DNA encoded nanoparticles with cytokine adjuvant and method of use thereof

Abstract

Disclosed herein are nanoparticle immunoadjuvant complexes and nucleic acid molecules encoding the same. Also disclosed herein is a method of treating or preventing a disease or disorder associated with an antigen presented on n nanoparticle immunoadjuvant complex in a subject in need thereof, by administering the antigen presenting nanoparticle immunoadjuvant complex, or encoding nucleic acid molecules, to the subject.

Description

[0001] DNA ENCODED NANOPARTICLES WITH CYTOKINE ADJUVANT AND METHOD OF USE THEREOF

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No.63 / 591,906, filed October 20, 2023, U.S. Provisional Patent Application No.63 / 592, 171, filed October 21, 2023, and U.S. Provisional Patent Application No.63 / 666.843, filed July 2, 2024, which are hereby incorporated by reference herein in their entirety.

[0004] REFERENCE TO A “SEQUENCE LISTING” SUBMITTED AS AN XML FILE

[0005] The present application hereby incorporates by reference the entire contents of the Sequence Listing written in the xml file named ‘'206193-0130-00WO Sequence Listing.xmT' created on October 21, 2024 that is 200,282 bytes in size.

[0006] BACKGROUND

[0007] Germinal centers are dynamic structures that form in secondary7lymphoid organs (e.g., lymph nodes, spleen). This is due to passive or active transport of foreign antigen (e.g.. a virus protein) to one of those sites. As such, a germinal center will form. Germinal centers result in B cells undergoing clonal expansion (multiplying) and somatic hypermutation (where they edit their B cell receptors on their surface to make a higher affinity - stronger -interaction with the target antigen). This is assisted by specialized helper T cells known as T follicular helper cells (Tfh). Tfh secrete / display numerous important "‘help” molecules to germinal center B cells (GC B) in this process. An alluring strategy for improving protective humoral immunity against infectious diseases is to directly target the germinal center (GC). Despite advances in formulation strategies and vaccine design, there remains a critical need for next generation approaches to elicit potent humoral immunity.

[0008] Thus, a need remains in the art for the development of therapeutic agents that augment germinal center responses for the treatment or prevention of a diseases or disorders. This invention addresses this unmet need. SUMMARY OF INVENTION

[0009] In some embodiments, the invention provides a composition comprising a cytokine nanoparticle immunoadjuvant fusion polypeptide comprising: a) an immunoadjuvant domain; b) an antigen domain; and c) an oligomerization domain.

[0010] In some embodiments, at least two cytokine nanoparticle immunoadjuvant fusion polypeptides form a cytokine nanoparticle immunoadjuvant complex (NIC).

[0011] In some embodiments, the immunoadjuvant domain is selected from the group consisting of IL-21, IL-6, IL-4, and TSLP. In some embodiments, the immunoadjuvant domain is IL-21 comprising SEQ ID NON or a fragment or variant thereof. In some embodiments, the immunoadjuvant domain is IL-6 comprising SEQ ID NO: 112 or a fragment or variant thereof. In some embodiments, the immunoadjuvant domain is IL-4 comprising SEQ ID NO: 114 or a fragment or variant thereof. In some embodiments, the immunoadjuvant domain is TSLP comprising SEQ ID NO: 116 or a fragment or variant thereof.

[0012] In some embodiments, the antigen is an immunogenic antigen. In some embodiments, the antigen is an HIV antigen, a SARS-CoV-2 antigen, or an influenza antigen. In some embodiments, the antigen is an HIV antigen comprising SEQ ID NO: 96 or a fragment or variant thereof. In some embodiments, the antigen is a SARS- CoV-2 antigen comprising an amino acid sequence selected from the group consisting of SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO 27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37. SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO 53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO 59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO 79, SEQ ID NO:81, SEQ ID NO:83. SEQ ID NO:85, and fragments or variants thereof. In some embodiments, the antigen is an influenza antigen comprising SEQ ID NO: 1 18 or a fragment or variant thereof. In some embodiments, the oligomerization domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14. SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, and fragments or variants thereof.

[0013] In some embodiments, the cytokine nanoparticle immunoadjuvant fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 108. SEQ ID NO: 111, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:98. SEQ ID NO:99, SEQ ID NO: 102. SEQ ID NO: 103. and fragments or variants thereof.

[0014] In some embodiments, the invention provides a nucleic acid molecule encoding a cytokine nanoparticle immunoadjuvant fusion polypeptide comprising: a) an immunoadjuvant domain; b) an antigen domain; and c) an oligomerization domain.

[0015] In some embodiments, the immunoadjuvant domain is selected from the group consisting of IL-21, IL-6, IL-4, and TSLP. In some embodiments, the nucleotide sequence encoding the IL-21 immunoadjuvant domain comprises SEQ ID NO:5. SEQ ID NO:6, or a fragment or variant thereof. In some embodiments, the nucleotide sequence encoding the IL-6 immunoadjuvant domain comprises SEQ ID NO: 113 or a fragment or variant thereof. In some embodiments, the nucleotide sequence encoding the IL-4 immunoadjuvant domain comprises SEQ ID NO: 115 or a fragment or variant thereof. In some embodiments, the nucleotide sequence encoding the TSLP immunoadjuvant domain comprises SEQ ID NO: 117 or a fragment or variant thereof.

[0016] In some embodiments, the antigen is an immunogenic antigen. In some embodiments, the antigen is an HIV antigen, a SARS-CoV-2 antigen, or an influenza antigen. In some embodiments, the nucleotide sequence encoding the HIV antigen comprises SEQ ID NO: 97 or a fragment or variant thereof. In some embodiments, the nucleotide sequence encoding the SARS-CoV-2 comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26. SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32. SEQ ID NO:34, SEQ ID NO 36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO 64, SEQ ID NO:66, SEQ ID NO:68. SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, and fragments or variants thereof. In some embodiments, the nucleotide sequence encoding the influenza antigen comprises SEQ ID NO: 119 or a fragment or variant thereof.

[0017] In some embodiments, the nucleic acid molecule encodes an oligomerization domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20. and fragments or variants thereof.

[0018] In some embodiments, the nucleic acid molecule encodes a cytokine nanoparticle immunoadjuvant fusion polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 108, SEQ ID NO: 111, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:98, SEQ ID NO:99. SEQ ID NO: 102, SEQ ID NO: 103, and fragments or variants thereof.

[0019] In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO:109, SEQ ID NO: 110, SEQ ID NO:87, SEQ ID NO 89, SEQ ID NO:90, SEQ ID NO:92. SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO: 100. SEQ ID NO: 101, SEQ ID NO: 104. SEQ ID NO: 105, and fragments or variants thereof.

[0020] In some embodiments, the invention provides an immunogenic composition comprising a nucleic acid molecule encoding a cytokine nanoparticle immunoadjuvant fusion polypeptide comprising: a) an immunoadjuvant domain; b) an antigen domain; and c) an oligomerization domain.

[0021] In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable excipient.

[0022] In some embodiments, the invention provides a method of inducing an immune response against a target antigen in a subject in need thereof, the method comprising administering a composition comprising a cytokine nanoparticle immunoadjuvant fusion polypeptide comprising: a) an immunoadjuvant domain; b) an antigen domain; and c) an oligomerization domain, a nucleic acid molecule encoding a cytokine nanoparticle immunoadjuvant fusion polypeptide comprising: a) an immunoadjuvant domain; b) an antigen domain; and c) an oligomerization domain, or an immunogenic composition comprising the nucleic acid molecule thereof to the subject. In some embodiments, administering includes at least one of electroporation and injection. In some embodiments, the target antigen is an HIV antigen, a SARS- CoV-2 antigen, or an influenza antigen.

[0023] In some embodiments, the invention provides a method of protecting a subject in need thereof from a disease or disorder associated with an antigen, the method comprising administering a composition comprising a cytokine nanoparticle immunoadjuvant fusion polypeptide comprising: a) an immunoadjuvant domain; b) an antigen domain; and c) an oligomerization domain, a nucleic acid molecule encoding a cytokine nanoparticle immunoadjuvant fusion polypeptide comprising: a) an immunoadjuvant domain; b) an antigen domain; and c) an oligomerization domain, or an immunogenic composition comprising the nucleic acid molecule thereof to the subject. In some embodiments, administering includes at least one of electroporation and injection.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure lA-Figure II depict the development and characterization of GT8- IL-21-NIC. Figure 1 A is a model depicting GT8 and IL-21 fusion generated using Rosetta; GT8 (blue), IL-21 (red), Timeline of immunization scheme. Mice were immunized once and GC responses were interrogated at Day 7 in the DLNs. Figure IB depicts frequency of activated Tfh cells. Pre-gated on live CD 19- CD4+ CD44+. Figure 1C depicts frequency of GC B cells. Pre-gated on live CD4- CD19+. Figure IE is a model depicting GT8-IL-21-NIC generated using Rosetta; GT8 (blue), IL-21 (red), lumazine synthase scaffold (grey). Figure IF depicts binding ELISA to IL-21R using recombinant GT8-60mer or GT8-IL-21-NIC. Figure 1G depicts size exclusion chromatography (SEC) trace of GT8-IL-21-NIC. Figure 1H depicts a representative contour plot of follicular B cells labelled with Cell Trace Yellow from a Blimp- 1 reporter mouse cultured in vitro with recombinant GT8-60mer, GT8-IL-21-NIC, or cocultured with IL-4 / 5. Plots show Blimp-1+ plasma cells at 72 hours post co-culture. Figure II depicts frequency of Blimp-1+ plasma cells. Bars show geometric mean with geometric standard deviation. Each dot represents one mouse (Figure IB-Figure ID) or one technical replicate (Figure 1H); n=8 mice per group representative of three independent experiments (Figure IB-Figure ID), n=3 technical replicates representative from two independent experiments. One-way ANOVA adjusted for multiple comparisons with Bonferroni correction used to compare groups (Figure IB-Figure ID and Figure II); * p<0.05; ** p<0.01; *** p<0.001; **** pO.OOOl.

[0026] Figure 2A-Figure 2K depict the results of example experiments demonstrating GT8-IL-21-NIC augments humoral immunity and germinal center B cell responses. Figure 2A depicts GT8-specific serum IgG titers by ELISA. Figure 2B depicts GT8-specific bone marrow antibody-secreting cells at 16 weeks post immunization by ELISpot. Figure 2C depicts representative FACS plot of GC B cells at Day 7 post immunization in the DLNs. Figure 2D depicts frequency of GC B cells. Pregated on live CD4- CD19+. Figure 2E depicts representative FACS plot of GT8-specific GC B cells. Figure 2F depicts frequency of GT8-specific GC B cells. Pre-gated on live CD4- CD19+ GL7+ Fas+. Figure 2G depicts representative FACS plot of zonal skewing in GT8-specific GC B cells. Figure 2H depicts frequency of GT8-specific LZ or DZ GC B cells as a frequency of GT8-specific GC B cells. Pre-gated on live CD4- CD19+ GL7+ Fas+ GT8-24mer++. Figure 21 depicts immunofluorescent microscopy images of iliac lymph nodes 7 days post immunization (Figure 2J and 2K) Quantification of GC numbers per lymph node (Figure 2J) and total area per GC in pm (Figure 2K). Bars show geometric mean with geometric standard deviation. Each dot represents one mouse (Figure 2B-2F, 2H, 2J); n=10 mice per group combined from 2 independent experiments (Figure 2A and 2B), n=13-14 mice per group combined from 3 independent experiments (Figure 2C-2F), n=8-9 mice per group combined from 2 independent experiments (Figure 2G and 2H). n=5-7 mice per group combined from 2 independent experiments (Figure 2J and 2K). Each dot represents one germinal center (Figure 2K). Nonparametric Mann Whitney U Test used to compare groups where indicated; * p<0.05; ** p<0.01; *** p<0.001; **** pO.OOOl.

[0027] Figure 3A- Figure 3F depict the results of example experiments demonstrating GT8-1L-21-NIC generates superior GC responses to monomeric IL-21 co-administration. Figure 3A depicts representative FACS plot of GC B cells in DLNs 7 days post immunization. Figure 3B depicts frequency of GC B cells. Pre-gated on live CD4- CD19+. Figure 3C depicts frequency of GT8-specific GC B cells. Pre-gated on live CD4- CD 19+ CD38- GL7+. Figure 3D depicts representative FACS plot of GT8- specific GC B cells in the LZ or DZ. Figure 3E-3F depict frequency quantification of GT8-specific GC B cells in the LZ (Figure 3E) or DZ (Figure 3F). Pre-gated on live CD4- CD 19+ CD38- GL7+ GT7-24mer++. Bars show geometric mean with geometric standard deviation. Each dot represents one mouse; n=8-9 mice per group combined form 2 independent experiments. Ordinary- one-way ANOVA adjusted for multiple comparisons with Bonferroni correction was used to compare groups; * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

[0028] Figure 4A- Figure 4H depict the results of example experiments demonstrating GT8-IL-21-NIC enhances frequency and effector function of Tfh and downregulates Tfr. Figure 4A depicts representative FACS plot of Tfh in DLNs 7 days post immunization. Figure 4B depicts frequency of activated Tfh. Pre-gated on live CD19- CD4+ CD44+. Figure 4C depicts representative FACS plot of CD40L+ Tfh. Figure 4D depicts frequency of CD40L+ Tfh. Pre-gated on live CD19- CD4+ CD44+ PD-lhi CXCR5+. Figure 4E depicts representative FACS plot of Foxp3 expression in Tfh. Figure 4F depicts frequency of Foxp3- (Tfh) or Foxp3+ (Tfr) cells. Pre-gated on live CD19- CD4+ CD44+ PD-lhi CXCR5+. Figure 4G depicts ratio of Foxp3- Tfh to Foxp3+ Tfr. Figure 4H depicts frequency of non-GC Foxp3+ Tregs in the DLN. Pregated on live CD 19- CD4+ Bcl6-. Bars show geometric mean with geometric standard deviation. Each dot represents one mouse; n=13-14 mice per group combined from 3 independent experiments (Figure 4A-4B, 4E-4H), n=4-9 mice per group combined from 2 independent experiments (Figure 4C-4D). Non-parametric Mann-Whitney U test (Figure 4A-4B. 4E-4H) or ordinary- one-way ANOVA adjusted for multiple comparisons with Bonferroni correction (Figure 4C-4D) was used to compare groups; * p<0.05; ** p<0.01; *** p<0.001; **** pO.OOOl.

[0029] Figure 5 A-Figure 5L depict the results of example experiments demonstrating GT8-1L-21-N1C engineers GC kinetics and promotes a pro-selection phenoty pe. Figure 5A depicts frequency of GC B cells 7-, 14-, or 28-days post immunization in the DLNs. Pre-gated on live CD4- CD19+. Figure 5B depicts frequency of GT8-specific GC B cells. Pre-gated on live CD4- CD 19+ GL7+ Fas+. Figure 5C and Figure 5D depicts Frequency of LZ (Figure 5C) and DZ (Figure 5D) GT8-specific GC B cells. Pre-gated on live CD4- CD19+ GL7+ Fas+ GT8-24mer++. Bars show geometric mean with geometric standard deviation. Figure 5E depicts graphical timeline of experiment. Mice were immunized with 0.5pg of GT8-60mer or GT8-1L-21-N1C and pooled antigen-specific GC B cells were sorted and subjected to single cell RNA sequencing via the lOx platform. Figure 5F depicts UMAP separated by sample colored by cluster. Figure 5G depicts frequency of clusters 1-5 in each sample. Figure 5H depicts UMAP density plot of indicated genes of interest. Figure 5I-5L depict upregulated IPA regulators in cluster 1 (LZ) (Figure 51), cluster 3 (DZ) (Figure 5J), and cluster 5 (activated / selected LZ) (Figure 5K) in GT8-IL-21-NIC relative to the GT8- 60mer. Figure 5L depicts Upregulated genes of interest in cluster 1 (LZ). Each dot represents one mouse (Figure 5A-5D); n=8-13 mice combined from 2-3 independent experiments (Figure 5A-5D). n=10 mice pooled (Figure 5E-5L). Non-parametric Mann- Whitney U test used to compare groups (Figure 5A-5D); Wilcoxon rank sum used to compare groups (Figure 5H).* p<0.05; ** p<0.01; *** p<0.001; **** pO.OOOl.

[0030] Figure 6A-Figure 6L depict the results of example experiments demonstrating the NIC platform is amenable to alternate antigen and cy tokine domains. Figure 6A is a model depicting NC99-IL-21-NIC generated using Rosetta; NC99 (yellow). IL-21 (red), lumazine synthase scaffold (grey). Figure 6B depicts NC99 HA- specific serum IgG titers expressed as AUC by ELISA. Figure 6C depicts serum IgG titers at week 12 post immunization for NC99, SI06, and Bris07 HAs. Figure 6D depicts NC99 HA-specific bone marrow antibody -secreting cells at 12 weeks post immunization by ELISpot. Figure 6E depicts representative FACS plot of GC B cells at 7 days post immunization with NC99-60mer or NC99-IL-21-NIC in the DLNs. Figure 6F depicts frequency of GC B cells. Pre-gated on live CD4- CD19+. Figure 6G depicts representative FACS plot of NC99 HA-specific GC B cells. Figure 6H depicts frequency of NC99 HA-specific GC B cells. Pre-gated on live CD4- CD19+ CD38- Fas+. Figure 61 depicts representative FACS plot of GC B cells at 7 days post immunization with GT8- 60mer or indicated NICs in the DLNs. Figure 6J depicts frequency of GC B cells. Pregated on live CD4- CD19+. Figure 6K depicts representative FACS plot of GT8- speicific GC B cells. Figure 6H depicts frequency of GT8-specific GC B cells. Pre-gated on live CD4- CD19+ CD38- Fas+. Each dot represents one mouse. N=5 mice per group representative of two independent experiments (Figure 6B-Figure 6D), n=9 mice per group combined from two independent experiments (Figure 6E-Figure 6H), n=7-9 mice per group combined from three independent experiments (Figure 6I-Figure 6L). Nonparametric Mann-Whitney U test (Figure 6B-Figure 6H) or ordinary one-way ANOVA adjusted for multiple comparisons with Bonferroni correction (Figure 6I-Figure 6L) was used to compare groups; * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

[0031] Figure 7 depicts data demonstrating that IL-21 NIC elicits superior antibody titers at ultra-low doses. An ELISA was performed as in Figure 2A. The line graph and scater plot quantify the serological response elicited by the GT8-IL-21-NIC (abbreviated IL-21 NIC) relative to the GT8-60mer at 0. Ipg to Ipg.

[0032] Figure 8A- Figure 8D depict results of example experiments demonstrating low dose of GT8-IL-21-NIC analogously improves GC responses. Figure 8A depicts representative FACS plot of GC B cells at Day 7 post immunization in the DLNs. Mice were immunized with 0. Ipg of plasmid encoding either the GT8-60mer or GT8-IL-21-NIC. Figure 8B depicts frequency of GC B cells. Pre-gated on live CD4- CD19+. Figure 8C depicts representative FACS plot of GT8-specific GC B cells. Figure 8D depicts frequency of GT8-specific GC B cells. Pre-gated on live CD4- CD 19+ CD38- Fas+. Bars show geometric mean with geometric standard deviation. Each dot represents one mouse; n=10 mice per group combined from 2 independent experiments. Non-parametric Mann Whitney U Test used to compare groups where indicated; * p<0.05; ** p<0.01; *** pO.OOl; **** pO.OOOl.

[0033] Figure 9A -Figure 9C depict data demonstrating that germinal center enhancement by the GT8-IL-21-NIC (abbreviated IL-21 NIC) is not strain specific. C57BL / 6 mice were immunized with 0.5pg of the GT8-60mer or GT8-IL-21-NIC and GCs were interrogated in the DLNs at 7 days post immunization. Figure 9A depicts scater plots quantifying total and effector Tfh. Figure 9B depicts scatter plots quantifying total and antigen-specific GC B cells. Figure 9C depicts scater plots quantifying the LZ and DZ phenotypes of antigen-specific GC B cells.

[0034] Figure lOA-Figure 10C depict results of example experiments demonstrating GT8-IL-21-NIC does not drive enhanced CD25. ICOS, or OX-40 expression in activated Tfh. Figure 10A depicts the frequency of CD25hi Tfh, Figure 10B depicts the frequency of ICOShi Tfh, and Figure 10C depicts the frequency of OX- 40hi Tfh pre-gated on live CD19- CD4+ CD44+ PD-lhi CXCR5+. Bars show geometric mean with geometric standard deviation. Each dot represents one mouse; n=10 mice per group combined from 2 independent experiments. Non-parametric Mann Whitney U Test used to compare groups where indicated; * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

[0035] Figure 11 A- Figure 1 ID depict results of example experiments demonstrating representative cluster markers and phase of GC B cells via scRNAseq. Figure 11 A depicts a heatmap showing top upregulated genes per cluster. Figure 1 IB depicts cell cycle phase of GC B cells combined from both samples. Figure 11C depicts representative pseudotime plot across clusters. Figure 11D depicts frequency of indicated clusters by sample. n=10 mice pooled.

[0036] Figure 12 depicts a Venn diagram illustrating the change in clonal repertoire with the GT8-IL-21-NIC.

[0037] Figure 13 A- Figure 13D depict results of example experiments demonstrating NC99-IL-21-NIC enhances frequency and effector profile of Tfh. Figure 13A depicts representative FACS plot of Tfh in DLNs 7 days post immunization. Figure 13B depicts frequency of activated Tfh. Pre-gated on live CD 19- CD4+ CD44+. Figure 13C depicts representative FACS plot of CD40L+ Tfh. Figure 13D depicts frequency of CD40L+ Tfh. Pre-gated on live CD19- CD4+ CD44+ PD-lhi CXCR5+. Bars show geometric mean with geometric standard deviation. Each dot represents one mouse; n=10 mice per group combined from 2 independent experiments (Figures 13 A and 13B), n=4-5 mice from one independent experiment (Figures 13C and 13D). Non-parametric Mann Whitney U Test used to compare groups (Figure 13B); One-way ANOVA adjusted for multiple comparisons with Bonferroni correction used to compare groups (Figure 13D); **** p<0.0001.

[0038] Figure 14A and Figure 14B depict results of example experiments demonstrating Tfh responses of NICs with alternate cytokine domains. Figure 14A depicts representative FACS plot of Tfh in DLNs 7 days post immunization. Figure 14B depicts frequency of activated Tfh. Pre-gated on live CD19- CD4+ CD44+. Bars show geometric mean with geometric standard deviation. Each dot represents one mouse; n=7- 9 mice per group representative of three independent experiments. One-way ANOVA adjusted for multiple comparisons with Bonferroni correction used to compare groups; * p<0.05.

[0039] Figure 15 depicts further characterization of the NIC platform with IL-6. Mice were immunized with 0.5ug of the GT8-60mer or GT8 IL-6 NIC and germinal centers were surveyed in DLNs 7 days post immunization using flow cytometry. (Top) Total germinal center B cell responses (GL7+ Fas+ B cells) expressed as a frequency of total B cells. Pre-gated on live CD4- CD19+ and GT8-specific germinal center B cell responses expressed as a frequency of GC B cells. (Bottom left) Total T follicular helper cell responses expressed as a frequency of activated CD4+ T cells. Pre-gated on live CD 19- CD4+ CD44+. (Bottom right) CD40L+ Tfh cells after 18 hours of peptide stimulation. Expressed as a frequency of activated Tfh. Pre-gated on live CD 19- CD4+CD44+ PD-lhi CXCR5+.

[0040] Figure 16 depicts the strategy of marrying nanoparticles and gene- encoded adjuvants.

[0041] Figure 17 depicts an illustration ofNICs and a graphical representation of the GC response.

[0042] Figure 18A-Figure 18G depict data demonstrating co-administration of plasmid-encoded IL-21 monomer enhances germinal center responses. Figure 18A depicts an illustration of a pVAX vector with an IL-21 sequence behind an IgE secretion signal sequence. Figure 18B depicts a western blot of plasmid-encoded IL-21 in culture migrating at the correct molecular weight. Figure 18C depicts a schematic representation of study. Mice were immunized with either 2ug of DNA encoding a model SARS-CoV- 2 Spike RBD multimer vaccine (RBD-24mer) or co-immunized with plasmid-encoded IL-21 (0.5ug). Popliteal and iliac draining lymph nodes (DLNs) were harvested 7 days post immunization. Figure 18D depicts representative flow cytometry plot of total germinal center B (GC B) cells. Pre-gated on live dump- CD4- CD19+. Figure 18E depicts frequency of total germinal center B cells. Figure 18F depicts frequency of wildtype RBD+ GC B. Figure 18G depicts frequency of wild-type RBD andOmicron BA.4 / 5 RBD cross-reactive GC B. Non-parametric Mann- Whitney U test used to compare groups. *p<0.05, **p<0.01.

[0043] Figure 19A through Figure 19D depict data demonstrating that IL-21 augments Tfh frequency and effector profile. Mice were immunized with 2 pg of RBD- g5.1-24mer with or without 0.5 pg of IL-21 (separate plasmid). Germinal centers were assessed in draining lymph nodes 7 days post immunization. Figure 19A depicts flow plots and a bar graph assessing the frequency of Tfh cells with IL-21 co-immunization. Figure 19B depicts a flow plot assessing the frequency of costimulalorv surface molecule CD40L. Figure 19C depicts a bar graph quantifying the frequency of costimulatory surface molecule CD40L-expressing Tfh cells. Figure 19D depicts a bar graph quantifying the frequency of costimulatory surface molecule ICOS-expressing Tfh cells. DETAILED DESCRIPTION OF THE INVENTION

[0044] The present invention relates to an immunogenic composition or a vaccine comprising a cytokine-Nanoparticle Immunoadjuvant Complex (NIC) comprising a self-assembling nanoparticle comprising a cytokine adjuvant, an antigen and an oligomerization or nanoparticle self-assembly domain, as well as an immunogenic composition or a vaccine comprising a nucleic acid molecule encoding the NIC or an NIC monomer. In some embodiments, the NIC comprises a cytokine adjuvant linked to an antigen linked to an oligomerization or nanoparticle self-assembly domain. In some embodiments, the cytokine is IL-21, IL-6, IL-4, or TSLP.

[0045] In one embodiment, the invention relates to an NIC or a nucleic acid molecule encoding an NIC comprising one or more disease-associated antigen. The NIC or the nucleic acid molecule encoding the NIC comprising a disease-associated antigen can be used treat or to prevent a disease or disorder associated with the antigen. Exemplary diseases include, but are not limited to bacterial diseases, viral diseases, or cancers. The NIC can elicit both humoral and cellular immune responses that target the disease-associated antigen. The NIC can elicit neutralizing antibodies and immunoglobulin G (IgG) antibodies that are reactive with the disease-associated antigen. The NIC can also elicit CD8+and CD4 T cell responses that are reactive to the disease-associated antigen and produce interferon-gamma (IFN-y), tumor necrosis factor alpha (TNF-a). and interleukin-2 (IL-2).

[0046] In one embodiment, the invention relates to a nucleic acid molecule encoding a NIC comprising one or more HIV antigen. Exemplar}' HIV antigens that can be incorporated into an NIC of the invention include, but are not limited to, the HIV antigens described in International Patent Publication No WO2021174132A2, the contents of which are incorporated herein in their entirety. In one embodiment, the HIV antigen comprises an engineered outer domain (eOD) immunogen of HIV gp!20 (GT8- 60). In one embodiment, the nucleic acid molecule encodes an NIC comprising the HIV GT8-60 antigen. In one embodiment, the nucleic acid molecule encodes an IL-21 NIC comprising the HIV GT8-60 antigen. In one embodiment, the nucleic acid molecule encodes an IL-6 NIC comprising the HIV GT8-60 antigen. In one embodiment, the nucleic acid molecule encodes an IL-4 NIC comprising the HIV GT8-60 antigen. In one embodiment, the nucleic acid molecule encodes a TSLP NIC comprising the HIV GT8- 60 antigen. The NIC comprising a HIV antigen can be used treat HIV infection or to prevent or treat a disease or disorder associated with HIV infection. In one embodiment, the disease or disorder associated with HIV infection is AIDS. The NIC can elicit both humoral and cellular immune responses that target the HIV antigen. The NIC can elicit neutralizing antibodies and immunoglobulin G (IgG) antibodies that are reactive with the GT8 antigen. The NIC can also elicit CD8+and CD4+T cell responses that are reactive to the GT8 antigen and produce interferon-gamma (IFN-y), tumor necrosis factor alpha (TNF-a), and interleukin-2 (IL-2).

[0047] In one embodiment, the invention relates to a nucleic acid molecule encoding an NIC comprising one or more SARS coronavirus 2 (SARS-CoV-2) antigen. In one embodiment, the nucleic acid molecule encodes a self-assembling nanoparticle comprising the receptor binding domain (RBD) of the SARS-CoV-2 spike antigen. In one embodiment the nucleic acid molecule encodes a self-assembling nanoparticle comprising a dimer of the RBD of the SARS-CoV-2 spike antigen. Exemplary SARS- CoV-2 antigens that can be incorporated into an NIC of the invention include, but are not limited to, the SARS-CoV-2 antigens described in International Patent Publication No WO2022226083A1 or International Patent Publication No WO2022098728A1, the contents of each of which are incorporated herein in their entirety. In one embodiment, the nucleic acid molecule encodes an NIC comprising an RBD antigen. In one embodiment, the nucleic acid molecule encodes an IL-21 NIC comprising an RBD antigen. In one embodiment, the nucleic acid molecule encodes an IL-6 NIC comprising an RBD antigen. In one embodiment, the nucleic acid molecule encodes an IL-4 NIC comprising an RBD antigen. In one embodiment, the nucleic acid molecule encodes a TSLP NIC comprising an RBD antigen. In one embodiment, the SARS-CoV-2 antigen comprises a RBD g5.1 24mer antigen. In one embodiment, the nucleic acid molecule encodes an IL-21 NIC comprising a RBD g5. 1 24mer antigen.

[0048] The NIC can be used treat SARS-CoV-2 infection or to prevent or treat a disease or disorder associated with SARS-CoV-2 infection. In one embodiment, the disease or disorder associated with SARS-CoV-2 infection is COVID- 19. The NIC can elicit both humoral and cellular immune responses that target the SARS-CoV-2 spike antigen. The NIC can elicit neutralizing antibodies and immunoglobulin G (IgG) antibodies that are reactive with the SARS-CoV-2 spike antigen. The NIC can also elicit CD8+and CD4+T cell responses that are reactive to the SARS-CoV-2 spike antigen and produce interferon-gamma (IFN-y), tumor necrosis factor alpha (TNF-a), and interleukin-2 (IL-2).

[0049] In one embodiment, the invention relates to a nucleic acid molecule encoding an NIC comprising one or more influenza antigens. In some embodiments, the influenza antigen is the head domain of influenza H1N1 A / New Caledonia / 20 / 1999 hemagglutinin (NC99) antigen. In one embodiment, the nucleic acid molecule encodes an NIC comprising the NC99 antigen. In one embodiment, the nucleic acid molecule encodes an IL-21 NIC comprising the NC99 antigen. In one embodiment, the nucleic acid molecule encodes an IL-6 NIC comprising the NC99 antigen. In one embodiment, the nucleic acid molecule encodes an IL-4 NIC comprising the NC99 antigen. In one embodiment, the nucleic acid molecule encodes a TSLP NIC comprising the NC99 antigen.

[0050] The NIC comprising an influenza antigen can be used treat influenza infection or to prevent or treat a disease or disorder associated with influenza infection. The NIC can elicit both humoral and cellular immune responses that target the influenza antigen. The NIC can elicit neutralizing antibodies and immunoglobulin G (IgG) antibodies that are reactive with the NC99 antigen. The NIC can also elicit CD8+and CD4+T cell responses that are reactive to the NC99 antigen and produce interferongamma (IFN-y), tumor necrosis factor alpha (TNF-a), and interleukin-2 (IL-2).

[0051] Definitions

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary' skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0053] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising.” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0054] “Adjuvant” as used herein means any molecule added to the vaccine described herein to enhance the immunogenicity of the antigen.

[0055] “Antibody” as used herein means an antibody of classes IgG, IgM, IgA, IgD or IgE. or fragments, fragments or derivatives thereof, including Fab, F(ab')2, Fd, and single chain antibodies, diabodies. bispecific antibodies, bifunctional antibodies and derivatives thereof. The antibody can be an antibody isolated from the serum sample of mammal, a polyclonal antibody, affinity7purified antibody, or mixtures thereof which exhibits sufficient binding specificity7to a desired epitope or a sequence derived therefrom.

[0056] “Coding sequence” or “encoding nucleic acid” as used herein means the nucleic acids (RNA or DNA molecule) that comprise a nucleotide sequence which encodes a protein. The coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered.

[0057] “Complement” or “complementary ” as used herein means Watson-Crick (e.g., A-T / U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules.

[0058] “Consensus” or “Consensus Sequence” as used herein may mean a synthetic nucleic acid sequence, or corresponding polypeptide sequence, constructed based on analysis of an alignment of multiple subtypes of a particular antigen. The sequence may be used to induce broad immunity against multiple subtypes, serotypes, or strains of a particular antigen. Synthetic antigens, such as fusion proteins, may be manipulated to generate consensus sequences (or consensus antigens).

[0059] “Electroporation,” “electro-permeabilization,” or “electro-kinetic enhancement” (“EP”) as used interchangeably herein means the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane; their presence allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and water to pass from one side of the cellular membrane to the other.

[0060] “Fragment” as used herein means a nucleic acid sequence or a portion thereof that encodes a polypeptide capable of eliciting an immune response in a mammal. The fragments can be DNA fragments selected from at least one of the various nucleotide sequences that encode protein fragments set forth below.

[0061] “Fragment” or “immunogenic fragment” with respect to polypeptide sequences means a polypeptide capable of eliciting an immune response in a mammal that cross reacts with a full length wild type strain SARS-CoV-2 antigen. Fragments of consensus proteins can comprise at least 10%. at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of a consensus protein. In some embodiments, fragments of consensus proteins can comprise at least 20 amino acids or more, at least 30 amino acids or more, at least 40 amino acids or more, at least 50 amino acids or more, at least 60 amino acids or more, at least 70 amino acids or more, at least 80 amino acids or more, at least 90 amino acids or more, at least 100 amino acids or more, at least 1 10 amino acids or more, at least 120 amino acids or more, at least 130 amino acids or more, at least 140 amino acids or more, at least 150 amino acids or more, at least 160 amino acids or more, at least 170 amino acids or more, at least 180 amino acids or more, at least 190 amino acids or more, at least 200 amino acids or more, at least 210 amino acids or more, at least 220 amino acids or more, at least 230 amino acids or more, or at least 240 amino acids or more of a consensus protein.

[0062] As used herein, the term “genetic construct” refers to the DNA or RNA molecules that comprise a nucleotide sequence which encodes a protein. The coding sequence includes initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered. As used herein, the term “expressible form” refers to gene constructs that contain the necessary regulatory elements operable linked to a coding sequence that encodes a protein such that when present in the cell of the individual, the coding sequence will be expressed.

[0063] “Identical” or “identity” as used herein in the context of two or more nucleic acids or polypeptide sequences, means that the sequences have a specified percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0064] “Immune response” as used herein means the activation of a host’s immune system, e.g., that of a mammal, in response to the introduction of antigen. The immune response can be in the form of a cellular or humoral response, or both.

[0065] “Nucleic acid” or “oligonucleotide” or “polynucleotide” as used herein means at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

[0066] Nucleic acids can be single stranded or double stranded, or can contain portions of both double stranded and single stranded sequence. The nucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid can contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids can be obtained by chemical synthesis methods or by recombinant methods.

[0067] “Operably linked” as used herein means that expression of a gene is under the control of a promoter with which it is spatially connected. A promoter can be positioned 5' (upstream) or 3' (downstream) of a gene under its control. The distance between the promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance can be accommodated without loss of promoter function. A “peptide,” “protein,” or “polypeptide” as used herein can mean a linked sequence of amino acids and can be natural, synthetic, or a modification or combination of natural and synthetic.

[0068] “Promoter” as used herein means a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter can comprise one or more specific transcriptional regulatory sequences to further enhance expression and / or to alter the spatial expression and / or temporal expression of same. A promoter can also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter can regulate the expression of a gene component constitutively or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operatorpromoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter and the CMV IE promoter.

[0069] “Signal peptide” and “leader sequence” are used interchangeably herein and refer to an amino acid sequence that can be linked at the amino terminus of a S ARS- CoV-2 protein set forth herein. Signal peptides / leader sequences typically direct localization of a protein. Signal peptides / leader sequences used herein preferably facilitate secretion of the protein from the cell in which it is produced. Signal peptides / leader sequences are often cleaved from the remainder of the protein, often referred to as the mature protein, upon secretion from the cell. Signal peptides / leader sequences are linked at the N terminus of the protein.

[0070] “Subject” as used herein can mean a mammal that wants to or is in need of being immunized with the herein described vaccine. The mammal can be a human, chimpanzee, dog, cat, horse, cow, mouse, or rat.

[0071] “Substantially identical” as used herein can mean that a first and second amino acid sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%.or 99% over a region of 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, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400. 500, 600, 700. 800, 900, 1000, 1100 or more amino acids. Substantially identical can also mean that a first nucleic acid sequence and a second nucleic acid sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% over a region of 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, 30, 35, 40, 45, 50. 55. 60. 65, 70, 75, 80, 85, 90, 95, 100. 200, 300. 400, 500, 600. 700, 800. 900, 1000. 1100 or more nucleotides.

[0072] “Treatment'’ or “treating,” as used herein can mean protecting of an animal from a disease through means of preventing, suppressing, repressing, or completely eliminating the disease. Preventing the disease involves administering a vaccine of the present invention to an animal prior to onset of the disease. Suppressing the disease involves administering a vaccine of the present invention to an animal after induction of the disease but before its clinical appearance. Repressing the disease involves administering a vaccine of the present invention to an animal after clinical appearance of the disease.

[0073] “Variant” used herein with respect to a nucleic acid means (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto.

[0074] Variant can further be defined as a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Representative examples of “biological activity” include the ability to be bound by a specific antibody or to promote an immune response. Variant can also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e g., hydrophilicity’, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions can be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.

[0075] A variant may be a nucleic acid sequence that is substantially identical over the full length of the full gene sequence or a fragment thereof. The nucleic acid sequence may be 80%, 81%, 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% identical over the full length of the gene sequence or a fragment thereof. A variant may be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or fragment thereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%. 98%. 99%. or 100% identical over the full length of the amino acid sequence or a fragment thereof.

[0076] “Vector” as used herein means a nucleic acid sequence containing an origin of replication. A vector can be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self-replicating extrachromosomal vector, and preferably, is a DNA plasmid.

[0077] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0078] Nanoparticle-Immunoadjuvant Complex

[0079] In one embodiment, the invention relates to a composition comprising a self-assembling nanoparticle comprising an immunoadjuvant domain, an oligomerization domain and further comprising an antigen, a fragment thereof, a variant thereof, or a combination thereof, referred to herein as a nanoparticle immunoadjuvant complex (NIC).

[0080] In some embodiments, the invention provides an NIC comprising an IL- 21 immunoadjuvant domain. In some embodiments, the invention provides an NIC comprising an IL-6 immunoadjuvant domain. In some embodiments, the invention provides an NIC comprising an IL-4 immunoadjuvant domain. In some embodiments, the invention provides an NIC comprising a TSLP immunoadjuvant domain.

[0081] In some embodiments, the IL-21 immunoadjuvant domain comprises the amino acid sequence of SEQ ID NO: 1, or a fragment or variant thereof. In some embodiments, the IL-21 immunoadjuvant domain comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity7over an entire length of the amino acid sequence set forth in SEQ ID NO: 1. Immunogenic fragments of SEQ ID NO: 1 can be incorporated into the IL-21 nanoparticle-immunoadjuvant complex of the invention. Immunogenic fragments can comprise at least 60%, at least 65%, 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% or at least 99% of SEQ ID NO:1.

[0082] In some embodiments, the IL-21 NIC comprises a fragment of SEQ ID NO: 1 lacking the IgE leader sequence, or a variant thereof. In some embodiments, the fragment of SEQ ID NO: 1 lacking the IgE leader sequence comprises SEQ ID NO:4, or a fragment or variant thereof. In some embodiments, the IL-21 immunoadjuvant domain comprises an amino acid sequence having at least about 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NON. Immunogenic fragments of SEQ ID NON can be incorporated into the IL-21 nanoparticle-immunoadjuvant complex of the invention. Immunogenic fragments can comprise at least 60%, at least 65%, 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% or at least 99% of SEQ ID NON.

