V203G, L206F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, F456L, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, or Y505H relative to SEQ ID NO: 1.

[0282] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, or more) of the following mutations: T19I, A24-26, A27S, V83A, G142D, A145, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, or N969K relative to SEQ ID NO: 1.

[0283] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or more) of the following mutations: T19I, A24-26, A27S, V83A, G142D, A145, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, or Y505H relative to SEQ ID NO: 1.

[0284] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, 60, 61, 62, 63, 64, 65, 66, or 67 or more of the following list of mutations: insl6MPLF, T19I, R21T, T22N, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, F456L, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, V1104L, and P1143L.

[0285] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, or more of the following list of mutations: insl6MPLF, T19I, R21T, T22N, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, F456L, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, and Y505H.12947033vl Page 39 of 526Attorney Docket No.: 2013237-1485

[0286] In some embodiments, a fragment of a GS sequence comprises (G4S)1 (SEQ ID NO: 488), (GRS)2 (SEQ ID NO: 489), (G4S)3 (SEQ ID NO: 490), or (G4S)4 (SEQ ID NO 491) sequence or wherein the GS sequence comprises a sequence of GGSSGG (SEQ ID NO: 848).

[0287] In some embodiments, a transmembrane domain comprises an amino acid sequence of SEQ ID NO: 89 (EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT), or an amino acid sequence that is at least 70%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 89.

[0288] In some embodiments, an RNA comprises a nucleotide sequence that encodes a polypeptide comprising amino acids 1-528 of SEQ ID NO: 1, 14-528 of SEQ ID NO: 1, or 1-537 of SEQ ID NO: 1.

[0289] In some embodiments, an SI polypeptide comprises:(a) amino acids 1-528 of SEQ ID NO: 1, 14-528 of SEQ ID NO: 1, 1-537 of SEQ ID NO: 1, or 14-537 of SEQ ID NO: 1, or a corresponding region of any of the foregoing from the S protein of a SARS-CoV-2 variant;(b)QCVMPLFNLITTTQSYTNSFTRGVYYPDKVFRSSVLHLTQDLFLPFFSNVTWFHAISGTNGTKRFDNPVLPFNDGVYFASTEKSNIIR GWIFGTTLDSKTQSLLIVNNATNVFIKVCEFQFCNDPFLDVYHKNNKSWMESESGVYSSANNCTFEYVSQPFLMDLEGKQGNFKNL REFVFKNIDGYFKIYSKHTPIIGRDFPQGFSALEPLVDLPIGINITRFQTLLALNRSYLTPGDSSSGWTAGAADYYVGYLQPRTFLLKYN ENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNVTNLCPFHEVFNATTFASVYAWNRTRISNCVADYSVL YNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIKGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKHSGNYDYWYRS LRKSKLKPFERDISTEIYQAGNKPCKGKGPNCYFPLQSYGFRPTYGVGHQPYRVWLSFELLHAPATVCGPK (SEQ ID NO: 664), or a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; and / or(c)QCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPALPFNDGVYFASTEKSNIIRG WIFGrrTLDSKTQSLLIVNNATNWIKVCEFQFCNDPFLDVYQKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKEGNFKNLRE FVFKNIDGYFKIYSKHTPINLERDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPVDSSSGWTAGAAAYYVGYLQPRTFLLKYNE NGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYN FAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRK SKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVWLSFELLHAPATVCGPK (SEQ ID NO: 656), or a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0290] In some embodiments, an RNA comprises a 5' cap, a cap proximal sequence, a 5' UTR sequence, a 3' UTR sequence, and a polyA sequence.

[0291] In some embodiments, an RNA comprises:(i) a 5' cap comprising a Capl structure;(ii) a 5'-UTR sequence comprising a modified human alpha-globin 5'-UTR;(iii) a 3'-UTR sequence comprising a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA;(iv) a polyA sequence comprising at least 100 A nucleotides; or(v) a combination of any one of (i)-(iv).

[0292] In some embodiments, a 5' cap comprises a Capl structure, and the Capl structure comprises m7(3'OMeG)(5')ppp(5')(2'OMeAl)pG2, wherein Al is position +1 of the RNA, and G2 is position +2 of the RNA.12947033vl Page 40 of 526Attorney Docket No.: 2013237-1485

[0293] In some embodiments, a cap proximal sequence comprises Al and G2 of the Capl structure, and a sequence comprising: A3N4N5 at positions +3, +4 and +5 respectively of the RNA, wherein N4 and N5 are each independently selected from A, G, C, and U.

[0294] In some embodiments, a polyA sequence comprises an interrupted sequence of A nucleotides, optionally wherein the interrupted sequence comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30 adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence.

[0295] In some embodiments, a 5'-UTR sequence comprises SEQ ID NO: 112 or 113, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 112 or 113.

[0296] In some embodiments, a 3'-UTR sequence comprises SEQ ID NO: 118 or 647, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 118, 647, or 648.

[0297] In some embodiments, a interrupted polyA tail sequence comprises SEQ ID NO: 114, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 114.

[0298] In some embodiments, a sequence at the 5' end of the 3'UTR sequence (e.g., the sequence immediately adjacent to a sequence encoding an antigenic polypeptide) is CUCGAG (SEQ ID NO: 817) or GGAUCCGAU (SEQ ID NO: 818).

[0299] In some embodiments, a RNA is saRNA, self-amplifying RNA, trans-amplifying RNA (taRNA), or mRNA.

[0300] In some embodiments, a RNA is unmodified RNA or wherein the RNA comprises one or modified uridines in place of one or more uridines.

[0301] In some embodiments, a RNA comprises a single modified uridine in place of each uridine.

[0302] In some embodiments, a modified uridine is Nl-methyl-pseudouridine.

[0303] Among other things, the present disclosure describes composition comprising an RNA of described herein.

[0304] In some embodiments, a RNA is formulated in a nanoparticle.

[0305]

[0189] In some embodiments, a nanoparticle is a lipid nanopartide, a polyplex (PLX), a lipidated polyplex (LPLX), a liposome, or a polysaccharide nanopartide.

[0306] In some embodiments, a nanopartide is a lipid nanopartide.

[0307] In some embodiments, a lipid nanopartide comprises a cation ically ionizable lipid, a sterol, a neutral lipid, and a polymer-conjugated lipid.

[0308] In some embodiments, a polymer-conjugated lipid comprises a PEG-conjugated lipid.

[0309] In some embodiments, a RNA is encapsulated in a lipid nanopartide (LNP), preferably wherein the LNP comprises molar ratios of 20-60% ionizable cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG- modified lipid.

[0310] In some embodiments, nanopartides have an average diameter of about 50-150 nm.

[0311] In some embodiments, a composition comprises a cryoprotectant, optionally wherein the cryoprotectant is or comprises sucrose.

[0312] In some embodiments, a composition comprises an aqueous buffered solution, optionally wherein the aqueous buffered solution comprises one or more of Tris base, Tris HCI, NaCI, KCI, Na2HPO4, and KH2PO4.12947033vl Page 41 of 526Attorney Docket No.: 2013237-1485

[0313] In some embodiments, a composition comprises about 10 mM Tris buffer and about 10% sucrose.

[0314] Among other things, the present disclosure describes a pharmaceutical composition comprising (i) anRNA or a composition described herein and (ii) a pharmaceutically acceptable excipient.

[0315] In some embodiments, a pharmaceutical composition is formulated as a multi-dose formulation in a vial, a single-dose formulation in a vial, or a prefilled syringe.

[0316] In some embodiments, a pharmaceutical composition is formulated to provide a dose of about 100 pg or less (e.g., about 90 pg or less) of total RNA.

[0317] In some embodiments, a pharmaceutical composition is formulated to provide a dose of about 90 pg, about 60 pg, about 30 pg, about 25 pg, about 20 pg, about 10 pg, about 6 pg, about 5 pg, or about 3 pg of total RNA.

[0318] Among other things, the present disclosure describes a method that comprises administering a composition that comprises and / or delivers an RNA described herein, a composition described herein, or a pharmaceutical composition described herein.

[0319] In some embodiments, a method comprises administering about 100 pg or less (e.g., about 90 pg or less) of total RNA.

[0320] In some embodiments, a method comprises administering about 90 pg, about 60 pg, about 30 pg, about 25 pg, about 20 pg, about 10 pg, about 6 pg, about 5 pg, or about 3 pg of total RNA.

[0321] In some embodiments,(i) a subject is 12 years or older, and a method comprises administering about 30 pg, about 20 pg, about 10 pg, or about 5 pg of the RNA,(ii) a subject is 5 years to less than 12 years old, and a method comprises administering about 10 pg, about 6.6 pg, about 3.3 pg, or about 1.6 pg of the RNA, or(iii) a subject is 6 months to less than 5 years old, and a method comprises administering about 3 pg, about 2 pg, about 1 pg, or about 0.5 pg of the RNA.

[0322] In some embodiments, a composition is administered in a volume of about 200 pL to about 300 pL.

[0323] In some embodiments, a subject (a) has not previously been administered a SARS-CoV-2 vaccine and / or wherein a subject has not previously been determined to have been infected with SARS-CoV-2 (e.g., as determined using a PCR or antigen test), or (b) has previously been administered one or more doses of a SARS-CoV- 2 vaccine (e.g., at least about 2 months before being administered the RNA of any one of embodiments 1-276, the composition of any one of embodiments 277-287, or the pharmaceutical composition of any one of embodiments 288-291).

[0324] In some embodiments, a method comprises administering a single dose of the RNA, composition, or pharmaceutical composition to a subject.

[0325] In some embodiments, a method comprises administering two or more doses of an RNA, composition, or pharmaceutical composition to a subject, optionally wherein the two doses are administered about 21 days apart.

[0326] In some embodiments, RNA, composition, or pharmaceutical composition is administered three times to the subject, optionally wherein the first and the second dose are administered about 21 days apart, and the third dose is administered about 28 days after the second dose.

[0327] In some embodiments, a method comprises administering a further dose of RNA, composition, or pharmaceutical composition, at least about 2 months after the first dose of RNA, composition, or pharmaceutical12947033vl Page 42 of 526Attorney Docket No.: 2013237-1485 composition (e.g., 2-12 months, 2-10 months, 2-8 months, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months).

[0328] In some embodiments, a subject has previously been exposed to a SARS-CoV-2 antigen (e.g., by vaccination and / or by infection).

[0329] In some embodiments, a subject was previously administered one or more doses of a SARS-CoV-2 vaccine.

[0330] In some embodiments, a subject was previously administered a complete dosing regimen of a SARS- CoV-2 vaccine.

[0331] In some embodiments, a subject was previously administered a first dose and a second dose of a vaccine that delivers a full-length SARS-CoV-2 S protein (e.g., a composition comprising LNP-formulated RNA encoding a SARS-CoV-2 S protein), wherein the first dose and the second dose were administered about 21 days apart, and optionally wherein the subject was previously administered as a booster dose a monovalent or bivalent vaccine that delivers a SARS-CoV-2 S protein of one or more variants (e.g., (i) an S protein of a Wuhan strain and an S protein of an Omicron BA.4 / 5 strain, (ii) an S protein of an XBB.1.5 variant, (iii) an S protein of a KP.2 variant, (iv) an S protein of a JN.l variant, (v) an S protein of a JN.l.2 variant, (vi) an S protein of a JN.l.6 variant, (vii) an S protein of a KP.3 variant, (viii) an S protein of a XEC variant, (ix) an S protein of a JN.l.7 variant, (x) an S protein of a MV.l variant, (xi) an S protein of a MC.l variant, (xii) an S protein of a LP.8.1 variant, (xiii) an S protein of an LF.7 variant, and / or (xiv) an S protein of a BA.3.2 variant).

[0332] In some embodiments, a method described herein further comprises administering one or more vaccines against a non-SARS-CoV-2 disease, optionally wherein the one or more vaccines comprises an RSV vaccine, an influenza vaccine, or a combination thereof.

[0333] In some embodiments, a method is a method of preventing or reducing the chances of being infected with a SARS-CoV-2 virus and / or treating a SARS-CoV-2 infection.

[0334] In some embodiments, a method is a method of preventing SARS-CoV-2 infection, reducing the chance of SARS-CoV-2 infection, preventing or reducing the change of deleterious symptoms associated with SARS-CoV-2 infection (which can result, e.g., in a reduced change of hospitalization), increase the change of experiencing an asymptomatic SARS-CoV-2 infection, and / or treating a SARS-CoV-2 infection.

[0335] Among other things, an RNA, composition, or pharmaceutical composition described herein can be used for promoting an immune response in a subject. Among other things, the present disclosure describes a method of manufacturing an RNA described herein, optionally wherein the method of manufacturing comprises in vitro transcription of an RNA described herein.Brief Description of the Figures

[0336] FIG. 1 illustrates SARS-CoV-2 imprinting in subjects. Subjects administered a vaccine that delivers a wild-type (WT) antigen produce antibodies and form memory B cells. As new Variants of Concern (VOC) arise, VOC-adapted booster shots are administered. Certain VOCs have high immune escape potential and comprise mutations at neutralization epitopes in hypervariable domains (represented by the portions of12947033vl Page 43 of 526Attorney Docket No.: 2013237-1485 the antigen with different colors in the Figure). Subjects exposed to a VOC-adapted vaccine have a predisposition to activate memory B cells formed in response to the initial WT vaccine rather than produce de novo responses that recognize epitopes unique to the VOC (i.e., memory B cells that recognize conserved epitopes in the VOC antigen are more likely to be activated and naive B cells recognizing unique epitopes are less likely to be activated). So long as at least some of the neutralization epitopes in the WT antigen are preserved, administering a VOC-adapted vaccine will increase induction of neutralization antibodies against the VOC. As VOCs continue to evolve, however, and acquire further mutations at neutralization epitopes, neutralization responses induced by VOC-adapted vaccines become less efficacious. Further discussion of the imprinting phenomenon in the SARS-CoV-2 context can be found in Wheatley et al., Trends Immunol, 2021, the contents of which are incorporated by reference herein in their entirety. Immune imprinting is expected to be an issue for vaccine updates that addressing virus strains comprising a number of mutations at neutralization sensitive sites, i.e., exhibit close to no conserved neutralizing epitopes.