[0083] In some embodiments, the IL-21 immunoadjuvant domain is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:2, or a fragment or variant thereof. In some embodiments, the IL-21 immunoadjuvant domain is encoded by a nucleic acid molecule comprising a nucleotide sequence having at least about 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO:2. Immunogenic fragments of SEQ ID NO: 2 can be incorporated into the nucleic acid molecule encoding the IL-21 nanoparti cle-immunoadjuv ant complex of the invention. Immunogenic fragments can comprise at least 60%, at least 65%, 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% or at least 99% of SEQ ID NO:2.

[0084] In some embodiments, the IL-21 immunoadjuvant domain is encoded by a nucleic acid molecule comprising a fragment of SEQ ID NO:2 lacking the sequence encoding the IgE leader sequence. In some embodiments, the fragment of SEQ ID NO: 2 lacking the sequence encoding the IgE leader sequence comprises SEQ ID NO:5, or a fragment or variant thereof. In some embodiments, the IL-21 immunoadjuvant domain is encoded by a nucleic acid molecule comprising a nucleotide sequence having at least about 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO:5. Immunogenic fragments of SEQ ID NO: 5 can be incorporated into the nucleic acid molecule encoding the IL-21 nanoparti cle-immunoadjuv ant complex of the invention. Immunogenic fragments can comprise at least 60%, at least 65%, 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% or at least 99% of SEQ ID NO:5.

[0085] In some embodiments, the IL-21 immunoadjuvant domain is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:3, or a fragment or variant thereof. In some embodiments, the IL-21 immunoadjuvant domain is encoded by a nucleic acid molecule comprising a nucleotide sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO:3. Immunogenic fragments of SEQ ID NO: 3 can be incorporated into the nucleic acid molecule encoding the IL-21 nanoparti cle-immunoadjuv ant complex of the invention. Immunogenic fragments can comprise at least 60%, at least 65%, 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% or at least 99% of SEQ ID NO:3.

[0086] In some embodiments, the IL-21 immunoadjuvant domain is encoded by a nucleic acid molecule comprising a fragment of SEQ ID NO:3 lacking the sequence encoding the IgE leader sequence. In some embodiments, the fragment of SEQ ID NO:3 lacking the sequence encoding the IgE leader sequence comprises SEQ ID NO:6. or a fragment or variant thereof. In some embodiments, the IL-21 immunoadjuvant domain is encoded by a nucleic acid molecule comprising a nucleotide sequence having at least about 90%, 91%, 92%. 93%. 94%. 95%. 96%. 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO:6. Immunogenic fragments of SEQ ID NO: 6 can be incorporated into the nucleic acid molecule encoding the IL-21 nanoparti cle-immunoadjuv ant complex of the invention. Immunogenic fragments can comprise at least 60%, at least 65%, 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% or at least 99% of SEQ ID NO:6.

[0087] In some embodiments, the IL-6 immunoadjuvant domain comprises the amino acid sequence of SEQ ID NO: 112, or a fragment or variant thereof. In some embodiments, the IL-6 immunoadjuvant domain comprises an amino acid sequence having at least about 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO: 1 12. Immunogenic fragments of SEQ ID NO: 112 can be incorporated into the IL-6 nanoparti cle-immunoadjuv ant complex of the invention. Immunogenic fragments can comprise at least 60%, at least 65%, 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% or at least 99% of SEQ ID NO: 112.

[0088] In some embodiments, the IL-6 immunoadjuvant domain is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 113, or a fragment or variant thereof. In some embodiments, the IL-6 immunoadjuvant domain is encoded by a nucleic acid molecule comprising a nucleotide sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO: 113. Immunogenic fragments of SEQ ID NO: 113 can be incorporated into the nucleic acid molecule encoding the IL-6 nanoparticle-immunoadjuvant complex of the invention. Immunogenic fragments can comprise at least 60%, at least 65%, 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% or at least 99% of SEQ ID NO: 113.

[0089] In some embodiments, the IL-4 immunoadjuvant domain comprises the amino acid sequence of SEQ ID NO: 114, or a fragment or variant thereof. In some embodiments, the IL-4 immunoadjuvant domain comprises an amino acid sequence having at least about 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO: 1 14. Immunogenic fragments of SEQ ID NO: 114 can be incorporated into the IL-4 nanoparticle-immunoadjuvant complex of the invention. Immunogenic fragments can comprise at least 60%, at least 65%, 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% or at least 99% of SEQ ID NO: 114.

[0090] In some embodiments, the IL-4 immunoadjuvant domain is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 115, or a fragment or variant thereof. In some embodiments, the IL-4 immunoadjuvant domain is encoded by a nucleic acid molecule comprising a nucleotide sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity’ over an entire length of the amino acid sequence set forth in SEQ ID NO: 115. Immunogenic fragments of SEQ ID NO: 115 can be incorporated into the nucleic acid molecule encoding the IL-4 nanoparticle-immunoadjuvant complex of the invention. Immunogenic fragments can comprise at least 60%, at least 65%, 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% or at least 99% of SEQ ID NO: 115.

[0091] In some embodiments, the TSLP immunoadjuvant domain comprises the amino acid sequence of SEQ ID NO: 116, or a fragment or variant thereof. In some embodiments, the TSLP immunoadjuvant domain comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO: 1 16. Immunogenic fragments of SEQ ID NO: 1 16 can be incorporated into the TSLP nanoparticle-immunoadjuvant complex of the invention. Immunogenic fragments can comprise at least 60%, at least 65%, 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% or at least 99% of SEQ ID NO: 116. In some embodiments, the TSLP immunoadjuvant domain is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 117, or a fragment or variant thereof. In some embodiments, the TSLP immunoadjuvant domain is encoded by a nucleic acid molecule comprising a nucleotide sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO: 117. Immunogenic fragments of SEQ ID NO: 117 can be incorporated into the nucleic acid molecule encoding the TSLP nanoparti cle-immunoadjuv ant complex of the invention. Immunogenic fragments can comprise at least 60%, at least 65%, 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% or at least 99% of SEQ ID NO: 117.

[0092] Oligomerization Domain

[0093] In one embodiment, the invention relates to a composition comprising a self-assembling nanoparticle comprising an immunoadjuvant domain, an oligomerization domain and further comprising an antigen, a fragment thereof, a variant thereof, or a combination thereof, referred to herein as a nanoparticle immunoadjuvant complex (NIC). In one embodiment, the invention exploits ferritin, a ubiquitous iron storage protein, that self-assembles into spherical nanoparticles and serves as a scaffold to express a heterologous protein. Therefore, in one embodiment, the oligomerization domain comprises ferritin, or a fragment or variant thereof.

[0094] In one embodiment, the oligomerization domain comprises a sequence as set forth in SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO:20. In some embodiments, the oligomerization domain comprises an amino acid sequence having at least about 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO:20.

[0095] In some embodiments, the nucleic acid molecule encoding the oligomerization domain can comprise a nucleotide sequence that encodes the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 14. SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO:20. In some embodiments, the nucleic acid molecule encoding the oligomerization domain can comprise a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%. 97%. 98%. 99% or 100% identity over an entire length of the nucleic acid sequence set forth in SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19. In some embodiments, the nucleotide sequence encoding the oligomerization domain can be operably linked to a sequence encoding at least one linker sequence, such as an LS3 or GGS linker sequence.

[0096] Immunogenic fragments of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO:20 can be provided. Immunogenic fragments can comprise at least 60%, at least 65%, 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% or at least 99% of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16. SEQ ID NO: 18 or SEQ ID NO:20. Immunogenic fragments of proteins with amino acid sequences homologous to immunogenic fragments of SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO:20 can be provided. Such immunogenic fragments can comprise at least 60%, at least 65%, 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% or at least 99% identity to SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO:20.

[0097] Some embodiments relate to immunogenic fragments of SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 15. SEQ ID NO: 17 or SEQ ID NO: 19. Immunogenic fragments can be at least 60%. at least 65%. 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% or at least 99% of the full length of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO: 17 or SEQ ID NO:19. Immunogenic fragments can comprise at least 95%, at least 96%, at least 97% at least 98% or at least 99% identity to fragments of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19.

[0098] Leader Sequence

[0099] In some embodiments, the nucleic acid molecule encoding the NIC of the invention is operably linked to at least one signal peptide or leader sequence. "Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that can be linked at the amino terminus of a protein set forth herein. Signal peptides / leader sequences ty pically direct localization of a protein. Signal peptides / leader sequences used herein preferably facilitate secretion of the protein from the cell in which it is produced. Signal peptides / leader sequences are often cleaved from the remainder of the protein, often referred to as the mature protein, upon secretion from the cell. Signal peptides / leader sequences are linked at the N terminus of the protein.

[0100] In one embodiment, the leader sequence is the IgE leader sequence comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments therefore, the leader sequence in the disclosed expressible nucleic acid sequence comprises a sequence encoding SEQ ID NO:21.

[0101] Linker Sequence

[0102] In some embodiments, the sequences encoding each domain of the NIC of the invention are operably linked to at least one linker sequence. For example, in some embodiments the NIC sequences comprise a linker between the leader sequence and the immunoadjuvant domain, between the immunoadjuvant domain and the antigen domain, between the antigen domain and the oligomerization domain, or any combination thereof. A linker can be either flexible or rigid or a combination thereof. In one embodiment, the linker is a (GGS)nrepeat wherein, the GGS is repeated at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more than 10 times.

[0103] In some embodiments, the expressible nucleic acid sequence comprises at least one nucleic acid sequence comprising at least 70% sequence identity to SEQ ID NO:7. SEQ ID NO:7 is a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 8. In some embodiments, the at least one nucleic acid sequence, encoding a linker, encodes a sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity' to SEQ ID NO: 8 or a fragment thereof. In some embodiments, the at least one nucleic acid sequence, encoding a linker, comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 7 or a fragment thereof.

[0104] In some embodiments, the expressible nucleic acid sequence comprises at least one nucleic acid sequence encoding an LS3 linker comprising at least 70% sequence identity to SEQ ID NO:9 or a fragment thereof. SEQ ID NO:9 is a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 10. In some embodiments, the at least one nucleic acid sequence, encoding a linker, encodes a sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity' to SEQ ID NO: 10 or a fragment thereof. In some embodiments, the at least one nucleic acid sequence, encoding a linker, comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity' to SEQ ID NO:9 or a fragment thereof. Antigens

[0105] In one embodiment, the invention relates to a composition comprising a self-assembling nanoparticle comprising an immunoadjuvant domain, an oligomerization domain and further comprising an antigen, a fragment thereof, a variant thereof, or a combination thereof, referred to herein as a nanoparticle immunoadjuvant complex (NIC).

[0106] In one embodiment, the antigen comprises a polypeptide or peptide associated with a pathogen, such that the antigen induces an adaptive immune response against the antigen, and therefore the pathogen. In one embodiment, the antigen comprises a fragment of a polypeptide or peptide associated with a pathogen, such that the antigen induces an adaptive immune response against the pathogen.

[0107] In certain embodiments, the antigen comprises an amino acid sequence that is substantially homologous to the amino acid sequence of an antigen described herein and retains the immunogenic function of the original amino acid sequence. For example, in certain embodiments, the ammo acid sequence of the antigen has a degree of identity with respect to the original amino acid sequence of at least 60%, of at least 70%, of at least 85%, or of at least 95%.

[0108] Viral Antigens - In one embodiment, the antigen comprises a viral antigen, or fragment thereof, or variant thereof. In certain embodiments, the viral antigen is from a virus from one of the following families: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae. Picomaviridae, Poxviridae. Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae. In certain embodiments, the viral antigen is from papilloma viruses, for example, human papilloma virus (HPV), human immunodeficiency virus (HIV), polio virus, hepatitis B virus, hepatitis C virus, smallpox virus (Variola major and minor), vaccinia virus, influenza virus, rhinoviruses, dengue fever virus, equine encephalitis viruses, rubella virus, yellow fever virus. Norwalk virus, hepatitis A virus, human T-cell leukemia virus (HTLV-I), hairy cell leukemia vims (HTLV-II), California encephalitis vims, Hanta vims (hemorrhagic fever), rabies vims, Ebola fever virus, Marburg virus, measles virus, mumps virus, respiratory syncytial vims (RSV), herpes simplex 1 (oral herpes), herpes simplex 2 (genital herpes), herpes zoster (varicella-zoster. a.k.a., chickenpox), cytomegalovirus (CMV), for example human CMV, Epstein-Ban virus (EBV), flavivirus, foot and mouth disease virus, chikungunya virus, lassa virus, arenavirus, or cancer causing virus.

[0109] SARS-CoV-2 Antigen - In one embodiment, the antigen comprises an SARS-CoV-2 antigen or fragment thereof, or variant thereof. The SARS-CoV-2 antigens are those capable of eliciting an adaptive immune response in a mammal against one or more SARS-CoV-2 strain. In certain embodiments, the antigen comprises the full length spike protein, a variant thereof, or a fragment thereof. In certain embodiments, the fragment of the spike protein comprises the receptor binding domain (RBD).

[0110] In one embodiment, the SARS-CoV-2 antigen contains at least one antigenic epitope that can be effective against particular SARS-CoV-2 immunogens against which an immune response can be induced. In certain embodiments, the antigen may provide an entire repertoire of immunogenic sites and epitopes present in an intact SARS-CoV-2 virus.

[0111] Hepatitis Antigen - In one embodiment, the antigen comprises a hepatitis virus antigen (i.e., hepatitis antigen), or fragment thereof, or variant thereof. In certain embodiments, the hepatitis antigen comprises an antigen or immunogen from hepatitis A virus (HAV). hepatitis B virus (HBV). hepatitis C virus (HCV). hepatitis D virus (HDV), and / or hepatitis E virus (HEV). In certain embodiments, the hepatitis antigen is full-length or immunogenic fragments of full-length proteins.

[0112] In one embodiment, the hepatitis antigen comprises an antigen from HAV. For example, in certain embodiments, the hepatitis antigen comprises a HAV capsid protein, a HAV non-structural protein, a fragment thereof, a variant thereof, or a combination thereof.

[0113] In one embodiment, the hepatitis antigen comprises an antigen from HCV. For example, in certain embodiments, the hepatitis antigen comprises a HCV nucleocapsid protein (i.e., core protein), a HCV envelope protein (e.g., El and E2). a HCV non-structural protein (e.g., NS1, NS2, NS3, NS4a, NS4b, NS5a. and NS5b). a fragment thereof, a variant thereof, or a combination thereof.

[0114] In one embodiment, the hepatitis antigen comprises an antigen from HDV. For example, in certain embodiments, the hepatitis antigen comprises a HDV delta antigen, fragment thereof, or variant thereof. In one embodiment, the hepatitis antigen comprises an antigen from HEV. For example, in certain embodiments, the hepatitis antigen comprises a HEV capsid protein, fragment thereof, or variant thereof.

[0115] In one embodiment, the hepatitis antigen comprises an antigen from HBV. For example, in certain embodiments, the hepatitis antigen comprises a HBV core protein, a HBV surface protein, a HBV DNA polymerase, a HBV protein encoded by gene X, fragment thereof, variant thereof, or combination thereof. In certain embodiments, the hepatitis antigen comprises a HBV genotype A core protein, a HBV genotype B core protein, a HBV genotype C core protein, a HBV genotype D core protein, a HBV genotype E core protein, a HBV genotype F core protein, a HBV genotype G core protein, a HBV genotype H core protein, a HBV genotype A surface protein, a HBV genotype B surface protein, a HBV genotype C surface protein, a HBV genotype D surface protein, a HBV genotype E surface protein, a HBV genotype F surface protein, a HBV genoty pe G surface protein, a HBV genotype H surface protein, fragment thereof, variant thereof, or combination thereof.

[0116] Human Papilloma Virus (HPV) Antigen - In one embodiment, the antigen comprises a human papilloma virus (HPV) antigen, or fragment thereof, or variant thereof. For example, in certain embodiments, the antigen comprises an antigen from HPV types 16, 18, 31, 33, 35, 45, 52, and 58, which cause cervical cancer, rectal cancer, and / or other cancers. In one embodiment, the antigen comprises an antigen from HPV types 6 and 1 1, which cause genital warts, and are known to be causes of head and neck cancer. For example, in certain embodiments, the HPV antigen comprises a HPV E6 or E7 domain, or fragments, or variant thereof from any HPV type.

[0117] RSV Antigen - In one embodiment, the antigen comprises an RSV antigen or fragment thereof, or variant thereof. For example, in certain embodiments, the RSV antigen comprises a human RSV fusion protein (also referred to herein as “RSV F”, “RSV F protein” and “F protein”), or fragment or variant thereof. In one embodiment, the human RSV fusion protein is conserved between RSV subtypes A and B. In certain embodiments, the RSV antigen comprises a RSV F protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23994. 1). In one embodiment, the RSV antigen comprises a RSV F protein from the RSV A2 strain (GenBank AAB59858.1), or a fragment or variant thereof. In certain embodiments, the RSV antigen is a monomer, a dimer or trimer of the RSV F protein, or a fragment or variant thereof. According to the invention, in certain embodiments, the RSV F protein is in a prefusion form or a postfusion form.

[0118] In one embodiment, the RSV antigen comprises a human RSV attachment glycoprotein (also referred to herein as “RSV G”, “RSV G protein” and “G protein”), or fragment or variant thereof. The human RSV G protein differs between RSV subtypes A and B. In one embodiment, the antigen comprises a RSV G protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23993). In one embodiment, the RSV antigen comprises RSV G protein from: the RSV subtype B isolate H5601, the RSV subtype B isolate H1068, the RSV subtype B isolate H5598, the RSV subtype B isolate Hl 123, or a fragment or variant thereof.

[0119] In other embodiments, the RSV antigen comprises a human RSV non- structural protein 1 (“NS1 protein”), or fragment or variant thereof. For example, in one embodiment, the RSV antigen comprises RSV NS1 protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23987.1). In one embodiment, the RSV antigen comprises RSV non-structural protein 2 (“NS2 protein”), or fragment or variant thereof. For example, in one embodiment, the RSV antigen comprises RSV NS2 protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23988.1). In one embodiment, the RSV antigen comprises human RSV nucleocapsid (“N”) protein, or fragment or variant thereof. For example, in one embodiment, the RSV antigen is RSV N protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23989. 1 ). In one embodiment, the RSV antigen comprises human RSV Phosphoprotein (“P”) protein, or fragment or variant thereof. For example, in one embodiment, the RSV antigen comprises RSV P protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23990. 1). In one embodiment, the RSV antigen comprises human RSV Matrix protein (“M”) protein, or fragment or variant thereof. For example, in one embodiment, the RSV antigen comprises RSV M protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23991.1).

[0120] In still other embodiments, the RSV antigen comprises human RSV small hydrophobic (“SH”) protein, or fragment or variant thereof. For example, in one embodiment, the RSV antigen comprises RSV SH protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23992.1). In one embodiment, the RSV antigen comprises human RSV Matrix protein2-l (“M2-1”) protein, or fragment or variant thereof. For example, in one embodiment, the RSV antigen comprises RSV M2- 1 protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23995. 1). In one embodiment, the RSV antigen comprises RSV Matrix protein 2-2 (“M2-2”) protein, or fragment or variant thereof. For example, in one embodiment, the RSV antigen comprises RSV M2-2 protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23997.1). In one embodiment, the RSV antigen comprises RSV Polymerase L C'L") protein, or fragment or variant thereof. For example, in one embodiment, the RSV antigen comprises RSV L protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23996. 1).

[0121] Influenza Antigen - In one embodiment, the antigen comprises an influenza antigen or fragment thereof, or variant thereof. The influenza antigens are those capable of eliciting an adaptive immune response in a mammal against one or more influenza serotypes. In certain embodiments, the antigen comprises the full-length translation product Hemagglutinin (HA)0, subunit HA1, subunit HA2, a variant thereof, a fragment thereof or a combination thereof. In certain embodiments, the influenza hemagglutinin antigen is derived from one or more strains of influenza A serotype Hl, influenza A serotype H2. or influenza B.

[0122] In one embodiment, the influenza antigen contains at least one antigenic epitope that can be effective against particular influenza immunogens against which an immune response can be induced. In certain embodiments, the antigen may provide an entire repertoire of immunogenic sites and epitopes present in an intact influenza virus.

[0123] In some embodiments, the influenza antigen comprises Hl HA, H2 HA, H3 HA, H5 HA, or a BHA antigen. In certain embodiments, the influenza antigen comprises neuraminidase (NA), matrix protein, nucleoprotein, M2 ectodomain-nucleo- protein (M2e-NP), a variant thereof, a fragment thereof, or combinations thereof.

[0124] In some embodiments, the influenza antigen is the head domain of influenza H1N1 A / New Caledonia / 20 / 1999 hemagglutinin (NC99).

[0125] Human Immunodeficiency Virus (HIV) Antigen - In one embodiment, the antigen comprises an HIV antigen or fragment thereof, or variant thereof.

[0126] In certain embodiments, the HIV antigen comprises an envelope (Env) protein or fragment or variant thereof. For example, in certain embodiments, the HIV antigen comprises an Env protein selected from gpI20, gp41, or a combination thereof, or a fragment thereof. In some embodiments, the fragment comprises GT8-60.

[0127] In certain embodiments, the HIV antigen comprises at least one of nef, gag, pol, vif, vpr, vpu, tat, rev, or a fragment of variant thereof. The HIV antigen may be derived from any strain of HIV. For example, in certain embodiments the HIV antigen comprises an antigen from HIV groups M, N, O. and P, and subtype A, HIV subtype B, HIV subtype C, HIV subtype D, subtype E, subtype F, subtype G, subtype H, subtype J, or subtype K.

[0128] Parasite Antigens - In certain embodiments, the antigen comprises a parasite antigen or fragment or variant thereof. In certain embodiments, the parasite is a protozoa, helminth, or ectoparasite. In certain embodiments, the helminth (i.e., worm) is a flatworm (e.g., flukes and tapeworms), a thomy-headed worm, or a round worm (e.g., pinworms). In certain embodiments, the ectoparasite is lice, fleas, ticks, and mites.

[0129] In certain embodiments, the parasite is any parasite causing the following diseases: Acanthamoeba keratitis, Amoebiasis, Ascariasis, Babesiosis, Balantidiasis, Baylisascariasis, Chagas disease, Clonorchiasis, Cochliomyia, Cryptosporidiosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Giardiasis, Gnathostomiasis, Hymenolepiasis, Isosporiasis, Katayama fever, Leishmaniasis, Lyme disease, Malaria, Metagonimiasis, Myiasis, Onchocerciasis. Pediculosis, Scabies, Schistosomiasis. Sleeping sickness. Strongyloidiasis, Taeniasis, Toxocariasis, Toxoplasmosis, Trichinosis, and Trichuriasis.

[0130] In certain embodiments, the parasite is Acanthamoeba, Anisakis, Ascaris lumbricoides, Botfly, Balantidium coli, Bedbug, Cestoda (tapeworm), Chiggers, Cochliomyia hominivorax. Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, Hookworm. Leishmania, Linguatula serrata, Liver fluke, Loa loa, Paragonimus - lung fluke, Pinworm, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis, Mite, Tapeworm, Toxoplasma gondii, Trypanosoma, Whipworm, or Wuchereria bancrofti.

[0131] Malaria Antigen - In one embodiment, the antigen comprises a malaria antigen (i.e., PF antigen or PF immunogen), or fragment thereof, or variant thereof. For example, in one embodiment, the antigen comprises an antigen from a parasite causing malaria. In one embodiment, the malaria causing parasite is Plasmodium falciparum.

[0132] In some embodiments, the malaria antigen comprises one or more of P. falciparum immunogens CS; LSA1; TRAP; CelTOS; and Amal. The immunogens may be full length or immunogenic fragments of full-length proteins.

[0133] Bacterial Antigens - In one embodiment, the antigen comprises a bacterial antigen or fragment or variant thereof. In certain embodiments, the bacterium is from any one of the following phyla: Acidobacteria, Actinobacteria, Aquificae. Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae, and Verrucomicrobia.

[0134] In certain embodiments, the bacterium is a gram-positive bacterium or a gram negative bacterium. In certain embodiments, the bacterium is an aerobic bacterium or an anaerobic bacterium. In certain embodiments, the bacterium is an autotrophic bacterium or a heterotrophic bacterium. In certain embodiments, the bacterium is a mesophile, a neutrophile, an extremophile, an acidophile, an alkaliphile, a thermophile, psychrophile, halophile, or an osmophile.

[0135] In certain embodiments, the bacterium is an anthrax bacterium, an antibiotic resistant bacterium, a disease-causing bacterium, a food poisoning bacterium, an infectious bacterium, Salmonella bacterium, Staphylococcus bacterium, Streptococcus bacterium, or tetanus bacterium. In certain embodiments, bacterium is a mycobacteria, Clostridium tetani, Yersinia pestis, Bacillus anthracis, methicillin- resistant Staphylococcus aureus (MRS A), or Clostridium difficile.

[0136] Mycobacterium tuberculosis Antigens - In one embodiment, the antigen comprises a Mycobacterium tuberculosis antigen (i.e., TB antigen or TB immunogen), or fragment thereof, or variant thereof. The TB antigen can be from the Ag85 family of TB antigens, for example, Ag85A and Ag85B. The TB antigen can be from the Esx family of TB antigens, for example, Esx A, EsxB, EsxC, EsxD, EsxE, EsxF, EsxH, EsxO, EsxQ, EsxR, EsxS, EsxT, EsxU, EsxV, and EsxW.

[0137] Fungal Antigens - In one embodiment, the antigen comprises a fungal antigen or fragment or variant thereof. In certain embodiments, the fungus is Aspergillus species, Blastomyces dermatitidis, Candida yeasts (e.g., Candida albicans), Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, dermatophyte, Fusarium species, Histoplasma capsulatum, Mucoromycotina, Pneumocystis jirovecii, Sporothrix schenckii, Exserohilum, or Cladosporium.

[0138] Tumor Antigens - In certain embodiments, the antigen comprises a tumor antigen, including for example a tumor-associated antigen or a tumor-specific antigen. In the context of the present invention, “tumor antigen” or “hyperporoliferative disorder antigen” or “antigen associated with a hyperproliferative disorder” refer to antigens that are common to specific hyperproliferative disorders. In certain aspects, the hyperproliferative disorder antigens of the present invention are derived from cancers including, but not limited to, primary' or metastatic melanoma, mesothelioma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkins lymphoma, leukemias, uterine cancer, cervical cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like.

[0139] Tumor antigens are proteins that are produced by tumor cells that elicit an immune response, particularly T-cell mediated immune responses. In one embodiment, the tumor antigen of the present invention comprises one or more antigenic cancer epitopes immunogenically recognized by tumor infdtrating lymphocytes (TIL) derived from a cancer tumor of a mammal. The selection of the antigen will depend on the particular type of cancer to be treated or prevented by way of the composition of the invention.

[0140] Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), P-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF. prostase. prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

[0141] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor. Malignant tumors express a number of proteins that can serve as target antigens for an immune attack. These molecules include but are not limited to tissue-specific antigens such as MART-1, tyrosinase and GP 100 in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation- related molecules such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are onco-fetal antigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma the tumor-specific idiotype immunoglobulin constitutes a truly tumorspecific immunoglobulin antigen that is unique to the individual tumor. B-cell differentiation antigens such as CD19, CD20 and CD37 are other candidates for target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD 19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.

[0142] The type of tumor antigen referred to in the invention may also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). A TSA is unique to tumor cells and does not occur on other cells in the body. A TAA associated antigen is not unique to a tumor cell and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen. The expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells but which are expressed at much higher levels on tumor cells.

[0143] Non-limiting examples of TSA or TAA antigens include the following: Differentiation antigens such as MART-l / MelanA (MART-I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL. E2A-PRL. H4-RET. 1GH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (EIPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3. c-met, nm-23Hl, PSA, TAG-72, CA 19-9. CA 72-4. CAM 17.1, NuMa, K-ras, beta-Catenin. CDK4. Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15- 3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB / 70K, NY- CO-1, RCAS1. SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

[0144] Exemplary antigens that can be presented using the IL-21 NICs of the invention include, but are not limited to, a SARS-CoV-2 Spike antigen, SARS-CoV-2 Spike protein receptor binding domain (RBD), a hemagglutinin antigen (HA), HIV GT8 antigen and the like. SARS Coronavirus 2 (SARS-CoV-2) Antigen

[0145] Provided herein are NICs, comprising a SARS coronavirus 2 (SARS- CoV-2) antigen, a fragment thereof, a variant thereof, or a combination thereof. The NIC can be used to treat SARS-CoV-2 infection, thereby treating, preventing, and / or protecting against SARS-CoV-2 based pathologies. In one embodiment, the SARS- CoV-2 based pathology is COVID-19. The NIC can significantly induce an immune response of a subject administered the vaccine, thereby protecting against and treating SARS-CoV-2 infection.

[0146] In one embodiment, the SARS-CoV-2 antigen comprises the receptor binding domain of the SARS-CoV-2 spike protein, or a fragment thereof. In one embodiment, the SARS-CoV-2 antigen comprises a dimer of the receptor binding domain of the SARS-CoV-2 spike protein.

[0147] As described above, in one embodiment, the invention relates to a vaccine comprising a SARS-CoV-2 antigen, a fragment thereof, a variant thereof, or a combination thereof. Coronaviruses, including SARS-CoV-2. are encapsulated by a membrane and have a type 1 membrane glycoprotein known as spike (S) protein, which forms protruding spikes on the surface of the coronavirus. The spike protein facilitates binding of the coronavirus to proteins located on the surface of a cell, for example, the metalloprotease amino peptidase N, and mediates cell-viral membrane fusion. In particular, the spike protein contains an S 1 subunit that facilitates binding of the coronavirus to cell surface proteins and thus comprises a receptor binding domain (RBD). Accordingly, the SI subunit of the spike protein controls which cells are infected by the coronavirus. In one embodiment, the SARS-CoV-2 antigen of the invention can comprise one or more SARS-CoV-2 spike protein RBD.

[0148] The SARS-CoV-2 antigen can be a SARS-CoV-2 spike protein RBD. a fragment thereof, a variant thereof, or a combination thereof. In one embodiment, the composition of the invention comprises a dimer of the SARS-CoV-2 spike protein RBD.

[0149] In one embodiment, the composition of the invention is capable of eliciting an immune response in a mammal against one or more SARS-CoV-2 strains. The SARS-CoV-2 antigen can comprise an epitope(s) that makes it particularly effective as an immunogen against which an anti-SARS-CoV-2 immune response can be induced.

[0150] The SARS-CoV-2 spike protein RBD can be a consensus sequence derived from two or more strains of SARS-CoV-2. The SARS-CoV-2 spike antigen can comprise a consensus sequence and / or modification(s) for improved expression. Modification can include codon optimization, RNA optimization, addition of a kozak sequence for increased translation initiation, and / or the addition of an immunoglobulin leader sequence to increase the immunogenicity of the one or more SARS-CoV-2 spike protein RBD. The one or more SARS-CoV-2 spike protein RBD can comprise a signal peptide such as an immunoglobulin signal peptide, for example, but not limited to, an immunoglobulin E (IgE) or immunoglobulin (IgG) signal peptide.

[0151] The SARS-CoV-2 RBD can have an amino acid sequence of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO 29 or SEQ ID NO:31, or a fragment or variant thereof. In some embodiments, the SARS-CoV-2 RBD can be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29 or SEQ ID NO:31.

[0152] The nucleic acid molecule encoding the SARS-CoV-2 RBD antigen can comprise the nucleic acid sequence of SEQ ID NO:22, SEQ ID NO:24. SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30, or a fragment or variant thereof. In some embodiments, the nucleic acid molecule encoding the SARS-CoV-2 RBD antigen can comprise a nucleotide sequence that encodes the amino acid sequence having at least about 80%. 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%, 89%, 90%, 91%, 92%, 93%, 94%. 95%. 96%. 97%. 98%. 99%. or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29 or SEQ ID NO:31. In some embodiments, the nucleic acid molecule encoding the SARS-CoV-2 RBD antigen can comprise a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%, 96%, 97%, 98%, 99% or 100% identity over an entire length of the nucleic acid sequence set forth in SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30. In some embodiments, the SARS-CoV-2 RBD antigen can be operably linked to an IgE leader sequence.

[0153] Immunogenic fragments of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29 or SEQ ID NO:31 can be provided. Immunogenic fragments can comprise at least 60%, at least 65%, 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% or at least 99% of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27. SEQ ID NO:29 or SEQ ID NO:31. In some embodiments, immunogenic fragments include a leader sequence, such as for example an immunoglobulin leader, such as the IgE leader. In some embodiments, immunogenic fragments are free of a leader sequence.

[0154] Immunogenic fragments of proteins with amino acid sequences homologous to immunogenic fragments of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29 or SEQ ID NO:31 can be provided. Such immunogenic fragments can comprise at least 60%, at least 65%, 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% or at least 99% identity to SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29 or SEQ ID NO:31. In some embodiments, immunogenic fragments include a leader sequence, such as for example an immunoglobulin leader, such as the IgE leader. In some embodiments, immunogenic fragments are free of a leader sequence.

[0155] Some embodiments relate to immunogenic fragments of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30. Immunogenic fragments can be at least 60%, at least 65%, 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% or at least 99% of the full length of SEQ ID NO:22, SEQ ID NO:24. SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30. Immunogenic fragments can comprise at least 95%, at least 96%, at least 97% at least 98% or at least 99% identity7to fragments of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26. SEQ ID NO:28, or SEQ ID NO:30. In some embodiments, immunogenic fragments include sequences that encode a leader sequence, such as for example an immunoglobulin leader, such as the IgE leader. In some embodiments, fragments are free of coding sequences that encode a leader sequence.

[0156] In some embodiments the NIC comprises an immunoadjuvant domain operably linked to SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39. SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71. SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO 77, SEQ ID NO:79, SEQ ID NO:81. SEQ ID NO: 83 or SEQ ID NO: 85, or a fragment or variant thereof comprising at least the RBD domain. Exemplary fragments and variants of SEQ ID NO:33, SEQ ID NO 35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO 51, SEQ ID NO:53, SEQ ID NO:55. SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID N0:81, SEQ ID NO:83 or SEQ ID NO: 85 include, but are not limited to fragments lacking the IgE leader sequence.

[0157] In some embodiments the nucleotide sequence encoding the NIC comprises a nucleotide sequence encoding an immunoadjuvant domain operably linked to SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44. SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56. SEQ ID NO:58, SEQ ID NO:60. SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO: 82 or SEQ ID NO: 84, or a fragment or variant thereof encoding at least the RED domain. Exemplary fragments and variants of SEQ ID NO:32, SEQ ID NO:34. SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66. SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76. SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82 or SEQ ID NO:84 include, but are not limited to, fragments lacking the nucleotide sequence encoding the IgE leader sequence.

[0158] In some embodiments, the NIC comprises an IL-21 immunoadjuvant domain and a SARS-Cov-2 antigen. In some embodiments, the NIC comprises an IL-6 immunoadjuvant domain and a SARS-Cov-2 antigen. In some embodiments, the NIC comprises an IL-4 immunoadjuvant domain and a SARS-Cov-2 antigen. In some embodiments, the NIC comprises a TSLP immunoadjuvant domain and a SARS-Cov-2 antigen. In some embodiments, the NIC comprises an IL-21 immunoadjuvant domain and an RBD antigen. In some embodiments, the NIC comprises an IL-6 immunoadjuvant domain and an RBD antigen. In some embodiments, the NIC comprises an IL-4 immunoadjuvant domain and an RBD antigen. In some embodiments, the NIC comprises a TSLP immunoadjuvant domain and an RBD antigen.

[0159] Human Immunodeficiency Virus (HIV) Antigen

[0160] Provided herein are NICs, comprising a human immunodeficiency vims (HIV) antigen, a fragment thereof, a variant thereof, or a combination thereof. The NIC can be used to treat HIV infection, thereby treating, preventing, and / or protecting against HIV based pathologies. In one embodiment, the HIV based pathology' is AIDS. The NIC can significantly induce an immune response of a subject administered the vaccine, thereby protecting against and treating HIV infection.

[0161] The HIV antigen can have an amino acid sequence of SEQ ID NO:96, or a fragment or variant thereof. In some embodiments, the HIV antigen can be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ IDNO:96.

[0162] The nucleic acid molecule encoding the HIV antigen can comprise the nucleic acid sequence of SEQ ID NO:97, or a fragment or variant thereof.

[0163] In some embodiments, the nucleic acid molecule encoding the HIV antigen can comprise a nucleotide sequence that encodes the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO:96. In some embodiments, the nucleic acid molecule encoding the HIV antigen can comprise a nucleotide sequence having at least about 80%. 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over an entire length of the nucleic acid sequence set forth in SEQ ID NO:97.

[0164] Immunogenic fragments of SEQ ID NO:96 can be provided. Immunogenic fragments can comprise at least 60%. at least 65%. 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% or at least 99% of SEQ ID NO:96. In some embodiments, immunogenic fragments include a leader sequence, such as for example an immunoglobulin leader, such as the IgE leader. In some embodiments, immunogenic fragments are free of a leader sequence.

[0165] Immunogenic fragments of proteins with amino acid sequences homologous to immunogenic fragments of SEQ ID NO:96 can be provided. Such immunogenic fragments can comprise at least 60%, at least 65%, 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% or at least 99% identity to SEQ ID NO:96. In some embodiments, immunogenic fragments include a leader sequence, such as for example an immunoglobulin leader, such as the IgE leader. In some embodiments, immunogenic fragments are free of a leader sequence. Some embodiments relate to immunogenic fragments of SEQ ID NO:97. Immunogenic fragments can be at least 60%, at least 65%, 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% or at least 99% of the full length of SEQ ID NO:97. Immunogenic fragments can comprise at least 95%, at least 96%, at least 97% at least 98% or at least 99% identity to fragments of SEQ ID NO: 97. In some embodiments, immunogenic fragments include sequences that encode a leader sequence, such as for example an immunoglobulin leader, such as the IgE leader. In some embodiments, fragments are free of coding sequences that encode a leader sequence.

[0166] In some embodiments, the NIC comprising an HIV antigen is an IL-21 NIC. In some embodiments, the NIC is a GT8 IL-21 NIC. In some embodiments the GT8 IL-21 NIC comprises an amino acid sequence of SEQ ID NO: 108, SEQ ID NO: 111, SEQ ID NO: 96 or a fragment or variant thereof. In some embodiments, the IL-6 NIC comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO: 108, SEQ ID NO: 111. or SEQ ID NO:96.

[0167] In some embodiments the nucleotide sequence encoding the GT8 IL-21 NIC comprises a nucleotide sequence of SEQ ID NO:106, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO:97, or a fragment or variant thereof. In some embodiments, the GT8 IL-21 NIC is encoded by a nucleic acid molecule comprising a nucleotide sequence having at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the nucleotide sequence set forth in SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 110, or SEQ ID NO:97.

[0168] In some embodiments, the NIC comprising an HIV antigen is an IL-6 NIC. In some embodiments, the NIC is a GT8 IL-6 NIC. In some embodiments the GT8 IL-6 NIC comprises an amino acid sequence of SEQ ID NO:91, SEQ NO: 93, SEQ ID NO: 96 or a fragment or variant thereof. In some embodiments, the GT8 IL-6 NIC comprises an amino acid sequence having at least about 90%. 91%. 92%. 93%. 94%. 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO: 91 or SEQ ID NO: 93, or SEQ ID NO: 96.

[0169] In some embodiments the nucleotide sequence encoding the GT8 IL-6 NIC comprises a nucleotide sequence of SEQ ID NO:92, SEQ ID NO: 94, SEQ ID NO:95, SEQ ID NO: 97, or a fragment or variant thereof. In some embodiments, the GT8 IL-6 NIC is encoded by a nucleic acid molecule comprising a nucleotide sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99%. or 100% identity over an entire length of the nucleotide sequence set forth in SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:95, or SEQ ID NO:97.

[0170] In some embodiments, the NIC comprising an HIV antigen is an IL-4 NIC. In some embodiments, the NIC is a GT8 IL-4 NIC In some embodiments, the GT8 IL-4 NIC comprises an amino acid sequence of SEQ ID NO:98, SEQ ID NO:99. SEQ ID NO:96, or a fragment or variant thereof. In some embodiments, the GT8 IL-4 NIC comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO:98, SEQ ID NO:99. or SEQ ID NO:96.

[0171] In some embodiments, the nucleotide sequence encoding the GT8 IL-4 NIC comprises a nucleotide sequence of SEQ ID NO:100, SEQ ID NO: 101, SEQ ID NO: 97, or a fragment or variant thereof. In some embodiments, the IL-4 NIC is encoded by a nucleic acid molecule comprising anucleotide sequence having at least about 90%, 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% identity over an entire length of the nucleotide sequence set forth in SEQ ID NO: 100, SEQ ID NO: 101, or SEQ ID NO:97.

[0172] In some embodiments, the NIC comprising an HIV antigen is a TSLP NIC. In some embodiments, the TSLP NIC comprises an amino acid sequence of SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO:96, or a fragment or variant thereof. In some embodiments, the TSLP NIC comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO: 102, SEQ ID NO: 103, or SEQ ID NO:96.