[0337] FIGs. 2A-2B. Exemplary analysis tools for assessing memory B cell responses, which can be useful for evaluating immune imprinting. FIG. 2A shows a schematic of one-dimensial flow-cytometry analysis of memory B cell (BMEM) phenotyping using fluorochrome-labeled Spike (or subdomain) tetramers. BMEM specificity can be assessed by labelling with Spike (or subdomains) of different SARS-CoV-2 variants. FIG. 2B depicts serological analysis after depletion of immune serum with Spike (or subdomain bait). Serum samples collected from a subject exposed to a SARS-CoV-2 S protein (e.g., via prior infection and / or vaccination) are incubated with a Spike protein or domain thereof (e.g., RBD or SI domain) immobilized on a support (e.g., a magnetic bead as shown in the figure). Isolation of the bead removes antibodies that bind the bait, and the remaining serum is analyzed to determine the specificity of antibodies in the serum sample (e.g., in the Figure, sera samples are incubated with Wild-Type (Wuhan) Spike immobilized on magnetic beads, the magnetic beads are removed, and any antibodies remaining in the sample that bind to a variant are variant-specific antibodies. Beads lacking bait can be used as a negative control (e.g., as shown in the Figure).

[0338] FIG. 3. Experimental protocol for characterizing variant specific antibodies. Sera samples were collected from subjects administered 2 or 3 doses of a vaccine delivering a SARS-CoV-2 S protein (e.g., BNT162b2) and (i) infected with an SARS-CoV-2 Omicron BA.l variant, (ii) infected with an Omicron BA.l SARS-CoV- 2 variant and subsequently administered an Omicron BA.l-adapted vaccine (BNT162b2(omi), or (iii) two doses of an Omicron BA.l-adpated vaccine.

[0339] FIGs. 4A-4B. BA.l-induced broad neutralization is likely mediated by expansion of responses against conserved epitopes ( / .e. recall responses). Provided is data demonstrating that, while exposure to a new variant of a infectious agent (Omicron BA.l in the Figure) can induce a broad immune response, in some embodiments, that broad immune response is driven by the activation of memory B cells and recognition of conserved epitopes, rather than generation of new antibodies that recognize epitopes unique to the new variant. Pseudovirus neutralization assays and FACS analysis of BMEM cells using fluorochrome-labeled Spike or RBD tetramers were performed on sera samples collected from the patient groups summarized in FIG. 3. FIG. 4A shows pseudovirus neutralization assay results. Pseudovirus neutralization titers (pVNso) titers (shown on the Y-axis) were collected for pseudoviruses comprising S proteins of various SARS-CoV-2 variants and strains (variants and strains indicated along X-axis). Assay results demonstrate that Omicron BA.l infection augments broadly neutralizing activity against Omicron variants, especially against BA.l. FIG. 4B shows representative FACS plots, using flourescently labeled S proteins or RBDs of a full-length S protein or RBD. FACS results show that majority of12947033vl Page 44 of 526Attorney Docket No.: 2013237-1485 memory B cells bind epitopes that are common to the Wuhan S protein and the Omicron BA.l S protein, or that are unique to the Wuhan S protein, but very few are specific to Omicron BA.l. These results suggest that a first exposure to wild-type S has imprinted against novel BMEM responses recognizing BA.l specific epitopes. BNT162b23corresponds to sera samples collected from subjects administered three doses of BNT162b2 and who showed no evidence of subsequent SARS-CoV-2 infection. BNT162b22+ Omi corresponds to sera samples collected from patients administered two doses of BNT162b2 who subsequently experienced a breakthrough SARS-CoV-2 infection at a time of high Omicron BA.1 prevalence. BNT162b23+ Omi corresponds to sera samples collected from patients administered three doses of BNT162b2 who subsequently experienced a breakthrough infection at a time of high Omicron BA.l prevalence. Blood drawn 1 month after last vaccination (SARS-CoV-2 naive) or infection (BA.l breakthrough). Data also shown and described in Quandt and Muik et al., 2022, the contents of which are hereby incorporated by reference in their entirety.

[0340] FIGs. 5A-5B. One exposure to BA.l, BA.2, and BA.4 / BA.5 may not recall XBB crossneutralizing immunity. Sera samples were collected from subjects (i) administered three doses of BNT162b2 ("BNT162b23") and showing no evidence of prior SARS-CoV-2 infection, (ii) administered four doses of BNT162b2 ("BNT162b24") and showing no evidence of prior SARS-CoV-2 infection, (iii) administered three doses of an RNA vaccine and who experienced a subsequent Omicron BA.l breakthrough infection ("mRNA-Vax3 + BA.l"), (iv) administered three doses of an RNA vaccine and who experienced a subsequent Omicron BA.2 breakthrough infection ("mRNA-Vax3+ BA.2"), or (v) administered three doses of an RNA vaccine and who experienced a subsequent Omicron BA.4 / 5 breakthrough infection ("mRNA-Vax3+ BA.4 / 5"). FIG. 5A shows pseudovirus neutralization titers. As shown in the figure, sera from mRNA-Vax experienced individuals with BA.l, BA.2 or BA.4 / 5 breakthrough infection showed limited neutralizing activity against the current, most immune escaping variants like XBB. FIG. 5B shows percent conservation of HLA class I and II T-cell epitopes and neutralizing B-cell epitopes for a number of SARS-CoV-2 variants. XBB displayed the lowest conservation of neutralizing B-cell epitopes across VOCs characterized. Data reproduced from Muik, Alexander, et al. "Progressive loss of conserved spike protein neutralizing antibody sites in Omicron sublineages is balanced by preserved T-cell recognition epitopes." bioRxiv (2022): 2022-12, the contents of which are incorporated by reference herein in their entirety.

[0341] FIGs. 6A-6B. Imprinting may not effectively be overcome by two subsequent exposures to Omicron BA.l. Sera samples were depleted using the indicated bait protein (e.g., using an assay similar to that depicted in FIG. 2B), and then screened in a pseudovirus neutralization assay comprising a Wuhan Spike protein (Wuhan-pVNT) (FIG. 6A) or an Omicron BA.l Spike protein (Omicron BA.l-pVNT) (FIG. 6B). Of the 13 individuals screened, only one showed an Omicron BA.l-specific neutralization response (indicated by arrow).

[0342] FIG. 7. Imprinting can limit build-up of private epitope-specific B cell memory even after two subsequent exposures to Omicron BA.l. Sera samples from subjects administered a booster dose of an RNA vaccine encoding an Omicron BA.l S protein (Omi BA.l Booster), an RNA vaccine encoding a SARS-CoV-2 Wuhan strain (BNT162b2 Booster), or no booster were collected, memory B cells isolated, and analyzed via depletion assays. Sera samples were collected on the day a booster dose was administered (VI), 7 days after a booster dose was administered (V2), and 1 month after a booster dose (V3). Memory B cells (BMEM) were stained for Spike binding, RBD binding, or NTD (N terminal domain) binding for each of Wuhan and Omicron BA.l. Indicated are the percent of screened B cells positive for the indicated probe. BMEM cells binding BA.l specific epitopes in the RBD12947033vl Page 45 of 526Attorney Docket No.: 2013237-1485 were not observed. A small population of BMEM cells specific to the BA.1 NTD were observed 1 week after administering an Omicron BA.l-adapted booster. The slight increase of full-length BA.l Spike binding BMEM cells in the BA.l adapted vaccine group most likely represents NTD-binders.

[0343] FIG. 8. Novel Spike antigen designs - SI and RBD-subdomain vaccines. Mutation density in new SARS-CoV-2 variants of concern (e.g., XBB) is highest in the Sl-fragment and especially in the RBD. Hence, in some embodiments, omitting the highly conserved S2 fragment can result in more efficient priming (e.g., by removing conserved epitopes that can activate BMEM cells and / or prevent activation of naive B cells). Shown are certain exemplary antigen designs, including (1) an RBD of an VOC attached to a trimerization domain (e.g., an RBD of XBB.1.5 attached to a T4 fibritin trimerization domain), (2) an SI domain of an VOC attached to a trimerization domain (e.g., an SI of XBB.1.5 attached to a T4 fibritin trimerization domain), (3) an RBD of an VOC attached to a trimerization domain and a transmembrane (TM) domain (e.g., an RBD of XBB.1.5 attached to a T4 fibritin trimerization domain and a TM domain of a SARS-CoV-2 S protein), and (4) an SI domain of an VOC attached to a trimerization domain and a transmembrane domain (e.g., an SI of XBB.1.5 attached to a T4 fibritin domain and a TM domain of a SARS-CoV-2 S protein)). Constructs (1) and (2) are soluble and secreted, whereas constructs (3) and (4) are TM-anchored.

[0344] FIG. 9. Exemplary Immunogenecity Study in Vaccine-Experienced mice. Mice are administered two doses of BNT162b2 (encoding an S protein of a Wuhan strain), or a composition comprising a first RNA that encodes a SARS-CoV-2 S protein of a Wuhan strain and a second RNA encoding a full-length S protein of an Omicron BA.4 / 5 variant (Bivalent b2 + BA.4 / 5), followed by a third dose of a candidate vaccine. Third doses include RNA encoding full-length Spike protein of a Wuhan strain (BNT162b2); RNA encoding a full-length S protein of an XBB.1.5 variant (BNT162b2 (XBB.1.5)); RNA encoding an RBD of an XBB.1.5 S protein comprising a secretory signal and a timerization domain (RBD (XBB.1.5)); RNA encoding an SI domain of an XBB.1.5 S protein comprising a timerization domain (SI (XBB.1.5)); RNA encoding an SI domain of an XBB.1.5 S protein comprising a timerization domain and a transmembrane domain (Sl-TM (XBB.1.5)); and RNA encoding an RBD of an XBB.1.5 S protein comprising a secretory signal, a timerization domain, and a transmembrane domain (RBD-TM (XBB.1.5)). Dot-filled cells indicate days on which sera sample are collected, solid black-filled cells indicate days on which vaccines are administered, and hatch-filled cells indicate days on which mice are sacrificed and final samples collected. The exemplary protocol can be used to characterize immune responses induced by compositions described herein.

[0345] FIG. 10. Exemplary Protocol for Characterizing Immune Cell (Including B Cell and T cell) Responses. A spleen sample and lymph nodes are collected and analyzed as shown in the Figure. Figure also summarizes analysis of blood samples collected throughout a study (including collection of viral neutralizing titers (pVNTs) and virus binding antibody titers (ELISA), which can be performed in parallel. The exemplary protocol can be used to characterize immune responses induced by compositions described herein.

[0346] FIG. 11. Exemplary Immunogenecity Study in Vaccine-Naive Mice. Mice are administered two doses of RNA encoding (i) a full-length Spike protein of a Wuhan strain (BNT162b2); (ii) RNA encoding a full-length S protein of an XBB.1.5 variant (BNT162b2 (XBB.1.5)); (iii) RNA encoding a full-length S protein of an XBB.1.5 variant and comprising a 19 amino acid C-terminal truncation (BNT162b2 (XBB.1.5) Cdl9); (iv) RNA encoding an RBD of an XBB.1.5 S protein comprising a secretory signal (SP19) and a timerization domain (RBD (XBB.1.5) (SP19)); (v) RNA encoding an SI domain of an XBB.1.5 S protein comprising a trimerization domain (SI (XBB.1.5)); (vi) RNA encoding an RBD of an XBB.1.5 S protein comprising a secretory signal (SP19), a timerization domain, and a transmembrane12947033vl Page 46 of 526Attorney Docket No.: 2013237-1485 domain (RBD-TM (XBB.1.5) (SP19)); (vii) RNA encoding an SI domain of an XBB.1.5 S protein comprising a timerization domain and a transmembrane domain (Sl-TM (XBB.1.5); and (viii) RNA encoding an RBD of an XBB.1.5 S protein comprising a secretory signal (SP16) and a timerization domain (RBD (XBB.1.5) (SP16)). Dot-filled cells indicate days on which sera sample are collected, solid black-filled cells indicate days on which vaccines are administered, and hatch-filled cells indicate days on which mice are sacrificed and final samples collected. The exemplary protocol can be used to characterize immune responses induced by compositions described herein.

[0347] FIG. 12. Exempalry Protocol for Characterizing Immune Cell (Including B Cell and T Cell) Responses. Spleen samples can be collected and analyzed as shown in the figure to characterize immune cell responses induced by compositions described herein (including, e.g., T cell and B cell immune responses).

[0348] FIG. 13. Exemplary Polypeptides Comprising an RBD or an NTD. "SP" stands for secretory peptide, "F" stands for a fibritin trimerization domain, "TM" stands for a transmembrane domain, and "CT' stands for a C-terminal sequence derived from a SARS-CoV-2 S protein.

[0349] FIGs. 14A-14B. Exemplary "Dumbbell" Constructs. FIG. 14A shows graphics summarizing certain "dumbbell" designs that are described herein (i.e., a polypeptide comprising one or more NTD polypeptides and / or one or more RBD polypeptides, and a multimerization domain such that multiple polypeptides associate in solution, forming a multimer of polypeptides, each polypeptide comprising two or more RBD polypeptides, two or more NTD polypeptides, and / or one or more RBD polypeptides and one or more NTD polypeptides). FIG. 14B depicts a model of the structure of one such dumbbell construct, comprising three polypeptides, each comprising two RBD polypeptides and a T4 fibritin trimerization domain, such that an oligomer comprising 6 RBD polypeptides is formed in solution.

[0350] FIG. 15. Exemplary Library for Characterizing Effect of N-terminal Secretory Signals onAntigen Expression. Indicated sequence corresponds to (SEQ ID NO: 717).

[0351] FIG. 16. Exemplary Experimental Protocol for Identifying Vaccine Constructs withImproved Expression. Shown is an exemplary in vitro protocol for testing extracellular expression of vaccine candidates in vitro. A cell line (HEK293) is transfected with a plasmid encoding a vaccine candidate. Cells are incubated with ACE2 or an antibody that binds RBD, followed by a secondary antibody, and then screened by flow cytometry to measure cell surface expression of RBD.

[0352] FIG. 17. Initial in vitro expression data. Shown is flow cytometry data obtained using BNT162b3, which comprises an RBD of an XBB.1.5 SARS-CoV-2 variant, and a secretory signal of a SARS-CoV-2 S protein (aa 1- 19) and a transmembrane domain. The amount of transfected plasmid was varied so as to identify a dynamic range (concentration of plasmid at which to transfect cells).

[0353] FIGs. 18A-18B. Effect of N-terminal Secretory Signal on in vitro Expression of VaccineCandidates. HEK293 were transfected with a library of vaccine candidates, comprising various N-terminal secretory signals (design shown in FIG. 15, specific sequences tested include those shown in Table 72 of the present disclosure). FIG. 18A shows background fluorescent signal (transfected cells incubated with 2° antibody but not 1° antibody). FIG. 18B shows intial results from a library of candidates. Significant background signal was observed but could be addressed by subtracting background signal. As shown in FIG. 18B, all signal peptides tested resulted in significantly increased cell surface expression of RBD as compared to a SARS-CoV-2 S protein secretory signal (aa 1-19).12947033vl Page 47 of 526Attorney Docket No.: 2013237-1485

[0354] FIGs. 19A-19B. Further Data Characterizing the Effect of Secretory Signals on Cell surface Expression of Antigens. A repeat of the experimental protocol shown in FIG. 16 was performed to confirm the results shown in FIG. 18. FIG. 19A shows background signal was observed, after incubated with secondary antibody only. FIG. 19B shows results from screeing for expression. Once again, all signal peptides tested resulted in significantly higher surface expression of antigen as compared to the SARS-CoV-2 spike signal peptide (aa 1-19).

[0355] FIGs. 20A-20B. Further Data Characterizing the Effect of Secretory Signals on Cell Surface Expression of Antigens (Anti-RBD Antibody + Human Secondary Antibody). A repeat of the experimental protocol shown in FIG. 16 was performed using a primary antibody that binds the RBD and human secondary antibodies. FIG. 20A shows background signal (cells not incubated with primary antibody). As shown, reduced background fluorescence was observed relative to mouse secondary antibody and ACE2 labelling. FIG. 20B shows RBD signal. Once again, all signal peptides tested resulted in significantly higher surface expression of RBD as compared to the SARS-CoV-2 spike signal peptide (aa 1-19).

[0356] FIGs. 21A-21C. Expression Achieved by Top 5 Secretory Signals. FIGs. 21A, 21B, and 21C show the results from FIGs. 18, 19, and 20, respectively for the top 5 secretory signals.

[0357] FIGs. 22A-22B. Effect of N-terminal Secretory Signals on Expression of RBD and Truncated SI Subdomain Polypeptides. FIG. 22A shows in vitro expression data from 5 different secretory signal peptides attached to a polypeptide comprising a SARS-CoV-2 RBD. Red arrow indicates improved expression of the topperforming candidate (RNA003) as compared to a polypeptide comprising an RBD linked to the SARS-CoV-2 S protein secretory signal peptide (RNA040). As shown in FIG. 22A, optimizing the secretory signal peptide resulted in a dose sparing effect of approximately 16-fold for polypeptides comprising an RBD (i.e., 16-fold less RNA was required to produce the same level of in vitro expression). FIG. 22B shows in vitro expression data from the top 5 performing secretory signal peptides when attached to a fragment of the SI subdomain of the SARS-CoV-2 S protein (comprising a sequence corresponding to amino acids 1-528 of SEQ ID NO: 1, with the N-terminal secretory signal peptide replaced in constructs comprising a heterologous secretory signal peptide). RNA035 comprises the native SARS-CoV- 2 secretory peptide. As shown in FIG. 22B, optimizing the secretory signal peptide was also found to improve expression of the fragment of the SI subdomain, resulting in an about 4-fold dose-sparing effect.

[0358] FIGs. 23A-23B. Omitting a Fibritin Trimerization Domain Improves Expression and Does Not Reduce Antigenicity of a Truncated 51 subdomain. FIG. 23A shows results from an in vitro experiment in which a cell line was transfected with nucleic acid encoding a construct with (RNA008) and without (RNA017) a fibritin trimerization domain. As shown in the FIG. 23A, omission of the fibritin trimerization domain was found to significantly improve antigen expression. FIG. 23B shows results from a mouse experiment in which mice were administered RNA encoding a polypeptide with (RNA008) and without (RNA017) a fibritin trimerization domain. As shown in the figure, omission of the trimerization domain did not interfere with the immune response.

[0359] FIGs. 24A-24D. Screening Transmembrane Domains for Effect on Antigen Expression. FIG. 24A shows results from an experiment in which the same antigen (corresponding to amino acids 1-528 of the S protein) was fused to different transmembrane domains. As shown in FIG. 24A, each of the RNAs tested resulted in antigen expression, and little variation in the amount of expression was observed. FIG. 24B provides expression results from the top 5 transmembrane domains in terms of expression identified in initial experiments. FIG. 24C and FIG. 24D provide results from a further confirmatory experiment, characterizing the same constructs plus a few newly identified constructs. FIG. 24D shows the results from the top candidates tested in the further confirmatory12947033vl Page 48 of 526Attorney Docket No.: 2013237-1485 experiment. Once again, it was concluded that the top performers provided about the same level of expression as those comprising the transmembrane domain of a SARS-CoV-2 S protein.

[0360] FIG. 25. Exemplary Experimental Protocol for Testing the Immunogenicity of Different Fragments of the SI Subdomain in Mice. Shown is an exemplary experimental protocol for testing the immunogenicity of different fragments of the S protein in mice. Dot filled cells indicate days on which serum samples were collected, solid black filled cells indicate days on which a vaccine was administered, and hatched cells indicate the final day of the experiment, on which mice were euthanized and final samples collected.

[0361] FIGs. 26A-26F. A Fragment of the SI Subdomain Induces a Strong Immune Response in Mice. FIG. 26A, FIG. 26B, FIG. 26C, FIG. 26D, and FIG. 26E show neutralization titers (pVNTso) on the day of vaccination, and 7, 14, 21, 28 and 35 days after vaccination, respectively; FIG. 26F shows a line graph of neutralization titers (pVNTso) for constructs across vaccination days. As shown in FIG. 26C, a statistically significant difference was observed at day 21 between (i) mice administered RNA encoding a fragment of the SI subdomain (RNA035) or RNA encoding an NTD and RBD connected via a GS linker (RNA038), and (ii) RNA encoding a full-length S protein.

[0362] FIG. 27. Exemplary Protocol for Investigating Immunogenicity of Improved SARS-CoV-2 Antigens in Mice. Dot filled cells indicate days on which serum samples were collected. Solid black filled cells indicate days on which the indicated compositions were administered. Hatch filled cells indicate the final day of the experiment, during which mice with euthanized and final samples were collected.

[0363] FIGs. 28A-28F. Mouse Immunogenicity Data Generated Using Improved SARS-CoV-2 Vaccines. FIG. 28A, FIG. 28B, FIG. 28C, FIG. 28D, and FIG. 28E show neutralization titers (pVNTso) 7, 15, 21, 28, and 34 days after vaccination, respectively, in an experiment performed in accordance with the protocol shown in FIG. 27. FIG. 28F provides a summary of the neutralization titers that were collected at different time points.

[0364] FIG. 29. Exemplary Protocol for Investigating Immunogenicity of Improved SARS-CoV-2 RNA Compositions in Mice. Dot filled cells indicate days on which serum samples were collected. Solid black filled cells indicate days on which the indicated compositions were administered. Hatch filled cells indicate the final day of the experiment, during which mice with euthanized and final samples were collected.

[0365] FIGs. 30A-30E. Mouse Immunogenicity Data Generated Using Improved SARS-CoV-2 Vaccines. FIG. 30A, FIG. 30B, FIG. 30C, and FIG. 30D show neutralization titers (pVNTso) 7, 14, 21, and 28 days after vaccination, respectively, in an experiment performed in accordance with the protocol shown in FIG. 29. FIG. 30E provides a summary of the neutralization titers that were collected at different time points.

[0366] FIG. 31. Exemplary Protocol for Investigating Immunogenicity of Improved SARS-CoV-2 Antigens in Mice. Dot filled cells indicate days on which serum samples were collected. Solid black filled cells indicate days on which the indicated compositions were administered. Hatch filled cells indicate the final day of the experiment, during which mice with euthanized and final samples were collected.

[0367] FIGs. 32A-32G. Mouse Immunogenicity Data Generated Using Improved SARS-CoV-2 Vaccines. FIG. 32A, FIG. 32B, FIG. 32C, and FIG. 32D show neutralization titers (pVNTso) 7, 14, 21, and 28 days after vaccination, respectively, in an experiment performed in accordance with the protocol shown in FIG. 31. FIG. 32E provides a summary of the neutralization titers that were collected at different time points. FIG. 32F and FIG. 32G provide a summary of the same data summarized in FIG. 32E, along with neutralization titers from a repeated12947033vl Page 49 of 526Attorney Docket No.: 2013237-1485 experiment, shown neutralizing titers for the original BNT162b2 construct (RNA041) and the optimized construct (RNA017).

[0368] FIGs. 33A-33E. Summary of Effects of Optimizing Secretory Signal, Multimerization Domain, Linker Domain, and Transmembrane Domain on Antigen Expression. FIG. 33A-FIG. 33D show the effects of optimizing the indicated domains on polypeptide expression (in the figure, a fragment of the SI polypeptide comprising a sequence corresponding to amino acids 1-528). Aside from the indicated domain (secretory signal, multimerization domain, linker, transmembrane domain), all other portions of the encoded polypeptide were identical in each of the panels. Arrow indicates improvement in expression relative to a reference. In FIG. 33D, "reference" refers to a polypeptide comprising the transmembrane domain of the SARS-CoV-2 S protein. FIG. 33E provides expression domains from polypeptides (a fragment of the SI domain comprising a sequence corresponding to amino acids 1-528 of SEQ ID NO: 1) comprising a combination of different optimized domains. As indicated in the figure, the optimized construct improved expression approximately 5- to 10-fold as compared to the original BNT162b2 product (encoding a full-length S protein), and approximately 2-fold as compared to other optimized constructs.

[0369] FIG. 34. Correlation of Surface Expression (in vitro) and Elicited nAb Titers (in vivo).Provided is an analysis showing the correlation between in vitro expression measurements and neutralizing antibody titers observed in mice (neutralization titers measured 28 days after administering a first dose of a composition to vaccine-immune mice). As shown in the figure, in vitro expression data showed a strong correlation with mouse neutralization titers, indicating that in vitro expression data was a good predictor for in vivo immunogenicity.

[0370] FIG. 35. Exemplary Protocol for Investigating Immunogenicity of Improved SARS-CoV-2 Antigens in Mice. Dot filled cells indicate days on which serum samples were collected. Solid black filled cells indicate days on which the indicated compositions were administered. Hatch filled cells indicate the final day of the experiment, during which mice with euthanized and final samples were collected.

[0371] FIGs. 36A-36E. Mouse Immunogenicity Data Generated Using Improved SARS-CoV-2 Vaccines. FIG. 36A, FIG. 36B, FIG. 36C, and FIG. 36D show neutralization titers (pVNTso) 7, 14, 21, and 28 days after vaccination, respectively, in an experiment performed in accordance with the protocol shown in FIG. 35. FIG. 36E provides a summary of the neutralization titers that were collected at different time points.

[0372] FIGs. 37A-37F. Mouse Immunogenicity Data Confirms Dose-Sparing Effect Provided by Vaccines Described Herein. Vaccine naive mice were administered 0.4, 0.1, or 0.025 pg of LNP-formulated RNA. 7, 14, 21, 28, and 35 days after administration, serum samples were collected and neutralization titers against an XBB.1.5-adapted pseudovirus were measured. Results are shown in FIG. 37A-37F. As shown in the figure, a clear dose sparing effect was observed by days 21, 28, and 35 for constructs described herein as compared to vaccines encoding a full-length SARS-CoV-2 S protein.

[0373] FIGs. 38A-38G. SARS-CoV-2 Vaccines Described Herein Induce Higher Neutralization Titers and Broader Cross Neutralization in Vaccine Naive Mice as Compared to RNA Encoding Full-length S Protein. Vaccine naive mice were administered a single dose of the indicated RNAs. Serum samples were collected 7, 14, 21, 28, and 35 days after administration of RNA. Results are shown in FIG. 38A, FIG. 38B, FIG. 38C, FIG. 38D, and FIG. 38E, respectively. FIG. 38F provides a summary of the neutralization titers collected at each time point. FIG. 38G shows neutralization titers against further SARS-CoV-2 strains and variants (Wuhan, Omicron BA.l, and Omicron BA.4 / 5), in sera sample collected 35 days after administration of RNA. As shown in each of FIGs. 38A-12947033vl Page 50 of 526Attorney Docket No.: 2013237-148538F, RNA described herein resulted in greatly increased neutralization titers against a matched SARS-CoV-2 variant as compared to RNA encoding a full-length S protein (titers ~10-fold higher by day 35). Cross-neutralization titers were also greatly increased, with neutralization titers of BA.4 / 5 being ~10-fold higher for RNA described herein as compared to RNA encoding full-length S protein.