[0173] In some embodiments, the nucleotide sequence encoding the TSLP comprises a nucleotide sequence of SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO:97 or a fragment or variant thereof. In some embodiments, the TSLP NIC is encoded by a nucleic acid molecule comprising a nucleotide sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the nucleotide sequence set forth in SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO:97. Influenza Antigen

[0174] Provided herein are NICs, comprising an influenza antigen, a fragment thereof, a variant thereof, or a combination thereof. The NIC can be used to treat influenza infection, thereby treating, preventing, and / or protecting against influenza based pathologies. The NIC can significantly induce an immune response of a subject administered the vaccine, thereby protecting against and treating influenza infection.

[0175] In some embodiments, the influenza antigen is the head domain of influenza H1N1 A / New Caledonia / 20 / 1999 hemagglutinin (NC99) antigen. The NC99 antigen can have an amino acid sequence of SEQ ID NO: 118, or a fragment or variant thereof. In some embodiments, the NC99 antigen can be an amino acid sequence having at least about 80%. 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO: 118.

[0176] The nucleic acid molecule encoding the NC99 antigen can comprise the nucleic acid sequence of SEQ ID NO: 119.or a fragment or variant thereof. In some embodiments, the nucleic acid molecule encoding the HIV antigen can comprise a nucleotide sequence that encodes the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO: 118. In some embodiments, the nucleic acid molecule encoding the NC99 antigen can comprise a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over an entire length of the nucleic acid sequence set forth in SEQ ID NO: 118.

[0177] Immunogenic fragments of SEQ ID NO: 118 can be provided. Immunogenic fragments can comprise at least 60%, at least 65%, 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% or at least 99% of SEQ ID NO: 118. In some embodiments, immunogenic fragments include a leader sequence, such as for example an immunoglobulin leader, such as the IgE leader. In some embodiments, immunogenic fragments are free of a leader sequence.

[0178] Immunogenic fragments of proteins with amino acid sequences homologous to immunogenic fragments of SEQ ID NO: 118 can be provided. Such immunogenic fragments can comprise at least 60%, at least 65%, 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% or at least 99% identity to SEQ ID NO: 118. In some embodiments, immunogenic fragments include a leader sequence, such as for example an immunoglobulin leader, such as the IgE leader. In some embodiments, immunogenic fragments are free of a leader sequence.

[0179] Some embodiments relate to immunogenic fragments of SEQ ID NO: 119. Immunogenic fragments can be at least 60%, at least 65%, 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% or at least 99% of the full length of SEQ ID NO: 119. Immunogenic fragments can comprise at least 95%. at least 96%. at least 97% at least 98% or at least 99% identity to fragments of SEQ ID NO: 1 19. In some embodiments, immunogenic fragments include sequences that encode a leader sequence, such as for example an immunoglobulin leader, such as the IgE leader. In some embodiments, fragments are free of coding sequences that encode a leader sequence.

[0180] In some embodiments, the NIC comprises an IL-21 immunoadjuvant domain and an influenza antigen. In some embodiments, the NIC comprises an IL-6 immunoadjuvant domain and an influenza antigen. In some embodiments, the NIC comprises an IL-4 immunoadjuvant domain and an influenza antigen. In some embodiments, the NIC comprises a TSLP immunoadjuvant domain and an influenza antigen. In some embodiments, the NIC comprises an IL-21 immunoadjuvant domain and an NC99 antigen. In some embodiments, the NIC comprises an IL-6 immunoadjuvant domain and an NC99 antigen. In some embodiments, the NIC comprises an IL-4 immunoadjuvant domain and an NC99 antigen. In some embodiments, the NIC comprises a TSLP immunoadjuvant domain and an NC99 antigen.

[0181] In some embodiments, the NIC comprising IL-21 and NC99 (NC99 IL- 21 NIC) comprises an amino acid sequence of SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO: 1 18, or a fragment or variant thereof. In some embodiments, the NC99 IL-21 NIC comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO:86, SEQ ID NO:88, or SEQ ID NO: 118.

[0182] In some embodiments the nucleotide sequence encoding the NC99 IL-21 NIC comprises a nucleotide sequence of SEQ ID NO:87, SEQ ID NO: 89, SEQ ID NO:90, SEQ ID NO: 97, SEQ ID NO: 119, or a fragment or variant thereof. In some embodiments, the NC99 IL-21 NIC is encoded by a nucleic acid molecule comprising a nucleotide sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the nucleotide sequence set forth in SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO:90, SEQ ID NO: 97, or SEQ ID NO: 119.

[0183] Cytokine NICs

[0184] Provided herein are cytokine NICs and nucleic acid molecules encoding a cytokine NIC monomer, comprising a disease-associated antigen or a nucleotide sequence encoding the same. The cytokine NIC can be used to treat, prevent, and / or protecting against a disease or disorder associated with the antigen displayed by the NIC. The NIC can significantly induce an immune response of a subject administered the NIC or a nucleic acid molecule encoding the NIC, thereby protecting against and treating a disease or disorder associated with the antigen displayed by the NIC.

[0185] The composition comprising the NIC or a nucleic acid molecule encoding the NIC can be a DNA vaccine, a peptide vaccine, a delivery vehicle comprising a nucleic acid molecule encoding the NIC, or a combination DNA and peptide vaccine. The composition can include a nucleic acid sequence encoding the NIC. The nucleic acid sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid sequence can also include additional sequences that encode linker, leader, or tag sequences that are linked to the disease-associated antigen by a peptide bond. The peptide vaccine can include a NIC monomer or an assembled / oligomerized NIC, a variant thereof, a fragment thereof, or a combination thereof.

[0186] NICs may be formed by the assembly of NIC monomers comprising a) a cytokine immunoadjuvant domain, b) at least one antigen and c) at least one protein oligomerization domain. Without limitation, the NICs of the invention may selfassemble into a tetrahedron, a cube, an octahedron, a dodecahedron, or an icosahedron. The NICs of the invention may be used as an efficient means for presenting multiple copies of a disease-associated antigen.

[0187] The composition comprising the NIC or a nucleic acid molecule encoding the NIC can induce a humoral immune response in the subject administered the composition comprising the NIC or a nucleic acid molecule encoding the NIC. The induced humoral immune response can be specific for the disease-associated antigen. The induced humoral immune response can be reactive with the disease-associated antigen. The humoral immune response can be induced in the subject administered the vaccine by about 1.5-fold to about 16-fold, about 2-fold to about 12-fold, or about 3-fold to about 10-fold. The humoral immune response can be induced in the subject administered the vaccine by at least about 1.5-fold, at least about 2.0-fold, at least about

[0188] 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5 -fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0- fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 1 l.O-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about

[0189] 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 15.5-fold, or at least about 16.0-fold.

[0190] The humoral immune response induced by the vaccine can include an increased level of neutralizing antibodies associated with the subject administered the vaccine as compared to a subject not administered the vaccine. The neutralizing antibodies can be specific for the disease-associated antigen. The neutralizing antibodies can be reactive with the disease-associated antigen. The neutralizing antibodies can provide protection against and / or treatment of a disease or disorder associated with the antigen and its associated pathologies in the subject administered the composition comprising the NIC or a nucleic acid molecule encoding the NIC.

[0191] The humoral immune response induced by the vaccine can include an increased level of IgG antibodies associated with the subject administered the vaccine as compared to a subject not administered the vaccine. These IgG antibodies can be specific for the disease associated antigen. These IgG antibodies can be reactive with the disease associated antigen. In some embodiments, the humoral response is cross-reactive against two or more strains of a pathogen. The level of IgG antibody associated with the subject administered the vaccine can be increased by about 1.5-fold to about 16-fold, about 2-fold to about 12-fold, or about 3-fold to about 10-fold as compared to the subject not administered the vaccine. The level of IgG antibody associated with the subject administered the vaccine can be increased by at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5- fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about l l.O-fold, at least about 1 1.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 15.5-fold, or at least about 16.0-fold as compared to the subject not administered the vaccine.

[0192] The vaccine can induce a cellular immune response in the subject administered the vaccine. The induced cellular immune response can be specific for the disease associated antigen. The induced cellular immune response can be reactive to the disease associated antigen. In some embodiments, the cellular response is cross-reactive against two or more strains of a pathogen. The induced cellular immune response can include eliciting a CD8+T cell response. The elicited CD8+T cell response can be reactive with the disease associated antigen. The elicited CD8+T cell response can be polyfunctional. The induced cellular immune response can include eliciting a CD8 T cell response, in which the CD8+T cells produce interferon-gamma (IFN-y), tumor necrosis factor alpha (TNF-a), interleukin-2 (IL-2), or a combination of IFN-y and TNF- a.

[0193] The induced cellular immune response can include an increased CD8+T cell response associated with the subject administered the vaccine as compared to the subject not administered the vaccine. The CD81T cell response associated with the subject administered the vaccine can be increased by about 2-fold to about 30-fold, about 3-fold to about 25-fold, or about 4-fold to about 20-fold as compared to the subject not administered the vaccine. The CD8+T cell response associated with the subject administered the vaccine can be increased by at least about 1.5-fold, at least about 2.0-fold, at least about 3.0-fold, at least about 4.0-fold, at least about 5.0-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5- fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about

[0194] 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 1 l.O-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 16.0-fold, at least about 17.0-fold, at least about 18.0-fold, at least about 19.0-fold, at least about 20.0-fold, at least about 21.0-fold, at least about 22.0-fold, at least about 23.0-fold, at least about 24.0-fold, at least about 25.0-fold, at least about 26.0-fold, at least about 27.0-fold, at least about 28.0-fold, at least about 29.0-fold, or at least about 30.0-fold as compared to the subject not administered the vaccine.

[0195] The induced cellular immune response can include an increased frequency of CD3+CD8+T cells that produce IFN-y. The frequency of CD3 CD8 IFN-y+T cells associated with the subject administered the vaccine can be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold. 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12- fold, 13-fold. 14-fold, 15-fold. 16-fold, 17-fold. 18-fold, 19-fold, or 20-fold as compared to the subject not administered the vaccine.

[0196] The induced cellular immune response can include an increased frequency of CD3+CD8+T cells that produce TNF-a. The frequency of CD3 CD8 TNF- a+T cells associated with the subject administered the vaccine can be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, or 14-fold as compared to the subject not administered the vaccine.

[0197] The induced cellular immune response can include an increased frequency of CD3+CD8+T cells that produce IL-2. The frequency of CD3+CD8+IL-2+T cells associated with the subject administered the vaccine can be increased by at least about 0.5-fold, l.O-fold, 1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, or 5.0-fold as compared to the subject not administered the vaccine.

[0198] The induced cellular immune response can include an increased frequency of CD31CD81T cells that produce both IFN-y and TNF-a. The frequency of CD3+CD8+IFN-y+TNF-a+T cells associated with the subject administered the vaccine can be increased by at least about 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 110-fold. 120-fold. 130-fold. 140-fold, 150-fold, 160-fold, 170-fold, or 180-fold as compared to the subject not administered the vaccine.

[0199] The cellular immune response induced by the vaccine can include eliciting a CD4+T cell response. The elicited CD4+T cell response can be reactive with the disease associated antigen. The elicited CD4+T cell response can be polyfunctional. The induced cellular immune response can include eliciting a CD4+T cell response, in which the CD4+T cells produce IFN-y, TNF-a, IL-2, or a combination of IFN-y and TNF-a.

[0200] The induced cellular immune response can include an increased frequency of CD3+CD4+T cells that produce IFN-y. The frequency of CD3+CD4+IFN-y+T cells associated with the subject administered the vaccine can be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12- fold, 13-fold. 14-fold, 15-fold. 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold as compared to the subject not administered the vaccine.

[0201] The induced cellular immune response can include an increased frequency of CD3+CD4+T cells that produce TNF-a. The frequency of CD3 CD4 TNF- a+T cells associated with the subject administered the vaccine can be increased by at least about 2-fold, 3-fold, 4-fold. 5-fold. 6-fold. 7-fold, 8-fold, 9-fold. 10-fold, 11-fold. 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, or 22 -fold as compared to the subject not administered the vaccine.

[0202] The induced cellular immune response can include an increased frequency of CD3+CD4+T cells that produce IL-2. The frequency of CD3+CD4+IL-2+T cells associated with the subject administered the vaccine can be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12- fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold. 21-fold, 22- fold, 23-fold. 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32- fold, 33-fold. 34-fold, 35-fold. 36-fold, 37-fold. 38-fold, 39-fold, 40-fold. 45-fold, 50- fold, 55-fold, or 60-fold as compared to the subject not administered the vaccine.

[0203] The induced cellular immune response can include an increased frequency of CD3+CD4+T cells that produce both IFN-y and TNF-a. The frequency of CD3 CD4 IFN-y TNF-a associated with the subject administered the vaccine can be increased by at least about 2-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0- fold, 5.5-fold, 6.0-fold, 6.5-fold, 7.0-fold, 7.5-fold, 8.0-fold, 8.5-fold, 9.0-fold, 9.5-fold, 10.0-fold, 10.5-fold, l l.O-fold. 11.5-fold, 12.0-fold, 12.5-fold, 13.0-fold, 13.5-fold, 14.0-fold, 14.5-fold, 15.0-fold. 15.5-fold, 16.0-fold, 16.5-fold, 17.0-fold, 17.5-fold, 18.0-fold, 18.5-fold, 19.0-fold, 19.5-fold, 20.0-fold, 21-fold, 22-fold, 23-fold 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31 -fold, 32-fold, 33-fold, 34-fold, or 35-fold as compared to the subject not administered the vaccine.

[0204] The vaccine of the present invention can have features required of effective vaccines such as being safe so the vaccine itself does not cause illness or death; is protective against illness resulting from exposure to live pathogens such as viruses or bacteria; induces neutralizing antibody to prevent invention of cells; induces protective T cells against intracellular pathogens; and provides ease of administration, few side effects, biological stability, and low cost per dose. The vaccine can further induce an immune response when administered to different tissues such as the muscle or skin. The vaccine can further induce an immune response when administered via electroporation, or injection, or subcutaneously, or intramuscularly.

[0205] Vector

[0206] In some embodiments, the invention provides nucleic acid molecules encoding the NICs of the invention. In some embodiments, the nucleic acid molecules can comprise one or more vectors that include a nucleotide sequence encoding an NIC monomer. The one or more vectors can be capable of expressing the NIC monomer. The vector can have a nucleic acid sequence containing an origin of replication. The vector can be a plasmid, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. The vector can be either a self-replicating extrachromosomal vector or a vector which integrates into a host genome.

[0207] The one or more vectors can be an expression construct, which is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation machinery ribosomal complexes. The plasmid is frequently engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector. The vectors of the present invention express large amounts of stable messenger RNA, and therefore proteins.

[0208] The vectors may have expression signals such as a strong promoter, a strong termination codon, adjustment of the distance between the promoter and the cloned gene, and the insertion of a transcription termination sequence and a PTIS (portable translation initiation sequence).

[0209] (1) Expression Vectors

[0210] The vector can be a circular plasmid or a linear nucleic acid. The circular plasmid and linear nucleic acid are capable of directing expression of a particular nucleotide sequence in an appropriate subject cell. The vector can have a promoter operably linked to the antigen-encoding nucleotide sequence, which may be operably linked to termination signals. The vector can also contain sequences required for proper translation of the nucleotide sequence. The vector comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter, which initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development.

[0211] (2) Circular and Linear Vectors

[0212] The vector may be a circular plasmid, which may transform a target cell by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication).

[0213] The vector can be pVAX, pcDNA3.0, or provax. or any other expression vector capable of expressing DNA encoding the antigen and enabling a cell to translate the sequence to an antigen that is recognized by the immune system.

[0214] Also provided herein is a linear nucleic acid vaccine, or linear expression cassette (“LEC”), that is capable of being efficiently delivered to a subject via electroporation and expressing one or more desired antigens. The LEC may be any linear DNA devoid of any phosphate backbone. The DNA may encode one or more antigens. The LEC may contain a promoter, an intron, a stop codon, and / or a poly adenylation signal. The expression of the antigen may be controlled by the promoter. The LEC may not contain any antibiotic resistance genes and / or a phosphate backbone. The LEC may not contain other nucleic acid sequences unrelated to the desired antigen gene expression.

[0215] (3) Promoter, Intron, Stop Codon, and Polyadenylation Signal

[0216] The vector may have a promoter. A promoter may be any promoter that is capable of driving gene expression and regulating expression of the isolated nucleic acid. Such a promoter is a cis-acting sequence element required for transcription via a DNA dependent RNA polymerase, which transcribes the antigen sequence described herein. Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application. The promoter may be positioned about the same distance from the transcription start in the vector as it is from the transcription start site in its natural setting. However, variation in this distance may be accommodated without loss of promoter function. The promoter may be operably linked to the nucleic acid sequence encoding the antigen and signals required for efficient poly adenylation of the transcript, ribosome binding sites, and translation termination. The promoter may be a CMV promoter, SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or another promoter shown effective for expression in eukaryotic cells.

[0217] The vector may include an enhancer and an intron with functional splice donor and acceptor sites. The vector may contain a transcription termination region dow nstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.

[0218] Excipients and other Components of the Vaccine

[0219] The vaccine may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be functional molecules such as vehicles, carriers, or diluents. The pharmaceutically acceptable excipient can be a transfection facilitating agent, which can include surface active agents, such as immune- stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, poly cations, or nanoparticles, or other known transfection facilitating agents.

[0220] The transfection facilitating agent is a polyanion, polycation, including poly-L-glutamate (LGS), or lipid. The transfection facilitating agent is poly-L- glutamate, and the poly-L-glutamate may be present in the vaccine at a concentration less than 6 mg / ml. The transfection facilitating agent may also include surface active agents such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid may also be used administered in conjunction with the genetic construct. The DNA plasmid vaccines may also include a transfection facilitating agent such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, as a DNA-liposome mixture (see for example W09324640). calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. The transfection facilitating agent is a polyanion, polycation, including poly -L -glutamate (LGS), or lipid. Concentration of the transfection agent in the vaccine is less than 4 mg / ml, less than 2 mg / ml, less than 1 mg / ml, less than 0.750 mg / ml, less than 0.500 mg / ml, less than 0.250 mg / ml, less than 0.100 mg / ml, less than 0.050 mg / ml, or less than 0.010 mg / ml.

[0221] The pharmaceutically acceptable excipient can be an adjuvant. The adjuvant can be other genes that are expressed in an alternative plasmid or are delivered as proteins in combination with the plasmid above in the vaccine. The adjuvant may be selected from the group consisting of: a-interferon(IFN- a), P-interferon (IFN- ), y- interferon, platelet derived growth factor (PDGF), TNFa, TNFp, GM-CSF, epidermal growth factor (EGF). cutaneous T cell -attracting chemokine (CTACK). epithelial thymus-expressed chemokine (TECK), mucosae-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 including IL-15 having the signal sequence deleted and optionally including the signal peptide from IgE. The adjuvant can be IL-12, IL- 15, IL-28, CTACK, TECK, platelet derived growth factor (PDGF), TNFa, TNFp, GM-CSF, epidermal growth factor (EGF). IL-1. IL-2. IL-4. IL-5, IL-6, IL-10, IL-12. IL- 18, TSLP or a combination thereof.

[0222] Other genes that can be useful as adjuvants include those encoding: MCP-1, MIP-la, MIP-lp, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1. Mac-1. pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF. DR4. DR5, KILLER, TRAIL-R2, TRICK2, DR6. Caspase ICE. Fos, c-jun. Sp-1. Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK. TRAF6, IkB, Inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, 0x40, 0x40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPI, TAP2 and functional fragments thereof.

[0223] The composition comprising a nucleic acid molecule encoding a NIC monomer may further comprise a genetic vaccine facilitator agent as described in U.S. Serial No. 021,579 filed April 1, 1994, which is fully incorporated by reference.

[0224] The composition comprising a nucleic acid molecule encoding a NIC monomer can be formulated according to the mode of administration to be used. An injectable vaccine pharmaceutical composition can be sterile, pyrogen free and particulate free. An isotonic formulation or solution can be used. Additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The vaccine can comprise a vasoconstriction agent. The isotonic solutions can include phosphate buffered saline. Vaccine can further comprise stabilizers including gelatin and albumin. The stabilizers can allow the formulation to be stable at room or ambient temperature for extended periods of time, including LGS or poly cations or polyanions.

[0225] Method of Vaccination

[0226] Also provided herein is a method of treating, protecting against, and / or preventing disease in a subject in need thereof by administering the NIC or a composition comprising a nucleic acid molecule encoding a NIC monomer to the subject. Administration of the NIC or a composition comprising a nucleic acid molecule encoding a NIC monomer to the subject can induce or elicit an immune response in the subject. The induced immune response can be used to treat, prevent, and / or protect against disease, for example, pathologies relating to infection. In one embodiment, the pathology relating to SARS-CoV-2 infection is COVID- 19. In one embodiment, the pathology relating to HIV infection is AIDS.

[0227] The induced immune response can include an induced humoral immune response and / or an induced cellular immune response. The humoral immune response can be induced by about 1.5-fold to about 16-fold, about 2-fold to about 12-fold, or about 3-fold to about 10-fold. The induced humoral immune response can include IgG antibodies and / or neutralizing antibodies that are reactive to the disease associated antigen. The induced cellular immune response can include a CD8+T cell response, which is induced by about 2-fold to about 30-fold, about 3-fold to about25-fold, or about 4-fold to about 20-fold.

[0228] The vaccine dose can be between 1 pg to 10 mg active component / kg body weight / time, and can be 20 pg to 10 mg component / kg body weight / time. The vaccine can be administered every 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. or 31 days. The number of vaccine doses for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Administration

[0229] The NIC or a composition comprising a nucleic acid molecule encoding a NIC monomer can be formulated in accordance with standard techniques well known to those skilled in the pharmaceutical art. Such compositions can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular subject, and the route of administration. The subject can be a mammal, such as a human, a horse, a cow, a pig, a sheep, a cat, a dog, a rat, or a mouse.

[0230] The vaccine can be administered prophylactically or therapeutically. In prophylactic administration, the vaccines can be administered in an amount sufficient to induce an immune response. In therapeutic applications, the vaccines are administered to a subject in need thereof in an amount sufficient to elicit a therapeutic effect. An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition of the vaccine regimen administered, the manner of administration, the stage and severity of the disease, the general state of health of the patient, and the judgment of the prescribing physician.

[0231] The vaccine can be administered by methods well known in the art as described in Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997)); Feigner et al. (U.S. Pat. No. 5,580.859, issued Dec. 3, 1996); Feigner (U.S. Pat. No. 5,703,055. issued Dec. 30, 1997); and Carson et al. (U.S. Pat. No. 5,679,647, issued Oct. 21, 1997), the contents of all of which are incorporated herein by reference in their entirety. The DNA of the vaccine can be complexed to particles or beads that can be administered to an individual, for example, using a vaccine gun. One skilled in the art would know that the choice of a pharmaceutically acceptable earner, including a physiologically acceptable compound, depends, for example, on the route of administration of the expression vector.

[0232] The NIC or a composition comprising a nucleic acid molecule encoding a NIC monomer can be delivered via a variety of routes. Typical delivery routes include parenteral administration, e.g., intradermal, intramuscular or subcutaneous deliver}’. Other routes include oral administration, intranasal, and intravaginal routes. For the DNA of the vaccine in particular, the vaccine can be delivered to the interstitial spaces of tissues of an individual (Feigner et al., U.S. Pat. Nos. 5,580,859 and 5.703,055, the contents of all of w hich are incorporated herein by reference in their entirety). The vaccine can also be administered to muscle, or can be administered via intradermal or subcutaneous injections, or transdermally. such as by iontophoresis. Epidermal administration of the vaccine can also be employed. Epidermal administration can involve mechanically or chemically irritating the outermost layer of epidermis to stimulate an immune response to the irritant (Carson et al., U.S. Pat. No. 5,679,647, the contents of which are incorporated herein by reference in its entirety).

[0233] The NIC or a composition comprising a nucleic acid molecule encoding NIC monomer can also be formulated for administration via the nasal passages. Formulations suitable for nasal administration, wherein the carrier is a solid, can include a coarse powder having a particle size, for example, in the range of about 10 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. The formulation can be a nasal spray, nasal drops, or by aerosol administration by nebulizer. The formulation can include aqueous or oily solutions of the NIC or a composition comprising a nucleic acid molecule encoding a NIC monomer.

[0234] The NIC or a composition comprising a nucleic acid molecule encoding a NIC monomer can be a liquid preparation such as a suspension, syrup or elixir. The NIC or a composition comprising a nucleic acid molecule encoding a NIC monomer can also be a preparation for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration), such as a sterile suspension or emulsion.

[0235] The NIC or a composition comprising a nucleic acid molecule encoding NIC monomer can be incorporated into lipid nanoparticles, liposomes, microspheres or other polymer matrices (Feigner et al., U.S. Pat. No. 5,703,055; Gregoriadis, Liposome Technology’, Vols. Ito III (2nd ed. 1993), the contents of which are incorporated herein by reference in their entirety). Liposomes or lipid nanoparticles can consist of phospholipids or other lipids, and can be nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

[0236] The NIC or a composition comprising a nucleic acid molecule encoding a NIC monomer can be administered via electroporation, such as by a method described in U.S. Patent No. 7,664,545, the contents of which are incorporated herein by reference. The electroporation can be by a method and / or apparatus described in U.S. Patent Nos. 6,302.874; 5,676,646; 6,241,701: 6,233,482; 6,216,034; 6.208,893; 6,192,270; 6,181,964; 6.150,148; 6,120.493; 6,096.020; 6,068,650; and 5,702,359. the contents of which are incorporated herein by reference in their entirety. The electroporation may be carried out via a minimally invasive device.

[0237] The minimally invasive electroporation device ("MID") may be an apparatus for injecting the vaccine described above and associated fluid into body tissue. The device may comprise a hollow needle, DNA cassette, and fluid deliver}7means, wherein the device is adapted to actuate the fluid delivery means in use so as to concurrently (for example, automatically) inject DNA into body tissue during insertion of the needle into the said body tissue. This has the advantage that the ability to inject the DNA and associated fluid gradually while the needle is being inserted leads to a more even distribution of the fluid through the body tissue. The pain experienced during injection may be reduced due to the distribution of the DNA being injected over a larger area.

[0238] The MID may inject the vaccine into tissue without the use of a needle. The MID may inject the vaccine as a small stream or jet with such force that the vaccine pierces the surface of the tissue and enters the underlying tissue and / or muscle. The force behind the small stream or jet may be provided by expansion of a compressed gas, such as carbon dioxide through a micro-orifice within a fraction of a second. Examples of minimally invasive electroporation devices, and methods of using them, are described in published U.S. Patent Application No. 20080234655; U.S. Patent No. 6,520.950; U.S. Patent No. 7.171,264; U.S. Patent No. 6,208,893; U.S. Patent NO. 6,009.347; U.S. Patent No. 6, 120,493; U.S. Patent No. 7,245,963; U.S. Patent No. 7,328,064; and U.S. Patent No. 6,763,264, the contents of each of which are herein incorporated by reference.

[0239] The MID may comprise an injector that creates a high-speed jet of liquid that painlessly pierces the tissue. Such needle-free injectors are commercially available. Examples of needle-free injectors that can be utilized herein include those described in U.S. Patent Nos. 3,805,783; 4,447,223; 5,505,697; and 4,342,310, the contents of each of which are herein incorporated by reference.

[0240] A desired vaccine in a form suitable for direct or indirect electrotransport may be introduced (e.g., injected) using a needle-free injector into the tissue to be treated, usually by contacting the tissue surface with the injector so as to actuate delivery7of a jet of the agent, with sufficient force to cause penetration of the vaccine into the tissue. For example, if the tissue to be treated is mucosa, skin or muscle, the agent is projected towards the mucosal or skin surface with sufficient force to cause the agent to penetrate through the stratum comeum and into dermal layers, or into underlying tissue and muscle, respectively.

[0241] Needle-free injectors are well suited to deliver vaccines to all types of tissues, particularly to skin and mucosa. In some embodiments, a needle-free injector may be used to propel a liquid that contains the vaccine to the surface and into the subject's skin or mucosa. Representative examples of the various types of tissues that can be treated using the invention methods include pancreas, larynx, nasopharynx, hypopharynx, oropharynx, lip, throat, lung, heart, kidney, muscle, breast, colon, prostate, thymus, testis, skin, mucosal tissue, ovary, blood vessels, or any combination thereof.

[0242] The MID may have needle electrodes that electroporate the tissue. By pulsing between multiple pairs of electrodes in a multiple electrode array, for example set up in rectangular or square patterns, provides improved results over that of pulsing between a pair of electrodes. Disclosed, for example, in U.S. Patent No. 5,702,359 entitled “Needle Electrodes for Mediated Delivery’ of Drugs and Genes” is an array of needles wherein a plurality of pairs of needles may be pulsed during the therapeutic treatment. In that application, which is incorporated herein by reference as though fully set forth, needles were disposed in a circular array, but have connectors and switching apparatus enabling a pulsing between opposing pairs of needle electrodes. A pair of needle electrodes for delivering recombinant expression vectors to cells may be used. Such a device and system is described in U.S. Patent No. 6,763,264, the contents of which are herein incorporated by reference. Alternatively, a single needle device may be used that allows injection of the DNA and electroporation with a single needle resembling a normal injection needle and applies pulses of lower voltage than those delivered by presently used devices, thus reducing the electrical sensation experienced by the patient.

[0243] The MID may comprise one or more electrode arrays. The arrays may comprise two or more needles of the same diameter or different diameters. The needles may be evenly or unevenly spaced apart. The needles may be between 0.005 inches and 0.03 inches, between 0.01 inches and 0.025 inches; or between 0.015 inches and 0.020 inches. The needle may be 0.0175 inches in diameter. The needles may be 0.5 mm, 1.0 mm, 1.5 mm. 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, or more spaced apart.

[0244] The MID may consist of a pulse generator and a two or more-needle vaccine injectors that deliver the vaccine and electroporation pulses in a single step. The pulse generator may allow for flexible programming of pulse and injection parameters via a flash card operated personal computer, as well as comprehensive recording and storage of electroporation and patient data. The pulse generator may deliver a variety of volt pulses during short periods of time. For example, the pulse generator may deliver three 15 volt pulses of 100 ms in duration. An example of such a MID is the Eigen 1000 system by Inovio Biomedical Corporation, which is described in U.S. Patent No. 7,328,064, the contents of which are herein incorporated by reference.

[0245] The MID may be a CELLECTRA (Inovio Pharmaceuticals, Blue Bell PA) device and system, which is a modular electrode system, that facilitates the introduction of a macromolecule, such as a DNA, into cells of a selected tissue in a body or plant. The modular electrode system may comprise a plurality of needle electrodes; a hypodermic needle; an electrical connector that provides a conductive link from a programmable constant-current pulse controller to the plurality of needle electrodes; and a power source. An operator can grasp the plurality of needle electrodes that are mounted on a support structure and firmly insert them into the selected tissue in a body or plant. The macromolecules are then delivered via the hypodermic needle into the selected tissue. The programmable constant-current pulse controller is activated and constant-current electrical pulse is applied to the plurality of needle electrodes. The applied constant-current electrical pulse facilitates the introduction of the macromolecule into the cell between the plurality of electrodes. Cell death due to overheating of cells is minimized by limiting the power dissipation in the tissue by virtue of constant-current pulses. The Cellectra device and system is described in U.S. Patent No. 7,245,963, the contents of which are herein incorporated by reference.

[0246] The MID may be an Eigen 1000 system (Inovio Pharmaceuticals). The Eigen 1000 system may comprise device that provides a hollow needle; and fluid delivery means, wherein the apparatus is adapted to actuate the fluid delivery means in use so as to concurrently (for example automatically) inject fluid, the described vaccine herein, into body tissue during insertion of the needle into the said body tissue. The advantage is the ability to inject the fluid gradually while the needle is being inserted leads to a more even distribution of the fluid through the body tissue. It is also believed that the pain experienced during injection is reduced due to the distribution of the volume of fluid being injected over a larger area. In addition, the automatic injection of fluid facilitates automatic monitoring and registration of an actual dose of fluid injected. This data can be stored by a control unit for documentation purposes if desired.

[0247] It will be appreciated that the rate of injection could be either linear or non-linear and that the injection may be carried out after the needles have been inserted through the skin of the subject to be treated and while they are inserted further into the body tissue.

[0248] Suitable tissues into which fluid may be injected by the apparatus of the present invention include tumor tissue, skin or liver tissue but may be muscle tissue.

[0249] The apparatus further comprises needle insertion means for guiding insertion of the needle into the body tissue. The rate of fluid injection is controlled by the rate of needle insertion. This has the advantage that both the needle insertion and injection of fluid can be controlled such that the rate of insertion can be matched to the rate of injection as desired. It also makes the apparatus easier for a user to operate. If desired means for automatically inserting the needle into body tissue could be provided.

[0250] A user could choose when to commence injection of fluid. Ideally however, injection is commenced when the tip of the needle has reached muscle tissue and the apparatus may include means for sensing when the needle has been inserted to a sufficient depth for injection of the fluid to commence. This means that injection of fluid can be prompted to commence automatically when the needle has reached a desired depth (which will normally be the depth at which muscle tissue begins). The depth at which muscle tissue begins could for example be taken to be a preset needle insertion depth such as a value of 4 mm which would be deemed sufficient for the needle to get through the skin layer.

[0251] The sensing means may comprise an ultrasound probe. The sensing means may comprise a means for sensing a change in impedance or resistance. In this case, the means may not as such record the depth of the needle in the body tissue but will rather be adapted to sense a change in impedance or resistance as the needle moves from a different type of body tissue into muscle. Either of these alternatives provides a relatively accurate and simple to operate means of sensing that injection may commence. The depth of insertion of the needle can further be recorded if desired and could be used to control injection of fluid such that the volume of fluid to be injected is determined as the depth of needle insertion is being recorded. The apparatus may further comprise: a base for supporting the needle: and a housing for receiving the base therein, wherein the base is moveable relative to the housing such that the needle is retracted within the housing when the base is in a first rearward position relative to the housing and the needle extends out of the housing when the base is in a second forw ard position within the housing. This is advantageous for a user as the housing can be lined up on the skin of a patient, and the needles can then be inserted into the patient's skin by moving the housing relative to the base.

[0252] As stated above, it is desirable to achieve a controlled rate of fluid injection such that the fluid is evenly distributed over the length of the needle as it is inserted into the skin. The fluid delivery means may comprise piston driving means adapted to inject fluid at a controlled rate. The piston driving means could for example be activated by a servo motor. However, the piston driving means may be actuated by the base being moved in the axial direction relative to the housing. It will be appreciated that alternative means for fluid delivery7could be provided. Thus, for example, a closed container which can be squeezed for fluid delivery7at a controlled or non-controlled rate could be provided in the place of a syringe and piston system.

[0253] The apparatus described above could be used for any type of injection. It is how ever envisaged to be particularly useful in the field of electroporation and so it may further comprises means for applying a voltage to the needle. This allows the needle to be used not only for injection but also as an electrode during, electroporation. This is particularly advantageous as it means that the electric field is applied to the same area as the injected fluid. There has traditionally been a problem with electroporation in that it is very difficult to accurately align an electrode with previously injected fluid and so users have tended to inject a larger volume of fluid than is required over a larger area and to apply an electric field over a higher area to attempt to guarantee an overlap between the injected substance and the electric field. Using the present invention, both the volume of fluid injected and the size of electric field applied may be reduced while achieving a good fit between the electric field and the fluid.

[0254] Kit

[0255] Provided herein is a kit, which can be used for treating a subject using the method of vaccination described above. In one embodiment, the kit can comprise an IL- 21 NIC or a nucleic acid molecule encoding a IL-21 NIC monomer. The kit can also comprise instructions for earn ing out the vaccination method described above and / or how to use the kit. Instructions included in the kit can be affixed to packaging material or can be included as a package insert. While instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g.. magnetic discs, tapes, cartridges), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” can include the address of an internet site which provides instructions.

[0256] The present invention has multiple aspects, illustrated by the following non-limiting examples.

[0257] Examples

[0258] Example 1- NANOPARTICLE IMMUNO ADJUVANT COMPLEXES AUGMENT ANTIGEN-SPECIFIC GERMINAL CENTER RESPONSES AND ENHANCE HUMORAL IMMUNITY

[0259] An alluring strategy for improving protective humoral immunity' against infectious diseases is to directly target the germinal center (GC). Robust GC responses are associated with high titer and high affinity antibody responses. Crucial Tfh-derived cognate help given to GC B cells includes costimulation (e.g., CD40L) and cytokine signals (e.g., IL-21). Despite advances in formulation strategies and vaccine design, there remains a critical need for next generation approaches to elicit potent humoral immunity . Multimeric nanoparticles offer enhanced immunogenicity compared with monomeric antigen partially due to potent B cell activation through B cell receptor clustering and increased antigen density on the nanoparticle. Here, whether GC signaling molecules could be scaffolded on nanoparticles to further enhance immune responses w as investigated. A chimeric nanoparticle w as designed using the GT8- 60mer, a germline targeting HIV immunogen, as a model to scaffold IL-21. IL-21 and GT8 scaffolded nanoparticles, termed GT8 IL-21 nanoparticle immunoadjuvant complexes (GT8-IL-21 -NIC), increased serum antibody titers, antigen-specific GC B cells, and functional Tfh cell responses relative to the antigen-only GT8-60mer. Additionally, GT8-IL-21-NICs reorganized antigen-specific GC B cell responses to the light zone (LZ) of the GC, the canonical site of antigen-driven selection. Single-cell RNA sequencing of antigen-specific GC B cells from GT8-IL-21-NIC immunized mice demonstrated upregulation of LZ and selection-associated gene signatures. The NIC platform was amenable to modulation through changing both the antigen domain as well as the cytokine domain to numerous GC-associated cytokines. Thus, the NIC platform may provide value for improving vaccine-induced immunity.

[0260] Germinal center (GC) responses are crucial for humoral immunity, resulting in robust antibody responses with high affinity for the target antigen. GCs are dynamic structures that form in B cell follicles of secondary lymphoid organs that feature organized collections of primarily B cells, T cells, and specialized stromal cells (Victora et al., Annual Review of Immunology7. 2022;40(l):413-42). After initial antigen encounter, cognate B cells migrate to the border between the T cell zone and B cell follicle (T:B border) and if interact productively with T cells migrate back to the follicle and expand, forming the basis of the GC (Haberman et al.. Immunological Reviews. 2019;288(l): 10-27).

[0261] GC dynamics canonically involve GC B cell recognition and processing of antigen from a network of follicular dendritic cells (FDCs) enables cognate interactions with T follicular helper (Tfh) cells in the light zone (LZ) (Nakagawa et al.. Frontiers in Immunology7. 2021;12). Productive Tfh signals to cognate B cells come in the form of costimulation (CD40L, ICOS, OX-40 among others) and cytokine signals (IL-21, IL-4 among others) (Crotty, Immunity. 2014;41(4):529-42; Crotty, Immunity. 2019:50(5): 1132-48). Clones that receive Tfh help in can enter the dark zone (DZ) for rounds of clonal expansion and somatic hypermutation; the amount of Tfh help dictates the amount of dark zone (DZ) activity of GC B cell clones, mediated through transcription factors such as Myc (Dominguez-Sola wt al., Nat Immunol.

[0262] 2012: 13(11): 1083-91). Increasing the potency ofhumoral responses through GC modulation remains critical for vaccine-induced immunity against infectious diseases.

[0263] Cytokine signals play an important role in GC dynamics influencing the differentiation and proliferation of both Tfh and GC B cells. IL-6 is a crucial GC cytokine for efficient Tfh differentiation through engendering the expression of Bcl6 and induces Tfh production of IL-21 (Crotty, Immunity. 2014;41(4):529-42; Crotty, Immunity. 2019;50(5): 1132-48). Among Tfh-derived help signals to GC B cells, IL-21 is a crucial cytokine implicated in the proliferation of GC B cells and their differentiation into effector fates (Zotos et al., J Exp Med. 2010;207(2):365-78). IL-21 can also signal in an autocrine fashion to promote T cell expansion and differentiation (Quast et al., Immunity7. 2022;55(8): 1414-30.e5). IL-4 has been recently described to have numerous roles within the GC, simultaneously functioning to promote and repress Bcl6 in a spatiotemporal manner, influencing selection and memory B cell (MBC) differentiation (Duan et al.. Immunity. 2021:54(10):2256-72.e6: Shehata et al., Immunity. 2024;57(4): 843-58. e5). More recently, the cytokine TSLP has been described to influence GC dynamics including upregulation of IRF4, a key transcription factor to promote GC responses (Domeier et al., Science Immunology. 2023;8(79):eadd9413). These among other cytokine and chemokine signaling mediators work in concert to promote GC initiation, maintenance, and exit.