[0374] FIGs. 39A-39G. RNA Described Herein Results in Improved in vitro Expression of XBB.1.5 and KP.2 SARS-CoV-2 Antigens. Cell surface expression was measured in cells transfected with RNA using fluorescently labeled ACE2 protein. FIG. 39A and FIG. 39B compare antigen expression for an RNA encoding a truncated SI subdomain (RNA017) and an RNA encoding a full-length S protein (RNA041), where the truncated SI subdomain and full-length S protein are each adapted to the XBB.1.5 SARS-CoV-2 variant. FIG. 39C and FIG. 39D compare expression of an additional RNA encoding a truncated SI subdomain (RNA043) to an RNA encoding a full- length S protein of an XBB.1.5 variant (RNA041). FIG. 39E and FIG. 39F compare antigen expression of an RNA encoding a polypeptide comprising a truncated SI subdomain of a KP.2 SARS-CoV-2 S protein (RNA042) to RNA encoding a full-length KP.2 S protein (RNA044). FIG. 39G compares antigen expression of an alternative RNA encoding a polypeptide comprising a truncated SI subdomain of a KP.2 SARS-CoV-2 S protein (RNA049) to RNA encoding a full-length KP.2 S protein (RNA044). In each experiment, RNA encoding a truncated SI subdomain was found to improve antigen expression as compared to RNA encoding a full-length S protein.

[0375] FIGs. 40A-40C. Phenotypic Characterization of Antigen-Specific B Cells. FIG. 40A provides an illustration showing the B cell maturation process and the various intermediary states a B cell transitions through as it transforms from a naive cell to a plasma cell. Also indicated are cell surface markers that are characteristic of the different B cell differentiation stages. FIG. 40B Lists the different mixtures of fluorescently labeled bait proteins that were used to phenotypically characterize antigen-specific B cells obtained from mice administered a composition described herein. FIG. 40C lists flow antibodies from the B-cell flow panel that was used in the experiment. FLS is an abbreviation for "Full-length Spike," and BC is an abbreviation for B Cell.

[0376] FIG. 41. An Example of a Protocol for Phenotypically Screening B cells. Cells are screened to identify single cell lymphocytes. Single cell lymphocytes are probed for cell surface expression of CD19 and CD20 to identify B cells, which can be screened for a variety of markers of interest and binding to various bait proteins. CD19+ / CD20+ B cells are screened for staining Spike-antigen bait positive. Antigen-specific B cells are then analyzed for cell surface expression of CD38 (activation marker), CD95 (Germinal Center marker), and CD273 / 80 (markers for memory B cells) and CD138 (a marker of plasma cells) for phenotypic characterization though a combinatorial / Boolean gating approach.

[0377] FIGs. 42A-42C. Representative Plots Showing Antigen-specificity of B Cells Obtained from Mice Administered Compositions Described Herein. FIG. 42A-FIG. 42C show exemplary plots of CD19+ / CD20+ cells probed for binding to full-length S protein, RBD, and NTD respectively. Each plot characterizes B cells obtained from a single mouse. As shown in FIG. 42C, an RNA encoding a truncated SI polypeptide (RNA017) was found to induce surprising high numbers of B cells capable of binding the NTD. Without wishing to be bound by theory, the improved immune response to NTD may result from increased accessibility of the NTD in the truncated SI subdomain as compared to a full-length S protein.

[0378] FIGs. 43A-43C. Summary of Phenotypic Characteristics of B Cells Obtained from Mice Administered Compositions Described Herein. FIG. 43A-FIG. 43C show the percent of CD19+ B cells that were found to bind full-length S protein, RBD, and NTD, respectively. As shown, RNA017 was found to produce12947033vl Page 51 of 526Attorney Docket No.: 2013237-1485 significantly higher numbers of B cells that bind the full-length S protein, RBD, and NTD at each concentration of RNA administered. For both the NTD and the RBD, B cell responses were ~4x higher for RNA encoding a truncated SI subdomain (RNA017) as compared to those induced by RNA encoding a full-length S protein (RNA041) for each concentration of RNA tested. The B cell response induced by 0.1 pg of RNA encoding a truncated SI subdomain was comparable to that induced by 0.4 pg of RNA encoding a full-length S protein, representing a ~4-fold dose sparing effect.

[0379] FIGs. 44A-44C. RNA Compositions Described Herein Induce a Prolonged Germinal Center Response. B cells found to bind a full-length S protein (FIG. 44A), an RBD (FIG. 44B), or an NTD (FIG. 44C) were probed for cell surface expression of germinal center markers. As shown, when administered at 0.4 pg, an RNA encoding a truncated SI subdomain (RNA017) produced a higher proportion of B cells showing a germinal center phenotype as compared to mice administered the same amount of RNA encoding a full-length S protein (RNA041). This observation suggests that RNA encoding a truncated SI subdomain produces a germinal center reaction that is extended as compared to RNA encoding a full-length S protein and is consistent with observations in other experiments described herein, in which neutralization titers induced by a truncated SI subdomain continued to increase for a longer period of time as compared to RNA encoding a full-length S protein.

[0380] FIGs. 45A-45E. Neutralizing Titers Induced in Vaccine-Naive Mice Administered Two Doses (~21 days apart) of RNA Compositions Described. FIG. 45A-FIG. 45B show geometric mean titers (GMTs) of antibodies that neutralize a KP.2 SARS-CoV-2 virus 7 and 14 days after administering a first dose of the indicated composition to vaccine naive mice. FIG. 45C-FIG. 45E show SARS-CoV-2 KP.2 neutralization titers 7, 14, and 21 days after administering a second dose of the indicated composition (28, 35, and 42 days after administering the first dose). As shown, by day 14, RNA encoding a truncated SI subdomain (RNA042) began to show increased neutralization titers as compared to RNA encoding a full-length SARS-CoV-2 S protein (RNA044), and by days 28-42, had produced neutralization titers that were over 2-fold higher than those induced by the same amount of RNA encoding a full-length S protein.

[0381] FIGs. 46A-46F. Neutralizing Titers Induced in Vaccine-Naive Mice Administered Two Doses (~28 days apart) of RNA Compositions Described. FIG. 46A-FIG. 46D show geometric mean titers (GMTs) of antibodies that neutralize a KP.2 SARS-CoV-2 virus 7, 14, 21, and 28 days after administering a first dose of the indicated composition to vaccine naive mice. FIG. 48E shows SARS-CoV-2 KP.2 neutralization titers 7 days after administering a second dose of the indicated composition (35 days after administering the first dose). FIG. 46F is a time plot, summarizing the data shown in FIG. 46A-FIG. 46E. The data again shows that RNA encoding a truncated SI subdomain (RNA042) begins to show increased neutralization titers as compared to RNA encoding a full-length SARS-CoV-2 S protein by day 14, which increases to be >4x those induced by RNA encoding a full-length SARS-CoV- 2 S protein by day 21 and ~8x higher by day 28. 35 days after the first dose, and 7 days after the second dose, titers had increased to be ~3x those induced by an RNA encoding a full-length S protein. Titers induced by a 0.1 pg dose of the RNA encoding a truncated SI subdomain were also found to be comparable to those induced by a 0.5 pg dose of RNA encoding a full-length S protein at each time point, indicating a ~5-fold dose-sparing effect.

[0382] FIGs. 47A-47D. Neutralization Titers Induced in Vaccine-Experienced Mice Administered RNA Compositions Described Herein. FIG. 47A-FIG. 47C show results from an experiment in which vaccine experienced mice were administered an RNA encoding a full-length S protein or RNA encoding a truncated SI. Prior to administering a candidate RNA, each mouse was first administered 2 doses of RNA encoding an S protein of a12947033vl Page 52 of 526Attorney Docket No.: 2013237-1485SARS-CoV-2 Wuhan strain (on days 0 and 21) and one dose of a bivalent composition comprising an RNA encoding an S protein of a SARS-CoV-2 Wuhan strain and an RNA encoding an S protein of a BA.4 / 5 Omicron SARS-CoV-2 variant (on day 49). 133 days after administering the first vaccine (dl33) candidate RNA were administered. (A), (B), and (C) show neutralization titers 0 days (dl33 of the experiment), 7 days (dl40 of the experiment), and 14 days (dl47 of the experiment) after administering the indicated candidate. FIG. 47D provides Geometric Mean Fold Increases (GMFI) of neutralizing antibody titers at dl47 as compared to mice administered carrier solution. As shown in FIG. 47C, by dl47, absolute titers of neutralization antibodies induced by RNA encoding a truncated SI subdomain were ~2-fold higher than those induced by RNA encoding a full-length S protein. As shown in FIG. 47D, GMFI of neutralizing antibody titers were ~3-fold higher for the RNA encoding a truncated SI subdomain as compared to RNA encoding a full-length, prefusion-stabilized S protein.

[0383] FIG. 48 is a schematic of the various domains (RBD) of a coronavirus spike protein expressed in sequence with a trimerization domain, according to some aspects disclosed herein.

[0384] FIG. 49 is a schematic of the various domains (RBD, NTD) of a coronavirus spike protein expressed in sequence with a trimerization domain, as well as a transmembrane domain and C-terminal domain, according to some aspects disclosed herein.

[0385] FIG. 50 is a schematic of the various domains (RBD, NTD) of a coronavirus spike protein expressed in sequence with and without a trimerization domain, linked to a self-assembling protein domain, according to some aspects disclosed herein.

[0386] FIG. 51 shows the oligomeric state characterization using size-exclusion-chromatography for the secreted antigen constructs.

[0387] FIGs. 52A-52D are graphs showing in-vitro-measured (via flow cytometry) SARS-CoV-2 antigen expression as a function of transfected RNA quantity, according to an illustrative embodiment.

[0388] FIGs. 53A-53B. BA.3.2 variant escapes JN.l lineage-adapted vaccine-elicited immunity in naive mice. FIG. 53A shows a schematic of an exemplary dosing schedule. Vaccine naive mice were administered a single dose (0.5 pg) of the indicated RNAs on day 0 then dosed again (0.5 pg) with the indicated RNAs at day 21. Serum samples were collected at day 49. FIG. 53B is a bar graph showing the 50% neutralization titers (geometric mean ration of 50% neutralization titers) of collected serum samples in FIG. 53A as measured by a pseudovirus neutralization assay. N= 10 mice per vaccine group.

[0389] FIGs. 54A-54B. BA.3.2 variant escapes JN.l lineage-adapted vaccine-elicited immunity in vaccine-experienced mice. FIG. 54A shows a schematic of an exemplary dosing schedule. Vaccine naive mice were administered a single dose (0.5 pg) of Wuhan (WT) RNA on day 0 then dosed again (0.5 pg) with Wuhan (WT) RNA at day 21, and Wuhan (WT) and Omicron BA.4 / 5 variant RNA on day 49. Mice were dosed (0.5 pg) with the indicated RNAs at day 162 and serum samples were collected at day 189. FIG. 54B is a bar graph showing the 50% neutralization titers (geometric mean ration of 50% neutralization titers) of collected serum samples in FIG. 54A as measured by a pseudovirus neutralization assay. N= 10 mice per vaccine group.Certain Definitions

[0390] In general, terminology used herein is in accordance with its understood meaning in the art, unless clearly indicated otherwise. Explicit definitions of certain terms are provided below; meanings of these and other terms in particular instances throughout this specification will be clear to those skilled in the art from context.12947033vl Page 53 of 526Attorney Docket No.: 2013237-1485

[0391] In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0392] About. The term "about", when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by "about" in that context. For example, in some embodiments, the term "about" may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0393] Agent. As used herein, the term "agent", may refer to a physical entity or phenomenon. In some embodiments, an agent may be characterized by a particular feature and / or effect. In some embodiments, an agent may be a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof. In some embodiments, the term "agent" may refer to a compound, molecule, or entity that comprises a polymer. In some embodiments, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term "agent" may refer to a compound, molecule, or entity that is substantially free of a particular polymer or polymeric moiety. In some embodiments, the term may refer to a compound, molecule, or entity that lacks or is substantially free of any polymer or polymeric moiety.

[0394] Amino acid: In its broadest sense, as used herein, the term "amino acid" refers to a compound and / or substance that can be, is, or has been incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. "Standard amino acid" refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. "Nonstandard amino acid" refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and / or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.

[0395] Antibody agent. As used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses a polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding. For example, in some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some embodiments an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR12947033vl Page 54 of 526Attorney Docket No.: 2013237-1485(e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1-5 amino acid substitutions as compared with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody agent in or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art to correspond to CDRsl, 2, and 3 of an antibody variable domain; in some such embodiments, an antibody agent in or comprises a polypeptide or set of polypeptides whose amino acid sequence(s) together include structural elements recognized by those skilled in the art to correspond to both heavy chain and light chain variable region CDRs, e.g., heavy chain CDRs 1, 2, and / or 3 and light chain CDRs 1, 2, and / or 3. In some embodiments, an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain. In some embodiments, an antibody agent may be or comprise a polyclonal antibody preparation. In some embodiments, an antibody agent may be or comprise a monoclonal antibody preparation. In some embodiments, an antibody agent may include one or more constant region sequences that are characteristic of a particular organism, such as a camel, human, mouse, primate, rabbit, rat; in many embodiments, an antibody agent may include one or more constant region sequences that are characteristic of a human. In some embodiments, an antibody agent may include one or more sequence elements that would be recognized by one skilled in the art as a humanized sequence, a primatized sequence, a chimeric sequence, etc. In some embodiments, an antibody agent may be a canonical antibody (e.g., may comprise two heavy chains and two light chains). In some embodiments, an antibody agent may be in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals ("SMIPs™"); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins® ; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®;12947033vl Page 55 of 526Attorney Docket No.: 2013237-1485Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group (e.g., poly-ethylene glycol, etc.)).

[0396] Antigen-. Those skilled in the art, reading the present specification, will appreciate that the term "antigen" refers to a molecule that is recognized by the immune system, e.g., in particular embodiments, the adaptive immune system, such that it elicits an antigen-specific immune response. In some embodiments, an antigen-specific immune response may be or comprise generation of antibodies and / or antigen-specific T cells. In some embodiments, an antigen is a peptide or polypeptide that comprises at least one epitope against which an immune response can be generated. In one embodiment, an antigen is presented by cells of the immune system such as antigen presenting cells like dendritic cells or macrophages. In one embodiment, an antigen or a processed product thereof such as a T-cell antigen is bound by a T- or B-cell receptor, or by an immunoglobulin molecule such as an antibody. Accordingly, an antigen or a processed product thereof may react specifically with antibodies or T lymphocytes (T cells). In one embodiment, an antigen is a parasitic antigen. In accordance with the present disclosure, in some embodiments, an antigen may be delivered by RNA molecules as described herein. In some embodiments, a peptide or polypeptide antigen can be 2-100 amino acids, including for example, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids in length. In some embodiments, a peptide or polypeptide antigen can be greater than 50 amino acids. In some embodiments, a peptide or polypeptide antigen can be greater than 100 amino acids. In some embodiments, an antigen is recognized by an immune effector cell. In some embodiments, an antigen, if recognized by an immune effector cell, is able to induce in the presence of appropriate co-stimulatory signals, stimulation, priming and / or expansion of the immune effector cell carrying an antigen receptor recognizing the antigen. In the context of the embodiments of the present disclosure, in some embodiments, an antigen can be presented or present on the surface of a cell, e.g., an antigen presenting cell. In one embodiment, an antigen is presented by a diseased cell such as a virus- infected cell. In one embodiment, an antigen receptor is a TCR which binds to an epitope of an antigen presented in the context of MHC. In one embodiment, binding of a TCR when expressed by T cells and / or present on T cells to an antigen presented by cells such as antigen presenting cells results in stimulation, priming and / or expansion of said T cells. In one embodiment, binding of a TCR when expressed by T cells and / or present on T cells to an antigen presented on diseased cells results in cytolysis and / or apoptosis of the diseased cells, wherein said T cells preferably release cytotoxic factors, e.g., perforins and granzymes.

[0397] Associated-. Two events or entities are "associated" with one another, as that term is used herein, if the presence, level, degree, type and / or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of, susceptibility to, severity of, stage of, etc. the disease, disorder, or condition {e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or12947033vl Page 56 of 526Attorney Docket No.: 2013237-1485 more entities that are physically associated with one another are not covalently linked to one another but are non- covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0398] Binding-. Those skilled in the art, reading the present specification, will appreciate that the term "binding" typically refers to a non-covalent association between or among entities or moieties. In some embodiments, binding data are expressed in terms of "IC50". As is understood in the art, IC50 is the concentration of an assessed agent in a binding assay at which 50% inhibition of binding of reference agent known to bind the relevant binding partner is observed. In some embodiments, assays are run under conditions in which {e.g., limiting binding target and reference concentrations), IC50 values approximate KD values. Assays for determining binding are well known in the art and are described in detail, for example, in PCT publications WO 94 / 20127 and WO 94 / 03205, and other publications such Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); and Sette, et al., Mol. Immunol. 31:813 (1994). Alternatively, binding can be expressed relative to binding by a reference standard peptide. For example, can be based on its IC50, relative to the IC50 of a reference standard peptide. Binding can also be determined using other assay systems including those using live cells (e.g., Ceppellini et al., Nature 339:392 (1989); Christnick et al., Nature 352:67 (1991); Busch et al., Int. Immunol. 2:443 (1990); Hill et al., J. Immunol. 147:189 (1991); del Guercio et al., J. Immunol. 154:685 (1995)), cell free systems using detergent lysates (e.g., Cerundolo et al., J. Immunol 21:2069 (1991)), immobilized purified MHC (e.g., Hill et al., J. Immunol. 152, 2890 (1994); Marshall et al., J. Immunol. 152:4946 (1994)), ELISA systems (e.g., Reay et al., EMBO J. 11:2829 (1992)), surface plasmon resonance (e.g., Khilko et al., J. Biol. Chem. 268:15425 (1993)); high flux soluble phase assays (Hammer et al., J. Exp. Med. 180:2353 (1994)), and measurement of class I MHC stabilization or assembly (e.g., Ljunggren et al., Nature 346:476 (1990); Schumacher et al., Cell 62:563 (1990); Townsend et al., Cell 62:285 (1990); Parker et al., J. Immunol. 149:1896 (1992)).

[0399] Cap As used herein, the term "cap" refers to a structure comprising or essentially consisting of a nudeoside-5 '-triphosphate that is typically joined to a 5'-end of an uncapped RNA {e.g., an uncapped RNA having a 5'- diphosphate). In some embodiments, a cap is or comprises a guanine nucleotide. In some embodiments, a cap is or comprises a naturally occurring RNA 5' cap, including, e.g., but not limited to a 7- methylguanosine cap, which has a structure designated as "m7G." In some embodiments, a cap is or comprises a synthetic cap analog that resembles an RNA cap structure and possesses the ability to stabilize RNA if attached thereto, including, e.g., but not limited to anti-reverse cap analogs (ARCAs) known in the art). Those skilled in the art will appreciate that methods for joining a cap to a 5' end of an RNA are known in the art. For example, in some embodiments, a capped RNA may be obtained by in vitro capping of RNA that has a 5' triphosphate group or RNA that has a 5' diphosphate group with a capping enzyme system (including, e.g., but not limited to vaccinia capping enzyme system or Saccharomyces cerevisiae capping enzyme system). Alternatively, a capped RNA can be obtained by in vitro transcription (IVT) of a singlestranded DNA template in the presence of a dinucleotide or trinucleotide cap analog.

[0400] Cell-mediated immunity. "Cell-mediated immunity," "cellular immunity," "cellular immune response," or similar terms are meant to include a cellular response directed to cells characterized by expression of an antigen, in particular characterized by presentation of an antigen with class I or class II MHC. A cellular response relates to immune effector cells, in particular to T cells or T lymphocytes which act as either "helpers" or "killers." The helper T cells (also termed CD4+T cells or CD4 T cells) play a central role by regulating the immune response and the killer12947033vl Page 57 of 526Attorney Docket No.: 2013237-1485 cells (also termed cytotoxic T cells, cytolytic T cells, CD8+T cells, CD8 T cells, or CTLs) kill diseased cells such as virus-infected cells, preventing the production of more diseased cells.

[0401] Co-administration-. As used herein, the term "co-administration" refers to use of a pharmaceutical composition {e.g., immunogenic composition, e.g., vaccine) described herein and an additional therapeutic agent. The combined use of a pharmaceutical composition {e.g., immunogenic composition, e.g., vaccine) described herein and an additional therapeutic agent may be performed concurrently or separately {e.g., sequentially in any order). In some embodiments, a pharmaceutical composition {e.g., immunogenic composition, e.g., vaccine) described herein and an additional therapeutic agent may be combined in one pharmaceutically acceptable carrier, or they may be placed in separate carriers and delivered to a target cell or administered to a subject at different times. Each of these situations is contemplated as falling within the meaning of "co-administration" or "combination," provided that a pharmaceutical composition {e.g., immunogenic composition, e.g., vaccine) described herein and an additional therapeutic agent are delivered or administered sufficiently close in time that there is at least some temporal overlap in biological effect(s) generated by each on a target cell or a subject being treated.

[0402] Codon-optimized. As used herein, the term "codon-optimized" refers to alteration of codons in a coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism without preferably altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, in some embodiments coding regions are codon-optimized for optimal expression in a subject to be treated using the RNA molecules described herein. In some embodiments, codon -optimization may be performed such that codons for which frequently occurring tRNAs are available are inserted in place of "rare codons." In some embodiments, codonoptimization may include increasing guanosine / cytosine (G / C) content of a coding region of RNA described herein as compared to the G / C content of the corresponding coding sequence of a wild type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence.

[0403] Combination therapy. As used herein, the term "combination therapy" refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens {e.g., two or more therapeutic agents). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially {e.g., all "doses" of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, "administration" of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition.

[0404] Comparable: As used herein, the term "comparable" refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances,12947033vl Page 58 of 526Attorney Docket No.: 2013237-1485 individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0405] Corresponding ta As used herein, the term "corresponding to" refers to a relationship between two or more entities. For example, the term "corresponding to" may be used to designate the position / identity of a structural element in a compound or composition relative to another compound or composition (e.g., to an appropriate reference compound or composition). For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as "corresponding to" a residue in an appropriate reference polymer. For example, those of ordinary skill will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid "corresponding to" a residue at position 190, for example, need not actually be the 190thamino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify "corresponding" amino acids. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify "corresponding" residues in polypeptides and / or nucleic acids in accordance with the present disclosure. Those of skill in the art will also appreciate that, in some instances, the term "corresponding to" may be used to describe an event or entity that shares a relevant similarity with another event or entity (e.g., an appropriate reference event or entity). To give but one example, a gene or protein in one organism may be described as "corresponding to" a gene or protein from another organism in order to indicate, in some embodiments, that it plays an analogous role or performs an analogous function and / or that it shows a particular degree of sequence identity or homology or shares a particular characteristic sequence element.

[0406] As those skilled in the art are aware, sequence alignment strategies enable consideration, for example, of "gaps" in sequences, and / or of "repeated" residues. Moreover, those skilled in the art understand that in some cases, it is not possible to unambiguously determine the exact location of a sequence change relative to a reference sequence. For example, when a reference sequence includes a stretch of two or more contiguous identical residues, and a changed sequence has one fewer of the residues, it is not possible to assign a particular singular residue in the reference sequence as the one that was deleted, as deletion of any one of the identical contiguous residues would generate the same changed sequence. Those skilled in the art therefore appreciate the convention of arbitrarily assigning one of the reference residue positions as the deleted residue. To give a specific example, SEQ ID NO: 1 is a polypeptide sequence in which two adjacent Y residues are present at positions 144 and 145. If one of these amino acid residues is deleted, a person of skill in the art will not be able to determine whether amino acid 144 or 145 has been deleted in the changed sequence. They will understand, however, that either deletion describes the same polypeptide sequence, and therefore will be able to unambiguously determine the sequence of a polypeptide described as having a deletion at position 144 or 145 of SEQ ID NO: 1 (i.e., they will understand that a polypeptide described as having a deletion at a position corresponding to position 144 of SEQ ID NO: 1 and a polypeptide12947033vl Page 59 of 526Attorney Docket No.: 2013237-1485 described as having a deletion at a position corresponding to position 145 of SEQ ID NO: 1 have the same amino acid sequence).

[0407] Derived-. In the context of an amino acid sequence (peptide or polypeptide) "derived from" a designated amino acid sequence (peptide or polypeptide), refers to a structural analogue of a designated amino acid sequence. In some embodiments, an amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof. Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof. For example, it will be understood by one of ordinary skill in the art that the antigens suitable for use herein may be altered such that they vary in sequence from the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences.

[0408] Designed: As used herein, the term "designed" refers to an agent (i) whose structure is or was selected by the hand of man; (ii) that is produced by a process requiring the hand of man; and / or (iii) that is distinct from natural substances and other known agents.

[0409] Dosing regimen-. Those skilled in the art will appreciate that the term "dosing regimen" may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (Z , is a therapeutic dosing regimen).

[0410] Encode. As used herein, the term "encode" or "encoding" refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e.g., mRNA) or a defined sequence of amino acids. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a gene, a cDNA, or an RNA molecule (e.g., an mRNA) encodes a polypeptide if transcription and translation of RNA (e.g., mRNA) corresponding to that gene produces the polypeptide in a cell or other biological system. In some embodiments, a coding region of an RNA molecule encoding a target antigen refers to a coding strand, the nucleotide sequence of which is identical to the RNA (e.g., mRNA) sequence of such a target antigen. In some embodiments, a coding region of an RNA molecule encoding a target antigen refers to a non-coding strand of such a target antigen, which may be used as a template for transcription of a gene or cDNA.

[0411] Endogenous. As used herein, the term "endogenous", with respect to a secretory signal or a transmembrane region operably linked to a polypeptide (e.g., spike protein variant, or an immunogenic portion thereof), refers to a secretory signal or transmembrane region from virus or viral strain that is the same as the virus12947033vl Page 60 of 526Attorney Docket No.: 2013237-1485 or viral strain from which the polypeptide (e.g., spike protein variant, or an immunogenic portion thereof) was derived.

[0412] Engineered: In general, the term "engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to be "engineered" when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and / or when a particular residue in a polynucleotide is non-naturally occurring and / or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature.

[0413] Epitope. As used herein, the term "epitope" refers to a moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. For example, an epitope may be recognized by a T cell, a B cell, or an antibody. In some embodiments, an epitope is comprised of a plurality of chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen adopts a relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically near to each other in space when the antigen adopts such a conformation. In some embodiments, at least some such chemical atoms are groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized). Accordingly, in some embodiments, an epitope of an antigen may include a continuous or discontinuous fragment of the antigen. In some embodiments, an epitope is or comprises a T cell epitope. In some embodiments, an epitope may have a length of about 5 to about 30 amino acids, or about 10 to about 25 amino acids, or about 5 to about 15 amino acids, or about 5 to 12 amino acids, or about 6 to about 9 amino acids.

[0414] Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the generation of a gene product from the nucleic acid sequence. In some embodiments, a gene product can be a transcript. In some embodiments, a gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc.); (3) translation of an RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein.

[0415] Five prime untranslated region-. As used herein, the terms "five prime untranslated region" or "5' UTR" refer to a sequence of an RNA (e.g., mRNA) molecule between a transcription start site and a start codon of a coding region of an RNA. In some embodiments, "5' UTR" refers to a sequence of an RNA (e.g., mRNA) molecule that begins at a transcription start site and ends one nucleotide (nt) before a start codon (usually AUG) of a coding region of an RNA molecule, e.g., in its natural context.