[0264] Multimeric nanoparticle antigens have been shown to elicit potent GC responses and can increase the breadth of humoral immune responses through recruitment of lower affinity B cell clones compared to monomeric antigen (Kato et al., Immunity. 2020;53(3):548-63.e8). Generally, high antigen valency can promote potent B cell activation through B cell receptor (BCR) clustering and alternate antigen trafficking and uptake dynamics (Kelly et al., Expert Review of Vaccines. 2019:18(3):269-80; Puffer et al.. ACS Chem Biol. 2007;2(4):252-62; Ingale et al., Cell Rep. 2016; 15(9): 1986-99). DNA-launched multimeric nanoparticle vaccines (DNA nanovaccines) that assemble in vivo, are dose sparing, and induce robust humoral immune responses relative to traditional DNA vaccines have recently been described (Xu et al., Advanced Science. 2020;7(8): 1902802; Xu et al., Cancer Immunology Research. 2020;8(11): 1354-64; Konrath et al.. Cell Rep. 2022;38(5): 110318; Tursi et al.. Frontiers in Immunology. 2023;14; Tursi et al., WIREs Nanomedicine and Nanobiotechnology7. 2023;15(4):el880). Additionally, DNA nanovaccines have been demonstrated to efficiently deposit onto FDCs (Xu et al., Advanced Science. 2020:7(8): 1902802). The DNA platform is also amenable to co-delivery of molecular adjuvants to augment immune responses, such as enzymes, cytokines, and chemokines among others (Gary et al., Vaccine. 2020;38(22):382I-31; Gary et al.. Cell Rep Med. 2022;3(7): 100693; Gary et al., Frontiers in Immunology . 2023; 14; De Rosa et al., JCI Insight. 2020;5(13)).

[0265] Here, it is shown that linkage of cytokine to antigen drives more potent GC responses. Using DNA nanovaccines as a model, engineering multivalent nanoparticles scaffolding both antigen as well as a signaling domain such as a cytokine was tested as a strategy to enhance GC reactions, termed nanoparticle-immunoadjuvant complex (NIC). A chimeric nanoparticle was designed using the GT8-60mer, a germline targeting HIV immunogen, as a model to scaffold IL-21, termed GT8-IL-21-NIC. Immunization with the GT8-IL-21-NIC led to improved long lived serum antibody responses as well as augmented germinal center responses skewed toward the LZ, the canonical site of antigen-driven selection. Frequency and LZ reorganization was dependent on IL-21 fusion to the nanoparticle, as co-delivery of IL-21 monomer resulted in only a modest increase in antigen-specific GC B cell responses. In the T cell compartment, GT8-IL-21-NIC improved functional Tfh cell responses as determined through activation-induced marker staining and suppressed T follicular regulatory (Tfr) cells. GT8-IL-21-NIC altered GC kinetics with dramatic increases in frequency observed only early in the response at 7 days post immunization. However, zonal reorganization persisted throughout the response for as long as 28 days. In the serum compartment, immunization with low dose GT8-IL-21-NIC enhanced serum antibody titers and bone marrow antibody-secreting cell responses. To further the mechanism of GC modulation, single cell RNA sequencing was performed (scRNAseq) which showed GT8-IL-21-NIC enhanced genes and pathways associated with GC B cell formation and selection. The NIC platform was extended using an alternate antigen- influenza H1N1 A / New Caledonia / 20 / 1999 hemagglutinin (NC99 HA) head domain - to generate the NC99-IL-21-NIC. Compared to antigen only NC99-60mer, the NC99-IL-21-NIC drove improved serum and GC responses. Similarly, changing IL-21 on the NIC for other GC associated cytokines IL-4, IL-6, or TSLP analogously drove improved GC responses with varying degrees of potency and cell-types impacted. Thus, the NIC platform is a versatile approach for generating robust antigen-specific GC responses to promote adaptive immunity.

[0266] Results

[0267] Linking antigen and cytokine via genetic fusion drives superior GC responses: Direct linkage of antigen to cytokine as a molecular adjuvant was tested to generate more potent responses than cytokine adjuvant delivered separately. eOD-GT8 (herein abbreviated GT8), a germline targeting HIV immunogen, was utilized as a model antigen that we have characterized previously using a DNA vaccine approach (Xu et al.. Advanced Science. 2020:7(8): 1902802). For a molecular adjuvant, IL-21 was utilized as a key GC cytokine implicated in both Tfh and GC B cell proliferation and differentiation. DNA plasmid cassettes were designed expressing naive mouse IL-21 monomer as a molecular adjuvant alone as well as a GT8-IL-21 combination gene via genetic fusion using glycine-serine linkers (Fig. 1 A). To determine whether cytokine directly linked to antigen drives superior responses relative to traditional cytokine adjuvant co-delivery, mice were immunized once with plasmid DNA encoding Ipg GT8, Ipg GT8 co-formulated with Ipg IL-21, or I pg GT8-IL-21 linked together and GC responses were interrogated in the draining lymph nodes (DLNs) 7 days post immunization. Genetic linkage of GT8 and IL-21 led to improved activated Tfh cell responses relative to both GT8 alone or GT8 delivered with IL-21 separately (Fig. IB). While a trend toward increased total GC B cell responses with linked IL-21 (Fig. 1C) were observed, significantly increased GT8-specific GC B cells were observed when antigen was linked to cytokine relative to low levels with unlinked cytokine and absent in the antigen only group (Fig. ID). These data demonstrate that antigen linked to a cytokine adjuvant can drive improved antigen-specific immune responses relative to monomeric antigen alone or in the context of a soluble cytokine adjuvant.

[0268] Multimeric GT8-IL-21-NIC drives B cell differentiation in vitro:

[0269] Considering the potency of genetic linkage of cytokine adjuvant IL-21 to antigen relative to IL-21 monomer co-delivery, it was next sought to engineer NICs, nanoparticulate variants of antigen-cytokine fusions. To investigate whether a cytokine can be scaffolded on the surface of an antigen nanoparticle, the GT8-60mer, a nanoparticle displaying 60 copies of GT8, was utilized as a model to scaffold IL-21 (Fig. IE). It was first demonstrated that the antigen-cytokine fused nanoparticle, GT8-IL-21- NIC, assembled in vitro using size exclusion chromatography (Fig. IF). Next, it was determined that IL-21 and GT8 were indeed co-displayed in a conformationally correct manner on the GT8-IL-21-NIC using an ELISA-based method to interrogate binding to both mouse IL-21 receptor (IL-21 R) and VRC01, a broadly neutralizing antibody recognizing eOD-GT8 (Fig. 1G). Additionally, using VRC01 enabled us to demonstrate the key germline-targeting epitope on GT8 is not occluded on the surface of the GT8-IL- 21-NIC. To determine if IL-21 is able to signal when co-displayed with antigen on the NIC, isolated follicular B cells from B6. Blimp 1+ / GFP reporter mice were stimulated in the presence of recombinant mouse IL-4, IL-5, and GT8-60mer or GT8-IL-21-NIC. Robust induction of plasma cells were observed with the GT8-IL-21-NIC relative to the GT8-60mer alone, indicating IL-21 is presented in its native capacity and is capable of signaling (Fig. 1H and II). Taken together, these data demonstrate that multimeric NICs are capable of being engineered to display antigen and cytokine in their native conformation and drive robust signaling in B cells capable of plasma cell differentiation. GT8-IL-21-NIC generates robust humoral immune responses and light zone skewed GC B cells: Generating durable antibody titers is a crucial facet of humoral immunity. To understand the impact of GT8-IL-21-NICs on serum antibody titers, mice were immunized once with 0. Ipg doses of plasmid DNA encoding either GT8-60mer or GT8-IL-21-NIC. Mice immunized the GT8-IL-21-NIC generated superior GT8-specific IgG antibody titers relative to the GT8-60mer alone (Fig. 2A). Both immunogens elicited durable antibody titers out to at least 4 months, with the GT8- IL-21-NIC eliciting titers approximately one log higher than GT8-60mer beginning two weeks post immunization. Long lived plasma cells (LLPCs) are critical mediators of durable serum titers through antibody production over time. It was next determined whether GT8-IL-21-NIC generated improved antibody-secreting cell (ASC) responses in the bone marrow (BM) of immunized mice 16 weeks post vaccination. An increase in BM IgG+ ASC responses with GT8-IL-21-NIC immunization compared to the GT8- 60mer was observed (Fig. 2B). Antibody response augmentation was not observed at Ipg, illustrating the potency of the NIC platform at low doses such as 0.1 pg (Fig. 7).

[0270] To investigate the impact of GT8-IL-21-NIC immunization on resulting germinal centers, mice were immunized with 0.5pg of plasmid encoding the antigen- only GT8-60mer or GT8-IL-21-NIC. Immunization with the GT8-IL-21-NIC resulted in a significant increase in total GC B cell responses (Fig. 2C and 2D) and GT8-specific GC B cell responses (Fig. 2E and 2F) in the draining lymph nodes (DLNs) 7 days post immunization. Similarly, immunization with a low dose of 0.1 pg of the GT8-IL-21-NIC drove superior total and GT8-speciifc GC responses than the GT8-60mer alone, albeit to a lesser magnitude (Fig. 8A-8D). IL-21 signaling in GC B cells regulates specific zonal localization of GC B cells; IL-21R signaling promotes a DZ phenotype where IL-21R deficient GC B cells demonstrated skewed localization to the LZ of the GC (Quast et al.. Immunity. 2022;55(8):1414-30.e5). Strikingly, in contrast with these results, GT8-IL- 21 -NIC immunization resulted in a statistically significant increase in GT8-specific GC B cells in the LZ (and a decrease in DZ) (Fig. 2G and 2H). Thus, GT8-IL-21-NIC immunization results in a significant increase in antigen-specific GC B cells that localize in the LZ.

[0271] To determine whether the improvements observed with the NIC platform could be seen in additional mouse strains, C57BL / 6 mice were immunized as above with 0.5pg of either the GT8-60mer or GT8-IL-21-NIC. The same pattern was observed, with IL-21 NIC increasing total and effector (CD40L+) Tfh (Fig. 9A), total and GT8-specific GC B cells (Fig. 9B) and resulted in a LZ skewed antigen-specific GC B cell phenotype (Fig. 9C).

[0272] It was next sought to interrogate the architecture of induced GCs in vivo using immunofluorescent microscopy. Anatomically, the GC exists within a B cell follicle of IgD+ naive B cells, outside of the T cell zone; zonal distribution can be inferred through the location of CR2 / CR1+ FDCs, which will localize in the light zone (Allen et al., Nature Immunology. 2004;5(9):943-52). Mice were immunized with either the GT8-60mer or GT8-IL-21-NIC and iliac lymph nodes were harvested 7 days post immunization. There was a significant increase in the number of GCs per lymph node with GT8-IL-21-NIC immunization compared with the GT8-60mer (Fig. 21 and 2J). However, there was no difference in the average GC area for individual GCs (Fig. 2K), demonstrating the increases in GCs seen by flow cytometry after immunization with GT8-IL-21-NIC are due to de novo GC formation in the lymph node.

[0273] Next, the capacity of GT8-IL-21-NIC to augment germinal center responses relative to monomeric plasmid-encoded IL-21 as a molecular adjuvant was determined. To this end. mice were immunized with the GT8-60mer. co-immunized with a low (0.5pg) or high (5 pg) dose of plasmid-encoded monomeric IL-21, or the GT8-IL-21-NIC. Trends toward increases in both total (Fig. 3 A) and GT8-specific (Fig. 3B) GC B cells were observed with increasing amounts of plasmid-encoded IL-21, w ith the highest frequencies observed in the IL-21 NIC. Additionally, to further dissect the GC B cell response, the zonal localization of GT8-specific GC B cells were determined with this regimen. A dose-dependent increase in the frequency of GT8-specific GC B cells in the LZ (and a decrease in the DZ) was observed, with effect size magnified using the NIC platform (Fig. 3C and 3D). Thus, increasing exogenous IL-21 through nanoparticulate display increases antigen-specific GC B cell responses and zonal reorganization in a dose-dependent manner.

[0274] GT8-IL-21-NIC increases the frequency and quality of GC Tfh: Tfh- derived cognate help is crucial for GC B cell proliferation and differentiation, and IL-21 is functionally implicated in Tfh proliferation and differentiation (Quast et al., Immunity. 2022;55(8): 1414-30.e5). The Tfh response after IL-21 NIC immunization relative to the GT8-60mer w as next observed. GT8-IL-21-NIC immunized mice had a significant increase in the frequency of activated (CD44+) Tfh cells at 7 days post immunization in the DLNs (Fig. 4A and 4B). Cognate help signals include the presence of costimulatory molecules on the Tfh cell surface as well as secreted cytokines. To determine whether GT8-IL-21-NICs modulate the functional capacity of induced Tfh, lymph node suspensions were stimulated with peptides spanning the GT8 and lumazine synthase domains and looked for the presence of various activation-induced markers. GT8-IL-21-NIC immunization drove a significant increase in the frequency of CD40L+ Tfh cells relative to GT8-60mer alone (Fig. 4C and 4D). CD40L was distinct among activation-induced markers, as differences in CD25. OX-40, or ICOS expression were not observed between GT8-60mer and GT8-IL-21-NIC (Fig. 10A-10C). Thus, these data demonstrate that GT8-IL-21-NICs increase both frequency and CD40L expression on induced Tfh.

[0275] T follicular regulatory' (Tfr) cells are suppressive to Tfh in the germinal center, and co-express Foxp3 alongside Tfh-like surface expression of PD-1 and CXCR5. IL-21 has been demonstrated to be restrictive to Tfr proliferation (Jandl et al., Nature Communications. 2017;8(l): 14647). To determine whether IL-21 NICs negatively regulate Tfr cells in the germinal center, intracellular transcription factor staining was used to discriminate Foxp3+ Tfr cells from Foxp3- Tfh cells within the heterogeneous population of PD-1 and CXCR5 co-expressing cells 7 days post immunization. There was a significant decrease in the frequency of Tfr with GT8-IL-21- NIC immunization relative to the GT8-60mer (Fig. 4E and 4F). As such, GT8-IL-21- NICs significantly increased the ratio of Tfh (Foxp3-) to Tfr (Foxp3+) (Fig. 4G). However, a significant change in the frequency of total non-GC regulatory T cells (Tregs) was not observed between the two treatment groups (Fig. 4H). Together, these data show that GT8-IL-21-NICs specifically regulate the Tfh-Tfr axis in a pro-Tfh manner.

[0276] GT8-IL-21-NIC differentially regulates GC kinetics and zonal segregation: Natural infection or vaccines that generate a T-dependent GC response in secondary lymphoid organs peak approximately 14- to 21 -days post immunization and subsequently wane (De Silva et al., Nat Rev Immunol. 2015;15(3): 137-48; Allen et al., Immunity. 2007;27(2): 190-202; Yewdell et al., Cell Rep. 2021;37(6):109961). To understand the kinetics of the GC response after GT8-IL-21-NIC immunization, we sequentially immunized mice and examined the DLNs 7-, 14-, and 28-days post immunization. Timepoints beyond 7-days post immunization do not exhibit a dramatic difference between the GT8-60mer and GT8-IL-21-NIC in the frequency of total (Fig. 5 A) or GT8-specific GC B cell responses (Fig. 5B). Beyond the increases observed at Day 7, there was a significant increase in the frequency of total but not GT8-specific GC B cells at Day 28. However, among antigen-specific GC B cells, the zonal reorganization phenotype with the GT8-IL-21-NIC persists through 28-days post immunization, demonstrating a statistically significant increase in antigen-specific GC B cells in the LZ and decrease in the DZ (Fig. 5C and 5D). Thus, immunization with the GT8-IL-21-NIC augments the magnitude of early antigen-specific GC responses but imparts changes to GC organization throughout the duration of the GC response.

[0277] Single cell RNA sequencing demonstrates GT8-IL-21-NIC alters GC B cell gene expression: Considering the early GC modulation after immunization with NICs, the mechanism was investigated using single cell RNA sequencing (scRNAseq) on GT8-specific GC B cells 7 days post immunization (Fig. 5E). Paired transcriptomics with BCR sequencing was utilized to elucidate both gene expression dynamics and clonal recruitment to the germinal center. Seurat clustering resulted in 9 distinct clusters containing heterogeneous subpopulations of GC B cells (Fig. 5F). Clusters were identified using cell cycle state, pseudotime analysis, and top upregulated genes in each cluster (Fig. 11 A-l 1C). Based on gene expression profiles, top upregulated genes were identified such as I14i 1 and Cd83 belonging to a LZ signature in cluster 1, and a DZ signature in cluster 3 with upregulated genes such as Mki67 (Fig. 1 1A) (Victora et al., Cell. 2010;143(4):592-605; Victora et al., Blood. 2012;120(l l):2240-8). Additionally, numerous clusters were observed (cluster 2, 4. 5) with a heterogeneous gene expression profile in numerous phases of the cell cycle. Among DZ-like clusters, one primarily in G2 / M (cluster 3) and one primarily in S phase (cluster 2) were observed with active DNA replication genes. Cluster 4 had a DZ signature with cells primarily in Gl, indicative of exit from the DZ and active cell division. In contrast, Cluster 5 had a gene signature / cell cycle profile of cells entering active cell division and thus into the DZ. Clusters 6-9 were primarily in Gl and represented either pre- or post-GC B cell fates. Cluster 6 was defined as pre-memory due to robust expression of Ccr6, Sell, and Gprl83 (Inoue, International Immunology. 2023). Cluster 7 had high expression of Ighd, thus was defined as naive B cells. Cluster 8 had upregulation of genes associated with an activated B cell / pre-GC B program (Bach2, Pax5. and Zbtb20). Cluster 9 had high expression of specific immunoglobulin variable genes, Irf4, and Jchain, indicative of plasma cells (Xu et al., eLife. 2020;9:e59850; Klein et al., Nature Immunology. 2006:7(7):773-82).

[0278] In line with flow cytometry data, an increased frequency of cells in the LZ (cluster 1) and decreased frequency of cells in the DZ (clusters 2 and 3) with GT8- IL-21-NIC immunization relative to the GT8-60mer (Fig. 5G) were observed. Interestingly, an increase in cells in an activated / selected LZ B cell (cluster 5) with GT8- IL-21-NIC immunization was observed; this cluster co-localizes with Myc expression, a transcription factor transiently induced after GC B cell selection canonically from Tfh (Dominguez-Sola wt al., Nat Immunol. 2012; 13(11): 1083-91) (Fig. 5G and 5H). Additional density plots of gene expression further confirmed GC vs. pre- or post-GC states. Decreases in the frequency of cells in differentiated states were also observed, such as pre-memory (cluster 6) and plasma cell (cluster 9) but an increase in pre-GC like cells (cluster 8), indicating a pro-GC transcriptional landscape within the GC. (Fig. 11D).

[0279] To further understand pathways of interest within select GC B clusters, differentially expressed genes (DEGs) and pathways upregulated after immunization with GT8-IL-21-NIC relative to the GT8-60mer alone were examined. In the LZ (cluster 1), Ingenuity Pathway Analysis (IP A) of regulators demonstrated upregulation of genes / signaling pathways implicated in GC B cell commitment and maintenance such as SMARCA4, IRF8. and POU2F2 (Schmiedel et al., Front Immunol. 2021;12:705848; Wang et al., Proceedings of the National Academy of Sciences. 2019;! 16(19): 9511-20; Laidlaw et al., Nature Reviews Immunology7. 2021;21(4):209-20), activation including TNFRSF8 (CD30) (Sperling et al.. Blood. 2019;133(24):2597-609), and pathway s associated with T cell help such as IL-4, and CD40 (Gonzalez et al., J Immunol. 2018;201(12):3569-79; Elgueta et al., Immunol Rev. 2009;229(l): 152-72) (Fig. 51). The LZ also had two Myc-associated regulators upregulated compared with the control GT8- 60mer, indicative of a pro-selection / activation phenoty pe. Interesting, cells in the DZ (cluster 3) after GT8-IL-21-NIC immunization had IPA regulators activated reminiscent of a LZ signature; strong Myc (and related Myc effector) activation and STAT3 induction (Fike et al., Cell Rep. 2023;42(5): 112512) (Fig. 5J). Additionally7, PI3K related regulators and downstream AKT1 were both upregulated, signaling cascades known to negatively regulate Foxol, a transcription factor crucial for DZ identity (Sander et al., Immunity. 2015;43(6): 1075-86; Zhu et al., Life Sci Alliance. 2019;2(6); Inoue et al., J Exp Med. 2017;214(4): 1181-98). In line with the increase in activating transitory7B cells (Entering DZ / Cell cycle, cluster 5), upregulation in Myc or My c- related regulators was observed using IPA, in addition to other regulators associated with transcriptional activation and BCR signaling (Fig. 5K). Further examination of individual DEGs in the LZ (cluster 1) demonstrated upregulation of genes associated with GC formation (Plcg2, Id3, Irf8, Pou2f2), maintenance (Bach2, Pax5), and T cell help (Cd40) after GT8-IL-21-NIC immunization in line with our IPA analysis (Fig. 5L). Increased Ccnd3 upregulation was also observed, which was demonstrated to be important for GC B cell cycling between light and dark zones (Pae et al., J Exp Med. 2021;218(4)). Together, these data indicate that immunization with GT8-IL-21-NIC generates a pro-GC maintenance and activated transcriptional phenotype, supporting zonal remodeling toward the selection-associated light zone.

[0280] The clonal repertoire after immunization with GT8-IL-21-NICs was also modulated relative to immunization with the antigen-only GT8-60mer. A shift in the clonal repertoire was observed, illustrating that different GC B cell clones are getting recruited to the GC (Fig. 12).

[0281] NIC with alternative antigen domain analogously enhance GC responses: To expand upon the initial findings by expanding the NIC platform to alternative antigen and cytokine domains, a NIC scaffolding IL-21 and an alternative infectious disease glycoprotein were developed. A DNA-launched 60mer nanoparticle vaccine scaffolding the head domain of influenza H1N1 A / New Caledonia / 20 / 1999 hemagglutinin (NC99-60mer) was previously described (Xu et al., Advanced Science. 2020;7(8): 1902802). An NC99-IL-21-NIC w as engineered by genetic fusion of IL-21 to the antigen domain (Fig. 6A). To investigate whether the NC99-IL-21-NIC generated humoral immune responses, mice were immunized with 0.1 pg plasmid DNA encoding the NC99-60mer or the NC99-IL-21 -NIC. Mice immunized with the NC99-IL-21 -NIC generated enhanced NC99 HA-specific IgG titers relative to antigen-only NC99-60mer (Fig. 6B). At week 12 post immunization, binding to related H1N1 Has w as assessed, and the NC99-IL-21-NIC generated trends toward significantly enhanced titers to these unmatched HAs, suggestive of improved breadth (Fig. 6C). Additionally, the formation of LLPCs in the BM were assessed for, where w e observed significantly increased IgG+ ASCs with NC99-IL-21-NIC immunization relative to the NC99-60mer at week 12 (Fig. 6D).

[0282] To determine whether the NC99-IL-21-NIC could augment GC responses, mice were immunized with 0.5pg of plasmid encoding the NC99-60mer or NC99-IL-21-NIC and assessed GC responses at Day 7 in the DLNs. Immunization with NC99 IL-21 NIC led to a significant increase in the frequency of total (Fig. 6E and 6F) and antigen-specific (Fig. 6G and 6H) GC B cells compared to the NC99-60mer alone. A significant increase in the frequency of total activated Tfh cells was also observed at the same timepoint with the NC99-IL-21-NIC (Fig. 13A and 13B). Using an activation- induced marker stimulation assay to determine the quality of Tfh help, a significant increase in CD40L+ Tfh was observed with the NC99 IL-21 NIC relative to the NC99- 60mer alone (Fig. 13C and 13D). Taken together, these data demonstrate that NIC platform is amenable to numerous antigens and can augment GC responses and humoral immunity.

[0283] NICs with alternative GC-enhancing cytokine domains analogously enhance GC responses: Considering that the NIC platform is amenable to numerous antigen domains, it was next determined whether additional cytokine domains could be scaffolded to enhance immune responses. Alternative NICs were designed with GT8 and GC cytokines IL-4 (GT8-IL-4-NIC). IL-6 (GT8-IL-6-NIC). and TSLP (GT8-TSLP- NIC). To interrogate whether these additional NICs could augment immune responses, GC responses were measured in the DLNs 7 days post immunization. Relative to the GT8-60mer alone, increases in total GC B cells were observed with the GT8-IL-21-NIC and GT8-TSLP-NIC, while trends with the GT8-IL-4-NIC and GT8-IL-6-NIC (Fig. 61 and 6J) were observed. Notably, in the antigen-specific GC B cell compartment, significant increases were observed with all tested cytokine variant NICs (Fig. 6K and 6L). Potency between the GT8-IL-21-NIC and GT8-TSLP-NIC was similar, with the GT8-IL-4-NIC and GT8-IL-6-NIC having attenuated potency relative to the others. Interestingly, immunization with the GT8-IL-4-NIC only impacted the antigen-specific GC B cell compartment directly, with no differences in activated Tfh responses compared to the GT8-60mer (Fig. 14A and 14B). However, both the GT8-IL-6-NIC and GT8-TSLP-NIC showed a trend toward or significant increase in activated Tfh responses respectively. Further characterization of the GT8-IL-6-NIC demonstrated improvements in CD40L+ effector Tfh (Fig. 15). Taken together, these data show that the NIC platform is amenable to scaffolding numerous cytokines to enhance adaptive immune responses. The NIC platform is superior to monomeric gene-encoded cytokine adjuvants and serves to modulate GC responses by increasing their magnitude, their quality, and speeding up their kinetics (Fig. 16 and 17).

[0284] Discussion

[0285] Germinal center reactions are crucial to humoral immunity against T dependent antigens. Manipulating GCs via vaccination remains a key target for generating responses against complex pathogens. Strategies employing germline targeting immunogens have tailored germinal center responses to elicit broadly neutralizing antibody (bnAbs) precursors to the CD4 binding site and V3 glycan patch. Additionally, sequential immunization utilizing heterologous germline targeting immunogens has demonstrated the ability to “walk” antibody lineages toward bnAbs (Escolano et al., Cell. 2016;166(6): 1445-58. el2; Escolano et al., Nature. 2019;570(7762):468-73; Jardine et al.. Science. 2015;349(6244): 156-61; Briney et al., Cell. 2016;166(6): 1459-70.el l). Compared with sequential immunization, it would be beneficial to simplify dosing schema that can elicit potent and broadly neutralizing humoral responses. Here, a potent platform was demonstrated to induce strong priming responses, particularly in the germinal center, exemplified using a germline targeting immunogen. Utilizing crucial GC cytokines, the NIC platform was demonstrated to robustly increase antigen-specific GC B cell and functional Tfh cell responses. These enhancements were elicited after a single vaccination with low doses of plasmid DNA; enhancements of the NICs relative to antigen only nanoparticles were reflected in both the GC as well as serum antibody responses.

[0286] Vaccine platforms that generate robust GC response, such as lipid nanoparticle-formulated mRNA, display altered GC kinetics with B cell responses peaking 7-days post immunization (Li et al., Nature Immunology7. 2022;23(4):543-55; Lederer et al., Immunity. 2020;53(6): 1281-95. e5). Immunization with GT8-IL-21-NICs led to dramatically enhanced antigen-specific GC B cell responses 7-days post immunization, that contract to levels comparable with antigen alone at 14 days post immunization and after. Responses were skewed toward the LZ, where Tfh-driven selection occurs. This is in contrast to prior studies suggesting IL-21 is crucial for DZ localization using IL-21R KO mice (Zotos et al., J Exp Med. 2010;207(2):365-78; Quast et al.. Immunity. 2022;55(8): 14I4-30.e5; Dvorscek et al., EMBO reports. 2022;23(9):e54677; Zotos et al., Nature Communications. 2021;12(l):7160). The data presented herein suggests that additional exogenous IL-21 drives a unique LZ skewed program in GC B cells, indicating the value of exploring the mechanism of cytokine signaling through this platform. However. DNA delivery has unique kinetics of protein production as compared to a recombinant protein bolus (Parzych et al., Nature Communications. 2022;13(l):5886); as such, additional studies to understand the immunogenicity of NICs using other delivery mechanisms such as recombinant protein or mRNA would be of interest. GT8-IL-21-NIC also had an impact on the T cell compartment, through expanding the frequency of total and effector Tfh after immunization. In its native state, IL-21 has an independent role on Tfh cells through paracrine signaling mechanisms to promote proliferation and differentiation (Quast et al., Immunity. 2022;55(8):1414- 3O.e5; Vogelzang et al., Immunity. 2008;29(l): 127-37; McGuire et al., The Journal of Immunology. 2015; 195(11 ):5123-35). Additionally, there is a role for IL-21 in suppressing Tfr (Jandl et al.. Nature Communications. 2017;8(l): 14647, Tian et al., Trends Immunol. 2016;37(8):557-68). The GT8-IL-21-NIC was demonstrated to selectively enhances Tfh and negatively regulates Tfr, with no impact on the non-GC Treg compartment. This, paired with dramatic increases in GT8-specific GC B and antibody responses, shows local regulation of the GC response to promote antigenspecific immunity.

[0287] Immunization with GT8-IL-21-NICs led to a pro-selection and pro-GC fate in GC B cells. The transcription factor Myc is a key mediator of the LZ to DZ transition; increased Myc expression in LZ B cells leads to increased cell divisions in the DZ and is proportional to T cell help (Dominguez-Sola wt al., Nat Immunol. 2012;13(l l): 1083-91; Finkin et al., Immunity. 2019;51(2):324-36.e5; Calado et aL Nat Immunol. 2012;13(l 1): 1092-100). LZ skewing was recapitulated through transcriptomics, with a cluster containing Myc expression indicative of LZ to DZ transition being upregulated with GT8-IL-21-NIC immunization relative to GT8-60mer alone in numerous clusters. Additionally, genes associated with the cell cycle, such as Ccnd3, were significantly upregulated in numerous clusters with GT8-IL-21-NIC immunization. Ccnd3 has been shown to be crucial for inertial cell cycling of GC B cells in response to potent Tfh help (Pae et al., J Exp Med. 2021 ;218(4)). Additionally, a gene program in DZ GC B cells w as observed reminiscent of the LZ in mice immunized with GT8-IL-21-NIC; this could be due to direct interaction with the immunogen and B cells. Dendritic cells are capable of presenting unprocessed antigen to B cells within the germinal center, with the potential for direct DC-B cell interaction at the follicle border (Bergtold et al., Immunity. 2005;23(5):503-14; Qi et al., Science. 2006;312(5780): 1672- 6). Further studies to visualize GT8-IL-21-NIC distribution within the B cell follicle are warranted.

[0288] It was demonstrated that the NIC platform could be modulated to incorporate additional antigens or cytokines. Additionally, using plasmid DNA as a vector enables for rapid replacement of specific domains through mutagenesis or subcloning, reducing the purification constraints of recombinant nanoparticle platforms. The NICs generated in this work scaffolded GC associated cytokines IL-21, IL-4, IL-6, and TSLP to specifically target the GC reaction. Various cytokines led to variable increases in the GC response; further work characterizing each cytokine and relative doses and / or ratios that may be optimal for inducing GC responses remains warranted. Additionally, further exploration of additional cytokines, chemokines, costimulatory molecules, or other signaling molecules scaffolded as a NIC is of interest.

[0289] In summary, co-display of antigen and GC-associated cytokine as a NIC induces potent adaptive immune responses at low doses. Additional characterization of this platform with additional antigens and signaling molecules is of interest.

[0290] Methods

[0291] Design of antigen-cytokine NIC immunogens - Plasmid DNA-encoded GT8-60mer and NC99-60mer was developed in a previous work (Xu et al., Advanced Science. 2020;7(8): 1902802). GT8 NICs were designed by genetic fusion of mouse IL- 21 (Umprot E9PX58; GT8-IL-21-NIC). IL-4 (Umprot P07750; GT8-IL-4-NIC), IL-6 (Uniprot P08505; GT8-IL-6-NIC), and TSLP (Umprot Q9JIE6; GT8-TSLP-NIC) to the C terminus of GT8. NC99-IL-21-NIC was designed similarly with genetic fusion of IL- 21 on the C terminus of the NC99 HA head.

[0292] Modeling - Models of each NIC nanoparticle were created using LS (PDB ID: 1HQK) as the seeding base. The relevant sequence of NC99, GT8, and IL-21 were predicted using ColabFold, an online accessible version of Alphafold (Jumper, Mirdita) and predictions were verified using the closest crystal structure, if available (Jumper et al., Nature. 2021;596(7873):583-9; Mirdita et al., Nature Methods. 2022; 19(6):679-82). A single subunit of the relevant combinations of antigen and cytokine were created with GS linkers, and then copied to the biological assembly of LS to create 60-mers. Multiple geometries were tested and each final nanoparticle versions consist of subunits sampled from two different geometries. The nanoparticle core, antigens, and cytokines are all shown as surface filled models and GS linkers are shown in cartoon representation.

[0293] In vitro production and size exclusion chromatography - Expi293F cells (Invitrogen) were transfected with plasmid encoding the GT8-IL-21-NIC with PEI (Sigma Aldrich) / OPTI-MEM (Invitrogen) and harvested 6 days post-transfection. Transfection supernatant was first purified with affinity chromatography using the AKTA pure 25 system and Protein G column (GE Healthcare). The eluate fractions from the affinity purification were pooled, concentrated with Ami con Ultra- 15 Centrifugal Filter Unit with 30kDa cut-off (Milipore), and dialyzed into IX PBS buffer before being loaded onto the Superose 6 Increase 10 / 300 GL size-exclusion chromatography (SEC) column (GE healthcare) for purification. Identified eluate fractions were then collected and concentrated to 1 mg / mL in PBS.

[0294] In vitro stimulation of isolated follicular B cells - Spleens were harvested from B6.BlimpU7GFP (PrdmU7GFP) mice by originally provided by Stephen Nutt (Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia), minced between frosted glass slides, and lysed with ACK lysis buffer to remove red blood cells. Single cell suspensions were labeled with Cell Trace Yellow according to manufacturer’s protocol. Follicular B cells were purified by CD23 positive selection using MACS columns (Miltenyi). Purified cells were plated in 96-well plates at 2x105 cells per well with culture media containing RPMI 1640 supplemented with 10% Fetal Bovine Serum, 1% penicillin streptomycin, L-glutamine, HEPES, non-essential amino acids, sodium pyruvate, and 0. 1% 2-mercaptoethanol and gentamicin. Cells were stimulated with indicated concentrations of GT8-60mer or GT8-IL-21-NIC protein alone or in combination with IL-4 (100 ng / mL), IL-5 (100 ng / mL), IL-21 (lOOng / mL), and / or hamster anti-mouse CD40 (clone HM40-3) for 72 hours incubated at 5% CO2 and 37 C. Stimulation conditions were plated at culture initiation. Downstream assessment was performed by harvesting cells from 96-well plate using PBS + 0.1% BSA + ImM EDTA and staining with BUV395 anti-CD80 (Clone 16-10A1, BD) and BV421 anti-CD86 (Clone GL1, Biolegend) at 1:200 for 30 minutes at 4 C, washing, and resuspending in IX ToPro-3 solution. Flow cytometry was performed on a BD Symphony A3 Lite and analyzed using FlowJo analysis software.

[0295] Animal studies - All animal studies were conducted under protocols approved by the Wistar Institute Institutional Animal Care and Use Committee (IACUC). Six-to-twelve-week-old BALB / cJ (Jackson Laboratory) were housed in the Wistar Institute Animal Facility. Mice were immunized with indicated plasmid DNA constructs at 0. 1 pg or 0.5pg in the tibialis anterior muscle formulated in water followed by in vivo adaptive electroporation using the CELLECTRA 3P device (Inovio Pharmaceuticals). For serological experiments, mice were bled weekly or biw eekly by submandibular bleed. For germinal center experiments, lymph nodes were harvested at indicated timepoints post vaccination. Tissue processing - Mice were euthanized using CO2 at timepoints where indicated and popliteal and iliac draining lymph nodes were harvested.

[0296] Microscopy: Iliac lymph nodes (LNs) were placed into 10% formalin for 1 hour at RT. Iliac LNs were subsequently placed in 15% sucrose 0.1% sodium azide (NaN3) in PBS for 6 hours at 4C before final incubation in 30% sucrose 0.1% NaN3 in PBS overnight (ON) at 4C. The following day, lymph nodes were washed once in PBS before freezing in Optimal Cutting Temperature (OCT) compound on dry ice. OCT blocks were subsequently sectioned by microtome at lOpM thickness onto slides before subsequent staining. Flow cytometry: Popliteal and iliac LNs were pooled into RPMI media containing 10% FBS and 1% Penicillin / Streptomycin (R10). LNs were then mechanically dissociated through a 40pm strainer before washing with R10. Samples were plated for subsequent flow cytometry staining or downstream assays.

[0297] Immunofluorescent microscopy - Iliac lymph nodes sections were blocked in 5% donkey serum in PBS-Tween-20 for 30 minutes at RT with FcR block, followed by staining with anti-mouse IgD. anti-mouse CD3, anti-mouse CD21 / 35, and anti-mouse GL7 for 1 hour at RT. Slides were washed and mounted using ProLongTMDiamond antifade (Invitrogen) and imaged using a Zeiss LSM 980 Confocal microscope. Images were analyzed using Fiji.

[0298] Flow cytometry - Germinal centers: LNs were resuspended in Fixable Viability Dye eF780 (eBioscience) in 0.2% BSA in PBS (FACS) for 10 min at RT. Cells were next washed before resuspension with Biotin anti-mouse CXCR5 (Clone SPRCL5, eBioscience) or Biotin anti-mouse CXCR4 (Clone 2B11, eBioscience) for 30min at RT in FACS. Cells were subsequently washed and resuspended with a surface stain cocktail for 30min at RT containing a subset of the antibodies or proteins listed in Supplementary Table 1. Probes were made via direct conjugation of fluorochromes to recombinant ferritin-scaffolded GT8 (GT8-24mer) made in house as in (Xu et al., Advanced Science. 2020;7(8): 1902802) or influenza NC99 HA (Cat# 11683-V08B1, Sino Biologicals). Lightning Link Kits for FITC (abl02884) or PE (abl02918) were used according to manufacturer’s protocol. Cells were then washed and resuspended in FACS buffer before acquiring on a FACSymphony A3 or A5 SE analyzer (BD). FCS files were exported and analyzed using FlowJo (Treestar).

[0299] Activation induced marker stimulation: LNs were first incubated with a pool of overlapping peptides spanning the GT8 and lumazine synthase domains (GT8- based immunogens) or NC99 RBD and lumazine domains (NC99-based immunogens) for 18 hours at 37°C with APC anti-mouse CD40L (Clone MR1, Biolegend). DMSO and Cell Stimulation Cocktail (eBioscience) were used as positive and negative controls, respectively. After stimulation, cells were first washed in FACS before incubation with Fixable Viability Dye eF780 (eBioscience) for lOmin at RT. Cells were then washed in FACS before incubation with a surface stain cocktail for 30min at RT containing a subset of antibodies or proteins. After incubation, cells were then washed in FACS before acquisition on a FACSymphony A3 or A5 SE analyzer (BD). FCS files were exported and analyzed using FlowJo (Treestar).

[0300] ELISA - 96-well half area plates (Cat# 3690, Coming) were coated at Ipg / mL with anti -His capture antibody (Cat#A01857. Genscript) overnight at 4°C. The following day, plates were blocked with 5% non-fat dry milk in PBS for 1 hour at RT. Plates were incubated with Ipg / mL His-tagged GT8 monomer (in house, see (Xu et al.. Advanced Science. 2020;7(8): 1902802)) for 1 hour at RT. Mouse samples were then added in serial dilutions for 2 hours at RT. Plates were then incubated with HRP- conjugated goat anti-mouse IgG heavy+light chain (Cat# A90-516A, Bethyl) for 1 hour at RT. Plates were then developed with TMB Ultra Substrate (Thermo) for 5 min at RT before quenching with IN H2SO4. Washing occurred between each step using 0.05% Tween-20 in PBS. Plates were read on a Biotek Synergy plate reader at 450nm and 570nm.