[0416] Fragment. The term "fragment" as used herein in the context of a nucleic acid sequence (e.g., RNA sequence) or an amino acid sequence may typically be a fragment of a reference sequence. In some embodiments, a reference sequence is a full-length sequence of e.g., a nucleic acid sequence or an amino acid sequence. Accordingly, a fragment, typically, refers to a sequence that is identical to a corresponding stretch within a reference sequence. In some embodiments, a fragment comprises a continuous stretch of nucleotides or amino acid residues that corresponds to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the total length of a reference sequence from which the fragment is derived. In some embodiments, the term "fragment", with reference to an amino acid sequence (peptide or polypeptide), relates to a part of an amino acid sequence, e.g., a sequence which represents the amino acid sequence shortened at the N-terminus and / or C-terminus. In some embodiments, a12947033vl Page 61 of 526Attorney Docket No.: 2013237-1485 fragment of an amino acid sequence comprises at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from an amino acid sequence.

[0417] Heterologous: As used herein, the term "heterologous", with respect to a secretory signal or a transmembrane region operably linked to a polypeptide (e.g., spike protein variant, or an immunogenic portion thereof), refers to a secretory signal or transmembrane region from a different virus or viral strain from which the polypeptide (e.g., spike protein variant, or an immunogenic portion thereof) was derived.

[0418] Homology: As used herein, the term "homology" or "homolog" refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be "homologous" to one another if their sequences are at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions). For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as similar to one another as "hydrophobic" or "hydrophilic" amino acids, and / or as having "polar" or "non-polar" side chains. Substitution of one amino acid for another of the same type may often be considered a "homologous" substitution.

[0419] Humoral immunity: As used herein, the term "humoral immunity" or "humoral immune response" refers to antibody production and the accessory processes that accompany it, including: Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell generation. It also refers to the effector functions of antibodies, which include pathogen neutralization, classical complement activation, and opsonin promotion of phagocytosis and pathogen elimination.

[0420] Identity: As used herein, the term "identity" refers to the overall relatedness between polynucleotide molecules {e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polynucleotide molecules {e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules are considered to be "substantially identical" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.12947033vl Page 62 of 526Attorney Docket No.: 2013237-1485For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller, 1989, which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.

[0421] Immunologically equivalent: The term "immunologically equivalent" means that an immunologically equivalent molecule such as the immunologically equivalent amino acid sequence exhibits the same or essentially the same immunological properties and / or exerts the same or essentially the same immunological effects, e.g., with respect to the type of the immunological effect. In the context of the present disclosure, in some embodiments, the term "immunologically equivalent" is used with respect to the immunological effects or properties of antigens or antigen variants used for immunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if said amino acid sequence when exposed to the immune system of a subject induces an immune reaction having a specificity of reacting with the reference amino acid sequence.

[0422] In one embodiment, an antigen receptor is an antibody or B cell receptor which binds to an epitope of an antigen. In one embodiment, an antibody or B cell receptor binds to native epitopes of an antigen.

[0423] Increased, Induced, or Reduced: As used herein, these terms or grammatically comparable comparative terms, indicate values that are relative to a comparable reference measurement. For example, in some embodiments, an assessed value achieved with a provided pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) may be "increased" relative to that obtained with a comparable reference pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine). Alternatively or additionally, in some embodiments, an assessed value achieved in a subject may be "increased" relative to that obtained in the same subject under different conditions (e.g., prior to or after an event; or presence or absence of an event such as administration of a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) as described herein, or in a different, comparable subject (e.g., in a comparable subject that differs from the subject of interest in prior exposure to a condition, e.g., absence of administration of a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) as described herein.). In some embodiments, comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and / or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and / or prevalence of difference that is required or sufficient to achieve such statistical significance. In some embodiments, the term "reduced", or equivalent terms refers to a reduction in the level of an assessed value by at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or higher, as compared to a comparable reference. In some embodiments, the term "reduced", or equivalent terms refers to a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero. In some embodiments, the term "increased" or "induced" refers to an increase in the level of an assessed value by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or higher, as compared to a comparable reference.

[0424] Ionizable. The term "ionizable" refers to a compound or group or atom that is charged at a certain pH. In the context of an ionizable amino lipid, such a lipid or a function group or atom thereof bears a positive charge at a certain pH. In some embodiments, an ionizable amino lipid is positively charged at an acidic pH. In some embodiments, an ionizable amino lipid is predominately neutral at physiological pH values, e.g., in some12947033vl Page 63 of 526Attorney Docket No.: 2013237-1485 embodiments about 7.0-7.4, but becomes positively charged at lower pH values. In some embodiments, an ionizable amino lipid may have a pKa within a range of about 5 to about 7.

[0425] Isolated: The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated", but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated". An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0426] Lipid. As used herein, the terms "lipid" and "lipid-like material" are broadly defined as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also typically denoted as amphiphiles.

[0427] RNA lipid nanoparticie. As used herein, the term "RNA lipid nanoparticle" refers to a nanoparticle comprising at least one lipid and RNA molecule(s). In some embodiments, an RNA lipid nanoparticie comprises at least one ionizable amino lipid. In some embodiments, an RNA lipid nanoparticie comprises at least one ionizable amino lipid, at least one helper lipid, and at least one polymer-conjugated lipid (e.g., PEG-conjugated lipid). In various embodiments, RNA lipid nanoparticles as described herein can have an average size {e.g., Z-average) of about 100 nm to 1000 nm, or about 200 nm to 900 nm, or about 200 nm to 800 nm, or about 250 nm to about 700 nm. In some embodiments of the present disclosure, RNA lipid nanopartides can have a particle size (e.g., Z- average) of about 30 nm to about 200 nm, or about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, an average size of lipid nanopartides is determined by measuring the particle diameter. In some embodiments, RNA lipid nanopartides may be prepared by mixing lipids with RNA molecules described herein.

[0428] Lipidoid: As used herein, a "lipidoid" refers to a lipid-like molecule. In some embodiments, a lipoid is an amphiphilic molecule with one or more lipid-like physical properties. In the context of the present disclosure, the term lipid is considered to encompass lipidoids.

[0429] Nanoparticie: As used herein, the term "nanoparticie" refers to a particle having an average size suitable for parenteral administration. In some embodiments, a nanoparticie has a longest dimension (e.g., a diameter) of less than 1,000 nanometers (nm). In some embodiments, a nanoparticie may be characterized by a longest dimension (e.g., a diameter) of less than 300 nm. In some embodiments, a nanoparticie may be characterized by a longest dimension (e.g., a diameter) of less than 100 nm. In many embodiments, a nanoparticie may be characterized by a longest dimension between about 1 nm and about 100 nm, or between about 1 pm and about 500 nm, or between about 1 nm and 1,000 nm. In many embodiments, a population of nanopartides is characterized by an average size (e.g., longest dimension) that is below about 1,000 nm, about 500 nm, about 100 nm, about 50 nm, about 40 nm, about 30 nm, about 20 nm, or about 10 nm and often above about 1 nm. In many embodiments, a nanoparticie may be substantially spherical so that its longest dimension may be its diameter. In some embodiments, a nanoparticie has a diameter of less than 100 nm as defined by the National Institutes of Health.

[0430] Naturally occurring: The term "naturally occurring" as used herein refers to an entity that can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and can be12947033vl Page 64 of 526Attorney Docket No.: 2013237-1485 isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.

[0431] Neutralization: i s used herein, the term "neutralization" refers to an event in which binding agents such as antibodies bind to a biological active site of a virus such as a receptor binding protein, thereby inhibiting the parasitic infection of cells. In some embodiments, the term "neutralization" refers to an event in which binding agents eliminate or significantly reduce ability of infecting cells.

[0432] Nucleic acid particle. A "nucleic acid particle" can be used to deliver nucleic acid to a target site of interest (e.g., cell, tissue, organ, and the like). A nucleic acid particle may comprise at least one cationic or cationically ionizable lipid or lipid-like material, at least one cationic polymer such as protamine, or a mixture thereof and nucleic acid. In some embodiments, a nucleic acid particle is a lipid nanoparticle. In some embodiments, a nucleic acid particle is a lipoplex particle.

[0433] Nucleic acid / Polynucleotide. As used herein, the term "nucleic acid" refers to a polymer of at least 10 nucleotides or more. In some embodiments, a nucleic acid is or comprises DNA. In some embodiments, a nucleic acid is or comprises RNA. In some embodiments, a nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, a nucleic acid is or comprises a single stranded nucleic acid. In some embodiments, a nucleic acid is or comprises a double-stranded nucleic acid. In some embodiments, a nucleic acid comprises both single and doublestranded fragments. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N-phosphoramidite linkages and / or one or more peptide bonds, e.g., as in a "peptide nucleic acid". In some embodiments, a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises on or more, or all, non-natural residues. In some embodiments, a non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 - propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, 6-O-methylguanine, 2-th iocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that comprises one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides long.12947033vl Page 65 of 526Attorney Docket No.: 2013237-1485

[0434] Nucleotide: As used herein, the term "nucleotide" refers to its art- recognized meaning. When a number of nucleotides is used as an indication of size, e.g., of a polynucleotide, a certain number of nucleotides refers to the number of nucleotides on a single strand, e.g., of a polynucleotide.

[0435] Patient: As used herein, the term "patient" refers to any organism who is suffering or at risk of a disease or disorder or condition. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, nonhuman primates, and / or humans). In some embodiments, a patient is a human. In some embodiments, a patient is suffering from or susceptible to one or more diseases or disorders or conditions. In some embodiments, a patient displays one or more symptoms of a disease or disorder or condition. In some embodiments, a patient has been diagnosed with one or more diseases or disorders or conditions. In some embodiments, a disease or disorder or condition that is amenable to provided technologies is or includes a HSV infection. In some embodiments, a patient is receiving or has received certain therapy to diagnose and / or to treat a disease, disorder, or condition. In some embodiments, a patient is a patient suffering from or susceptible to a HSV infection.

[0436] PEG-conjugated iipid. The term "PEG-conjugated lipid" refers to a molecule comprising a lipid portion and a polyethylene glycol portion.

[0437] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for parenteral administration, for example, by subcutaneous, intramuscular, or intravenous injection as, for example, a sterile solution or suspension formulation.

[0438] Pharmaceutically effective amount: The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of the treatment of a particular disease, a desired reaction in some embodiments relates to inhibition of the course of the disease. In some embodiments, such inhibition may comprise slowing down the progress of a disease and / or interrupting or reversing the progress of the disease. In some embodiments, a desired reaction in a treatment of a disease may be or comprise delay or prevention of the onset of a disease or a condition. An effective amount of pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) described herein will depend, for example, on a disease or condition to be treated, the severity of such a disease or condition, individual parameters of the patient, including, e.g., age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, doses of pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[0439] Poiy(A) sequence: As used herein, the term "poly(A) sequence" or "poly-A tail" refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3'-end of an RNA molecule. Poly(A) sequences are known to those of skill in the art and may follow the 3'-UTR in the RNAs described herein. An uninterrupted poly(A) sequence is characterized by consecutive adenylate residues. In nature, an uninterrupted poly(A) sequence is typical. RNAs disclosed herein can have a poly(A) sequence attached to the free12947033vl Page 66 of 526Attorney Docket No.: 2013237-14853'-end of the RNA by a template-independent RNA polymerase after transcription or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0440] Polypeptide: As used herein, the term "polypeptide" refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide's N-terminus, at the polypeptide's C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications comprise acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and / or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and / or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term "polypeptide" may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and / or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and / or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and / or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and / or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide.

[0441] Prevent: As used herein, the term "prevent" or "prevention" when used in connection with the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing the disease, disorder and / or condition and / or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time.12947033vl Page 67 of 526Attorney Docket No.: 2013237-1485

[0442] Recombinant: The term "recombinant" in the context of the present disclosure means "made through genetic engineering". In some embodiments, a "recombinant" entity such as a recombinant nucleic acid in the context of the present disclosure is not naturally occurring.

[0443] Reference: As used herein, the term "reference" describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control.

[0444] Ribonucleic acid (RNA): As used herein, the term "RNA" refers to a polymer of ribonucleotides. In some embodiments, an RNA is single stranded. In some embodiments, an RNA is double stranded. In some embodiments, an RNA comprises both single and double stranded fragments. In some embodiments, an RNA can comprise a backbone structure as described in the definition of "Nucleic acid I Polynucleotide" above. An RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA). In some embodiments where an RNA is a mRNA. In some embodiments where an RNA is a mRNA, an RNA typically comprises at its 3' end a poly(A) region. In some embodiments where an RNA is a mRNA, an RNA typically comprises at its 5' end an art-recognized cap structure, e.g., for recognizing and attachment of a mRNA to a ribosome to initiate translation. In some embodiments, an RNA is a synthetic RNA. Synthetic RNAs include RNAs that are synthesized in vitro (e.g., by enzymatic synthesis methods and / or by chemical synthesis methods).

[0445] Ribonucleotide: As used herein, the term "ribonucleotide" encompasses unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides may include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g. , replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 21position or 41position) or replacement of the sugar, and (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. The term "ribonucleotide" also encompasses ribonucleotide triphosphates including modified and non-modified ribonucleotide triphosphates.

[0446] Risk: As will be understood from context, "risk" of a disease, disorder, and / or condition refers to a likelihood that a particular individual will develop the disease, disorder, and / or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%. In some embodiments risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples. In some embodiments, a reference sample or group of reference samples have a known risk of a disease, disorder, condition and / or event. In some embodiments a reference sample or group of reference samples are from individuals comparable to a particular individual. In some embodiments, relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In some embodiments, risk may reflect one or more genetic attributes, e.g.,12947033vl Page 68 of 526Attorney Docket No.: 2013237-1485 which may predispose an individual toward development (or not) of a particular disease, disorder and / or condition. In some embodiments, risk may reflect one or more epigenetic events or attributes and / or one or more lifestyle or environmental events or attributes.

[0447] RNA lipoplex particle, As used herein, the term "RNA lipoplex particle" refers to a complex comprising liposomes, in particular cationic liposomes, and RNA molecules. Without wishing to bound by a particular theory, electrostatic interactions between positively charged liposomes and negatively charged RNA results in complexation and spontaneous formation of RNA lipoplex particles. In some embodiments, positively charged liposomes may comprise a cationic lipid, such as in some embodiments DOTMA, and additional lipids, such as in some embodiments DOPE. In one embodiment, an RNA lipoplex particle is a nanoparticle.

[0448] Selective or specific: The term "selective" or "specific", when used herein in reference to an agent having an activity, is understood by those skilled in the art to mean that the agent discriminates between potential target entities, states, or cells. For example, in some embodiments, an agent is said to bind "specifically" to its target if it binds preferentially with that target in the presence of one or more competing alternative targets. In many embodiments, specific interaction is dependent upon the presence of a particular structural feature of the target entity (e.g., an epitope, a cleft, a binding site). It is to be understood that specificity need not be absolute. In some embodiments, specificity may be evaluated relative to that of a target-binding moiety for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to that of a reference specific binding moiety. In some embodiments, specificity is evaluated relative to that of a reference non-specific binding moiety.

[0449] Stable: As used herein, the term "stable" in the context of the present disclosure refers to a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) as a whole and / or components thereof (e.g., RNA, including, e.g., LNP-formulated RNA) meeting or exceeding pre-determined acceptance criteria. For example, in some embodiments, a stable pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) exhibits no unacceptable levels of microbial growth, and substantially no or no breakdown or degradation of the active biological molecule component(s). In some embodiments, a stable pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) refers to the integrity of RNA molecules being maintained at least above 90% or more when stored under certain conditions for a certain amount of time.. In some embodiments, a stable pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) refers to at least 90% or more (including, e.g., at least 95%, at least 96%, at least 97%, or more) of RNA molecules being maintained to be encapsulated within lipid nanoparticles. In some embodiments, a stable pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) refers to a formulation that remains capable of eliciting a desired immunologic response when administered to a subject. In some embodiments, a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) remains stable for a specified period of time under certain conditions.

[0450] Subject. As used herein, the term "subject" refers to an organism to be administered with a composition described herein, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans. In some embodiments, a subject is a human subject. In some embodiments, a subject is suffering from a disease, disorder, or condition (e.g., a HSV infection). In some embodiments, a subject is susceptible to a disease, disorder, or condition (e.g., a HSV infection). In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder, or condition (e.g., a HSV infection). In some embodiments, a subject displays12947033vl Page 69 of 526Attorney Docket No.: 2013237-1485 one or more non-specific symptoms of a disease, disorder, or condition (e.g., a HSV infection). In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition (e.g., a HSV infection). In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition (e.g., a HSV infection). In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0451] Suffering from-. An individual who is "suffering from" a disease, disorder, and / or condition has been diagnosed with and / or displays one or more symptoms of a disease, disorder, and / or condition.

[0452] Susceptible ter. An individual who is "susceptible to" a disease, disorder, and / or condition is one who has a higher risk of developing the disease, disorder, and / or condition than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition may not have been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0453] Synthetic: As used herein, the term "synthetic" refers to an entity that is artificial, or that is made with human intervention, or that results from synthesis rather than naturally occurring. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule that is chemically synthesized, e.g., in some embodiments by solid-phase synthesis. In some embodiments, the term "synthetic" refers to an entity that is made outside of biological cells. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule (e.g., an RNA) that is produced by in vitro transcription using a template.

[0454] Therapy: The term "therapy" refers to an administration or delivery of an agent or intervention that has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect (e.g., has been demonstrated to be statistically likely to have such effect when administered to a relevant population). In some embodiments, a therapeutic agent or therapy is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, a therapeutic agent or therapy is a medical intervention (e.g., surgery, radiation, phototherapy) that can be performed to alleviate, relieve, inhibit, present, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition.

[0455] Three prime untranslated region-. As used herein, the terms "three prime untranslated region" or "3' UTR" refer to a sequence of an RNA (e.g., mRNA) molecule that begins following a stop codon of a coding region of an open reading frame sequence. In some embodiments, the 3' UTR begins immediately after a stop codon of a coding region of an open reading frame sequence, e.g., in its natural context. In other embodiments, the 3' UTR does not begin immediately after stop codon of the coding region of an open reading frame sequence, e.g., in its natural context.

[0456] Threshold level (e.g., acceptance criteria)-. As used herein, the term "threshold level" refers to a level that are used as a reference to attain information on and / or classify the results of a measurement, for example,12947033vl Page 70 of 526Attorney Docket No.: 2013237-1485 the results of a measurement attained in an assay. For example, in some embodiments, a threshold level means a value measured in an assay that defines the dividing line between two subsets of a population (e.g., a batch that satisfy quality control criteria vs. a batch that does not satisfy quality control criteria). Thus, a value that is equal to or higher than the threshold level defines one subset of the population, and a value that is lower than the threshold level defines the other subset of the population. A threshold level can be determined based on one or more control samples or across a population of control samples. A threshold level can be determined prior to, concurrently with, or after the measurement of interest is taken. In some embodiments, a threshold level can be a range of values.

[0457] Treat: As used herein, the term "treat," "treatment," or "treating" refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and / or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject at a later-stage of disease, disorder, and / or condition.

[0458] Vaccination-. As used herein, the term "vaccination" refers to the administration of a composition intended to generate an immune response, for example to a disease-associated (e.g., disease-causing) agent. In some embodiments, vaccination can be administered before, during, and / or after exposure to a disease-associated agent, and in certain embodiments, before, during, and / or shortly after exposure to the agent. In some embodiments, vaccination includes multiple administrations, appropriately spaced in time, of a vaccine composition. In some embodiments, vaccination generates an immune response to an infectious agent.

[0459] Vaccine: As used herein, the term "vaccine" refers to a composition that induces an immune response upon administration to a subject. In some embodiments, an induced immune response provides protective immunity.

[0460] Variant: As used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term "variant" refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a "variant" of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or12947033vl Page 71 of 526Attorney Docket No.: 2013237-1485 nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a "variant" of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions. Typically, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in a variant are substituted, inserted, or deleted, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues as compared to a reference. Often, a variant polypeptide or nucleic acid comprises a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional residues (i.e., residues that participate in a particular biological activity) relative to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises not more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly fewer than about 5, about 4, about 3, or about 2 additions or deletions as compared to the reference. In some embodiments, a reference polypeptide or nucleic acid is one found in nature.

[0461] Vector, as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." In some embodiments, known techniques may be used, for example, for generation or manipulation of recombinant DNA, for oligonucleotide synthesis, and for tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), which is incorporated herein by reference for any purpose.

[0462] All literature and similar material cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, and web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls. The section headings used12947033vl Page 72 of 526Attorney Docket No.: 2013237-1485 herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.Detailed Description

[0463] In some embodiments, the present provides technologies (e.g., compositions, pharmaceutical compositions, immunogenic compositions, vaccines, and methods) that can be used to induce an immune response against SARS-CoV-2. In some embodiments, technologies provided in the present disclosure can be used to mitigate immune imprinting effects and / or induce a stronger de novo immune response (e.g., as compared to other vaccination approaches).SARS-CoV-2 Overview

[0464] SARS-CoV-2 Spike (S) protein can be proteolytically cleaved into SI (685 aa) and S2 (588 aa) subunits. SI of SARS-CoV-2 comprises a receptor-binding domain (RBD), which mediates virus entry into host cells through the host angiotensin-converting enzyme 2 (ACE2) receptor.

[0465] The presentation of COVID-19 is generally with cough and fever, with chest radiography showing ground-glass opacities or patchy shadowing. However, many patients present without fever or radiographic changes, and infections may be asymptomatic which is relevant to controlling transmission. For symptomatic subjects, progression of disease may lead to acute respiratory distress syndrome requiring ventilation and subsequent multiorgan failure and death. Common symptoms in hospitalized patients (in order of highest to lowest frequency) include fever, dry cough, shortness of breath, fatigue, myalgias, nausea / vomiting or diarrhoea, headache, weakness, and rhinorrhoea. Anosmia (loss of smell) or ageusia (loss of taste) may be the sole presenting symptom in approximately 3% of individuals who have COVID-19.

[0466] All ages may present with the disease, but notably case fatality rates (CFR) are elevated in persons >60 years of age. Comorbidities are also associated with increased CFR, including cardiovascular disease, diabetes, hypertension, and chronic respiratory disease. Healthcare workers are overrepresented among COVID-19 patients due to occupational exposure to infected patients.

[0467] In most situations, a molecular test is used to detect SARS-CoV-2 and confirm infection. The reverse transcription polymerase chain reaction (RT-PCR) test methods targeting SARS-CoV-2 viral RNA is one method for diagnosing suspected cases of COVID-19. Samples to be tested are collected from the nose and / or throat with a swab.SARS-CoV-2 Variants

[0468] Since the initial discovery of SARS-CoV-2, a number of variants have arisen around the world. The emergence of these novel circulating variants of SARS-CoV-2 has raised significant concerns about geographic and temporal efficacy of vaccine interventions. The emergence of Omicron (B.l.1.529) variants, which comprise a number of mutations in the S protein, has been of particular concern. As used herein, a SARS-CoV-2 variant refers to a SARS-CoV-2 virus that has acquired one or more mutations that differentiate it from the Wuhan strain of SARS- CoV-2 that first emerged in 2019. Variants can be identified by virologists and / or health organizations using an appropriate classification system, including, e.g., the Pango or NextClade classification systems, examples of which are described herein.

[0469] In some embodiments, the present disclosure refers to a SARS-CoV-2 variant that is prevalent and / or rapidly spreading in a relevant jurisdiction. In some embodiments, such variants may be identified based on publicly12947033vl Page 73 of 526Attorney Docket No.: 2013237-1485 available data (e.g., data provided in the GISAID Initiative database and / or data provided by the World Health Organization WHO. In some embodiments, such a variant refers to a variant disclosed herein.

[0470] The Omicron BA.l variant was first reported to WHO on 24 November 24, 2021, and was detected in South Africa. Omicron and its sublineages have had a major impact on the epidemiological landscape of the COVID- 19 pandemic since their initial emergence (WHO Technical Advisory Group on SARS-CoV-2 Virus Evolution (TAG-VE): Classification of Omicron (B.l.1.259): SARS-CoV-2 Variant of Concern (2021); WHO Headquarters (HQ), WHO Health Emergencies Programme, Enhancing Response to Omicron SARS-CoV-2 variant: Technical brief and priority actions for Member States (2022)). Significant alterations in the spike (S) glycoprotein of the first Omicron variant BA.l resulted in the loss of many neutralizing antibody epitopes (M. Hoffmann et al., "The Omicron variant is highly resistant against antibody mediated neutralization: Implications for control of the COVID-19 pandemic", Cell 185, 447-456.ell (2022)) and rendered BA.l capable of partially escaping previously established SARS-CoV-2 wild-type strain (Wuhan-Hu-l)-based immunity (V. Servellita, et al., "Neutralizing immunity in vaccine breakthrough infections from the SARS-CoV-2 Omicron and Delta variants", Cell 185, 1539-1548.e5 (2022); Y. Cao et al., "Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies", Nature 602, 657-663 (2022)).

[0471] As a result, breakthrough infection of vaccinated individuals with Omicron is more common than with previous Variants of Concern (VOCs). While Omicron BA.l was displaced by the BA.2 variant in many countries around the globe, other variants such as BA.1.1 and BA.3 temporarily and / or locally gained momentum but did not become globally dominant (S. Xia et al., "Origin, virological features, immune evasion and intervention of SARS-CoV- 2 Omicron sublineages. Signal Transduct. Target. Ther. 7, 241 (2022); H. Gruell et al., "SARS-CoV-2 Omicron sublineages exhibit distinct antibody escape patterns, Cell Host Microbe 7, 241 (2022).). Omicron BA.2.12.1 subsequently displaced BA.2 to become dominant in the United States, whereas BA.4 and BA.5 displaced BA.2 in Europe, parts of Africa, and Asia / Pacific (H. Gruell et al., "SARS-CoV-2 Omicron sublineages exhibit distinct antibody escape patterns," Cell Host Microbe 7, 241 (2022); European Centre for Disease Prevention and Control, Weekly COVID-19 country overview -Country overview report: Week 31 2022 (2022); J. Hadfield et al., "Nextstrain: Realtime tracking of pathogen evolution," Bioinformatics 34, 4121-4123 (2018)). Currently, Omicron BA.5 is dominant globally, including in the United States (Centers for Disease Control and Prevention. COVID Data Tracker. Atlanta, GA: US Department of Health and Human Services, CDC; 2022, August 12. https: / / covid.cdc.gov / coviddata-tracker (2022)).

[0472] Omicron has acquired numerous alterations (amino acid exchanges, insertions, or deletions) in the S glycoprotein, among which some are shared between all Omicron VOCs while others are specific to one or more Omicron sublineages. Antigenically, BA.2.12.1 exhibits high similarity with BA.2 but not BA.l, whereas BA.4 and BA.5 differ considerably from their ancestor BA.2 and even more so from BA.l, in line with their genealogy (A. Z. Mykytyn et al., "Antigenic cartography of SARS-CoV-2 reveals that Omicron BA.l and BA.2 are antigenically distinct," Sci. Immunol. 7, eabq4450 (2022).). Major differences of BA.l from the remaining Omicron VOCs include A143-145, L212I, or ins214EPE in the S glycoprotein N-terminal domain and G446S or G496S in the receptor binding domain (RBD). Amino acid changes T376A, D405N, and R408S in the RBD are in turn common to BA.2 and its descendants but not found in BA.l. In addition, some alterations are specific for individual BA.2-descendant VOCs, including L452Q for BA.2.12.1 or L452R and F486V for BA.4 and BA.5 (BA.4 and BA.5 encode for the same S sequence). Most of these shared and VOC-specific alterations were shown to play an important role in immune escape from monoclonal antibodies and polyclonal sera raised against the wild-type S glycoprotein. In particular, the BA.4 / BA.5-12947033vl Page 74 of 526Attorney Docket No.: 2013237-1485 specific alterations are strongly implicated in immune escape of these VOCs (P. Wang et al., "Antibody resistance of SARS-CoV-2 variants B.1.351 and B.l.1.7. Nature 593, 130-135 (2021); Q. Wang et al., "Antibody evasion by SARS- CoV-2 Omicron subvariants BA.2.12.1, BA.4, & BA.5. Nature 608, 603-608 (2022)).