[0301] ELISpot - Bone marrow (BM) was flushed from femurs and tibia from each mouse using a 23G X 3 / 4” needle and syringe into FACS buffer and filtered through 63-micron Nitex mesh. Red blood cells were lysed in ACK lysis buffer for 5 min on ice. Resulting cells were counted using aNexcelom Cellometer Auto 2000 Cell Viability Counter Profiler (Nexcelom Bioscience LLC). MultiScreenHTS IP Filter Plate. 0.45 pm (Millipore Sigma), was coated with Ipg / mL anti-His (Cat#A01857, Genscript) followed by His-tagged GT8 monomer at Ipg / mL (GT8 immunogens) or with 0.5pg / mL A / New Caledonia / 20 / 1999 HA (Cat# 11683-V08H, Sino Biologicals) in sodium carbonate / sodium bicarbonate buffer (pH 9.6) (35 mM NaHCO3 and 15 mM Na2CO3) for 1 hour at 37°C. Plates were then washed with 200 pL PBS / well three times and blocked at 37°C in complete RPMI + 10% FBS for 30 min. BM cells were plated in eight halving dilutions beginning at 2.5 million cells per well and incubated overnight in complete RPMI + 10% FBS. Plates were then washed with wash buffer (lx PBS + 0.1% Tween 20) five times and incubated with biotinylated polyclonal goat anti-mouse anti- IgG detection antibody (SouthemBiotech) in PBS + 2% BSA at RT for 1 hour. Plates were once again washed five times, and streptavidin-alkaline phosphatase (1 :20,000 dilution in PBS + 2% BSA) was added prior to incubation at RT for 30 min. Plates were then washed five times with wash buffer, and 50 pL / well BCIP / NBT single solution (SigmaAldrich) was added for 5 min or until spots developed, at which time the reaction was quenched with 100 mL 1 M sodium phosphate monobasic solution. After plates were rinsed with dH2O and dried overnight, they were scanned and counted using Mabtech IRIS.

[0302] Single cell RNA sequencing - CellRanger suite (pipeline v7.0.0 support.10xgenomics.com) was used with refdata-gex-mml 0-2020-A transcriptome as a reference to map reads on the mouse genome and the CellRanger VDJ pipeline with default settings was used to align and assemble VD(J) transcripts per cell. The barcodes, genes, and counts were imported into R package Seurat (Hao et al., Cell. 2021;184(13):3573-87.e29) where quality control was performed by discarding cells with over 5% mitochondrial content and those with fewer than 250 genes with reads. R package SingleR (Aran et al., Nature Immunology. 2019;20(2): 163-72) was used to identify individual cell types with the ImmGen mouse dataset as a reference and around 1 10 cells that weren’t predicted as B cells were further discarded. After these steps, there were 7822 cells in the GT8_60mer sample and 8831 cells in the GT8_IL21_NIC sample. Seurat's shared nearest neighbor was then used to unbiasedly cluster the remaining cells and UMAPs were used for visualization. Sample integration wasn’t necessary as batch effect wasn’t observed. Germinal center B cells were subset, and reclustered to further identify7transitionary states. Slingshot was used for pseudotime analysis with the LZ cluster as the initial node (Street et al., BMC Genomics. 2018;19(l):477). Differential Expression analysis was performed using Wilcoxon Rank Sum Test and FDR < 5% was used to determine statistical significance. Qiagen IPA (QIAGEN Inc., digitalinsights.qiagen.com / IPA) was used to perform enrichment analysis to identify over-represented pathw ays and regulators. For the VD(J) analysis, only cells that had both heavy and light chains were considered to account for technical errors in capturing data. Unique clones were determined using CDR3 heavy chain sequences and a sequence-match rate of 90% as determined using Levenshtein distance (pypi.org / project / Levenshtein / ). Unique V-D-J gene combinations were also determined and visualized comparing relative prevalence in the tw o samples. Visualization w as done on RStudio with ggplot2 and scCustomize. Statistics - All statistical tests were performed using GraphPad Prism 10. Graphs and error bars represent geometric means ± geometric SD. Non-parametric Mann-Whitney U tests or one-way ANOVA adjusted for multiple comparisons with Bonferroni correction were used to compare groups where indicated. The number of samples in each graph is notated in the figure legend. In all datasets, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0303] Example 2- VACCINATION UTILIZZING IL-21 AS AN ADJUVANT AS A CYTOKINE MONOMER

[0304] This invention was developed with the goal to immunize with antigen and co-deliver IL-21 to determine if GC responses can be enhanced.

[0305] To determine whether IL-21 could be delivered as a molecule adjuvant, a pVAX vector with IL-21 behind an IgE signal sequence was designed (Fig. 18A). Successful expression was confirmed using transient transfection in vitro and Western blotting, where robust IL-21 expression was observed (Fig. 18B). To determine whether IL-21 co-delivery could augment GC responses, mice were immunized with either 2ug of DNA encoding a wild-ty pe SARS-CoV-2 Spike RBD multimer vaccine (RBD- 24mer) or co-immunized with plasmid-encoded IL-21 (0.5ug. Fig. 18C). GCs were assessed in the DLNs 7 days post immunization. In the DLNs, a significant increase in total GC B cell responses was observed with IL-21 co-immunization (Fig. 18D-E). Among SARS-CoV-2 Spike RBD-specific GC B cells, an increase was observed among both wild-type RBD-binding (matched) as well as Omicron BA.4 / 5 Spike RBD-binding (unmatched) B cells with IL-21 co-immunization, demonstrating increased breadth (Fig. 18F-G).

[0306] Another important effector population of cells within the GC are Tfh cells. To assess whether IL-21 co-immunization as a molecular adjuvant could improve Tfh responses, mice were immunized with 2pg of plasmid encoding the RBD-24mer or co-immunized with 0.5pg of plasmid-encoded IL-21. Germinal centers were assessed in the DLNs 7 days post immunization. An increased frequency of activated (CD44+) Tfh cells with IL-21 co-immunization was observed (Fig. 19A). Additionally, to assess whether the induced Tfh cells had an improved effector profile, the presence of important costimulatory surface molecules CD40L and ICOS was assessed. An increase in CD40L expression and a trend toward improved ICOS expression was observed on Tfh from IL-21 co-immunized mice relative to antigen alone (Fig. 19B-D). These data demonstrate that IL-21 co-immunization can drive increases in the frequency and effector profile of Tfh.

[0307] The methods used for the experiments are now described.

[0308] Plasmid production and expression

[0309] Mouse IL-21 (Uniprot E9PX58) was cloned into a modified pVAX vector as described above (Genscript). The RBD-24mer design was described in Konrath et al., Cell Rep. 2022;38(5): 110318. Briefly, Expi293F cells were transfected using the Expifectamine kit (Thermo Fisher) with plasmid-DNA encoding IL-21. Supernatants were harvested 3 days post transfection. SDS-PAGE was performed using a NuPAGE 4-12% Bis-Tris gel (Thermo Fisher). After separation and transfer to a PVDF membrane, probing for IL-21 protein was performed using an anti -mouse IL-21 antibody (R&D Sy stems) with the appropriate secondary7antibody (Goat anti-rat IRDye 800CW, LiCOR). The western blot was visualized using a LiCOR Odyssey.

[0310] Flow cytometry for germinal centers

[0311] Single cell suspensions from lymph nodes were washed and resuspended in Live / Dead Fixable eFluor780 Dye (Thermo Fisher) for 10 minutes at RT. Cells were washed in FACS before resuspension in anti-mouse CXCR5-Biotin (eBioscience) for 30 min at RT. Cells were washed in FACS and resuspended in surface stain cocktail in FACS buffer containing Streptavidin (SAV), PD-1 , CD4, CD44, CD19, Fas, CD38, GL7, and antigen probes. Cells were subsequently washed and resuspended in FACS buffer before acquisition on a FACSymphony A3 or A5. Cells were analyzed using FlowJo. Cells were gated as:

[0312] Tfh: lymphocytes single cells live CD 19- CD4+ CD44+ PD-1 hi CXCR5+ GC B: lymphocytes single cells live CD4- CD19+ CD38- GL7+ (or CD38- Fas+ or Fas+ GL7+)

[0313] SARS-CoV-2 specific GC B: same as above, except Wild-type RBD probes are on FITC, APC and BA.4 / 5 Omicron RBD probes are on PE

[0314] Activation induced marker assay

[0315] Draining lymph nodes were incubated with peptides spanning the antigen domains for 18 hours at 37C in the presence of APC conjugated anti-mouse CD40L (Biolegend). The following day, single cell suspensions from lymph nodes were washed and resuspended in Live / Dead Fixable eFluor780 Dye (Thermo Fisher) for 10 minutes at RT. Cells were washed in FACS before resuspension in anti-mouse CXCR5-Biotin (eBioscience) for 30 min at RT. Cells were washed in FACS and resuspended in surface stain cocktail in FACS buffer containing Streptavidin (SAV), anti-mouse PD-1, CD4, CD44, CD19, and ICOS. Cells were subsequently washed and resuspended in FACS buffer before acquisition on a FACSymphony A3 or A5. Cells were analyzed using FlowJo. Cells were gated as:

[0316] Tfh: lymphocytes single cells live CD 19- CD4+ CD44+ PD-1 hi CXCR5+ AIM+: CD40L+ or ICOShi of Tfh

[0317] SEQUENCES:

[0318]

[0319] SEQ ID NO: 1 (AA IL-21 immunoadjuvant domain with IgE)

[0320] MDWTWILFLVAAATRVHSHKSSPQGPDRLLIRLRHLIDIVEQLKIYENDLDPELLSAPQD

[0321] VKGHCEHAAFACFQKAKLKPSNPGNNKTFIIDLVAQLRRRLPARRGGKKQKHIAKCPSC DSYEKRTPKEFLERLKWLLQKMIHQHLS SEQ ID NO: 2 (DNA IL-21 immunoadjuvant domain with IgE)

[0322] ATGGATTGGACCTGGATACTGTTCCTGGTGGCCGCCGCCACAAGAGTGCACTCTCAC

[0323] AAGAGCAGCCCCCAGGGACCTGACAGGCTGCTGATCAGACTGCGGCACCTGATCGA

[0324] TATCGTGGAGCAGCTGAAGATCTACGAGAACGACCTGGACCCCGAGCTGCTGTCCG

[0325] CCCCACAGGACGTGAAGGGACACTGCGAGCACGCAGCCTTCGCCTGTTTTCAGAAG

[0326] GCCAAGCTGAAGCCATCTAATCCCGGCAACAATAAGACCTTCATCATCGATCTGGT

[0327] GGCCCAGCTGAGGAGACGGCTGCCAGCAAGGAGGGGCGGCAAGAAGCAGAAGCAC

[0328] ATCGCCAAGTGCCCCTCTTGTGATAGCTATGAGAAGCGCACACCTAAGGAGTTTCTG

[0329] GAGAGGCTGAAGTGGCTGCTGCAGAAGATGATCCACCAGCACCTGAGCT

[0330] SEQ ID NO: 3 (RNA IL-21 immunoadjuvant domain with IgE)

[0331] AUGGAUUGGACCUGGAUACUGUUCCUGGUGGCCGCCGCCACAAGAGUGCACUCUC

[0332] ACAAGAGCAGCCCCCAGGGACCUGACAGGCUGCUGAUCAGACUGCGGCACCUGAU

[0333] CGAUAUCGUGGAGCAGCUGAAGAUCUACGAGAACGACCUGGACCCCGAGCUGCU

[0334] GUCCGCCCCACAGGACGUGAAGGGACACUGCGAGCACGCAGCCUUCGCCUGUUUU

[0335] CAGAAGGCCAAGCUGAAGCCAUCUAAUCCCGGCAACAAUAAGACCUUCAUCAUCG

[0336] AUCUGGUGGCCCAGCUGAGGAGACGGCUGCCAGCAAGGAGGGGCGGCAAGAAGC

[0337] AGAAGCACAUCGCCAAGUGCCCCUCUUGUGAUAGCUAUGAGAAGCGCACACCUAA

[0338] GGAGUUUCUGGAGAGGCUGAAGUGGCUGCUGCAGAAGAUGAUCCACCAGCACCU GAGCU

[0339] SEQ ID NO: 4 (AA IL-21 immunoadjuvant domain without IgE)

[0340] HKSSPQGPDRLLIRLRHLIDIVEQLKIYENDLDPELLSAPQDVKGHCEHAAFACFQKAKL

[0341] KPSNPGNNKTFIIDLVAQLRRRLPARRGGKKQKHIAKCPSCDSYEKRTPKEFLERLKWL LQKMIHQHLS

[0342] SEQ ID NO: 5 (DNA IL-21 immunoadjuvant domain without IgE)

[0343] CACAAGAGCAGCCCCCAGGGACCTGACAGGCTGCTGATCAGACTGCGGCACCTGAT

[0344] CGATATCGTGGAGCAGCTGAAGATCTACGAGAACGACCTGGACCCCGAGCTGCTGT

[0345] CCGCCCCACAGGACGTGAAGGGACACTGCGAGCACGCAGCCTTCGCCTGTTTTCAG

[0346] AAGGCCAAGCTGAAGCCATCTAATCCCGGCAACAATAAGACCTTCATCATCGATCT

[0347] GGTGGCCCAGCTGAGGAGACGGCTGCCAGCAAGGAGGGGCGGCAAGAAGCAGAAG

[0348] CACATCGCCAAGTGCCCCTCTTGTGATAGCTATGAGAAGCGCACACCTAAGGAGTTT

[0349] CTGGAGAGGCTGAAGTGGCTGCTGCAGAAGATGATCCACCAGCACCTGAGCT

[0350] SEQ ID NO: 6 (RNA IL-21 immunoadjuvant domain without IgE)

[0351] CACAAGAGCAGCCCCCAGGGACCUGACAGGCUGCUGAUCAGACUGCGGCACCUGA

[0352] UCGAUAUCGUGGAGCAGCUGAAGAUCUACGAGAACGACCUGGACCCCGAGCUGC

[0353] UGUCCGCCCCACAGGACGUGAAGGGACACUGCGAGCACGCAGCCUUCGCCUGUUU

[0354] UCAGAAGGCCAAGCUGAAGCCAUCUAAUCCCGGCAACAAUAAGACCUUCAUCAUC

[0355] GAUCUGGUGGCCCAGCUGAGGAGACGGCUGCCAGCAAGGAGGGGCGGCAAGAAG

[0356] CAGAAGCACAUCGCCAAGUGCCCCUCUUGUGAUAGCUAUGAGAAGCGCACACCUA

[0357] AGGAGUUUCUGGAGAGGCUGAAGUGGCUGCUGCAGAAGAUGAUCCACCAGCACC UGAGCU

[0358] SEQ ID NO: 7 (DNA GGS linker)

[0359] GGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAGGC

[0360] SEQ ID NO: 8 (AA GGS linker)

[0361] GGSGGSGGSGGSGGG

[0362] SEQ ID NO: 9 (DNA LS3 linker)

[0363] TTGCGATTTGGTATTGTCGCTTCCCGCGCAAACCATGCGCTCGTGGGTGGTTCCGGT GGC SEQ ID NO: 10 (AA LS3 linker)

[0364] LRFGIVASRANHALVGGSGG

[0365] SEQ ID NO: 11 (nucleotide 180)

[0366] CTCTCAATTGCCCCAACGTTGATCAACCGGGACAAGCCATACACGAAAGAGGAACT

[0367] TATGGAGATATTGCGGTTGGCCATTATAGCTGAACTCGATGCAATTAATCTCTATGA

[0368] ACAAATGGCCCGCTATAGCGAAGACGAAAATGTGAGAAAGATCTTGTTGGACGTCG

[0369] CTAGGGAAGAGAAAGCACACGTAGGAGAGTTCATGGCTTTGTTGCTTAACCTCGAC

[0370] CCTGAGCAAGTCACAGAGCTGAAGGGCGGGTTCGAGGAAGTTAAAGAATTGACCG

[0371] GTATAGAAGCTCACATTAATGACAACAAGAAAGAGGAAAGTAATGTAGAGTATTTC

[0372] GAGAAGCTCAGATCTGCCTTGTTGGATGGAGTCAACAAGGGTCGCAGCTTGCTCAA

[0373] ACATCTGCCCGTTACAAGAATAGAAGGGCAGTCTTTTCGAGTAGACATCATCAAATT

[0374] TGAGGACGGCGTCCGAGTGGTTAAACAAGAGTATAAGCCTATACCCCTTCTTAAGA

[0375] AGAAGTTCTACGTCGGCATTCGAGAACTGAATGACGGGACATATGATGTCAGCATT

[0376] GCTACTAAAGCCGGTGAGCTGCTGGTTAAAGACGAAGAAAGTCTTGTGATCCGGGA

[0377] AATTTTGTCAACGGAAGGCATCAAGAAAATGAAATTGTCATCCTGGGACAATCCAG

[0378] AAGAAGCCCTGAATGATTTGATGAATGCGCTCCAAGAAGCTAGCAATGCTAGTGCT

[0379] GGCCCCTTCGGCCTTATTATCAATCCAAAGCGGTACGCCAAACTGCTGAAGATCTAT

[0380] GAAAAGTCAGGTAAGATGCTCGTAGAAGTACTCAAGGAAATCTTCCGGGGTGGAAT

[0381] AATCGTAACTCTTAATATCGACGAAAACAAAGTGATTATCTTCGCTAATACGCCCGC

[0382] CGTTCTGGACGTGGTGGTGGGTCAAGACGTTACGCTCCAGGAGCTTGGTCCGGAAG

[0383] GGGATGATGTCGCATTCCTGGTCAGTGAAGCCATTGGTATAAGAATCAAGAACCCG

[0384] GAAGCTATAGTTGTTCTCGAA

[0385] SEQ ID NO: 12 (AA 180)

[0386] LSIAPTLINRDKPYTKEELMEILRLAIIAELDAINLYEQMARYSEDENVRKILLDVAREEK

[0387] AHVGEFMALLLNLDPEQVTELKGGFEEVKELTGIEAHINDNKKEESNVEYFEKLRSALL

[0388] DGVNKGRSLLKHLPVTRIEGQSFRVDIIKFEDGVRVVKQEYKPIPLLKKKFYVGIRELND

[0389] GTYDVSIATKAGELLVKDEESLVIREILSTEGIKKMKLSSWDNPEEALNDLMNALQEAS

[0390] NASAGPFGLIINPKRYAKLLKIYEKSGKMLVEVLKEIFRGGIIVTLNIDENKVIIFANTPAV

[0391] LDVVVGQDVTLQELGPEGDDVAFLVSEAIGIRIKNPEAIVVLE

[0392] SEQ ID NO: 13 (nucleotide FR)

[0393] CTGTCCAAGGACATAATCAAACTGCTCAACGAACAGGTGAACAAGGAGATGCAATC

[0394] TTCTAACCTGTACATGAGCATGTCTAGCTGGTGTTATACTCACAGTCTCGACGGCGC

[0395] TGGCCTCTTTCTGTTCGATCACGCCGCTGAAGAATATGAGCACGCGAAGAAGCTTAT

[0396] AATCTTCCTGAACGAGAATAATGTTCCCGTCCAACTGACGTCCATTTCCGCCCCTGA

[0397] ACACAAGTTTGAGGGTCTGACTCAGATCTTTCAAAAGGCGTACGAGCACGAGCAGC

[0398] ATATCAGCGAGAGCATTAACAACATTGTCGATCACGCCATTAAAAGCAAAGACCAC

[0399] GCTACCTTCAACTTTCTCCAATGGTACGTCGCCGAACAGCATGAGGAAGAAGTATTG

[0400] TTCAAGGATATACTCGATAAGATTGAACTGATCGGAAATGAGAATCACGGCCTGTA

[0401] CCTGGCCGACCAATACGTCAAAGGCATTGCCAAGTCTAGGAAAAGC

[0402] SEQ ID NO: 14 (AA FR)

[0403] LSKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIF

[0404] LNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQ

[0405] WYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS

[0406] SEQ ID NO: 15 (nucleotide IMX)

[0407] AAAAAACAGGGGGATGCTGATGTCTGCGGAGAGGTGGCTTATATCCAGAGCGTGGT

[0408] GTCCGACTGCCACGTGCCAACCGCAGAGCTGCGGACACTGCTGGAGATCAGAAAAC

[0409] TGTTCCTGGAGATTCAGAAACTGAAAGTCGAGCTGCAGGGGCTGTCAAAAGAACTG

[0410] AGGTTCGGAATCGTCGCAAGTAGGGCTAATCACGCTCTGGTG

[0411] SEQ ID NO: 16 (AA IMX) KKQGDADVCGEVAYIQSVVSDCHVPTAELRTLLEIRKLFLEIQKLKVELQGLSKELRFGI VASRANHALV

[0412] SEQ ID NO: 17 (nucleotide IMX 2)

[0413] AAGAAACAAGGCGACGCCGATGTGTGTGGGGAAGTTGCCTACATTCAATCTGTCGT

[0414] CAGCGATTGTCATGTCCCCACAGCCGAATTGCGAACCCTTCTCGAAATTAGGAAGCT

[0415] CTTTCTTGAAATCCAAAAGCTCAAGGTGGAATTGCAAGGACTCAGTAAAGAGCTTA

[0416] GATTTGGCATAGTAGCGTCCAGAGCCAACCATGCCCTTGTC

[0417] SEQ ID NO: 18 (AA IMX 2)

[0418] KKQGDADVCGEVAYIQSVVSDCHVPTAELRTLLEIRKLFLEIQKLKVELQGLSKELRFGI

[0419] VASRANHALV

[0420] SEQ ID NO: 19 (nucleotide LS)

[0421] ATGCAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGC

[0422] AAGCCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAA

[0423] TTGTCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGC

[0424] TGGGAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGT

[0425] GATCGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTC

[0426] AGAAGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTT

[0427] TTGGCGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGC

[0428] CATGGAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTT

[0429] CAAATCTCTGCGA

[0430] SEQ ID NO:20 (AA LS)

[0431] MQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIVRHGGREEDITLVRVCGSWEI

[0432] PVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGLADLSLELRKPITFGVITADT

[0433] LEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLR

[0434] SEQ ID NO: 21 (AA IgE leader)

[0435] MDWTWILFLVAAATRVHS

[0436] SEQ ID NO: 22 (Nucleotide SARS-CoV-2_RBD)

[0437] AATATTACCAATCTTTGTCCCTTCGGCGAGGTGTTCAACGCCACGCGGTTCGCGTCT

[0438] GTATACGCCTGGAACCGCACCCGGATCAGCAACTGTACCGCTGACTACAGCGTTCT

[0439] GTACAACAGCAGCAGCTTTTCAACTTTTAAGTGTTACGGAGTCAACCCCACAAAACT

[0440] GAACGACCTGTGCTTCACCAATGTGTATGCAGACTCTTTTGTGATACGCGGAGATGA

[0441] AGTACGCCAAATTGCCCCCGGACAAACCGGCAAGATCGCGGACTACAACTACAAGC

[0442] TGCCCGATAACTTTACAGGTTGCGTGATCGCGTGGAACAGTAATAATCTGGATTCCA

[0443] AGGTTGGCGGGAATTATAATTACCTTTATCGCCTGTTTCGGAAGTCTAACCTGAGCC

[0444] CGTTCGAGAGAGACATCTCAACGGAAATTTATCAAGCCGGCAGTACCCCTTGCAAT

[0445] GGGACCGAAGGATTCAATTGTTATTTCCCACTCCAATCTTATGGCTTCCAACCGACA

[0446] AACGGTGTTGGTTATCAGCCCTATCGGGTGGTTGTGCTTTCTTTCGAAAACCTTAGC

[0447] GCTCCAGCTACCGTGTGCGGGCCCTAATGA

[0448] SEQ ID NO:23 (AA SARS-CoV-2_RBD)

[0449] NITNLCPFGEVFNATRFASVYAWNRTRISNCTADYSVLYNSSSFSTFKCYGVNPTKLND

[0450] LCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDNFTGCVIAWNSNNLDSKVGG

[0451] NYNYLYRLFRKSNLSPFERDISTEIYQAGSTPCNGTEGFNCYFPLQSYGFQPTNGVGYQP

[0452] YRVVVLSFENLSAPATVCGP

[0453] SEQ ID NO: 24 (nucleotide SARS-CoV-2 RBD 2)

[0454] AACATTACCAACCTTTGCCCCTTCGGCGAGGTCTTCAATGCCACAAGGTTTGCCAGT

[0455] GTATATGCATGGAACAGGACTCGCATCTCAAACTGCACCGCTGATTACTCTGTTCTG

[0456] TACAATTCAAGCAGCTTCTCCACATTCAAGTGTTACGGAGTCAATCCTACAAAACTG AACGACCTCTGCTTTACGAACGTCTACGCTGACTCCTTTGTCATACGGGGCGACGAG GTGAGGCAAATTGCGCCCGGCCAAACTGGGAAAATTGCGGACTATAATTACAAACT TCCTGATAACTTCACCGGTTGTGTGATCGCTTGGAATTCAAACAACCTGGACTCCAA GGTGGGCGGCAATTACAACTACCTTTATAGGCTCTTTCGGAAAAGCAACCTGAGTCC CTTCGAGCGAGATATTAGTACGGAGATCTACCAGGCCGGGTCTACGCCATGCAACG GTACGGAAGGTTTCAACTGTTATTTTCCGCTGCAGAGTTACGGATTCCAACCAACAA ATGGGGTGGGCTACCAACCTTATAGGGTGGTCGTCCTGTCCTTTGAGCTTAACCACA

[0457] GCCCTGCGACCGTATGTGGACCCTAATGA

[0458] SEQ ID NO:25 (AA SARS-CoV-2_RBD_2)

[0459] NITNLCPFGEVFNATRFASVYAWNRTRISNCTADYSVLYNSSSFSTFKCYGVNPTKLND LCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDNFTGCVIAWNSNNLDSKVGG

[0460] NYNYLYRLFRKSNLSPFERDISTEIYQAGSTPCNGTEGFNCYFPLQSYGFQPTNGVGYQP YRVVVLSFELNHSPATVCGP

[0461] SEQ ID NO:26 (Nucleotide SARS-CoV-2 RBD 3)

[0462] AATATTACCAATCTTTGTCCCTTCGGCGAGGTGTTCAACGCCACGCGGTTCGCGTCT GTATACGCCTGGAACCGCACCCGGATCAGCAACTGTACCGCTGACTACAGCGTTCT GTACAACAGCAGCAGCTTTTCAACTTTTAAGTGTTACGGAGTCAACCCCACAAAACT GAACGACCTGTGCTTCACCAATGTGTATGCAGACTCTTTTGTGATACGCGGAGATGA AGTACGCCAAATTGCCCCCGGACAAACCGGCAAGATCGCGGACTACAACTACAAGC TGCCCGATGACTTTACAGGTTGCGTGATCGCGTGGAACAGTAATAATCTGGATTCCA AGGTTGGCGGGAATTATAATTACCTTTATCGCCTGTTTCGGAAGTCTAACCTGAGCC

[0463] CGTTCGAGAGAGACAACTCAACGGAAATTTATCAAGCCGGCAGTACCCCTTGCAAT GGGACCGAAGGATTCAATTGTTATTTCCCACTCCAATCTTATGGCTTCCAACCGACA AACGGTGTTGGTTATCAGCCCTATCGGGTGGTTGTGCTTTCTTTCGAAAACCTTAGC GCTCCAGCTACCGTGTGCGGGCCCTAATGA

[0464] SEQ ID NO:27 (AA SARS-CoV-2_RBD_3)

[0465] NITNLCPFGEVFNATRFASVYAWNRTRISNCTADYSVLYNSSSFSTFKCYGVNPTKLND LCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGG NYNYLYRLFRKSNLSPFERDNSTEIYQAGSTPCNGTEGFNCYFPLQSYGFQPTNGVGYQ PYRVVVLSFENLSAPATVCGP

[0466] SEQ ID NO:28 (Nucleotide SARS-CoV-2_ RBD 4)

[0467] AACATTACCAACCTTTGCCCCTTCGGCGAGGTCTTCAATGCCACAAGGTTTGCCAGT GTATATGCATGGAACAGGACTCGCATCTCAAACTGCGTTGCTGATTACTCTGTTCTG TACAATTCAGCGAGCTTCTCCACATTCAAGTGTTACGGAGTCAATCCTACAAAACTG AACGACCTCTGCTTTACGAACGTCTACGCTGACTCCTTTGTCATACGGGGCGACGAG GTGAGGCAAATTGCGCCCGGCCAAACTGGGAAAATTGCGGACTATAATTACAAACT TCCTGATAACTTCACCGGTTGTGTGATCGCTTGGAATTCAAACAACCTGGACTCCAA GGTGGGCGGCAATTACAACTACCTTTATAGGCTCTTTCGGAAAAGCAACCTGAGTCC CTTCGAGCGAGATATTAGTACGGAGATCTACCAGGCCGGGTCTACGCCATGCAACG GTACGGAAGGTTTCAACTGTTATTTTCCGCTGCAGAGTTACGGATTCCAACCAACAA ATGGGGTGGGCTACCAACCTTATAGGGTGGTCGTCCTGTCCTTTGAGCTTCTCCACG

[0468] GCCCTGCGACCGTATGTGGACCCTAATGA

[0469] SEQ ID NO:29 (AA SARS-CoV-2_RBD_4)

[0470] NITNLCPFGEVFNATRFASVYAWNRTRISNCVADYSVLYNSASFSTFKCYGVNPTKLND LCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDNFTGCVIAWNSNNLDSKVGG NYNYLYRLFRKSNLSPFERDISTEIYQAGSTPCNGTEGFNCYFPLQSYGFQPTNGVGYQP YRVVVLSFELLHAPATVCGP

[0471] SEQ ID NO:30 (Nucleotide SARS-CoV-2_ RBD dimer) CGAGTCCAACCCACTGAGAGCATCGTAAGATTTCCGAACATTACCAACCTTTGCCCC

[0472] TTCGGCGAGGTCTTCAATGCCACAAGGTTTGCCAGTGTATATGCATGGAACAGGACT CGCATCTCAAACTGCGTTGCTGATTACTCTGTTCTGTACAATTCAGCGAGCTTCTCCA CATTCAAGTGTTACGGAGTCAATCCTACAAAACTGAACGACCTCTGCTTTACGAACG TCTACGCTGACTCCTTTGTCATACGGGGCGACGAGGTGAGGCAAATTGCGCCCGGC

[0473] CAAACTGGGAAAATTGCGGACTATAATTACAAACTTCCTGATAACTTCACCGGTTGT GTGATCGCTTGGAATTCAAACAACCTGGACTCCAAGGTGGGCGGCAATTACAACTA CCTTTATAGGCTCTTTCGGAAAAGCAACCTGAGTCCCTTCGAGCGAGATATTAGTAC GGAGATCTACCAGGCCGGGTCTACGCCATGCAACGGTACGGAAGGTTTCAACTGTT

[0474] ATTTTCCGCTGCAGAGTTACGGATTCCAACCAACAAATGGGGTGGGCTACCAACCTT

[0475] ATAGGGTGGTCGTCCTGTCCTTTGAGCTTCTCCACGCCCCTGCGACCGTATGTGGAC

[0476] CCAAGAAGTCTACAAACCTCGTAAAGAATAAGCGGGTACAGCCGACCGAATCAATA GTCCGATTCCCCAATATCACGAATCTGTGTCCTTTTGGGGAAGTGTTTAACGCTACG CGGTTCGCATCCGTGTACGCCTGGAATCGGACCCGAATTTCCAATTGTGTGGCAGAC TATAGCGTGCTTTATAACTCTGCTTCCTTTTCTACCTTTAAATGCTATGGGGTAAACC

[0477] CAACCAAGCTCAATGATCTGTGTTTCACCAATGTGTATGCGGATAGCTTCGTGATCC GAGGAGATGAAGTTCGACAGATCGCCCCGGGACAGACCGGCAAGATAGCTGATTAC AACTATAAGTTGCCCGACAATTTTACAGGCTGCGTCATTGCATGGAACTCTAATAAT TTGGATTCAAAAGTCGGTGGAAACTATAATTATCTGTACAGACTTTTCAGGAAGAGT AATCTGTCCCCTTTTGAAAGAGACATCAGCACCGAAATATATCAAGCGGGAAGCAC

[0478] CCCTTGTAATGGCACAGAGGGCTTTAATTGCTACTTCCCTTTGCAATCTTATGGTTTT

[0479] CAGCCCACCAACGGTGTCGGATATCAGCCCTACCGGGTAGTTGTATTGTCTTTCGAA CTCCTGCATGCTCCAGCCACTGTTTGCGGGCCTAAGAAGAGCACTAATTTGGTGAAG AACAAATAGTGA

[0480] SEQ ID NO:31 (AA SARS-CoV-2_RBD dimer)

[0481] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRTRISNCVADYSVLYNSASFSTFK

[0482] CYGVNPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDNFTGCVIAW

[0483] NSNNLDSKVGGNYNYLYRLFRKSNLSPFERDISTEIYQAGSTPCNGTEGFNCYFPLQSYG

[0484] FQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKRVQPTESIVRFPNITNLCP

[0485] FGEVFNATRFASVYAWNRTRISNCVADYSVLYNSASFSTFKCYGVNPTKLNDLCFTNV

[0486] YADSFVIRGDEVRQIAPGQTGKIADYNYKLPDNFTGCVIAWNSNNLDSKVGGNYNYLY RLFRKSNLSPFERDISTEIYQAGSTPCNGTEGFNCYFPLQSYGFQPTNGVGYQPYRVVVL SFELLHAPATVCGPKKSTNLVKNK

[0487] KEY for SEQ ID NOs: 32-85

[0488] Leader sequence Nanoparticle scaffold LS3 helper epitope RBD

[0489] SEQ ID NO: 32 (Nucleotide RBD 48mer)

[0490] ATGGACTGGACTTGGATTCTGTTCCTGGTCGCAGCAGCCACTCGGGTGC

[0491] ATAGCCGCGTGCAGCCCACTGAAAGCATTGTGAGATTCCCTAACATCACCAATCTGT

[0492] GCCCATTCGGCGAGGTGTTTAACGCCACACGGTTCGCCAGCGTGTACGCCTGGAAC

[0493] AGGAAGAGAATCTCCAATTGCGTGGCCGACTACTCTGTGCTGTATAATAGCGCCTCC

[0494] TTCTCTACCTTTAAGTGCTACGGCGTGTCTCCCACCAAGCTGAACGACCTGTGCTTC

[0495] ACAAACGTGTACGCCGACAGCTTTGTGATCAGGGGCGATGAGGTGAGACAGATCGC

[0496] ACCAGGACAGACCGGCAAGATCGCAGACTACAACTATAAGCTGCCCGACGATTTCA

[0497] CAGGCTGCGTGATCGCCTGGAATAGCAACAATCTGGATTCCAAAGTGGGCGGCAAC

[0498] TACAATTATCTGTACAGGCTGTTCAGAAAGAGCAACCTGAAGCCCTTTGAGCGGGA

[0499] CATCTCTACCGAGATCTACCAGGCCGGCAGCACACCTTGCAACGGCGTGGAGGGCT

[0500] TCAATTGTTACTTTCCACTGCAGTCTTATGGCTTCCAGCCCACAAACGGCGTGGGCT

[0501] ACCAGCCTTATCGCGTGGTGGTGCTGAGCTTTGAGCTGCTGCACGCACCAGCAACCG

[0502] TGTGCGGACCTAAGAAGAGCACAAACCTGGTGAAGAATAAGAGGGTGCAGCCTAC

[0503] CGAGTCCATCGTGAGATTCCCCAACATCACAAATCTGTGCCCTTTCGGCGAAGTGTT

[0504] TAACGCCACCCGCTTTGCCTCCGTGTACGCCTGGAACCGGAAGCGCATCAGTAACTG CGTGGCCGACTACTCCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTA CGGCGTGTCCCCAACTAAGCTGAACGACCTGTGCTTTACCAACGTGTACGCTGACTC TTTTGTGATCCGGGGCGATGAGGTGCGCCAGATCGCACCTGGCCAGACTGGCAAGA TCGCAGACTATAACTACAAGCTGCCAGATGATTTCACAGGATGCGTGATTGCTTGGA ATTCTAACAATCTGGATAGTAAAGTGGGCGGCAATTATAATTACCTGTATCGGCTGT TCCGCAAGTCCAACCTGAAGCCATTTGAGAGGGATATCTCCACTGAAATCTACCAG GCCGGCTCTACACCCTGTAATGGAGTCGAAGGCTTCAATTGCTATTTCCCTCTGCAG TCCTATGGCTTCCAGCAACCAACGGAGTGGGATACCAGCCTTATAGAGTCGTGGTG CTGAGCTTTGAGTTACTGCATGCCCCTGCCACCGTGTGCGGACCCAAGAAGTCCACA AATCTGGTCAAGAATAAGGCAAGCGGGGAAAGCCAGGTGCGACAGCAGTTCTCCA AAGACATCGAAAAGCTGCTGAATGAACAGGTCAACAAGGAAATGCAGAGCAGCAA CCTGTACATGTCCATGAGCTCCTGGTGCTATACCCACTCTCTGGACGGAGCAGGCCT GTTCCTGTTTGATCACGCCGCCGAGGAGTACGAGCACGCCAAGAAGCTGATCATCTT CCTGAATGAGAACAATGTGCCCGTGCAGCTGACCTCTATCAGCGCCCCTGAGCACA AGTTCGAGGGCCTGACACAGATCTTTCAGAAGGCCTACGAGCACGAGCAGCACATC TCCGAGTCTATCAACAATATCGTGGACCACGCCATCAAGTCCAAGGATCACGCCAC ATTCAACTTTCTGCAGTGGTACGTGGCCGAGCAGCACGAGGAGGAGGTGCTGTTTA AGGACATCCTGGATAAGATCGAGCTGATCGGCAACGAGAATCACGGGCTGTATCTG GCCGACCAGTATGTGAAGGGCATCGCTAAAAGCAGGAAATCAGGAAGCTGATAA

[0505] SEQ ID NO: 33 (AA RED 48mer)

[0506] MDWTWILFLVAAATRVHSRVOPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRI SNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGOTGKI ADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGS TPCNGVEGFNCYFPLOSYGFOPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVK NKRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFST FKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGOTGKIADYNYKLPDDFTGCVIA WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQS YGFQPTNGVGYOPYRVVVLSFELLHAPATVCGPKKSTNLVKNKASGESOVRQQFSKDI EKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNEN NVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYV AEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS**

[0507] SEQ ID NO: 34 (Nucleotide RBD_g5.1_180mer)

[0508] ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCCTC TCAATTGCCCCAACGTTGATCAACCGGGACAAGCCATACACGAAAGAGGAACTTAT GGAGATATTGCGGTTGGCCATTATAGCTGAACTCGATGCAATTAATCTCTATGAACA AATGGCCCGCTATAGCGAAGACGAAAATGTGAGAAAGATCTTGTTGGACGTCGCTA GGGAAGAGAAAGCACACGTAGGAGAGTTCATGGCTTTGTTGCTTAACCTCGACCCT GAGCAAGTCACAGAGCTGAAGGGCGGGTTCGAGGAAGTTAAAGAATTGACCGGTA TAGAAGCTCACATTAATGACAACAAGAAAGAGGAAAGTAATGTAGAGTATTTCGAG AAGCTCAGATCTGCCTTGTTGGATGGAGTCAACAAGGGTCGCAGCTTGCTCAAACA TCTGCCCGTTACAAGAATAGAAGGGCAGTCTTTTCGAGTAGACATCATCAAATTTGA GGACGGCGTCCGAGTGGTTAAACAAGAGTATAAGCCTATACCCCTTCTTAAGAAGA AGTTCTACGTCGGCATTCGAGAACTGAATGACGGGACATATGATGTCAGCATTGCT ACTAAAGCCGGTGAGCTGCTGGTTAAAGACGAAGAAAGTCTTGTGATCCGGGAAAT TTTGTCAACGGAAGGCATCAAGAAAATGAAATTGTCATCCTGGGACAATCCAGAAG AAGCCCTGAATGATTTGATGAATGCGCTCCAAGAAGCTAGCAATGCTAGTGCTGGC CCCTTCGGCCTTATTATCAATCCAAAGCGGTACGCCAAACTGCTGAAGATCTATGAA AAGTCAGGTAAGATGCTCGTAGAAGTACTCAAGGAAATCTTCCGGGGTGGAATAAT CGTAACTCTTAATATCGACGAAAACAAAGTGATTATCTTCGCTAATACGCCCGCCGT TCTGGACGTGGTGGTGGGTCAAGACGTTACGCTCCAGGAGCTTGGTCCGGAAGGGG ATGATGTCGCATTCCTGGTCAGTGAAGCCATTGGTATAAGAATCAAGAACCCGGAA GCTATAGTTGTTCTCGAAGGCGGGTCTGGTGGGAGCGGTGGTAGTGGTGGTTCTGGT

[0509] GGTGGTGGGTCAGGTGGCGGCTCAGGCGGCGGCTTGCGATTTGGTATTGTCGCTTCC CGCGCAAACCATGCGCTCGTGGGTGGTTCCGGTGGCAACATTACCAACCTTTGCCCC TTCGGCGAGGTCTTCAATGCCACAAGGTTTGCCAGTGTATATGCATGGAACAGGACT CGCATCTCAAACTGCGTTGCTGATTACTCTGTTCTGTACAATTCAGCGAGCTTCTCCA

[0510] CATTCAAGTGTTACGGAGTCAATCCTACAAAACTGAACGACCTCTGCTTTACGAACG TCTACGCTGACTCCTTTGTCATACGGGGCGACGAGGTGAGGCAAATTGCGCCCGGC CAAACTGGGAAAATTGCGGACTATAATTACAAACTTCCTGATAACTTCACCGGTTGT