[0473] In some embodiments, the present disclosure provides a polyribonucleotide encoding a SARS-CoV-2 Spike (S) protein variant, or an immunogenic portion thereof. In some embodiments, a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises one or more mutations (e.g., 1 to 200, 20 to 200, 40 to 200, 60 to 200, 80 to 200, 100 to 200, 120 to 200, 140 to 200, 160 to 200, 180 to 200, 1 to 180, 20 to 180, 40 to 180, 60 to 180, 80 to 180, 100 to 180, 120 to 180, 140 to 180, 160 to 180, 1 to 160, 20 to 160, 40 to 160, 60 to 160, 80 to 160, 100 to 160, 120 to 160, 140 to 160, 1 to 140, 20 to 140, 40 to 140, 60 to 140, 80 to 140, 100 to 140, 120 to 140, 1 to 120, 20 to 120, 40 to 120, 60 to 120, 80 to 120, 100 to 120, 1 to 100, 20 to 100, 40 to 100, 60 to 100, 80 to 100, 1 to 80, 20 to 80, 40 to 80, 60 to 80, 1 to 60, 20 to 60, 40 to 60, 1 to 40, 20 to 40, or 1 to 20 mutations) relative to the SARS-CoV-2 S protein of the Wuhan SARS-CoV-2 2 strain S protein (SEQ ID NO: 1).

[0474] In some embodiments, a polyribonucleotide encoding a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises one or more mutations originating in one or more SARS-CoV-2 variants listed in Table 1. In some embodiments, a polyribonucleotide encoding a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises one or more mutations originating in one or more of an XBB.1.5 variant, a KP.2 variant, a JN.l variant, an XEC variant, an LP.8.1 variant, and / or a BA.3.2 variant relative to the SARS-CoV-2 protein of the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1). In some embodiments, a polyribonucleotide encoding a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises one or more mutations originating in an XBB.1.5 variant relative to the SARS-CoV-2 protein of the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1). In some embodiments, a polyribonucleotide encoding a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises one or more mutations originating in an KP.2 variant relative to the SARS- CoV-2 protein of the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1). In some embodiments, a polyribonucleotide encoding a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises one or more mutations originating in an JN.l variant relative to the SARS-CoV-2 protein of the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1). In some embodiments, a polyribonucleotide encoding a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises one or more mutations originating in an XEC variant relative to the SARS- CoV-2 protein of the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1). In some embodiments, a polyribonucleotide encoding a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises one or more mutations originating in an LP.8.1 variant relative to the SARS-CoV-2 protein of the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1). In some embodiments, a polyribonucleotide encoding a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises one or more mutations originating in a BA.3.2 variant relative to the SARS- CoV-2 protein of the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1).

[0475] In some embodiments, a polyribonucleotide encoding a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises:(i) T19I, A24-26, A27S, V83A, G142D, A145, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, or any combination thereof;12947033vl Page 75 of 526Attorney Docket No.: 2013237-1485(ii) insl6MPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, M1229I, or any combination thereof;(iii) insl6MPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, R446T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, F456LN460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, M1229I, or any combination thereof;(iv) insl6MPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, or any combination thereof;(v) insl6MPLF, T19I, R21T, T22N, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, F456L, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, V1104L, P1143L, or any combination thereof;(vi) Insl6MPLF, T19I, R21T, L24del, P25del, P26del, A27S, S31del, S50L, H69del, V70del, V127F, G142D, Y144del, F157S, R158G, F186L, R190S, N211del, L212I, V213G, L216F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445R, G446S, N450D, L452W, L455S, F456L, N460K, S477N, T478K, N481K, V483del, E484K, F486P, Q493E, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, K1086R, V1104L, P1143L, or any combination thereof; and / or(vii) P9L, R21T, P26L, A67V, H69del, V70del, T95I, I101T, C136del, N137del, D138del, P139del, F140del, L141del, G142del, V143del, Y144del, Y145del, H146del, K147del, F157S, N164K, S172F, K187T, N211del, L212I, L242del, A243del, P251S, I326V, G339Y, A348P, S371F, S373P, S375F, R403K, D405N, T408S, K417N, A435S, N440R, V445A, G446D, L452W, N460K, S477N, T478N, E484K, G496S, Q498R, N501Y, K529N, A538-1210, or any combination thereof, wherein positions are relative to the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1).

[0476] In some embodiments, a polyribonucleotide encoding a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises: T19I, A24-26, A27S, V83A, G142D, A145, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, or any combination thereof, wherein positions are relative to the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1).

[0477] In some embodiments, a polyribonucleotide encoding a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises: insl6MPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F,12947033vl Page 76 of 526Attorney Docket No.: 2013237-1485T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, M1229I, or any combination thereof, wherein positions are relative to the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1).

[0478] In some embodiments, a polyribonucleotide encoding a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises: insl6MPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, R446T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, F456LN460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, M1229I, or any combination thereof, wherein positions are relative to the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1).

[0479] In some embodiments, a polyribonucleotide encoding a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises: insl6MPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, or any combination thereof, wherein positions are relative to the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1).

[0480] In some embodiments, a polyribonucleotide encoding a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises: insl6MPLF, T19I, R21T, T22N, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, F456L, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, V1104L, P1143L, or any combination thereof, wherein positions are relative to the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1).

[0481] In some embodiments, a polyribonucleotide encoding a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises: Insl6MPLF, T19I, R21T, L24del, P25del, P26del, A27S, S31del, S50L, H69del, V70del, V127F, G142D, Y144del, F157S, R158G, F186L, R190S, N211del, L212I, V213G, L216F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445R, G446S, N450D, L452W, L455S, F456L, N460K, S477N, T478K, N481K, V483del, E484K, F486P, Q493E, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, K1086R, V1104L, P1143L, or any combination thereof, wherein positions are relative to the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1).In some embodiments, a polyribonucleotide encoding a SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises: P9L, R21T, P26L, A67V, H69del, V70del, T95I, I101T, C136del, N137del, D138del, P139del, F140del, L141del, G142del, V143del, Y144del, Y145del, H146del, K147del, F157S, N164K, S172F, K187T, N211del, L212I, L242del, A243del, P251S, I326V, G339Y, A348P, S371F, S373P, S375F, R403K, D405N, T408S, K417N, A435S, N440R, V445A, G446...

Claims

1. Attorney Docket No.: 2013237-1485LISTING OF CLAIMSWHAT IS CLAIMED IS:

1. A polyribonucleotide comprising a nucleotide sequence that encodes a SARS-CoV-2 Spike (S) protein variant, or an immunogenic portion thereof, wherein the variant or the immunogenic portion thereof comprises from N-terminus to C-terminus:(i) an endogenous SARS-CoV-2 secretory signal;(ii) an N-terminal domain (NTD) of a SARS-CoV-2 S protein;(iii) a receptor-binding domain (RBD) of a SARS-CoV-2 S protein;(iv) a GS linker; and(v) an endogenous SARS-CoV-2 transmembrane domain (TMD), wherein all the uridines in the polyribonucleotide are replaced with Nl-methyl pseudouridines (mli ).

2. The polyribonucleotide of claim 1, wherein the SARS-CoV-2 S protein variant, or an immunogenic portion thereof, does not comprise a heterologous linker between the NTD and RBD.

3. The polyribonucleotide of claim 1 or 2, wherein the SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises one or more mutations relative to the NTD and RBD of the Wuhan SARS- CoV-2 strain S protein (SEQ ID NO: 1).

4. The polyribonucleotide of claim 3, wherein the one or more mutations comprise one or more mutations originating in one or more of an XBB.1.5 variant, a KP.2 variant, a JN.l variant, an XEC variant, an LP.8.1 variant, and / or a BA.3.2 variant relative to the NTD and RBD of the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1).

5. The polyribonucleotide of claim 3 or 4, wherein the one or more mutations comprise:(i) T19I, A24-26, A27S, V83A, G142D, A145, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, or any combination thereof;(ii) insl6MPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A201A211, L202IL212I, V203GV213G, L206FL216F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, or any combination thereof;(iii) insl6MPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, or any combination thereof;12947033vl Page 523 of 526Attorney Docket No.: 2013237-1485(iv) insl6MPLF, T19I, R21T, T22N, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, F456L, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, or any combination thereof;(v) Insl6MPLF, T19I, R21T, L24del, P25del, P26del, A27S, S31del, S50L, H69del, V70del, V127F, G142D, Y144del, F157S, R158G, F186L, R190S, N211del, L212I, V213G, L216F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445R, G446S, N450D, L452W, L455S, F456L, N460K, S477N, T478K, N481K, V483del, E484K, F486P, Q493E, Q498R, N501Y, Y505H, or any combination thereof; or(vi) P9L, R21T, P26L, A67V, H69del, V70del, T95I, I101T, C136del, N137del, D138del, P139del, F140del, L141del, G142del, V143del, Y144del, Y145del, H146del, K147del, F157S, N164K, S172F, K187T, N211del, L212I, L242del, A243del, P251S, G339Y, A348P, S371F, S373P, S375F, R403K, D405N, T408S, K417N, A435S, N440R, V445A, G446D, L452W, N460K, S477N, T478N, E484K, G496S, Q498R, N501Y, K529N, C538del, V539del, N540del, F541del, or any combination thereof, wherein the one or more mutations are relative to the NTD and RBD of the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1).

6. The polyribonucleotide of any one of claims 1 to 5, wherein the SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of the NTD and / or RBD of any one or more of SEQ ID NO: 125, SEQ ID NO: 1501, SEQ ID NO: 853, SEQ ID NO: 878, SEQ ID NO: 893, and / or SEQ ID NO: 903.

7. The polyribonucleotide of any one of claims 1 to 6, wherein the secretory signal comprises SEQ ID NO: 821, SEQ ID NO: 1750, SEQ ID NO: 44, SEQ ID NO: 15, SEQ ID NO: 822, SEQ ID NO: 9, SEQ ID NO: 16, or SEQ ID NO: 823.

8. The polyribonucleotide of any one of claims 1 to 7, wherein the GS linker comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of a GS linker listed in Table 8.

9. The polyribonucleotide of any one of claims 1 to 8, wherein the SARS-CoV-2 S protein variant, or an immunogenic portion thereof, comprises an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NO: 2015; SEQ ID NO: 2000; SEQ ID NO: 2003; SEQ ID NO: 2006; SEQ ID NO: 2009; or SEQ ID NO: 2012.

10. The polyribonucleotide of any one of claims 1 to 9, wherein the polyribonucleotide comprises a nucleotide sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%,12947033vl Page 524 of 526Attorney Docket No.: 2013237-1485 at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence according to any one of SEQ ID NO: 2016; SEQ ID NO: 2001; SEQ ID NO: 2002; SEQ ID NO: 2004; SEQ ID NO: 2005; SEQ ID NO: 2007; SEQ ID NO: 2008; SEQ ID NO: 2010; SEQ ID NO: 2011; SEQ ID NO: 2013; or SEQ ID NO: 2014.

11. The polyribonucleotide of any one of claims 1 to 10, wherein the GS linker comprises SEQ ID NO: 848.

12. The polyribonucleotide of any one of claims 1 to 11, wherein the NTD of the SARS-CoV-2 S protein comprises amino acids located at positions 1-209, 14-209, 15-209, 19-209, 20-209, 21-209, 1-305, 14-305, 15-305, 19-305, 20-305, 21-305, 1-311, 14-311, 15-311, 19-311, 20-311, 21-311, 1-318, 14-318, 15-318, 19-318, 20-318, 21-318, 1-302, 14-302, 15-302, 19-302, 20-302, or 21-302 of the SARS-CoV-2 S protein, wherein positions are relative to the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1).

13. The polyribonucleotide of any one of claims 1 to 12, wherein the RBD of the SARS-CoV-2 S protein comprises amino acids located at positions 319-528, 327-528, 328-528, 330-528, 319-530, 327-530, 328-530, 330- 530, 319-531, 327-531, 328-531, 330-531, 319-537, 327-537, 328-537, 330-537, 330-537, 319-541, 327-541, 328- 541, or 330-541 of the SARS-CoV-2 S protein, wherein positions are relative to the Wuhan SARS-CoV-2 strain S protein (SEQ ID NO: 1).

14. A composition comprising a polyribonucleotide of any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

15. The composition of claim 14, wherein the composition comprises lipid nanoparticles (LNPs), liposomes, lipoplexes (LPXs), or polyplexes (PLXs).

16. The composition of claim 15, wherein the polyribonucleotide is fully or partially encapsulated within the LNPs, liposomes, LPXs, or PLXs.

17. The composition of any one of claims 14 to 16, wherein the polyribonucleotide is encapsulated in a lipid nanoparticle (LNP).

18. A method comprising administering to a subject the polyribonucleotide of any one of claims 1 to 13, or composition of any one of claims 14 to 17.

19. A method of preventing or treating a SARS-CoV-2 infection in a subject comprising administering to the subject the polyribonucleotide of any one of claims 1 to 13, or composition of any one of claims 14 to 17.

20. A method of manufacturing a polyribonucleotide of any one of claims 1 to 13.

21. The method of claim 20, wherein the method comprises in vitro transcription of a DNA template.12947033vl Page 525 of 526

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