[0511] GTGATCGCTTGGAATTCAAACAACCTGGACTCCAAGGTGGGCGGCAATTACAACTA

[0512] CCTTTATAGGCTCTTTCGGAAAAGCAACCTGAGTCCCTTCGAGCGAGATATTAGTAC GGAGATCTACCAGGCCGGGTCTACGCCATGCAACGGTACGGAAGGTTTCAACTGTT ATTTTCCGCTGCAGAGTTACGGATTCCAACCAACAAATGGGGTGGGCTACCAACCTT

[0513] ATAGGGTGGTCGTCCTGTCCTTTGAGCTTCTCCACGCCCCTGCGACCGTATGTGGAC CCTAATGA

[0514] SEQ ID NO:35 (AA RBD_g5.1_180mer)

[0515] MDWTWILFLVAAATRVHSLSIAPTLINRDKPYTKEELMEILRLAIIAELDAINLYEQ

[0516] MARYSEDENVRKILLDVAREEKAHVGEFMALLLNLDPEQVTELKGGFEEVKELTGIEA

[0517] HINDNKKEESNVEYFEKLRSALLDGVNKGRSLLKHLPVTRIEGQSFRVDIIKFEDGVRVV

[0518] KQEYKPIPLLKKKFYVGIRELNDGTYDVSIATKAGELLVKDEESLVIREILSTEGIKKMKL

[0519] SSWDNPEEALNDLMNALQEASNASAGPFGLIINPKRYAKLLKIYEKSGKMLVEVLKEIF

[0520] RGGIIVTLNIDENKVIIFANTPAVLDVVVGQDVTLQELGPEGDDVAFLVSEAIGIRIKNPE

[0521] AIWLEGGSGGSGGSGGSGGGGSGGGSGGGIRFG / E4S7UAK4ZFGG5GGNITNLCPFGE

[0522] VFNATRFASVYAWNRTRISNCVADYSVLYNSASFSTFKCYGVNPTKLNDLCFTNVYAD

[0523] SFVIRGDEVRQIAPGQTGKIADYNYKLPDNFTGCVIAWNSNNLDSKVGGNYNYLYRLF

[0524] RKSNLSPFERDISTEIYQAGSTPCNGTEGFNCYFPLOSYGFOPTNGVGYQPYRVVVLSF ELLHAPATVCGP**

[0525] SEQ ID NO: 36 (Nucleotide RBD_g5.1_120mer (Twist))

[0526] ATGGATTGGACGTGGATATTGTTCCTGGTAGCCGCTGCGACTCGGGTCCATAGCATG

[0527] CAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAAG

[0528] CCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATTG TCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCTG GGAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTGA

[0529] TCGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCAG AAGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTTT GGCGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCCA

[0530] TGGAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCA

[0531] AATCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAGG CCGAGTCCAACCCACTGAGAGCATCGTAAGATTTCCGAACATTACCAACCTTTGCCC CTTCGGCGAGGTCTTCAATGCCACAAGGTTTGCCAGTGTATATGCATGGAACAGGA

[0532] CTCGCATCTCAAACTGCGTTGCTGATTACTCTGTTCTGTACAATTCAGCGAGCTTCTC

[0533] CACATTCAAGTGTTACGGAGTCAATCCTACAAAACTGAACGACCTCTGCTTTACGAA CGTCTACGCTGACTCCTTTGTCATACGGGGCGACGAGGTGAGGCAAATTGCGCCCG GCCAAACTGGGAAAATTGCGGACTATAATTACAAACTTCCTGATAACTTCACCGGTT

[0534] GTGTGATCGCTTGGAATTCAAACAACCTGGACTCCAAGGTGGGCGGCAATTACAAC

[0535] TACCTTTATAGGCTCTTTCGGAAAAGCAACCTGAGTCCCTTCGAGCGAGATATTAGT

[0536] ACGGAGATCTACCAGGCCGGGTCTACGCCATGCAACGGTACGGAAGGTTTCAACTG

[0537] TTATTTTCCGCTGCAGAGTTACGGATTCCAACCAACAAATGGGGTGGGCTACCAACC TTATAGGGTGGTCGTCCTGTCCTTTGAGCTTCTCCACGCCCCTGCGACCGTATGTGG ACCCAAGAAGTCTACAAACCTCGTAAAGAATAAGCGGGTACAGCCGACCGAATCAA

[0538] TAGTCCGATTCCCCAATATCACGAATCTGTGTCCTTTTGGGGAAGTGTTTAACGCTA

[0539] CGCGGTTCGCATCCGTGTACGCCTGGAATCGGACCCGAATTTCCAATTGTGTGGCAG

[0540] ACTATAGCGTGCTTTATAACTCTGCTTCCTTTTCTACCTTTAAATGCTATGGGGTAAA

[0541] CCCAACCAAGCTCAATGATCTGTGTTTCACCAATGTGTATGCGGATAGCTTCGTGAT CCGAGGAGATGAAGTTCGACAGATCGCCCCGGGACAGACCGGCAAGATAGCTGATT ACAACTATAAGTTGCCCGACAATTTTACAGGCTGCGTCATTGCATGGAACTCTAATA ATTTGGATTCAAAAGTCGGTGGAAACTATAATTATCTGTACAGACTTTTCAGGAAGA GTAATCTGTCCCCTTTTGAAAGAGACATCAGCACCGAAATATATCAAGCGGGAAGC ACCCCTTGTAATGGCACAGAGGGCTTTAATTGCTACTTCCCTTTGCAATCTTATGGTT TTCAGCCCACCAACGGTGTCGGATATCAGCCCTACCGGGTAGTTGTATTGTCTTTCG AACTCCTGCATGCTCCAGCCACTGTTTGCGGGCCTAAGAAGAGCACTAATTTGGTG AAGAACAAATAGTGA

[0542] SEQ ID NO: 37 (AA RBD_g5.1_120mer (Twist))

[0543] MDWTWILFLV AAA TRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIV RHGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKG LADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSG GSGGSGGSGGGRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRTRISNCVADYS VLYNSASFSTFKCYGVNPTKLNDLCFTNVYADSFVIRGDEVRQIAPGOTGKIADYNYKL PDNFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLSPFERDISTEIYQAGSTPCNGTE GFNCYFPLOSYGFOPTNGVGYOPYRVVVLSFELLHAPATVCGPKKSTNLVKNKRVOPT ESIVRFPNITNLCPFGEVFNATRFASVYAWNRTRISNCVADYSVLYNSASFSTFKCYGVN PTKLNDLCFTNVYADSFVIRGDEVRQIAPGOTGKIADYNYKLPDNFTGCVIAWNSNNLD SKVGGNYNYLYRLFRKSNLSPFERDISTEIYQAGSTPCNGTEGFNCYFPLOSYGFQPTN GVGYOPYRVVVLSFELLHAPATVCGPKKSTNLVKNK**

[0544] SEQ ID NO: 38 (Nucleotide RBD_g5.1_120mer (Genscript))

[0545] ATGGACTGGACTTGGATTCTGTTTCTGGTCGCCGCTGCCACTCGCGTGCATTCTATG CAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAAG CCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATTG TCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCTG GGAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTGA TCGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCAG AAGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTTT GGCGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCCA TGGAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCA AATCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAGG CAGGGTGCAGCCTACCGAGTCCATCGTGAGATTCCCTAACATCACAAATCTGTGCCC ATTCGGCGAGGTGTTTAACGCCACCAGGTTTGCCAGCGTGTACGCCTGGAACAGGA CAAGAATCTCTAATTGCGTGGCCGACTACAGCGTGCTGTATAACAGCGCCTCCTTCT CTACCTTTAAGTGCTACGGCGTGAACCCAACCAAGCTGAATGACCTGTGCTTCACAA ACGTGTACGCCGACAGCTTTGTGATCAGGGGCGATGAGGTGAGACAGATCGCACCA GGACAGACCGGCAAGATCGCAGACTACAACTATAAGCTGCCCGATAATTTCACAGG

[0546] CTGCGTGATCGCCTGGAACTCCAACAATCTGGACTCTAAAGTGGGCGGCAACTACA ATTATCTGTACAGGCTGTTCAGAAAGAGCAATCTGTCCCCCTTTGAGAGGGATATCA GCACCGAGATCTACCAGGCAGGCTCCACCCCTTGCAACGGAACAGAGGGCTTCAAT TGTTACTTTCCACTGCAGTCTTATGGCTTCCAGCCCACAAACGGCGTGGGCTACCAG CCTTATAGAGTGGTGGTGCTGAGCTTTGAGCTGCTGCACGCACCAGCAACCGTGTGC GGACCTAAGAAGTCCACAAACCTGGTGAAGAATAAGCGGGTGCAGCCCACCGAGTC TATCGTGCGCTTCCCCAATATTACAAATCTGTGCCCTTTCGGCGAAGTGTTTAACGC TACTAGATTCGCTAGCGTGTACGCTTGGAACCGGACACGCATCTCCAACTGCGTGGC TGACTACTCTGTGCTGTACAATAGCGCCTCCTTCTCTACCTTTAAGTGTTACGGCGTG AACCCTACTAAGCTGAATGACCTGTGCTTTACCAACGTGTACGCTGACTCCTTTGTG ATCCGGGGCGATGAGGTGCGCCAGATCGCACCTGGCCAGACTGGCAAGATCGCAGA CTATAACTACAAGCTGCCAGACAATTTCACAGGATGCGTGATTGCTTGGAACAGCA ACAATCTGGACTCCAAAGTGGGCGGCAACTATAATTACCTGTATCGGCTGTTCCGCA AGTCTAATCTGAGCCCATTTGAGCGGGATATCTCTACTGAAATCTACCAGGCAGGCA GCACCCCATGTAATGGCACCGAAGGCTTCAATTGCTACTTTCCACTGCAGTCCTACG GATTTCAGCCTACCAATGGAGTCGGCTACCAGCCTTATCGCGTCGTGGTGCTGAGCT TTGAGTTACTGCATGCCCCTGCCACCGTGTGCGGACCCAAGAAGTCCACCAACCTG GTCAAAAATAAGTGATAA SEQ ID NO:39 (AA RBD_g5.1_120mer (Genscript))

[0547] MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRLVEGAIDA IVRHGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSK GLADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGS GGSGGSGGSGGGRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRTRISNCVADY SVLYNSASFSTFKCYGVNPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYK LPDNFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLSPFERDISTEIYOAGSTPCNGTE GFNCYFPLQSYGFOPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKRVOPT

[0548] ESIVRFPNITNLCPFGEVFNATRFASVYAWNRTRISNCVADYSVLYNSASFSTFKCYGVN PTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDNFTGCVIAWNSNNLD SKVGGNYNYLYRLFRKSNLSPFERDISTEIYQAGSTPCNGTEGFNCYFPLQSYGFQPTN GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK**

[0549] SEQ ID NO: 40 (nucleotide RBD g5.1 60mer)

[0550] ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCAT GCAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAA GCCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATT GTCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCT GGGAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTG ATCGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCA GAAGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTT

[0551] TGGCGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCC ATGGAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTC AAATCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAG GCAACATTACCAACCTTTGCCCCTTCGGCGAGGTCTTCAATGCCACAAGGTTTGCCA GTGTATATGCATGGAACAGGACTCGCATCTCAAACTGCGTTGCTGATTACTCTGTTC TGTACAATTCAGCGAGCTTCTCCACATTCAAGTGTTACGGAGTCAATCCTACAAAAC TGAACGACCTCTGCTTTACGAACGTCTACGCTGACTCCTTTGTCATACGGGGCGACG AGGTGAGGCAAATTGCGCCCGGCCAAACTGGGAAAATTGCGGACTATAATTACAAA CTTCCTGATAACTTCACCGGTTGTGTGATCGCTTGGAATTCAAACAACCTGGACTCC

[0552] AAGGTGGGCGGCAATTACAACTACCTTTATAGGCTCTTTCGGAAAAGCAACCTGAG TCCCTTCGAGCGAGATATTAGTACGGAGATCTACCAGGCCGGGTCTACGCCATGCA ACGGTACGGAAGGTTTCAACTGTTATTTTCCGCTGCAGAGTTACGGATTCCAACCAA CAAATGGGGTGGGCTACCAACCTTATAGGGTGGTCGTCCTGTCCTTTGAGCTTCTCC ACGCCCCTGCGACCGTATGTGGACCCTAATAG

[0553] SEQ ID NO: 41 (AA RBD g5.1 60mer)

[0554] MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIV RHGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKG LADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSG GSGGSGGSGGGNITNLCPFGEVFNATRFASVYAWNRTRISNCVADYSVLYNSASFSTFK CYGVNPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDNFTGCVIAW NSNNLDSKVGGNYNYLYRLFRKSNLSPFERDISTEIYOAGSTPCNGTEGFNCYFPLOSY GFQPTNGVGYQPYRVVVLSFELLHAPATVCGP**

[0555] SEQ ID NO: 42 (nucleotide RBD g5.1 60mer short)

[0556] ATGGATTGGACGTGGATATTGTTCCTGGTAGCCGCTGCGACTCGGGTCCATAGCATG CAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAAG CCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATTG TCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCTG GGAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTGA TCGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCAG AAGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTTT GGCGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCCA TGGAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCA AATCTCTGCGAGGAGGGAGTGGAGGCTCAGGAGGAGGCAACATTACCAACCTTTGC CCCTTCGGCGAGGTCTTCAATGCCACAAGGTTTGCCAGTGTATATGCATGGAACAGG ACTCGCATCTCAAACTGCGTTGCTGATTACTCTGTTCTGTACAATTCAGCGAGCTTCT CCACATTCAAGTGTTACGGAGTCAATCCTACAAAACTGAACGACCTCTGCTTTACGA ACGTCTACGCTGACTCCTTTGTCATACGGGGCGACGAGGTGAGGCAAATTGCGCCC GGCCAAACTGGGAAAATTGCGGACTATAATTACAAACTTCCTGATAACTTCACCGG TTGTGTGATCGCTTGGAATTCAAACAACCTGGACTCCAAGGTGGGCGGCAATTACA

[0557] ACTACCTTTATAGGCTCTTTCGGAAAAGCAACCTGAGTCCCTTCGAGCGAGATATTA GTACGGAGATCTACCAGGCCGGGTCTACGCCATGCAACGGTACGGAAGGTTTCAAC TGTTATTTTCCGCTGCAGAGTTACGGATTCCAACCAACAAATGGGGTGGGCTACCAA CCTTATAGGGTGGTCGTCCTGTCCTTTGAGCTTCTCCACGCCCCTGCGACCGTATGTG GACCCTAGTGA

[0558] SEQ ID NO: 43 (AA RED g5.1 60mer short)

[0559] MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIV RHGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKG LADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSG GSGGGNITNLCPFGEVFNATRFASVYAWNRTRISNCVADYSVLYNSASFSTFKCYGVNP TKLNDLCFTNVYADSFVIRGDEVRQIAPGOTGKIADYNYKLPDNFTGCVIAWNSNNLDS KVGGNYNYLYRLFRKSNLSPFERDISTEIYQAGSTPCNGTEGFNCYFPLOSYGFOPTNG VGYOPYRVVVLSFELLHAPATVCGP**

[0560] SEQ ID NO:44 (Nucleotide RBD g5.1 48mer)

[0561] ATGGACTGGACTTGGATTCTGTTCCTGGTCGCAGCAGCCACTCGGGTGCATAGCCTG AGGTTCGGAATCGTCGCAAGTAGGGCTAATCACGCTCTGGTGGGCGGGTCCCGTGT GCAGCCGACAGAGAGCATCGTGCGCTTCCCAAACATTACCAACCTTTGCCCCTTCGG CGAGGTCTTCAATGCCACAAGGTTTGCCAGTGTATATGCATGGAACAGGACTCGCA TCTCAAACTGCGTTGCTGATTACTCTGTTCTGTACAATTCAGCGAGCTTCTCCACATT CAAGTGTTACGGAGTCAATCCTACAAAACTGAACGACCTCTGCTTTACGAACGTCTA CGCTGACTCCTTTGTCATACGGGGCGACGAGGTGAGGCAAATTGCGCCCGGCCAAA

[0562] CTGGGAAAATTGCGGACTATAATTACAAACTTCCTGATAACTTCACCGGTTGTGTGA TCGCTTGGAATTCAAACAACCTGGACTCCAAGGTGGGCGGCAATTACAACTACCTTT ATAGGCTCTTTCGGAAAAGCAACCTGAGTCCCTTCGAGCGAGATATTAGTACGGAG ATCTACCAGGCCGGGTCTACGCCATGCAACGGTACGGAAGGTTTCAACTGTTATTTT CCGCTGCAGAGTTACGGATTCCAACCAACAAATGGGGTGGGCTACCAACCTTATAG GGTGGTCGTCCTGTCCTTTGAGCTTCTCCACGCCCCTGCGACCGTATGTGGACCCAA GAAGAGCACGAACCTCGTGAAGAACAAGCGTGTGCAGCCGACAGAGAGCATTGTG

[0563] CGCTTCCCAAACATCACCAACCTCTGCCCCTTTGGAGAGGTCTTTAATGCCACCAGA TTTGCCTCTGTCTATGCCTGGAACCGCACACGAATCAGCAACTGTGTGGCGGACTAT TCTGTATTGTATAACAGTGCTTCGTTCTCTACCTTCAAGTGTTACGGTGTGAACCCCA CCAAGCTGAATGACCTGTGCTTCACCAATGTTTATGCAGATTCCTTTGTGATAAGAG GAGATGAAGTCCGGCAAATTGCCCCAGGGCAGACTGGGAAAATAGCAGACTACAA TTACAAACTACCAGATAACTTCACTGGTTGTGTCATTGCATGGAATTCAAACAACTT GGATTCCAAAGTGGGCGGCAACTACAACTATCTGTACAGGCTTTTCAGGAAAAGCA

[0564] ATTTAAGTCCTTTTGAAAGAGACATCTCAACTGAGATCTACCAGGCTGGATCAACAC CCTGCAATGGGACAGAAGGCTTTAACTGCTACTTCCCTCTCCAGTCCTATGGCTTCC AGCCTACAAATGGAGTTGGTTACCAACCTTACCGGGTAGTTGTCCTTAGTTTTGAGC TGCTGCACGCTCCAGCCACTGTGTGTGGCCCCAAGAAGAGCACGAATCTCGTGAAG AACAAGGCAAGCGGGGAAAGCCAGGTGCGACAGCAGTTCTCCAAAGACATCGAAA AGCTGCTGAATGAACAGGTCAACAAGGAAATGCAGAGCAGCAACCTGTACATGTCC ATGAGCTCCTGGTGCTATACCCACTCTCTGGACGGAGCAGGCCTGTTCCTGTTTGAT

[0565] CACGCCGCCGAGGAGTACGAGCACGCCAAGAAGCTGATCATCTTCCTGAATGAGAA CAATGTGCCCGTGCAGCTGACCTCTATCAGCGCCCCTGAGCACAAGTTCGAGGGCCT GACACAGATCTTTCAGAAGGCCTACGAGCACGAGCAGCACATCTCCGAGTCTATCA ACAATATCGTGGACCACGCCATCAAGTCCAAGGATCACGCCACATTCAACTTTCTGC AGTGGTACGTGGCCGAGCAGCACGAGGAGGAGGTGCTGTTTAAGGACATCCTGGAT AAGATCGAGCTGATCGGCAACGAGAATCACGGGCTGTATCTGGCCGACCAGTATGT GAAGGGCATCGCTAAAAGCAGGAAATCAGGAAGCTAGTGA

[0566] SEQ ID NO: 45 (AA RBD g5.1 48mer)

[0567] MDWTWILFLVAAATRVHS£RFG / G4Sfc4A7Fl£FGGSRVOPTESIVRFPNITNLCPFGEVF NATRFASVYAWNRTRISNCVADYSVLYNSASFSTFKCYGVNPTKLNDLCFTNVYADSF VIRGDEVRQIAPGOTGKIADYNYKLPDNFTGCVIAWNSNNLDSKVGGNYNYL YRLFRKSNLSPFERDISTEIYQAGSTPCNGTEGFNCYFPLOSYGFOPTNGVGYQPYRVVV LSFELLHAPATVCGPKKSTNLVKNKRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAW NRTRISNCVADYSVLYNSASFSTFKCYGVNPTKLNDLCFTNVYADSFVIRGDEVRQIAP GQTGKIADYNYKLPDNFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLSPFERDISTE lYQAGSTPCNGTEGFNCYFPLOSYGFOPTNGVGYQPYRVVVLSFELLHAPATVCGPKKS TNLVKNKASGESOVROQFSKDIEKLLNEOVNKEMOSSNLYMSMSSWCYTHSLDGAGL FLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINN IVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIA KSRKSGS**

[0568] SEQ ID NO: 46 (Nucleotide RBD g5.1 24mer (Twist))

[0569] ATGGATTGGACTTGGATCTTGTTCTTGGTCGCAGCGGCTACTAGAGTGCACAGTTTG CGATTTGGTATTGTCGCTTCCCGCGCAAACCATGCGCTCGTGGGTGGTTCCGGTGGC AACATTACCAACCTTTGCCCCTTCGGCGAGGTCTTCAATGCCACAAGGTTTGCCAGT GTATATGCATGGAACAGGACTCGCATCTCAAACTGCGTTGCTGATTACTCTGTTCTG TACAATTCAGCGAGCTTCTCCACATTCAAGTGTTACGGAGTCAATCCTACAAAACTG AACGACCTCTGCTTTACGAACGTCTACGCTGACTCCTTTGTCATACGGGGCGACGAG GTGAGGCAAATTGCGCCCGGCCAAACTGGGAAAATTGCGGACTATAATTACAAACT TCCTGATAACTTCACCGGTTGTGTGATCGCTTGGAATTCAAACAACCTGGACTCCAA GGTGGGCGGCAATTACAACTACCTTTATAGGCTCTTTCGGAAAAGCAACCTGAGTCC CTTCGAGCGAGATATTAGTACGGAGATCTACCAGGCCGGGTCTACGCCATGCAACG GTACGGAAGGTTTCAACTGTTATTTTCCGCTGCAGAGTTACGGATTCCAACCAACAA ATGGGGTGGGCTACCAACCTTATAGGGTGGTCGTCCTGTCCTTTGAGCTTCTCCACG CCCCTGCGACCGTATGTGGACCCGGCGGGAGCGGAGGGAGTGGCGGATCTGGCGGG AGCGGCGGAGGCCTTTCAAAAGATATTATTAAGCTCCTGAATGAGCAAGTCAATAA AGAAATGCAATCTTCTAACCTGTACATGAGCATGTCTAGCTGGTGTTATACTCACAG TCTCGACGGCGCTGGCCTCTTTCTGTTCGATCACGCCGCTGAAGAATATGAGCACGC GAAGAAGCTTATAATCTTCCTGAACGAGAATAATGTTCCCGTCCAACTGACGTCCAT TTCCGCCCCTGAACACAAGTTTGAGGGTCTGACTCAGATCTTTCAAAAGGCGTACGA GCACGAGCAGCATATCAGCGAGAGCATTAACAACATTGTCGATCACGCCATTAAAA GCAAAGACCACGCTACCTTCAACTTTCTCCAATGGTACGTCGCCGAACAGCATGAG GAAGAAGTATTGTTCAAGGATATACTCGATAAGATTGAACTGATCGGAAATGAGAA TCACGGCCTGTACCTGGCCGACCAATACGTCAAAGGCATTGCCAAGTCTAGGAAAA GCTAGTAG

[0570] SEQ ID NO: 47 (AA RBD g5. 1 24mer (Twist))

[0571] MDWTWILFLVAAATRVHS£R G / G4SR^AFM£FGGSGGNITNLCPFGEVFNATRFASVY AWNRTRISNCVADYSVLYNSASFSTFKCYGVNPTKLNDLCFTNVYADSFVIRGDEVRQI APGOTGKIADYNYKLPDNFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLSPFERDIS TEIYQAGSTPCNGTEGFNCYFPLOSYGFOPTNGVGYQPYRVVVLSFELLHAPATVCGPG GSGGSGGSGGSGGGLSKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFD HAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVD HAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKS RKS**

[0572] SEQ ID NO: 48 (Nucleotide RBD g5.1 24mer (Genscript)) ATGGATTGGACATGGATACTGTTCCTGGTGGCCGCCGCCACCAGAGTGCACTCTCTG AGGTTCGGCATCGTGGCCTCCAGGGCCAATCACGCCCTGGTGGGCGGCTCCGGCGG CAACATCACCAATCTGTGCCCTTTCGGCGAGGTGTTTAACGCCACCAGGTTTGCCTC CGTGTACGCCTGGAACCGCACAAGGATCAGCAACTGCGTGGCCGATTATAGCGTGC TGTACAATTCTGCCAGCTTCTCTACCTTTAAGTGCTACGGCGTGAACCCTACCAAGC

[0573] TGAACGATCTGTGCTTCACCAACGTGTATGCCGATAGCTTCGTGATCCGGGGCGATG AGGTGCGCCAGATCGCCCCTGGCCAGACAGGCAAGATCGCCGACTACAACTATAAG CTGCCAGATAATTTCACCGGCTGCGTGATCGCCTGGAATTCCAACAATCTGGACTCT AAGGTGGGCGGCAACTACAACTATCTGTACCGGCTGTTTCGCAAGTCCAACCTGTCT CCATTCGAGAGAGACATCTCCACAGAGATCTATCAGGCCGGCTCCACACCTTGTAA

[0574] CGGCACCGAGGGCTTCAACTGCTACTTCCCTCTGCAGAGCTATGGCTTCCAGCCTAC CAATGGCGTGGGCTATCAGCCCTACCGGGTGGTGGTGCTGTCTTTTGAGCTGCTGCA CGCCCCTGCCACAGTGTGCGGCCCTGGCGGCTCTGGCGGCTCCGGCGGCTCTGGCG GCAGCGGCGGCGGCCTGAGCAAGGATATCATCAAGCTGCTGAATGAACAGGTCAAC AAGGAAATGCAGAGCAGCAACCTGTACATGTCCATGAGCTCCTGGTGCTATACCCA

[0575] CTCTCTGGACGGAGCAGGCCTGTTCCTGTTTGATCACGCCGCCGAGGAGTACGAGC ACGCCAAGAAGCTGATCATCTTCCTGAATGAGAACAATGTGCCCGTGCAGCTGACC TCTATCAGCGCCCCTGAGCACAAGTTCGAGGGCCTGACACAGATCTTTCAGAAGGC CTACGAGCACGAGCAGCACATCTCCGAGTCTATCAACAATATCGTGGACCACGCCA

[0576] TCAAGTCCAAGGATCACGCCACATTCAACTTTCTGCAGTGGTACGTGGCCGAGCAG CACGAGGAGGAGGTGCTGTTTAAGGACATCCTGGATAAGATCGAGCTGATCGGCAA CGAGAATCACGGGCTGTATCTGGCCGACCAGTATGTGAAGGGCATCGCTAAAAGCA GGAAATCATGATAA

[0577] SEQ ID NO: 49 (AA RBD g5.1 24mer (Genscript))

[0578] MDWTWILFLVAAATRVHSZRFG / K4SR4Aa4£FGG,S,GGNITNLCPFGEVFNATRFASVY AWNRTRISNCVADYSVLYNSASFSTFKCYGVNPTKLNDLCFTNVYADSFVIRGDEVRQI APGQTGKIADYNYKLPDNFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLSPFERDIS TEIYQAGSTPCNGTEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPG GSGGSGGSGGSGGGLSKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFD

[0579] HAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVD HAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKS RKS**

[0580] SEQ ID NO:50 (Nucleotide RBD g5.1 7mer)

[0581] ATGGATTGGACTTGGATCTTGTTCTTGGTCGCAGCGGCTACTAGAGTGCACAGTTTG CGATTTGGTATTGTCGCTTCCCGCGCAAACCATGCGCTCGTGGGTGGTTCCGGTGGC AACATTACCAACCTTTGCCCCTTCGGCGAGGTCTTCAATGCCACAAGGTTTGCCAGT GTATATGCATGGAACAGGACTCGCATCTCAAACTGCGTTGCTGATTACTCTGTTCTG TACAATTCAGCGAGCTTCTCCACATTCAAGTGTTACGGAGTCAATCCTACAAAACTG

[0582] AACGACCTCTGCTTTACGAACGTCTACGCTGACTCCTTTGTCATACGGGGCGACGAG GTGAGGCAAATTGCGCCCGGCCAAACTGGGAAAATTGCGGACTATAATTACAAACT TCCTGATAACTTCACCGGTTGTGTGATCGCTTGGAATTCAAACAACCTGGACTCCAA GGTGGGCGGCAATTACAACTACCTTTATAGGCTCTTTCGGAAAAGCAACCTGAGTCC CTTCGAGCGAGATATTAGTACGGAGATCTACCAGGCCGGGTCTACGCCATGCAACG

[0583] GTACGGAAGGTTTCAACTGTTATTTTCCGCTGCAGAGTTACGGATTCCAACCAACAA ATGGGGTGGGCTACCAACCTTATAGGGTGGTCGTCCTGTCCTTTGAGCTTCTCCACG CCCCTGCGACCGTATGTGGACCCGGCGGGAGCGGAGGGAGTGGCGGATCTGGCGGG AGCGGCGGAGGCAAAAAACAGGGGGATGCTGATGTCTGCGGAGAGGTGGCTTATA TCCAGAGCGTGGTGTCCGACTGCCACGTGCCAACCGCAGAGCTGCGGACACTGCTG

[0584] GAGATCAGAAAACTGTTCCTGGAGATTCAGAAACTGAAAGTCGAGCTGCAGGGGCT GTCAAAAGAACTGAGGTTCGGAATCGTCGCAAGTAGGGCTAATCACGCTCTGGT GTAATAG

[0585] SEQ ID NO: 51 (AA RBD g5.1 7mer) MDWTWILFLVAAATRVHS£RFG / L4SF4Aa4LFGG,S,GGNITNLCPFGEVFNATRFASVY AWNRTRISNCVADYSVLYNSASFSTFKCYGVNPTKLNDLCFTNVYADSFVIRGDEVRQI

[0586] APGQTGKIADYNYKLPDNFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLSPFERDIS

[0587] TEIYQAGSTPCNGTEGFNCYFPLOSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPG GSGGSGGSGGSGGGKKQGDADVCGEVAYIQSVVSDCHVPTAELRTLLEIRKLFLEIQK LKVELQGLSKELRFGIVASRANHALV**

[0588] SEQ ID NO:52 (Nucleotide RBD g5.1 14mer trans)

[0589] ATGGATTGGACGTGGATATTGTTCCTGGTAGCCGCTGCGACTCGGGTCCATAGCCGA GTCCAACCCACTGAGAGCATCGTAAGATTTCCGAACATTACCAACCTTTGCCCCTTC GGCGAGGTCTTCAATGCCACAAGGTTTGCCAGTGTATATGCATGGAACAGGACTCG CATCTCAAACTGCGTTGCTGATTACTCTGTTCTGTACAATTCAGCGAGCTTCTCCACA TTCAAGTGTTACGGAGTCAATCCTACAAAACTGAACGACCTCTGCTTTACGAACGTC

[0590] TACGCTGACTCCTTTGTCATACGGGGCGACGAGGTGAGGCAAATTGCGCCCGGCCA

[0591] AACTGGGAAAATTGCGGACTATAATTACAAACTTCCTGATAACTTCACCGGTTGTGT GATCGCTTGGAATTCAAACAACCTGGACTCCAAGGTGGGCGGCAATTACAACTACC TTTATAGGCTCTTTCGGAAAAGCAACCTGAGTCCCTTCGAGCGAGATATTAGTACGG AGATCTACCAGGCCGGGTCTACGCCATGCAACGGTACGGAAGGTTTCAACTGTTATT TTCCGCTGCAGAGTTACGGATTCCAACCAACAAATGGGGTGGGCTACCAACCTTAT AGGGTGGTCGTCCTGTCCTTTGAGCTTCTCCACGCCCCTGCGACCGTATGTGGACCC AAGAAGTCTACAAACCTCGTAAAGAATAAGCGGGTACAGCCGACCGAATCAATAGT CCGATTCCCCAATATCACGAATCTGTGTCCTTTTGGGGAAGTGTTTAACGCTACGCG GTTCGCATCCGTGTACGCCTGGAATCGGACCCGAATTTCCAATTGTGTGGCAGACTA TAGCGTGCTTTATAACTCTGCTTCCTTTTCTACCTTTAAATGCTATGGGGTAAACCCA ACCAAGCTCAATGATCTGTGTTTCACCAATGTGTATGCGGATAGCTTCGTGATCCGA GGAGATGAAGTTCGACAGATCGCCCCGGGACAGACCGGCAAGATAGCTGATTACAA CTATAAGTTGCCCGACAATTTTACAGGCTGCGTCATTGCATGGAACTCTAATAATTT GGATTCAAAAGTCGGTGGAAACTATAATTATCTGTACAGACTTTTCAGGAAGAGTA ATCTGTCCCCTTTTGAAAGAGACATCAGCACCGAAATATATCAAGCGGGAAGCACC CCTTGTAATGGCACAGAGGGCTTTAATTGCTACTTCCCTTTGCAATCTTATGGTTTTC AGCCCACCAACGGTGTCGGATATCAGCCCTACCGGGTAGTTGTATTGTCTTTCGAAC

[0592] TCCTGCATGCTCCAGCCACTGTTTGCGGGCCTAAGAAGAGCACTAATTTGGTGAAGA ACAAAGGCGGGAGCGGAGGGAGTGGCGGATCTGGCGGGAGCGGCGGAGGCAAAA AACAGGGGGATGCTGATGTCTGCGGAGAGGTGGCTTATATCCAGAGCGTGGTGTCC GACTGCCACGTGCCAACCGCAGAGCTGCGGACACTGCTGGAGATCAGAAAACTGTT CCTGGAGATTCAGAAACTGAAAGTCGAGCTGCAGGGGCTGTCAAAAGAACTGAGG TTCGGAATCGTCGCAAGTAGGGCTAATCACGCTCTGGTGTAGTGA

[0593] SEQ ID NO: 53 (AA RBD g5.1 14mer trans)

[0594] MDWTWILFLVAAATRVHSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRT RISNCVADYSVLYNSASFSTFKCYGVNPTKLNDLCFTNVYADSFVIRGDEVRQIAPG QTGKIADYNYKLPDNFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLSPFERDIST EIYOAGSTPCNGTEGFNCYFPLOSYGFOPTNGVGYQPYRVVVLSFELLHAPATVCGPKK STNLVKNKRVOPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRTRISNCVADYSVLY NSASFSTFKCYGVNPTKLNDLCFTNVYADSFVIRGDEVROIAPGOTGKIADYNYKLPDN FTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLSPFERDISTEIYQAGSTPCNGTEGFNC YFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKGGSGGSGGS GGSGGGKKQGDADVCGEVAYIQSVVSDCHVPTAELRTLLEIRKLFLEIQKLKVELQGL SKELRFGI V ASRANH AL V* *

[0595] SEQ ID NO:54 (Nucleotide RBD g5.1 14mer cys)

[0596] ATGGATTGGACTTGGATCTTGTTCTTGGTCGCAGCGGCTACTAGAGTGCACAGTTTG CGATTTGGTATTGTCGCTTCCCGCGCAAACCATGCGCTCGTGGGTGGTTCCGGTGGC AACATTACCAACCTTTGCCCCTTCGGCGAGGTCTTCAATGCCACAAGGTTTGCCAGT GTATATGCATGGAACAGGACTCGCATCTCAAACTGCGTTGCTGATTACTCTGTTCTG TACAATTCAGCGAGCTTCTCCACATTCAAGTGTTACGGAGTCAATCCTACAAAACTG

[0597] AACGACCTCTGCTTTACGAACGTCTACGCTGACTCCTTTGTCATACGGGGCGACGAG

[0598] GTGAGGCAAATTGCGCCCGGCCAAACTGGGAAAATTGCGGACTATAATTACAAACT

[0599] TCCTGATAACTTCACCGGTTGTGTGATCGCTTGGAATTCAAACAACCTGGACTCCAA

[0600] GGTGGGCGGCAATTACAACTACCTTTATAGGCTCTTTCGGAAAAGCAACCTGAGTCC

[0601] CTTCGAGCGAGATATTAGTACGGAGATCTACCAGGCCGGGTCTACGCCATGCAACG

[0602] GTACGGAAGGTTTCAACTGTTATTTTCCGCTGCAGAGTTACGGATTCCAACCAACAA

[0603] ATGGGGTGGGCTACCAACCTTATAGGGTGGTCGTCCTGTCCTTTGAGCTTCTCCACG

[0604] CCCCTGCGACCGTATGTGGACCCGGCGGTTCCGGTGGATCAGGTGGGAGCGGTGGA

[0605] TCAGGGGGGGGTAAGAAACAAGGCGACGCCGATGTGTGTGGGGAAGTTGCCTACAT

[0606] TCAATCTGTCGTCAGCGATTGTCATGTCCCCACAGCCGAATTGCGAACCCTTCTCGA

[0607] AATTAGGAAGCTCTTTCTTGAAATCCAAAAGCTCAAGGTGGAATTGCAAGGACTCA

[0608] GTAAAGAGCTTAGATTTGGCATAGTAGCGTCCAGAGCCAACCATGCCCTTGTCGGT

[0609] GGCAGTGGCGGTTCCGGTGGTTCCGGTGGCAGTGGCGGTGGTAATATCACGAATCT

[0610] GTGTCCTTTTGGGGAAGTGTTTAACGCTACGCGGTTCGCATCCGTGTACGCCTGGAA

[0611] TCGGACCCGAATTTCCAATTGTGTGGCAGACTATAGCGTGCTTTATAACTCTGCTTC

[0612] CTTTTCTACCTTTAAATGCTATGGGGTAAACCCAACCAAGCTCAATGATCTGTGTTT

[0613] CACCAATGTGTATGCGGATAGCTTCGTGATCCGAGGAGATGAAGTTCGACAGATCG

[0614] CCCCGGGACAGACCGGCAAGATAGCTGATTACAACTATAAGTTGCCCGACAATTTT

[0615] ACAGGCTGCGTCATTGCATGGAACTCTAATAATTTGGATTCAAAAGTCGGTGGAAA

[0616] CTATAATTATCTGTACAGACTTTTCAGGAAGAGTAATCTGTCCCCTTTTGAAAGAGA

[0617] CATCAGCACCGAAATATATCAAGCGGGAAGCACCCCTTGTAATGGCACAGAGGGCT

[0618] TTAATTGCTACTTCCCTTTGCAATCTTATGGTTTTCAGCCCACCAACGGTGTCGGATA

[0619] T CAGCCCTACCGGGTAGTTGTATTGTCTTTCGAACTCCTGCATGCTCCAGCCACTGT

[0620] TTGCGGGCCTTAATAG

[0621] SEQ ID NO:55 (AA RBD g5.1 14mer cys)

[0622] MDWTWILFLVAAATRVHSZRFGZG4SR4Aa4LFGGS,GGNITNLCPFGEVFNATRFASVY

[0623] AWNRTRISNCVADYSVLYNSASFSTFKCYGVNPTKLNDLCFTNVYADSFVIRGDEVRQI

[0624] APGQTGKIADYNYKLPDNFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLSPFERDIS

[0625] TEIYOAGSTPCNGTEGFNCYFPLOSYGFOPTNGVGYQPYRVVVLSFELLHAPATVCGPG

[0626] GSGGSGGSGGSGGGKKQGDADVCGEVAYIQSVVSDCHVPTAELRTLLEIRKLFLEIQKL

[0627] KVELOGLSKELRFGIVASRANHALVGGSGGSGGSGGSGGGNITNLCPFGEVFNATRFAS

[0628] VYAWNRTRISNCVADYSVLYNSASFSTFKCYGVNPTKLNDLCFTNVYADSFVIRGDEV

[0629] RQIAPGOTGKIADYNYKLPDNFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLSPFER

[0630] DISTEIYOAGSTPCNGTEGFNCYFPLOSYGFOPTNGVGYOPYRVVVLSFELLHAPATVC

[0631] GP**

[0632] SEQ ID NO: 56 (Nucleotide RBD_g8.1_180mer)

[0633] ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCCTC

[0634] TCAATTGCCCCAACGTTGATCAACCGGGACAAGCCATACACGAAAGAGGAACTTAT

[0635] GGAGATATTGCGGTTGGCCATTATAGCTGAACTCGATGCAATTAATCTCTATGAACA

[0636] AATGGCCCGCTATAGCGAAGACGAAAATGTGAGAAAGATCTTGTTGGACGTCGCTA

[0637] GGGAAGAGAAAGCACACGTAGGAGAGTTCATGGCTTTGTTGCTTAACCTCGACCCT

[0638] GAGCAAGTCACAGAGCTGAAGGGCGGGTTCGAGGAAGTTAAAGAATTGACCGGTA

[0639] TAGAAGCTCACATTAATGACAACAAGAAAGAGGAAAGTAATGTAGAGTATTTCGAG

[0640] AAGCTCAGATCTGCCTTGTTGGATGGAGTCAACAAGGGTCGCAGCTTGCTCAAACA

[0641] TCTGCCCGTTACAAGAATAGAAGGGCAGTCTTTTCGAGTAGACATCATCAAATTTGA

[0642] GGACGGCGTCCGAGTGGTTAAACAAGAGTATAAGCCTATACCCCTTCTTAAGAAGA

[0643] AGTTCTACGTCGGCATTCGAGAACTGAATGACGGGACATATGATGTCAGCATTGCT

[0644] ACTAAAGCCGGTGAGCTGCTGGTTAAAGACGAAGAAAGTCTTGTGATCCGGGAAAT

[0645] TTTGTCAACGGAAGGCATCAAGAAAATGAAATTGTCATCCTGGGACAATCCAGAAG

[0646] AAGCCCTGAATGATTTGATGAATGCGCTCCAAGAAGCTAGCAATGCTAGTGCTGGC

[0647] CCCTTCGGCCTTATTATCAATCCAAAGCGGTACGCCAAACTGCTGAAGATCTATGAA

[0648] AAGTCAGGTAAGATGCTCGTAGAAGTACTCAAGGAAATCTTCCGGGGTGGAATAAT CGTAACTCTTAATATCGACGAAAACAAAGTGATTATCTTCGCTAATACGCCCGCCGT

[0649] TCTGGACGTGGTGGTGGGTCAAGACGTTACGCTCCAGGAGCTTGGTCCGGAAGGGG

[0650] ATGATGTCGCATTCCTGGTCAGTGAAGCCATTGGTATAAGAATCAAGAACCCGGAA

[0651] GCTATAGTTGTTCTCGAAGGCGGGTCTGGTGGGAGCGGTGGTAGTGGTGGTTCTGGT

[0652] GGTGGTGGGTCAGGTGGCGGCTCAGGCGGCGGCTTGCGATTTGGTATTGTCGCTTCC

[0653] CGCGCAAACCATGCGCTCGTGGGTGGTTCCGGTGGCAATATTACCAATCTTTGTCCC

[0654] TTCGGCGAGGTGTTCAACGCCACGCGGTTCGCGTCTGTATACGCCTGGAACCGCACC

[0655] CGGATCAGCAACTGTACCGCTGACTACAGCGTTCTGTACAACAGCAGCAGCTTTTCA

[0656] ACTTTTAAGTGTTACGGAGTCAACCCCACAAAACTGAACGACCTGTGCTTCACCAAT

[0657] GTGTATGCAGACTCTTTTGTGATACGCGGAGATGAAGTACGCCAAATTGCCCCCGG

[0658] ACAAACCGGCAAGATCGCGGACTACAACTACAAGCTGCCCGATGACTTTACAGGTT

[0659] GCGTGATCGCGTGGAACAGTAATAATCTGGATTCCAAGGTTGGCGGGAATTATAAT

[0660] TACCTTTATCGCCTGTTTCGGAAGTCTAACCTGAGCCCGTTCGAGAGAGACAACTCA

[0661] ACGGAAATTTATCAAGCCGGCAGTACCCCTTGCAATGGGACCGAAGGATTCAATTG

[0662] TTATTTCCCACTCCAATCTTATGGCTTCCAACCGACAAACGGTGTTGGTTATCAGCC CTATCGGGTGGTTGTGCTTTCTTTCGAAAACCTTAGCGCTCCAGCTACCGTGTGCGG GCCCTAATGA

[0663] SEQ ID NO: 57 (AA RBD_g8.1_180mer)

[0664] MDWTWILFLVAAATRVHSLSIAPTLINRDKPYTKEELMEILRLAIIAELDAINLYEQMA

[0665] RYSEDENVRKILLDVAREEKAHVGEFMALLLNLDPEQVTELKGGFEEVKELTGIEAHIN

[0666] DNKKEESNVEYFEKLRSALLDGVNKGRSLLKHLPVTRIEGQSFRVDIIKFEDGVRVVKQ

[0667] EYKPIPLLKKKFYVGIRELNDGTYDVSIATKAGELLVKDEESLVIREILSTEGIKKMKLSS

[0668] WDNPEEALNDLMNALQEASNASAGPFGLIINPKRYAKLLKIYEKSGKMLVEVLKEIFRG

[0669] GIIVTLNIDENKVIIFANTPAVLDVVVGQDVTLQELGPEGDDVAFLVSEAIGIRIKNPEAIV

[0670] VLEGGSGGSGGSGGSGGGGSGGGSGGGZR GJE45R4Aa4£KGGS,GGNITNLCPFGEVFN

[0671] ATRFASVYAWNRTRISNCTADYSVLYNSSSFSTFKCYGVNPTKLNDLCFTNVYADSFVI

[0672] RGDEVRQIAPGOTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSN LSPFERDNSTEIYOAGSTPCNGTEGFNCYFPLOSYGFOPTNGVGYOPYRVVVLSFENLS APATVCGP**

[0673] SEQ ID NO: 58 (Nucleotide RBD g8.1 60mer)

[0674] ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCAT

[0675] GCAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAA

[0676] GCCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATT

[0677] GTCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCT

[0678] GGGAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTG

[0679] ATCGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCA

[0680] GAAGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTT

[0681] TGGCGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCC

[0682] ATGGAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTC

[0683] AAATCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAG

[0684] GCAATATTACCAATCTTTGTCCCTTCGGCGAGGTGTTCAACGCCACGCGGTTCGCGT

[0685] CTGTATACGCCTGGAACCGCACCCGGATCAGCAACTGTACCGCTGACTACAGCGTTC

[0686] TGTACAACAGCAGCAGCTTTTCAACTTTTAAGTGTTACGGAGTCAACCCCACAAAAC

[0687] TGAACGACCTGTGCTTCACCAATGTGTATGCAGACTCTTTTGTGATACGCGGAGATG

[0688] AAGTACGCCAAATTGCCCCCGGACAAACCGGCAAGATCGCGGACTACAACTACAAG

[0689] CTGCCCGATGACTTTACAGGTTGCGTGATCGCGTGGAACAGTAATAATCTGGATTCC

[0690] AAGGTTGGCGGGAATTATAATTACCTTTATCGCCTGTTTCGGAAGTCTAACCTGAGC

[0691] CCGTTCGAGAGAGACAACTCAACGGAAATTTATCAAGCCGGCAGTACCCCTTGCAA

[0692] TGGGACCGAAGGATTCAATTGTTATTTCCCACTCCAATCTTATGGCTTCCAACCGAC AAACGGTGTTGGTTATCAGCCCTATCGGGTGGTTGTGCTTTCTTTCGAAAACCTTAG CGCTCCAGCTACCGTGTGCGGGCCCTAATAG

[0693] SEQ ID NO: 59 (AA RBD g8.1 60mer) MDWTWILFLV AAA TRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIV RHGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKG LADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSG GSGGSGGSGGGNITNLCPFGEVFNATRFASVYAWNRTRISNCTADYSVLYNSSSFSTFK CYGVNPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAW NSNNLDSKVGGNYNYLYRLFRKSNLSPFERDNSTEIYQAGSTPCNGTEGF NCYFPLQSYGFQPTNGVGYQPYRVVVLSFENLSAPATVCGP**

[0694] SEQ ID NO: 60 (Nucleotide RBD g8.1 24mer)

[0695] ATGGATTGGACTTGGATCTTGTTCTTGGTCGCAGCGGCTACTAGAGTGCACAGTTTG CGATTTGGTATTGTCGCTTCCCGCGCAAACCATGCGCTCGTGGGTGGTTCCGGTGGC AATATTACCAATCTTTGTCCCTTCGGCGAGGTGTTCAACGCCACGCGGTTCGCGTCT GTATACGCCTGGAACCGCACCCGGATCAGCAACTGTACCGCTGACTACAGCGTTCT GTACAACAGCAGCAGCTTTTCAACTTTTAAGTGTTACGGAGTCAACCCCACAAAACT GAACGACCTGTGCTTCACCAATGTGTATGCAGACTCTTTTGTGATACGCGGAGATGA AGTACGCCAAATTGCCCCCGGACAAACCGGCAAGATCGCGGACTACAACTACAAGC TGCCCGATGACTTTACAGGTTGCGTGATCGCGTGGAACAGTAATAATCTGGATTCCA AGGTTGGCGGGAATTATAATTACCTTTATCGCCTGTTTCGGAAGTCTAACCTGAGCC CGTTCGAGAGAGACAACTCAACGGAAATTTATCAAGCCGGCAGTACCCCTTGCAAT GGGACCGAAGGATTCAATTGTTATTTCCCACTCCAATCTTATGGCTTCCAACCGACA AACGGTGTTGGTTATCAGCCCTATCGGGTGGTTGTGCTTTCTTTCGAAAACCTTAGC GCTCCAGCTACAGTATGTGGTCCTGGCGGCTCCGGTGGCTCTGGCGGGAGTGGCGG ATCAGGTGGTGGACTGAGTAAGGACATCATCAAACTTCTCAACGAACAGGTGAACA AGGAGATGCAATCTTCTAACCTGTACATGAGCATGTCTAGCTGGTGTTATACTCACA GTCTCGACGGCGCTGGCCTCTTTCTGTTCGATCACGCCGCTGAAGAATATGAGCACG CGAAGAAGCTTATAATCTTCCTGAACGAGAATAATGTTCCCGTCCAACTGACGTCCA TTTCCGCCCCTGAACACAAGTTTGAGGGTCTGACTCAGATCTTTCAAAAGGCGTACG AGCACGAGCAGCATATCAGCGAGAGCATTAACAACATTGTCGATCACGCCATTAAA AGCAAAGACCACGCTACCTTCAACTTTCTCCAATGGTACGTCGCCGAACAGCATGA GGAAGAAGTATTGTTCAAGGATATACTCGATAAGATTGAACTGATCGGAAATGAGA ATCACGGCCTGTACCTGGCCGACCAATACGTCAAAGGCATTGCCAAGTCTAGGAAA AGCTAGTAG

[0696] SEQ ID NO: 61 (AA RBD g8.1 24mer)

[0697] MDWTWILFLVAAATRVHSZFFG / KdST AK RGGSGGNITNLCPFGEVFNATRFAS VYAWNRTRISNCTADYSVLYNSSSFSTFKCYGVNPTKLNDLCFTNVYADSFVIRGDEVR OIAPGOTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLSPFER DNSTEIYQAGSTPCNGTEGFNCYFPLOSYGFOPTNGVGYQPYRVVVLSFENLSAPATVC GPGGSGGSGGSGGSGGGLSKDIIKLLNEOVNKEMOSSNLYMSMSSWCYTHSLDGAGLF LFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNI VDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGI AKSRKS**

[0698] SEQ ID NO: 62 (Nucleotide RBD g8.1 7mer)

[0699] ATGGATTGGACTTGGATCTTGTTCTTGGTCGCAGCGGCTACTAGAGTGCACAGTTTG CGATTTGGTATTGTCGCTTCCCGCGCAAACCATGCGCTCGTGGGTGGTTCCGGTGGC AATATTACCAATCTTTGTCCCTTCGGCGAGGTGTTCAACGCCACGCGGTTCGCGTCT GTATACGCCTGGAACCGCACCCGGATCAGCAACTGTACCGCTGACTACAGCGTTCT GTACAACAGCAGCAGCTTTTCAACTTTTAAGTGTTACGGAGTCAACCCCACAAAACT GAACGACCTGTGCTTCACCAATGTGTATGCAGACTCTTTTGTGATACGCGGAGATGA AGTACGCCAAATTGCCCCCGGACAAACCGGCAAGATCGCGGACTACAACTACAAGC TGCCCGATGACTTTACAGGTTGCGTGATCGCGTGGAACAGTAATAATCTGGATTCCA AGGTTGGCGGGAATTATAATTACCTTTATCGCCTGTTTCGGAAGTCTAACCTGAGCC CGTTCGAGAGAGACAACTCAACGGAAATTTATCAAGCCGGCAGTACCCCTTGCAAT GGGACCGAAGGATTCAATTGTTATTTCCCACTCCAATCTTATGGCTTCCAACCGACA AACGGTGTTGGTTATCAGCCCTATCGGGTGGTTGTGCTTTCTTTCGAAAACCTTAGC GCTCCAGCTACCGTGTGCGGGCCCGGCGGGAGCGGAGGGAGTGGCGGATCTGGCGG GAGCGGCGGAGGCAAAAAACAGGGGGATGCTGATGTCTGCGGAGAGGTGGCTTAT

[0700] ATCCAGAGCGTGGTGTCCGACTGCCACGTGCCAACCGCAGAGCTGCGGACACTGCT GGAGATCAGAAAACTGTTCCTGGAGATTCAGAAACTGAAAGTCGAGCTGCAGGGGC TGTCAAAAGAACTGAGGTTCGGAATCGTCGCAAGTAGGGCTAATCACGCTCTGGT

[0701] GTAATAG

[0702] SEQ ID NO: 63 (AA RBD g8.1 7mer)

[0703] MDWTWILFLVAAATRVHSZR G / G4SR^A?Y4£FGGSGGNITNLCPFGEVFNATRFAS

[0704] VYAWNRTRISNCTADYSVLYNSSSFSTFKCYGVNPTKLNDLCFTNVYADSFVIRGDE

[0705] VRQIAPGOTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLS PFERDNSTEIYQAGSTPCNGTEGFNCYFPLOSYGFOPTNGVGYQPYRVVVLSFENLSAPA TVCGPGGSGGSGGSGGSGGGKKOGDADVCGEVAYIQSVVSDCHVPTAELRTLLEIRKL

[0706] FLEIQKLKVELQGLSKELRFGIVASRANHALV**

[0707] SEQ ID NO: 64 (Nucleotide RBD_g8.2_180mer)

[0708] ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCCTC

[0709] TCAATTGCCCCAACGTTGATCAACCGGGACAAGCCATACACGAAAGAGGAACTTAT

[0710] GGAGATATTGCGGTTGGCCATTATAGCTGAACTCGATGCAATTAATCTCTATGAACA AATGGCCCGCTATAGCGAAGACGAAAATGTGAGAAAGATCTTGTTGGACGTCGCTA GGGAAGAGAAAGCACACGTAGGAGAGTTCATGGCTTTGTTGCTTAACCTCGACCCT

[0711] GAGCAAGTCACAGAGCTGAAGGGCGGGTTCGAGGAAGTTAAAGAATTGACCGGTA TAGAAGCTCACATTAATGACAACAAGAAAGAGGAAAGTAATGTAGAGTATTTCGAG AAGCTCAGATCTGCCTTGTTGGATGGAGTCAACAAGGGTCGCAGCTTGCTCAAACA

[0712] TCTGCCCGTTACAAGAATAGAAGGGCAGTCTTTTCGAGTAGACATCATCAAATTTGA

[0713] GGACGGCGTCCGAGTGGTTAAACAAGAGTATAAGCCTATACCCCTTCTTAAGAAGA AGTTCTACGTCGGCATTCGAGAACTGAATGACGGGACATATGATGTCAGCATTGCT ACTAAAGCCGGTGAGCTGCTGGTTAAAGACGAAGAAAGTCTTGTGATCCGGGAAAT

[0714] TTTGTCAACGGAAGGCATCAAGAAAATGAAATTGTCATCCTGGGACAATCCAGAAG AAGCCCTGAATGATTTGATGAATGCGCTCCAAGAAGCTAGCAATGCTAGTGCTGGC CCCTTCGGCCTTATTATCAATCCAAAGCGGTACGCCAAACTGCTGAAGATCTATGAA

[0715] AAGTCAGGTAAGATGCTCGTAGAAGTACTCAAGGAAATCTTCCGGGGTGGAATAAT CGTAACTCTTAATATCGACGAAAACAAAGTGATTATCTTCGCTAATACGCCCGCCGT TCTGGACGTGGTGGTGGGTCAAGACGTTACGCTCCAGGAGCTTGGTCCGGAAGGGG

[0716] ATGATGTCGCATTCCTGGTCAGTGAAGCCATTGGTATAAGAATCAAGAACCCGGAA

[0717] GCTATAGTTGTTCTCGAAGGCGGGTCTGGTGGGAGCGGTGGTAGTGGTGGTTCTGGT

[0718] GGTGGTGGGTCAGGTGGCGGCTCAGGCGGCGGCTTGCGATTTGGTATTGTCGCTTCC CGCGCAAACCATGCGCTCGTGGGTGGTTCCGGTGGCAATATTACCAATCTTTGTCCC TTCGGCGAGGTGTTCAACGCCACGCGGTTCGCGTCTGTATACGCCTGGAACCGCACC

[0719] CGGATCAGCAACTGTACCGCTGACTACAGCGTTCTGTACAACAGCAGCAGCTTTTCA ACTTTTAAGTGTTACGGAGTCAACCCCACAAAACTGAACGACCTGTGCTTCACCAAT GTGTATGCAGACTCTTTTGTGATACGCGGAGATGAAGTACGCCAAATTGCCCCCGG

[0720] ACAAACCGGCAAGATCGCGGACTACAACTACAAGCTGCCCGATAACTTTACAGGTT

[0721] GCGTGATCGCGTGGAACAGTAATAATCTGGATTCCAAGGTTGGCGGGAATTATAAT TACCTTTATCGCCTGTTTCGGAAGTCTAACCTGAGCCCGTTCGAGAGAGACATCTCA ACGGAAATTTATCAAGCCGGCAGTACCCCTTGCAATGGGACCGAAGGATTCAATTG

[0722] TTATTTCCCACTCCAATCTTATGGCTTCCAACCGACAAACGGTGTTGGTTATCAGCC CTATCGGGTGGTTGTGCTTTCTTTCGAAAACCTTAGCGCTCCAGCTACCGTGTGCGG GCCCTAATGA

[0723] SEQ ID NO: 65 (AA RBD g8.2 _180mer)

[0724] MDWTWILFLVAAATRVHSLSIAPTLINRDKPYTKEELMEILRLAIIAELDAINLYEQMA

[0725] RYSEDENVRKILLDVAREEKAHVGEFMALLLNLDPEQVTELKGGFEEVKELTGIEAHIN

[0726] DNKKEESNVEYFEKLRSALLDGVNKGRSLLKHLPVTRIEGQSFRVDIIKFEDGVRVVKQ EYKPIPLLKKKFYVGIRELNDGTYDVSIATKAGELLVKDEESLVIREILSTEGIKKMKLSS WDNPEEALNDLMNALQEASNASAGPFGLIINPKRYAKLLKIYEKSGKMLVEVLKEIFRG GIIVTLNIDENKVIIFANTPAVLDVVVGQDVTLQELGPEGDDVAFLVSEAIGIRIKNPEAIV VLEGGSGGSGGSGGSGGGGSGGGSGGGZR GJE4S7 AH IKGG GG ITNLCPFGEVFN ATRFASVYAWNRTRISNCTADYSVLYNSSSFSTFKCYGVNPTKLNDLCFTNVYADSFVI RGDEVRQIAPGQTGKIADYNYKLPDNFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSN LSPFERDISTEIYQAGSTPCNGTEGFNCYFPLOSYGFQPTNGVGYQPYRVVVLSFENLS APATVCGP**

[0727] SEQ ID NO: 66 (Nucleotide RED g8.2 60mer)

[0728] ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCAT GCAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAA GCCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATT GTCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCT GGGAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTG ATCGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCA GAAGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTT TGGCGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCC ATGGAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTC AAATCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAG GCAATATTACCAATCTTTGTCCCTTCGGCGAGGTGTTCAACGCCACGCGGTTCGCGT CTGTATACGCCTGGAACCGCACCCGGATCAGCAACTGTACCGCTGACTACAGCGTTC TGTACAACAGCAGCAGCTTTTCAACTTTTAAGTGTTACGGAGTCAACCCCACAAAAC TGAACGACCTGTGCTTCACCAATGTGTATGCAGACTCTTTTGTGATACGCGGAGATG AAGTACGCCAAATTGCCCCCGGACAAACCGGCAAGATCGCGGACTACAACTACAAG CTGCCCGATAACTTTACAGGTTGCGTGATCGCGTGGAACAGTAATAATCTGGATTCC AAGGTTGGCGGGAATTATAATTACCTTTATCGCCTGTTTCGGAAGTCTAACCTGAGC

[0729] CCGTTCGAGAGAGACATCTCAACGGAAATTTATCAAGCCGGCAGTACCCCTTGCAA TGGGACCGAAGGATTCAATTGTTATTTCCCACTCCAATCTTATGGCTTCCAACCGAC AAACGGTGTTGGTTATCAGCCCTATCGGGTGGTTGTGCTTTCTTTCGAAAACCTTAG CGCTCCAGCTACCGTGTGCGGGCCCTAATAG

[0730] SEQ ID NO: 67 (AA RBD g8.2 60mer)

[0731] MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIV RHGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKG LADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSG GSGGSGGSGGGNITNLCPFGEVFNATRFASVYAWNRTRISNCTADYSVLYNSSSFSTFK CYGVNPTKLNDLCFTNVYADSFVIRGDEVRQIAPGOTGKIADYNYKLPDNFTGCVIAW NSNNLDSKVGGNYNYLYRLFRKSNLSPFERDISTEIYQAGSTPCNGTEGF NCYFPLQSYGFQPTNGVGYQPYRVVVLSFENLSAPATVCGP**

[0732] SEQ ID NO: 68 (Nucleotide RBD g8.2 24mer)

[0733] ATGGATTGGACTTGGATCTTGTTCTTGGTCGCAGCGGCTACTAGAGTGCACAGTTTG CGATTTGGTATTGTCGCTTCCCGCGCAAACCATGCGCTCGTGGGTGGTTCCGGTGGC AATATTACCAATCTTTGTCCCTTCGGCGAGGTGTTCAACGCCACGCGGTTCGCGTCT GTATACGCCTGGAACCGCACCCGGATCAGCAACTGTACCGCTGACTACAGCGTTCT GTACAACAGCAGCAGCTTTTCAACTTTTAAGTGTTACGGAGTCAACCCCACAAAACT GAACGACCTGTGCTTCACCAATGTGTATGCAGACTCTTTTGTGATACGCGGAGATGA AGTACGCCAAATTGCCCCCGGACAAACCGGCAAGATCGCGGACTACAACTACAAGC TGCCCGATAACTTTACAGGTTGCGTGATCGCGTGGAACAGTAATAATCTGGATTCCA AGGTTGGCGGGAATTATAATTACCTTTATCGCCTGTTTCGGAAGTCTAACCTGAGCC CGTTCGAGAGAGACATCTCAACGGAAATTTATCAAGCCGGCAGTACCCCTTGCAAT GGGACCGAAGGATTCAATTGTTATTTCCCACTCCAATCTTATGGCTTCCAACCGACA AACGGTGTTGGTTATCAGCCCTATCGGGTGGTTGTGCTTTCTTTCGAAAACCTTAGC GCTCCAGCTACCGTCTGTGGACCTGGTGGCAGTGGCGGCTCTGGTGGTTCAGGCGGC AGTGGTGGTGGGCTGTCCAAGGACATAATCAAACTGCTCAACGAACAGGTGAACAA

[0734] GGAGATGCAATCTTCTAACCTGTACATGAGCATGTCTAGCTGGTGTTATACTCACAG

[0735] TCTCGACGGCGCTGGCCTCTTTCTGTTCGATCACGCCGCTGAAGAATATGAGCACGC

[0736] GAAGAAGCTTATAATCTTCCTGAACGAGAATAATGTTCCCGTCCAACTGACGTCCAT

[0737] TTCCGCCCCTGAACACAAGTTTGAGGGTCTGACTCAGATCTTTCAAAAGGCGTACGA

[0738] GCACGAGCAGCATATCAGCGAGAGCATTAACAACATTGTCGATCACGCCATTAAAA

[0739] GCAAAGACCACGCTACCTTCAACTTTCTCCAATGGTACGTCGCCGAACAGCATGAG

[0740] GAAGAAGTATTGTTCAAGGATATACTCGATAAGATTGAACTGATCGGAAATGAGAA

[0741] TCACGGCCTGTACCTGGCCGACCAATACGTCAAAGGCATTGCCAAGTCTAGGAAAA

[0742] GCTAGTAG

[0743] SEQ ID NO: 69 (AA RBD g8.2 24mer)

[0744] MDWTWILFLVAAATRVHSZRFG / 4 7t4A?£4ZKGG GGNITNLCPFGEVFNATRFAS

[0745] VYAWNRTRISNCTADYSVLYNSSSFSTFKCYGVNPTKLNDLCFTNVYADSFVIRGDE

[0746] VRQIAPGQTGKIADYNYKLPDNFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLS

[0747] PFERDISTEIYQAGSTPCNGTEGFNCYFPLOSYGFQPTNGVGYQPYRVVVLSFENLSA

[0748] PATVCGPGGSGGSGGSGGSGGGLSKDIIKLLNEOVNKEMOSSNLYMSMSSWCYTHS

[0749] LDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEH

[0750] EQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYL

[0751] ADQYVKGIAKSRKS**

[0752] SEQ ID NO: 70 (Nucleotide RBD g8.2 7mer)

[0753] ATGGATTGGACTTGGATCTTGTTCTTGGTCGCAGCGGCTACTAGAGTGCACAGTTTG

[0754] CGATTTGGTATTGTCGCTTCCCGCGCAAACCATGCGCTCGTGGGTGGTTCCGGTGGC

[0755] AATATTACCAATCTTTGTCCCTTCGGCGAGGTGTTCAACGCCACGCGGTTCGCGTCT

[0756] GTATACGCCTGGAACCGCACCCGGATCAGCAACTGTACCGCTGACTACAGCGTTCT

[0757] GTACAACAGCAGCAGCTTTTCAACTTTTAAGTGTTACGGAGTCAACCCCACAAAACT

[0758] GAACGACCTGTGCTTCACCAATGTGTATGCAGACTCTTTTGTGATACGCGGAGATGA

[0759] AGTACGCCAAATTGCCCCCGGACAAACCGGCAAGATCGCGGACTACAACTACAAGC

[0760] TGCCCGATAACTTTACAGGTTGCGTGATCGCGTGGAACAGTAATAATCTGGATTCCA

[0761] AGGTTGGCGGGAATTATAATTACCTTTATCGCCTGTTTCGGAAGTCTAACCTGAGCC

[0762] CGTTCGAGAGAGACATCTCAACGGAAATTTATCAAGCCGGCAGTACCCCTTGCAAT

[0763] GGGACCGAAGGATTCAATTGTTATTTCCCACTCCAATCTTATGGCTTCCAACCGACA

[0764] AACGGTGTTGGTTATCAGCCCTATCGGGTGGTTGTGCTTTCTTTCGAAAACCTTAGC

[0765] GCTCCAGCTACCGTGTGCGGGCCCGGCGGGAGCGGAGGGAGTGGCGGATCTGGCGG

[0766] GAGCGGCGGAGGCAAAAAACAGGGGGATGCTGATGTCTGCGGAGAGGTGGCTTAT

[0767] ATCCAGAGCGTGGTGTCCGACTGCCACGTGCCAACCGCAGAGCTGCGGACACTGCT

[0768] GGAGATCAGAAAACTGTTCCTGGAGATTCAGAAACTGAAAGTCGAGCTGCAGGGGC

[0769] TGTCAAAAGAACTGAGGTTCGGAATCGTCGCAAGTAGGGCTAATCACGCTCTGGT GTAATAG

[0770] SEQ ID NO: 71 (AA RBD g8.2 7mer)

[0771] MDWTWILFLVAAATRVHSZRFG / G4S7t4A7 4£FGGSGGNITNLCPFGEVFNATRFASVY

[0772] AWNRTRISNCTADYSVLYNSSSFSTFKCYGVNPTKLNDLCFTNVYADSFVIRGDEVROI

[0773] APGQTGKIADYNYKLPDNFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLSPFERDIS

[0774] TEIYQAGSTPCNGTEGFNCYFPLQSYGFOPTNGVGYQPYRVVVLSFENLSAPATVCGPG

[0775] GSGGSGGSGGSGGGKKOGDADVCGEVAYIOSVVSDCHVPTAELRTLLEIRKLFLEIOK

[0776] LKVELQGLSKELRFGIVASRANHALV**

[0777] SEQ ID NO: 72 (Nucleotide RBD_g8.3_180mer)

[0778] ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCCTC

[0779] TCAATTGCCCCAACGTTGATCAACCGGGACAAGCCATACACGAAAGAGGAACTTAT

[0780] GGAGATATTGCGGTTGGCCATTATAGCTGAACTCGATGCAATTAATCTCTATGAACA

[0781] AATGGCCCGCTATAGCGAAGACGAAAATGTGAGAAAGATCTTGTTGGACGTCGCTA

[0782] GGGAAGAGAAAGCACACGTAGGAGAGTTCATGGCTTTGTTGCTTAACCTCGACCCT GAGCAAGTCACAGAGCTGAAGGGCGGGTTCGAGGAAGTTAAAGAATTGACCGGTA

[0783] TAGAAGCTCACATTAATGACAACAAGAAAGAGGAAAGTAATGTAGAGTATTTCGAG

[0784] AAGCTCAGATCTGCCTTGTTGGATGGAGTCAACAAGGGTCGCAGCTTGCTCAAACA

[0785] TCTGCCCGTTACAAGAATAGAAGGGCAGTCTTTTCGAGTAGACATCATCAAATTTGA

[0786] GGACGGCGTCCGAGTGGTTAAACAAGAGTATAAGCCTATACCCCTTCTTAAGAAGA

[0787] AGTTCTACGTCGGCATTCGAGAACTGAATGACGGGACATATGATGTCAGCATTGCT

[0788] ACTAAAGCCGGTGAGCTGCTGGTTAAAGACGAAGAAAGTCTTGTGATCCGGGAAAT

[0789] TTTGTCAACGGAAGGCATCAAGAAAATGAAATTGTCATCCTGGGACAATCCAGAAG AAGCCCTGAATGATTTGATGAATGCGCTCCAAGAAGCTAGCAATGCTAGTGCTGGC CCCTTCGGCCTTATTATCAATCCAAAGCGGTACGCCAAACTGCTGAAGATCTATGAA

[0790] AAGTCAGGTAAGATGCTCGTAGAAGTACTCAAGGAAATCTTCCGGGGTGGAATAAT

[0791] CGTAACTCTTAATATCGACGAAAACAAAGTGATTATCTTCGCTAATACGCCCGCCGT

[0792] TCTGGACGTGGTGGTGGGTCAAGACGTTACGCTCCAGGAGCTTGGTCCGGAAGGGG

[0793] ATGATGTCGCATTCCTGGTCAGTGAAGCCATTGGTATAAGAATCAAGAACCCGGAA

[0794] GCTATAGTTGTTCTCGAAGGCGGGTCTGGTGGGAGCGGTGGTAGTGGTGGTTCTGGT

[0795] GGTGGTGGGTCAGGTGGCGGCTCAGGCGGCGGCTTGCGATTTGGTATTGTCGCTTCC

[0796] CGCGCAAACCATGCGCTCGTGGGTGGTTCCGGTGGCAACATTACCAACCTTTGCCCC

[0797] TTCGGCGAGGTCTTCAATGCCACAAGGTTTGCCAGTGTATATGCATGGAACAGGACT

[0798] CGCATCTCAAACTGCACCGCTGATTACTCTGTTCTGTACAATTCAAGCAGCTTCTCC

[0799] ACATTCAAGTGTTACGGAGTCAATCCTACAAAACTGAACGACCTCTGCTTTACGAAC

[0800] GTCTACGCTGACTCCTTTGTCATACGGGGCGACGAGGTGAGGCAAATTGCGCCCGG

[0801] CCAAACTGGGAAAATTGCGGACTATAATTACAAACTTCCTGATAACTTCACCGGTTG

[0802] TGTGATCGCTTGGAATTCAAACAACCTGGACTCCAAGGTGGGCGGCAATTACAACT

[0803] ACCTTTATAGGCTCTTTCGGAAAAGCAACCTGAGTCCCTTCGAGCGAGATATTAGTA

[0804] CGGAGATCTACCAGGCCGGGTCTACGCCATGCAACGGTACGGAAGGTTTCAACTGT TATTTTCCGCTGCAGAGTTACGGATTCCAACCAACAAATGGGGTGGGCTACCAACCT TATAGGGTGGTCGTCCTGTCCTTTGAGCTTAACCACAGCCCTGCGACCGTATGTGG

[0805] ACCCTAATGA

[0806] SEQ ID NO:73 (AA RBD_g8.3_180mer)

[0807] MDWTWILFLVAAATRVHSLSIAPTLINRDKPYTKEELMEILRLAIIAELDAINLYEQMA

[0808] RYSEDENVRKILLDVAREEKAHVGEFMALLLNLDPEQVTELKGGFEEVKELTGIEAHIN

[0809] DNKKEESNVEYFEKLRSALLDGVNKGRSLLKHLPVTRIEGQSFRVDIIKFEDGVRVVKQ

[0810] EYKPIPLLKKKFYVGIRELNDGTYDVSIATKAGELLVKDEESLVIREILSTEGIKKMKLSS

[0811] WDNPEEALNDLMNALQEASNASAGPFGLIINPKRYAKLLKIYEKSGKMLVEVLKEIFRG

[0812] GIIVTLNIDENKVIIFANTPAVLDVVVGQDVTLQELGPEGDDVAFLVSEAIGIRIKNPEAIV

[0813] VLEGGSGGSGGSGGSGGGGSGGGSGGGZ7^GJE45R4Aa4£KGG GGNITNLCPFGEVFN

[0814] ATRFASVYAWNRTRISNCTADYSVLYNSSSFSTFKCYGVNPTKLNDLCFTNVYADSFVI RGDEVRQIAPGOTGKIADYNYKLPDNFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSN LSPFERDISTEIYOAGSTPCNGTEGFNCYFPLOSYGFQPTNGVGYQPYRVVVLSFELNH

[0815] SPATVCGP**

[0816] SEQ ID NO: 74 (Nucleotide RBD g8.3 60mer)

[0817] ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCAT

[0818] GCAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAA

[0819] GCCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATT

[0820] GTCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCT

[0821] GGGAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTG

[0822] ATCGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCA

[0823] GAAGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTT

[0824] TGGCGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCC

[0825] ATGGAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTC

[0826] AAATCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAG GCAACATTACCAACCTTTGCCCCTTCGGCGAGGTCTTCAATGCCACAAGGTTTGCCA GTGTATATGCATGGAACAGGACTCGCATCTCAAACTGCACCGCTGATTACTCTGTTC TGTACAATTCAAGCAGCTTCTCCACATTCAAGTGTTACGGAGTCAATCCTACAAAAC TGAACGACCTCTGCTTTACGAACGTCTACGCTGACTCCTTTGTCATACGGGGCGACG AGGTGAGGCAAATTGCGCCCGGCCAAACTGGGAAAATTGCGGACTATAATTACAAA CTTCCTGATAACTTCACCGGTTGTGTGATCGCTTGGAATTCAAACAACCTGGACTCC AAGGTGGGCGGCAATTACAACTACCTTTATAGGCTCTTTCGGAAAAGCAACCTGAG TCCCTTCGAGCGAGATATTAGTACGGAGATCTACCAGGCCGGGTCTACGCCATGCA ACGGTACGGAAGGTTTCAACTGTTATTTTCCGCTGCAGAGTTACGGATTCCAACCAA CAAATGGGGTGGGCTACCAACCTTATAGGGTGGTCGTCCTGTCCTTTGAGCTTAACC ACAGCCCTGCGACCGTATGTGGACCCTAATAG

[0827] SEQ ID NO:75 (AA RBD g8.3 60mer)

[0828] MDWTWILFLV AAA TRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIV RHGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKG LADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSG GSGGSGGSGGGNITNLCPFGEVFNATRFASVYAWNRTRISNCTADYSVLYNSSSFSTFK CYGVNPTKLNDLCFTNVYADSFVIRGDEVRQIAPGOTGKIADYNYKLPDNFTGCVIAW NSNNLDSKVGGNYNYLYRLFRKSNLSPFERDISTEIYQAGSTPCNGTEGFNCYFPLQSY GFQPTNGVGYQPYRVVVLSFELNHSPATVCGP**

[0829] SEQ ID NO: 76 (Nucleotide RBD g8.3 24mer)

[0830] ATGGATTGGACTTGGATCTTGTTCTTGGTCGCAGCGGCTACTAGAGTGCACAGTTTG CGATTTGGTATTGTCGCTTCCCGCGCAAACCATGCGCTCGTGGGTGGTTCCGGTGGC AACATTACCAACCTTTGCCCCTTCGGCGAGGTCTTCAATGCCACAAGGTTTGCCAGT GTATATGCATGGAACAGGACTCGCATCTCAAACTGCACCGCTGATTACTCTGTTCTG TACAATTCAAGCAGCTTCTCCACATTCAAGTGTTACGGAGTCAATCCTACAAAACTG AACGACCTCTGCTTTACGAACGTCTACGCTGACTCCTTTGTCATACGGGGCGACGAG GTGAGGCAAATTGCGCCCGGCCAAACTGGGAAAATTGCGGACTATAATTACAAACT TCCTGATAACTTCACCGGTTGTGTGATCGCTTGGAATTCAAACAACCTGGACTCCAA GGTGGGCGGCAATTACAACTACCTTTATAGGCTCTTTCGGAAAAGCAACCTGAGTCC CTTCGAGCGAGATATTAGTACGGAGATCTACCAGGCCGGGTCTACGCCATGCAACG GTACGGAAGGTTTCAACTGTTATTTTCCGCTGCAGAGTTACGGATTCCAACCAACAA ATGGGGTGGGCTACCAACCTTATAGGGTGGTCGTCCTGTCCTTTGAGCTTAACCACA GCCCTGCGACCGTATGTGGACCCGGCGGGAGCGGAGGGAGTGGCGGATCTGGCGG

[0831] GAGCGGCGGAGGCCTTTCAAAAGATATTATTAAGCTCCTGAATGAGCAAGTCAATA AAGAAATGCAATCTTCTAACCTGTACATGAGCATGTCTAGCTGGTGTTATACTCACA GTCTCGACGGCGCTGGCCTCTTTCTGTTCGATCACGCCGCTGAAGAATATGAGCACG CGAAGAAGCTTATAATCTTCCTGAACGAGAATAATGTTCCCGTCCAACTGACGTCCA TTTCCGCCCCTGAACACAAGTTTGAGGGTCTGACTCAGATCTTTCAAAAGGCGTACG AGCACGAGCAGCATATCAGCGAGAGCATTAACAACATTGTCGATCACGCCATTAAA AGCAAAGACCACGCTACCTTCAACTTTCTCCAATGGTACGTCGCCGAACAGCATGA

[0832] GGAAGAAGTATTGTTCAAGGATATACTCGATAAGATTGAACTGATCGGAAATGAGA ATCACGGCCTGTACCTGGCCGACCAATACGTCAAAGGCATTGCCAAGTCTAGGAAA AGCTAGTAG

[0833] SEQ ID NO: 77 (AA RBD g8.3 24mer)

[0834] MDWTWILFLVAAATRVHSZR G / F SR^AFMFFGGSGGNITNLCPFGEVFNATRFAS VYAWNRTRISNCTADYSVLYNSSSFSTFKCYGVNPTKLNDLCFTNVYADSFVIRGDE VROIAPGOTGKIADYNYKLPDNFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLS PFERDISTEIYQAGSTPCNGTEGFNCYFPLOSYGFOPTNGVGYQPYRVVVLSFELNHS PATVCGPGGSGGSGGSGGSGGGLSKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHS LDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEH

[0835] EQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLY L ADQY VKGI AKSRKS * *

[0836] SEQ ID NO: 78 (Nucleotide RBD g8.3 7mer) ATGGATTGGACTTGGATCTTGTTCTTGGTCGCAGCGGCTACTAGAGTGCACAGTTTG CGATTTGGTATTGTCGCTTCCCGCGCAAACCATGCGCTCGTGGGTGGTTCCGGTGGC AACATTACCAACCTTTGCCCCTTCGGCGAGGTCTTCAATGCCACAAGGTTTGCCAGT GTATATGCATGGAACAGGACTCGCATCTCAAACTGCACCGCTGATTACTCTGTTCTG TACAATTCAAGCAGCTTCTCCACATTCAAGTGTTACGGAGTCAATCCTACAAAACTG AACGACCTCTGCTTTACGAACGTCTACGCTGACTCCTTTGTCATACGGGGCGACGAG GTGAGGCAAATTGCGCCCGGCCAAACTGGGAAAATTGCGGACTATAATTACAAACT TCCTGATAACTTCACCGGTTGTGTGATCGCTTGGAATTCAAACAACCTGGACTCCAA GGTGGGCGGCAATTACAACTACCTTTATAGGCTCTTTCGGAAAAGCAACCTGAGTCC CTTCGAGCGAGATATTAGTACGGAGATCTACCAGGCCGGGTCTACGCCATGCAACG GTACGGAAGGTTTCAACTGTTATTTTCCGCTGCAGAGTTACGGATTCCAACCAACAA ATGGGGTGGGCTACCAACCTTATAGGGTGGTCGTCCTGTCCTTTGAGCTTAACCACA GCCCTGCGACCGTATGTGGACCCGGCGGGAGCGGAGGGAGTGGCGGATCTGGCGG GAGCGGCGGAGGCAAAAAACAGGGGGATGCTGATGTCTGCGGAGAGGTGGCTTAT ATCCAGAGCGTGGTGTCCGACTGCCACGTGCCAACCGCAGAGCTGCGGACACTGCT GGAGATCAGAAAACTGTTCCTGGAGATTCAGAAACTGAAAGTCGAGCTGCAGGGGC TGTCAAAAGAACTGAGGTTCGGAATCGTCGCAAGTAGGGCTAATCACGCTCTGGT GTAATAG

[0837] SEQ ID NO: 79 (AA RBD g8.3 7mer)

[0838] MDWTWILFLVAAATRVHSZRFG / G4S7 A7MLEGGSGGNITNLCPFGEVFNATRFASVY AWNRTRISNCTADYSVLYNSSSFSTFKCYGVNPTKLNDLCFTNVYADSFVIRGDEVROI APGQTGKIADYNYKLPDNFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLSPFERDIS TEIYQAGSTPCNGTEGFNCYFPLOSYGFOPTNGVGYQPYRVVVLSFELNHSPATVCGPG GSGGSGGSGGSGGGKKQGDADVCGEVAYIQSVVSDCHVPTAELRTLLE IRKLFLEIQKLKVELQGLSKELRFGIVASRANHALV**

[0839] SEQ ID NO: 80 (Nucleotide RBD g5.1 SA 24mer)

[0840] ATGGATTGGACATGGATACTGTTCCTGGTGGCCGCCGCCACCAGAGTGCACTCTC TGAGGTTCGGCATCGTGGCCTCCAGGGCCAATCACGCCCTGGTGGGCGGCTCCGG CGGCAACATCACCAATCTGTGCCCTTTCGGCGAGGTGTTTAACGCCACCAGGTTT GCCTCCGTGTACGCCTGGAACCGCACAAGGATCAGCAACTGCGTGGCCGATTATA GCGTGCTGTACAATTCTGCCAGCTTCTCTACCTTTAAGTGCTACGGCGTGAACCCT ACCAAGCTGAACGATCTGTGCTTCACCAACGTGTATGCCGATAGCTTCGTGATCC GGGGCGATGAGGTGCGCCAGATCGCCCCTGGCCAGACAGGCAACATCGCCGACT ACAACTATAAGCTGCCAGATAATTTCACCGGCTGCGTGATCGCCTGGAATTCCAA CAATCTGGACTCTAAGGTGGGCGGCAACTACAACTATCTGTACCGGCTGTTTCGC AAGTCCAACCTGTCTCCATTCGAGAGAGACATCTCCACAGAGATCTATCAGGCCG GCTCCACACCTTGTAACGGCACCAAGGGCTTCAACTGCTACTTCCCTCTGCAGAG CTATGGCTTCCAGCCTACCTACGGCGTGGGCTATCAGCCCTACCGGGTGGTGGTG CTGTCTTTTGAGCTGCTGCACGCCCCTGCCACAGTGTGCGGCCCTGGCGGCTCTGG CGGCTCCGGCGGCTCTGGCGGCAGCGGCGGCGGCCTGAGCAAGGATATCATCAA GCTGCTGAATGAACAGGTCAACAAGGAAATGCAGAGCAGCAACCTGTACATGTC CATGAGCTCCTGGTGCTATACCCACTCTCTGGACGGAGCAGGCCTGTTCCTGTTTG ATCACGCCGCCGAGGAGTACGAGCACGCCAAGAAGCTGATCATCTTCCTGAATG AGAACAATGTGCCCGTGCAGCTGACCTCTATCAGCGCCCCTGAGCACAAGTTCGA GGGCCTGACACAGATCTTTCAGAAGGCCTACGAGCACGAGCAGCACATCTCCGA GTCTATCAACAATATCGTGGACCACGCCATCAAGTCCAAGGATCACGCCACATTC AACTTTCTGCAGTGGTACGTGGCCGAGCAGCACGAGGAGGAGGTGCTGTTTAAG GACATCCTGGATAAGATCGAGCTGATCGGCAACGAGAATCACGGGCTGTATCTG GCCGACCAGTATGTGAAGGGCATCGCTAAAAGCAGGAAATCATGATAA

[0841] SEQ ID NO: 81 (AA RBD g5.1 SA 24mer)

[0842] MDWTWILFLVAAATRVHSZRFG / G4SR^AFL4£FGGSGG ITNLCPFGEVFNATRFASVY AWNRTRISNCVADYSVLYNSASFSTFKCYGVNPTKLNDLCFTNVYADSFVIRGDEVRQI APGQTGNIADYNYKLPDNFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLSPFERDIS

[0843] TEIYOAGSTPCNGTKGFNCYFPLOSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPG

[0844] GSGGSGGSGGSGGGLSKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFD

[0845] HAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVD

[0846] HAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKS

[0847] RKS**

[0848] SEQ ID NO: 82 (Nucleotide RBD g5.1 UK 24mer)

[0849] ATGGACTGGACATGGATACTGTTTCTGGTGGCCGCCGCCACAAGGGTGCACTCCCTG

[0850] AGATTTGGCATCGTGGCCTCTAGGGCCAACCACGCCCTGGTGGGCGGCTCTGGCGG

[0851] CAATATCACAAACCTGTGCCCATTCGGCGAGGTGTTCAACGCCACCAGGTTTGCCTC

[0852] CGTGTACGCCTGGAATAGGACCAGAATCAGCAACTGCGTGGCCGATTATTCCGTGC

[0853] TGTATAACTCTGCCTCCTTCAGCACCTTTAAGTGTTACGGCGTGAATCCTACCAAGC

[0854] TGAATGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGGGGCGAT

[0855] GAGGTGCGCCAGATCGCCCCAGGCCAGACAGGCAAGATCGCCGACTACAATTATAA

[0856] GCTGCCAGATAACTTCACAGGCTGCGTGATCGCCTGGAACAGCAATAACCTGGACT

[0857] CCAAGGTGGGCGGCAACTATAACTACCTGTATCGGCTGTTTAGGAAGAGCAACCTG

[0858] TCTCCTTTCGAGAGGGATATCTCCACCGAGATCTACCAGGCCGGCTCTACACCATGC

[0859] AACGGCACCAAGGGCTTCAACTGCTACTTCCCTCTGCAGTCCTATGGCTTCCAGCCT

[0860] ACCTACGGCGTGGGCTACCAGCCATATAGGGTGGTGGTGCTGAGCTTTGAGCTGCT

[0861] GCACGCCCCAGCCACAGTGTGCGGCCCAGGCGGCAGCGGCGGCTCCGGCGGCAGCG

[0862] GCGGCTCTGGCGGCGGCCTGAGCAAGGACATCATCAAGCTGCTGAACGAGCAGGTG

[0863] AACAAGGAGATGCAGTCTTCCAACCTGTACATGTCCATGTCCTCTTGGTGTTACACC

[0864] CACTCCCTGGATGGCGCCGGCCTGTTCCTGTTTGATCACGCCGCCGAGGAGTATGAG

[0865] CACGCCAAGAAGCTGATCATCTTTCTGAACGAGAACAACGTGCCAGTGCAGCTGAC

[0866] CTCTATCTCCGCCCCTGAGCACAAGTTCGAGGGCCTGACCCAGATCTTCCAGAAGGC

[0867] CTATGAGCACGAGCAGCACATCTCTGAGAGCATCAATAACATCGTGGATCACGCCA

[0868] TCAAGTCCAAGGATCACGCCACATTCAACTTTCTGCAGTGGTATGTGGCCGAGCAGC

[0869] ACGAGGAGGAGGTGCTGTTCAAGGATATCCTGGACAAGATCGAGCTGATCGGCAAT

[0870] GAGAACCACGGCCTGTACCTGGCCGACCAGTATGTGAAGGGCATCGCCAAGTCTAG

[0871] GAAGTCCTGATAA

[0872] SEQ ID NO: 83 (AA RBD g5.1 UK 24mer)

[0873] MDWTWILFLVAAATRVHSZRFG / K4S 4AfK4£FGGSGGNITNLCPFGEVFNATRFASVY

[0874] AWNRTRISNCVADYSVLYNSASFSTFKCYGVNPTKLNDLCFTNVYADSFVIRGDEVRQI

[0875] APGOTGKIADYNYKLPDNFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLSPFERDIS

[0876] TEIYOAGSTPCNGTKGFNCYFPLOSYGFOPTYGVGYOPYRVVVLSFELLHAPATVCGPG

[0877] GSGGSGGSGGSGGGLSKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFD

[0878] HAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVD

[0879] HAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLY

[0880] L ADQ Y VKGI AKS RKS * *

[0881] SEQ ID NO: 84 (Nucleotide RBD g5.1 BR 24mer)

[0882] ATGGACTGGACATGGATACTGTTTCTGGTGGCCGCCGCCACAAGGGTGCACTCCCTG

[0883] AGATTTGGCATCGTGGCCTCTAGGGCCAACCACGCCCTGGTGGGCGGCTCTGGCGG

[0884] CAATATCACAAACCTGTGCCCATTCGGCGAGGTGTTCAACGCCACCAGGTTTGCCTC

[0885] CGTGTACGCCTGGAATAGGACCAGAATCAGCAACTGCGTGGCCGATTATTCCGTGC

[0886] TGTATAACTCTGCCTCCTTCAGCACCTTTAAGTGTTACGGCGTGAATCCTACCAAGC

[0887] TGAATGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGGGGCGAT

[0888] GAGGTGCGCCAGATCGCCCCAGGCCAGACAGGCACCATCGCCGACTACAATTATAA

[0889] GCTGCCAGATAACTTCACAGGCTGCGTGATCGCCTGGAACAGCAATAACCTGGACT

[0890] CCAAGGTGGGCGGCAACTATAACTACCTGTATCGGCTGTTTAGGAAGAGCAACCTG

[0891] TCTCCTTTCGAGAGGGATATCTCCACCGAGATCTACCAGGCCGGCTCTACACCATGC

[0892] AACGGCACCAAGGGCTTCAACTGCTACTTCCCTCTGCAGTCCTATGGCTTCCAGCCT

[0893] ACCTACGGCGTGGGCTACCAGCCATATAGGGTGGTGGTGCTGAGCTTTGAGCTGCT GCACGCCCCAGCCACAGTGTGCGGCCCAGGCGGCAGCGGCGGCTCCGGCGGCAGCG GCGGCTCTGGCGGCGGCCTGAGCAAGGACATCATCAAGCTGCTGAACGAGCAGGTG AACAAGGAGATGCAGTCTTCCAACCTGTACATGTCCATGTCCTCTTGGTGTTACACC CACTCCCTGGATGGCGCCGGCCTGTTCCTGTTTGATCACGCCGCCGAGGAGTATGAG CACGCCAAGAAGCTGATCATCTTTCTGAACGAGAACAACGTGCCAGTGCAGCTGAC CTCTATCTCCGCCCCTGAGCACAAGTTCGAGGGCCTGACCCAGATCTTCCAGAAGGC CTATGAGCACGAGCAGCACATCTCTGAGAGCATCAATAACATCGTGGATCACGCCA TCAAGTCCAAGGATCACGCCACATTCAACTTTCTGCAGTGGTATGTGGCCGAGCAGC ACGAGGAGGAGGTGCTGTTCAAGGATATCCTGGACAAGATCGAGCTGATCGGCAAT GAGAACCACGGCCTGTACCTGGCCGACCAGTATGTGAAGGGCATCGCCAAGTCTAG GAAGTCCTGATAA

[0894] SEQ ID NO: 85 (AA RBD g5.1 BR 24mer)

[0895] MDWTWILFLVAAATRVHSZ7?FG7G4. Aa4IEGG,S,GGNITNLCPFGEVFNATRFASVY AWNRTRISNCVADYSVLYNSASFSTFKCYGVNPTKLNDLCFTNVYADSFVIRGDEVRQI APGQTGTIADYNYKLPDNFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLSPFERDIS TEIYOAGSTPCNGTKGFNCYFPLQSYGFQPTYGVGYOPYRVVVLSFELLHAPATVCGPG GSGGSGGSGGSGGGLSKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFD HAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVD HAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYL ADQYVKGIAKSRKS**

[0896] SEQ ID NO:86 (AA NC99 IL-21 NIC with IgE)

[0897] MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIVR HGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGL ADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGG SGGSGGSGGGAPLQLGNCSVAGWILGNPECELLISKESWSYIVETPNPENGTCFPGNFSD YEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLY PNLSKSYNNTKEKEVLVLWGVHHPPNIGNQRALYHTENAYVSVVSSHYSRRFTPNISKR PKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPWYAFALSRGNGSGGSGSGGGSHKSS PQGPDRLLIRLRHLIDIVEQLKIYENDLDPELLSAPQDVKGHCEHAAFACFQKAKLKPSN PGNNKTFIIDLVAQLRRRLPARRGGKKQKHIAKCPSCDSYEKRTPKEFLERLKWLLQKM IHQHLS**

[0898] SEQ ID NO: 87 (NC99 IL-21 NIC with IgE nucleotide): atggactggacttggattctgtttctggtcgccgctgccactcgcgtgcattctatgcagatctacgaaggaaaactgaccgctgagggactg aggttcggaattgtcgcaagccgcgcgaatcacgcactggtggataggctggtggaaggcgctatcgacgcaattgtccggcacggcgg gagagaggaagacatcacactggtgagagtctgcggcagctgggagattcccgtggcagctggagaactggctcgaaaggaggacatc gatgccgtgatcgctattggggtcctgtgccgaggagcaactcccagcttcgactacatcgcctcagaagtgagcaaggggctggctgat ctgtccctggagctgaggaaacctatcacttttggcgtgattactgccgacaccctggaacaggcaatcgaggcggccggcacctgccatg gaaacaaaggctgggaagcagccctgtgcgctattgagatggcaaatctgttcaaatctctgcgaggaggctccggaggatctggaggg agtggaggctcaggaggaggcgcccctctgcagctgggaaactgcagcgtggcaggatggattctgggcaatccagagtgtgagctgct gatctccaaggagtcctggtcttacatcgtggagaccccaaaccccgagaatggcacatgctttcccggcaacttctctgactatgaggagc tgagggagcagctgagctccgtgtctagcttcgagagatttgagatcttccctaaggagtcctcttggccaaatcacaccgtgacaggcgtg agcgcctcctgttctcacaacggcaagagctccttttacaggaatctgctgtggctgaccggcaagaacggcctgtaccctaatctgagcaa gtcctataacaatacaaaggagaaggaggtgctggtgctgtggggcgtgcaccaccctcccaacatcggcaatcagagggccctgtacc acaccgagaacgcctacgtgagcgtggtgtctagccactactctaggagattcacacccaacatcagcaagaggcctaaggtgcgcgac caggagggacggatcaattactattggaccctgctggagccaggcgatacaatcatctttgaggccaacggcaatctgatcgccccctggt atgccttcgccctgtctcgcggcaacggcagcggcggcagcggctctggcggcggctctcacaagagcagcccccagggacctgaca ggctgctgatcagactgcggcacctgatcgatatcgtggagcagctgaagatctacgagaacgacctggaccccgagctgctgtccgccc cacaggacgtgaagggacactgcgagcacgcagccttcgcctgttttcagaaggccaagctgaagccatctaatcccggcaacaataag accttcatcatcgatctggtggcccagctgaggagacggctgccagcaaggaggggcggcaagaagcagaagcacatcgccaagtgcc cctcttgtgatagctatgagaagcgcacacctaaggagtttctggagaggctgaagtggctgctgcagaagatgatccaccagcacctgag c SEQ ID NO: 88 (NC99 IL-21 NIC without IgE amino acid)

[0899] MQIYEGKLTAEGLRFGIVASRANHAL VDRL VEGAID Al VRHGGREEDITLVRVCGSWEI PVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGLADLSLELRKPITFGVITADT LEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGGSGGSGGSGGGAPLQLGNCS VAGWILGNPECELLISKESWSYIVETPNPENGTCFPGNFSDYEELREQLSSVSSFERFEIFP KESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYNNTKEKEVLVL WGVHHPPNIGNQRALYHTENAYVSVVSSHYSRRFTPNISKRPKVRDQEGRINYYWTLL EPGDTIIFEANGNLIAPWYAFALSRGNGSGGSGSGGGSHKSSPQGPDRLLIRLRHLIDIVE QLKIYENDLDPELLSAPQDVKGHCEHAAFACFQKAKLKPSNPGNNKTFIIDLVAQLRRR LPARRGGKKQKHIAKCPSCDSYEKRTPKEFLERLKWLLQKMIHQHLS**

[0900] SEQ ID NO: 89 (NC99 IL-21 NIC without IgE nucleotide) atgcagatctacgaaggaaaactgaccgctgagggactgaggttcggaattgtcgcaagccgcgcgaatcacgcactggtggataggct ggtggaaggcgctatcgacgcaattgtccggcacggcgggagagaggaagacatcacactggtgagagtctgcggcagctgggagatt cccgtggcagctggagaactggctcgaaaggaggacatcgatgccgtgatcgctattggggtcctgtgccgaggagcaactcccagcttc gactacatcgcctcagaagtgagcaaggggctggctgatctgtccctggagctgaggaaacctatcacttttggcgtgattactgccgacac cctggaacaggcaatcgaggcggccggcacctgccatggaaacaaaggctgggaagcagccctgtgcgctattgagatggcaaatctgt tcaaatctctgcgaggaggctccggaggatctggagggagtggaggctcaggaggaggcgcccctctgcagctgggaaactgcagcgt ggcaggatggattctgggcaatccagagtgtgagctgctgatctccaaggagtcctggtcttacatcgtggagaccccaaaccccgagaat ggcacatgctttcccggcaacttctctgactatgaggagctgagggagcagctgagctccgtgtctagcttcgagagatttgagatcttccct aaggagtcctcttggccaaatcacaccgtgacaggcgtgagcgcctcctgttctcacaacggcaagagctccttttacaggaatctgctgtg gctgaccggcaagaacggcctgtaccctaatctgagcaagtcctataacaatacaaaggagaaggaggtgctggtgctgtggggcgtgc accaccctcccaacatcggcaatcagagggccctgtaccacaccgagaacgcctacgtgagcgtggtgtctagccactactctaggagat tcacacccaacatcagcaagaggcctaaggtgcgcgaccaggagggacggatcaattactattggaccctgctggagccaggcgataca atcatctttgaggccaacggcaatctgatcgccccctggtatgccttcgccctgtctcgcggcaacggcagcggcggcagcggctctggc ggcggctctcacaagagcagcccccagggacctgacaggctgctgatcagactgcggcacctgatcgatatcgtggagcagctgaagat ctacgagaacgacctggaccccgagctgctgtccgccccacaggacgtgaagggacactgcgagcacgcagccttcgcctgttttcaga aggccaagctgaagccatctaatcccggcaacaataagaccttcatcatcgatctggtggcccagctgaggagacggctgccagcaagg aggggcggcaagaagcagaagcacatcgccaagtgcccctcttgtgatagctatgagaagcgcacacctaaggagtttctggagaggct gaagtggctgctgcagaagatgatccaccagcacctgagc

[0901] SEQ ID NO: 90 (NC99 IL-21 NIC full vector nucleotide)

[0902] Gctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcc catatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacg tatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgt atcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctac ttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacgg ggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccc cattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggctt atcgaaattaatacgactcactatagggagacccaagctggctagcgtttaaacttaagcttggtaccgagctcggatccgccaccatggac tggacttggattctgtttctggtcgccgctgccactcgcgtgcattctatgcagatctacgaaggaaaactgaccgctgagggactgaggttc ggaattgtcgcaagccgcgcgaatcacgcactggtggataggctggtggaaggcgctatcgacgcaattgtccggcacggcgggagag aggaagacatcacactggtgagagtctgcggcagctgggagattcccgtggcagctggagaactggctcgaaaggaggacatcgatgc cgtgatcgctattggggtcctgtgccgaggagcaactcccagcttcgactacatcgcctcagaagtgagcaaggggctggctgatctgtcc ctggagctgaggaaacctatcacttttggcgtgattactgccgacaccctggaacaggcaatcgaggcggccggcacctgccatggaaac aaaggctgggaagcagccctgtgcgctattgagatggcaaatctgttcaaatctctgcgaggaggctccggaggatctggagggagtgga ggctcaggaggaggcgcccctctgcagctgggaaactgcagcgtggcaggatggattctgggcaatccagagtgtgagctgctgatctc caaggagtcctggtcttacatcgtggagaccccaaaccccgagaatggcacatgctttcccggcaacttctctgactatgaggagctgagg gagcagctgagctccgtgtctagcttcgagagatttgagatcttccctaaggagtcctcttggccaaatcacaccgtgacaggcgtgagcgc ctcctgttctcacaacggcaagagctccttttacaggaatctgctgtggctgaccggcaagaacggcctgtaccctaatctgagcaagtccta taacaatacaaaggagaaggaggtgctggtgctgtggggcgtgcaccaccctcccaacatcggcaatcagagggccctgtaccacaccg agaacgcctacgtgagcgtggtgtctagccactactctaggagattcacacccaacatcagcaagaggcctaaggtgcgcgaccaggag ggacggatcaattactattggaccctgctggagccaggcgatacaatcatctttgaggccaacggcaatctgatcgccccctggtatgcctt cgccctgtctcgcggcaacggcagcggcggcagcggctctggcggcggctctcacaagagcagcccccagggacctgacaggctgct gatcagactgcggcacctgatcgatatcgtggagcagctgaagatctacgagaacgacctggaccccgagctgctgtccgccccacagg acgtgaagggacactgcgagcacgcagccttcgcctgttttcagaaggccaagctgaagccatctaatcccggcaacaataagaccttcat catcgatctggtggcccagctgaggagacggctgccagcaaggaggggcggcaagaagcagaagcacatcgccaagtgcccctcttgt gatagctatgagaagcgcacacctaaggagtttctggagaggctgaagtggctgctgcagaagatgatccaccagcacctgagctgataa ctcgagtctagagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttc cttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggg gtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctactgg gcggttttatggacagcaagcgaaccggaattgccagctggggcgccctctggtaaggttgggaagccctgcaaagtaaactggatggct ttcttgccgccaaggatctgatggcgcaggggatcaagctctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggatt gcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttc cggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggcagcgcggcta tcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtg ccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatcc ggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatct ggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgac ccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgct atcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgct cccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaattattaacgcttacaatttcctgatgcggtattttctccttacgcat ctgtgcggtatttcacaccgcatcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtat ccgctcatgagacaataaccctgataaatgcttcaataatagcacgtgctaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttg ataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatccttttt ttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaa ggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcc tacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccg gataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctac agcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagag cgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgct cgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctt

[0903] SEQ ID NO:91 (GT8 IL-6 NIC with IgE Amino Acid)

[0904] MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRLVEGAIDAIVR HGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGL ADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGG SGGSGGSGGGDTITLPCRPAPPPHCSSNITGLILTRQGGYSNDNTVIFRPSGGDWRDIARC QIAGTVVSTQLFLNGSLAEEEVVIRSEDWRDNAKSICVQLNTSVEINCTGAGHCNISRAK WDNTLKQIASKLREQYGNKTIIFKPSSGGDPEFVNHSFNCGGEFFYCDSTQLFDSTWFDS TGGSGSGGGSFPTSQVRRGDFTEDTTPNRPVYTTSQVGGLITHVLWEIVEMRKELCNGN SDCMNNDDALAENNLKLPEIQRNDGCYQTGYNQEICLLKISSGLLEYHSYLEYMKNNL KDNKKDKARVLQRDTETLIHIFNQEVKDLHKIVLPTPISNALLTDKLESQKEWLRTKTIQ FILKSLEEFLKVTLRSTRQT* *

[0905] SEQ ID NO: 92 (GT8 IL-6 NIC with IgE nucleotide) atggattggacctggatactgttcctggtggccgccgccacaagagtgcactctatgcagatctacgagggcaagctgaccgccgagggc ctgagattcggcatcgtggcctctagagccaaccacgccctggtggatagactggtggagggcgccatcgacgccatcgtgcggcacgg cggcagagaggaggacatcaccctggtgcgggtgtgcggctcttgggagatccctgtggccgccggcgagctggccagaaaggagga catcgatgccgtgatcgccatcggcgtgctgtgcagaggcgccacaccatctttcgactatatcgcctctgaggtgagcaagggcctggcc gatctgagcctggagctgagaaagcctatcacatttggcgtgatcaccgccgatacactggagcaggccatcgaggccgccggcacatgt cacggcaataagggctgggaggccgccctgtgcgccatcgagatggccaatctgttcaagtccctgcggggcggctccggcggctctg gcggctccggcggcagcggcggcggcgacaccatcaccctgccatgcaggccagccccacctcctcactgttctagcaacatcacagg cctgatcctgacaagacagggcggctactccaacgacaataccgtgatctttcggccatccggcggcgattggagagacatcgccaggtg tcagatcgccggcaccgtggtgtccacccagctgttcctgaacggctctctggccgaggaggaggtggtaatccggtctgaggattggcg cgacaatgccaagtccatctgcgtgcagctgaataccagcgtggagatcaactgcacaggcgccggccactgcaacatcagcagagcca agtgggacaataccctgaagcagatcgccagcaagctgcgcgagcagtatggcaacaagaccatcatcttcaagccaagctctggcggc gaccctgagttcgtgaaccactctttcaactgtggcggcgagttcttttactgcgatagcacccagctgttcgatagcacctggtttgattctac aggcggcagcggctctggcggcggctctttccctaccagccaggtgaggagaggcgactttacagaggataccacaccaaaccggccc gtgtataccacatcccaagtgggcggcctgatcacccacgtgctgtgggagatcgtggagatgcgcaaggagctgtgcaacggcaattct gactgtatgaacaatgacgatgccctggccgagaacaatctgaagctgcccgagatccagaggaacgatggctgctatcagacaggctac aatcaggagatctgtctgctgaagatcagctccggcctgctggagtaccactcctatctggagtacatgaagaacaatctgaaggacaataa gaaggataaggccagggtgctgcagagagacaccgagacactgatccacatcttcaaccaggaggtgaaggatctgcacaagatcgtgc tgcccacccctatctctaatgccctgctgacagacaagctggagagccagaaggagtggctgaggaccaagacaatccagttcatcctga agtccctggaggagtttctgaaggtgaccctgcggtctacccgccagaca

[0906] SEQ ID NO: 93 (GT8 IL-6 NIC without IgE amino acid)

[0907] MQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIVRHGGREEDITLVRVCGSWEI PVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGLADLSLELRKPITFGVITADT LEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGGSGGSGGSGGGDTITLPCRP APPPHCSSNITGLILTRQGGYSNDNTVIFRPSGGDWRDIARCQIAGTVVSTQLFLNGSLAE EEVVIRSEDWRDNAKSICVQLNTSVEINCTGAGHCNISRAKWDNTLKQIASKLREQYGN KTIIFKPSSGGDPEFVNHSFNCGGEFFYCDSTQLFDSTWFDSTGGSGSGGGSFPTSQVRR GDFTEDTTPNRPVYTTSQVGGLITHVLWEIVEMRKELCNGNSDCMNNDDALAENNLKL PEIQRNDGCYQTGYNQEICLLKISSGLLEYHSYLEYMKNNLKDNKKDKARVLQRDTET LIHIFNQEVKDLHKIVLPTPISNALLTDKLESQKEWLRTKTIQFILKSLEEFLKVTLRSTRQ T**

[0908] SEQ ID NO: 94 (GT8 IL-6 NIC without IgE nucleotide) atgcagatctacgagggcaagctgaccgccgagggcctgagattcggcatcgtggcctctagagccaaccacgccctggtggatagact ggtggagggcgccatcgacgccatcgtgcggcacggcggcagagaggaggacatcaccctggtgcgggtgtgcggctcttgggagat ccctgtggccgccggcgagctggccagaaaggaggacatcgatgccgtgatcgccatcggcgtgctgtgcagaggcgccacaccatctt tcgactatatcgcctctgaggtgagcaagggcctggccgatctgagcctggagctgagaaagcctatcacatttggcgtgatcaccgccga tacactggagcaggccatcgaggccgccggcacatgtcacggcaataagggctgggaggccgccctgtgcgccatcgagatggccaat ctgttcaagtccctgcggggcggctccggcggctctggcggctccggcggcagcggcggcggcgacaccatcaccctgccatgcagg ccagccccacctcctcactgttctagcaacatcacaggcctgatcctgacaagacagggcggctactccaacgacaataccgtgatctttcg gccatccggcggcgattggagagacatcgccaggtgtcagatcgccggcaccgtggtgtccacccagctgttcctgaacggctctctggc cgaggaggaggtggtaatccggtctgaggattggcgcgacaatgccaagtccatctgcgtgcagctgaataccagcgtggagatcaact gcacaggcgccggccactgcaacatcagcagagccaagtgggacaataccctgaagcagatcgccagcaagctgcgcgagcagtatg gcaacaagaccatcatcttcaagccaagctctggcggcgaccctgagttcgtgaaccactctttcaactgtggcggcgagttcttttactgcg atagcacccagctgttcgatagcacctggtttgattctacaggcggcagcggctctggcggcggctctttccctaccagccaggtgaggag aggcgactttacagaggataccacaccaaaccggcccgtgtataccacatcccaagtgggcggcctgatcacccacgtgctgtgggagat cgtggagatgcgcaaggagctgtgcaacggcaattctgactgtatgaacaatgacgatgccctggccgagaacaatctgaagctgcccga gatccagaggaacgatggctgctatcagacaggctacaatcaggagatctgtctgctgaagatcagctccggcctgctggagtaccactcc tatctggagtacatgaagaacaatctgaaggacaataagaaggataaggccagggtgctgcagagagacaccgagacactgatccacat cttcaaccaggaggtgaaggatctgcacaagatcgtgctgcccacccctatctctaatgccctgctgacagacaagctggagagccagaa ggagtggctgaggaccaagacaatccagttcatcctgaagtccctggaggagtttctgaaggtgaccctgcggtctacccgccagaca

[0909] SEQ ID NO: 95 (GT8 IL-6 NIC full vector) gctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagccc atatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgt atgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgta tcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctact tggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggg gatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgcccc attgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggctta tcgaaattaatacgactcactatagggagacccaagctggctagcgtttaaacttaagcttggtaccgagctcggatccgccaccatggattg gacctggatactgttcctggtggccgccgccacaagagtgcactctatgcagatctacgagggcaagctgaccgccgagggcctgagatt cggcatcgtggcctctagagccaaccacgccctggtggatagactggtggagggcgccatcgacgccatcgtgcggcacggcggcaga gaggaggacatcaccctggtgcgggtgtgcggctcttgggagatccctgtggccgccggcgagctggccagaaaggaggacatcgatg ccgtgatcgccatcggcgtgctgtgcagaggcgccacaccatctttcgactatatcgcctctgaggtgagcaagggcctggccgatctgag cctggagctgagaaagcctatcacatttggcgtgatcaccgccgatacactggagcaggccatcgaggccgccggcacatgtcacggca ataagggctgggaggccgccctgtgcgccatcgagatggccaatctgtcaagtccctgcggggcggctccggcggctctggcggctcc ggcggcagcggcggcggcgacaccatcaccctgccatgcaggccagccccacctcctcactgttctagcaacatcacaggcctgatcct gacaagacagggcggctactccaacgacaataccgtgatctttcggccatccggcggcgattggagagacatcgccaggtgtcagatcg ccggcaccgtggtgtccacccagctgttcctgaacggctctctggccgaggaggaggtggtaatccggtctgaggattggcgcgacaatg ccaagtccatctgcgtgcagctgaataccagcgtggagatcaactgcacaggcgccggccactgcaacatcagcagagccaagtggga caataccctgaagcagatcgccagcaagctgcgcgagcagtatggcaacaagaccatcatcttcaagccaagctctggcggcgaccctg agttcgtgaaccactctttcaactgtggcggcgagttcttttactgcgatagcacccagctgttcgatagcacctggtttgattctacaggcgg cagcggctctggcggcggctctttccctaccagccaggtgaggagaggcgactttacagaggataccacaccaaaccggcccgtgtata ccacatcccaagtgggcggcctgatcacccacgtgctgtgggagatcgtggagatgcgcaaggagctgtgcaacggcaattctgactgta tgaacaatgacgatgccctggccgagaacaatctgaagctgcccgagatccagaggaacgatggctgctatcagacaggctacaatcag gagatctgtctgctgaagatcagctccggcctgctggagtaccactcctatctggagtacatgaagaacaatctgaaggacaataagaagg ataaggccagggtgctgcagagagacaccgagacactgatccacatcttcaaccaggaggtgaaggatctgcacaagatcgtgctgccc acccctatctctaatgccctgctgacagacaagctggagagccagaaggagtggctgaggaccaagacaatccagttcatcctgaagtcc ctggaggagtttctgaaggtgaccctgcggtctacccgccagacatgataactcgagtctagagggcccgtttaaacccgctgatcagcct cgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaat aaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattggg aagacaatagcaggcatgctggggatgcggtgggctctatggcttctactgggcggttttatggacagcaagcgaaccggaattgccagct ggggcgccctctggtaaggttgggaagccctgcaaagtaaactggatggctttcttgccgccaaggatctgatggcgcaggggatcaagc tctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctatt cggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaag accgacctgtccggtgccctgaatgaactgcaagacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgt gctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgcc gagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgca tcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactg ttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaat ggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaaga gcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacga gttcttctgaattattaacgcttacaattcctgatgcggtattttctcctacgcatctgtgcggtatttcacaccgcatcaggtggcactttcgg ggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataat agcacgtgctaaaactcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttc cactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccac cgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactg ttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgct gccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttc gtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaag ggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtat ctttatagtcctgtcgggttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagc aacgcggccttttacggttcctggccttttgctggccttttgctcacatgttctt

[0910] SEQ ID NO: 98 (GT8-IL-4-NIC (With IgE) amino acid)

[0911] MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIVR HGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGL ADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGG SGGSGGSGGGDTITLPCRPAPPPHCSSNITGLILTRQGGYSNDNTVIFRPSGGDWRDIARC QIAGTVVSTQLFLNGSLAEEEVVIRSEDWRDNAKSICVQLNTSVEINCTGAGHCNISRAK WDNTLKQIASKLREQYGNKTIIFKPSSGGDPEFVNHSFNCGGEFFYCDSTQLFDSTWFDS TGGSGSGGGSHIHGCDKNHLREIIGILNEVTGEGTPCTEMDVPNVLTATKNTTESELVCR ASKVLRIFYLKHGKTPCLKKNSSVLMELQRLFRAFRCLDSSISCTMNESKSTSLKDFLES LKSIMQMDYS

[0912] SEQ ID NO: 99 (GT8-I...

Claims

CLAIMSWhat is claimed is:

1. A composition comprising a cytokine nanoparticle immunoadjuvant fusion polypeptide comprising: a) an immunoadjuvant domain; b) an antigen domain; and c) an oligomerization domain.

2. The composition of claim 1, wherein at least two cytokine nanoparticle immunoadjuvant fusion polypeptides form a cytokine nanoparticle immunoadjuvant complex (NIC).

3. The composition of claim 1 or 2, wherein the immunoadjuvant domain is selected from the group consisting of IL-21, IL-6, IL-4, and TSLP.

4. The composition of claim 3, wherein the immunoadjuvant domain is IL-21 comprising SEQ ID NO:4 or a fragment or variant thereof.

5. The composition of claim 3, wherein the immunoadjuvant domain is IL-6 comprising SEQ ID NO: 112 or a fragment or variant thereof.

6. The composition of claim 3, wherein the immunoadjuvant domain is IL-4 comprising SEQ ID NO: 114 or a fragment or variant thereof.

7. The composition of claim 3, wherein the immunoadjuvant domain is TSLP comprising SEQ ID NO: 116 or a fragment or variant thereof.

8. The composition of any one of claims 1-7, wherein the antigen is an immunogenic antigen.

9. The composition of claim 8, wherein the antigen is an HIV antigen, a SARS- CoV-2 antigen, or an influenza antigen.

10. The composition of claim 9, wherein the antigen is an HIV antigen comprising SEQ ID NO:96 or a fragment or variant thereof.

11. The composition of claim 9, wherein the antigen is a SARS-CoV-2 antigen comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

47. SEQ ID NO:49, SEQ ID NO:23, SEQ ID NO:

25. SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:

63. SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, and fragments or variants thereof.

12. The composition of claim 9, wherein the antigen is an influenza antigen comprising SEQ ID NO: 118 or a fragment or variant thereof.

13. The composition of any one of claims 1-12, wherein the oligomerization domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:

16. SEQ ID NO: 18, SEQ ID NO:

20. and fragments or variants thereof.

14. The composition of claim 1, wherein the cytokine nanoparticle immunoadjuvant fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 108, SEQ ID NO: 111, SEQ ID NO: 86, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO: 102, SEQ ID NO: 103, and fragments or variants thereof.

15. A nucleic acid molecule encoding a cytokine nanoparticle immunoadjuvant fusion polypeptide comprising: a) an immunoadjuvant domain; b) an antigen domain; and c) an oligomerization domain.

16. The nucleic molecule of claim 15, wherein the immunoadjuvant domain is selected from the group consisting of IL-21, IL-6, IL-4, and TSLP.

17. The nucleic acid molecule of claim 16, wherein the nucleotide sequence encoding the IL-21 immunoadjuvant domain comprises SEQ ID NO:5, SEQ ID NO:6, or a fragment or variant thereof.

18. The nucleic acid molecule of claim 16, wherein the nucleotide sequence encoding the IL-6 immunoadjuvant domain comprises SEQ ID NO: 113 or a fragment or variant thereof.

19. The nucleic acid molecule of claim 16, wherein the nucleotide sequence encoding the IL-4 immunoadjuvant domain comprises SEQ ID NO: 115 or a fragment or variant thereof.

20. The nucleic acid molecule of claim 16, wherein the nucleotide sequence encoding the TSLP immunoadjuvant domain comprises SEQ ID NO: 117 or a fragment or variant thereof.

21. The nucleic acid molecule of any one of claims 15-20, wherein the antigen is an immunogenic antigen.

22. The nucleic acid molecule of claim 21. wherein the antigen is an HIV antigen, a SARS-CoV-2 antigen, or an influenza antigen.

23. The nucleic acid molecule of claim 21, wherein the nucleotide sequence encoding the HIV antigen comprises SEQ ID NO: 97 or a fragment or variant thereof.

24. The nucleic acid molecule of claim 21, wherein the nucleotide sequence encoding the SARS-CoV-2 comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:

26. SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO 42, SEQ ID NO:44, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:

60. SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ IDNO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ IDNO:80, SEQ ID NO:82, SEQ ID NO:84, and fragments or variants thereof.

25. The nucleic acid molecule of claim 2E wherein the nucleotide sequence encoding the influenza antigen comprises SEQ ID NO: 119 or a fragment or variant thereof.

26. The nucleic acid molecule of any one of claims 15-25. wherein the nucleic acid molecule encodes an oligomerization domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, and fragments or variants thereof.

27. The nucleic acid molecule of claim 15. wherein the nucleic acid molecule encodes a cytokine nanoparticle immunoadjuvant fusion polypeptide compnsing an amino acid sequence selected from the group consisting of SEQ ID NO: 108, SEQ ID NO: 111, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO: 102, SEQ ID NO:

103. and fragments or variants thereof.

28. The nucleic acid molecule of claim 15 or 27, wherein the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO:

110. SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:100, SEQ ID NO: 101, SEQ ID NO: 104, SEQ ID NO: 105, and fragments or variants thereof.

29. An immunogenic composition comprising a nucleic acid molecule of any one of claims 15-28.

30. The immunogenic composition of claim 29, further comprising a pharmaceutically acceptable excipient.

31. A method of inducing an immune response against a target antigen in a subject in need thereof, the method comprising administering a composition of claim 1, a nucleic acid molecule of claim 15, or a composition of claim 29 to the subject.

32. The method of claim 31, wherein administering includes at least one of electroporation and injection.

33. The method of claim 31, wherein the target antigen is an HIV antigen, a SARS-CoV-2 antigen, or an influenza antigen.

34. A method of protecting a subject in need thereof from a disease or disorder associated with an antigen, the method comprising administering a composition of claim 1, a nucleic acid molecule of claim 15, or a composition of claim 29 to the subject.

35. The method of claim 34, wherein administering includes at least one of electroporation and injection.

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