Viral subunit vaccines

Abstract

Described herein is an engineered viral subunit vaccine encoding ASFV capsid proteins (e.g., P72 and Penton) for use in pigs. The vaccine with either P72 or Penton tested in mice elicited significantly higher levels of antigen-specific IgM and IgG, increased frequency of antibody-secreting cells in the bone marrow, and higher quality germinal centers in the spleens of immunized mice. Enhanced T cell responses were also observed, with higher levels of IFN-γ and TNF-α in splenocytes. Immunogenicity assessments in pigs further supported these findings, with both MB-P72 and MB-PN180Q inducing robust B cell and T cell responses. These findings show that expression of capsid proteins P72 and Penton in membrane-bound forms via mRNA preserves their native multimeric structure and enhances their immunogenicity and provides a basis for an effective ASFV vaccine.

Description

VIRAL SUBUNIT VACCINESCROSS REFRENCE TO RELATED APPLICATION

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 663,736, filed June 25, 2024, the entire contents of which is incorporated herein by reference.REFERENCE TO A SEQUENCE LISTING

[0002] The content of the electronic sequence listing (M065670558WO00-SEQ-EAS.xml; Size: 129,344 bytes; and Date of Creation: June 21, 2025) is herein incorporated by reference in its entirety.BACKGROUND

[0003] African swine fever (ASF) is a highly contagious and lethal viral disease that affects both domestic and feral swine across all age groups. Presently, the United States remains ASF free, but African Swine Fever Virus (ASFV) infection has documented in the Caribbean countries. ASF leads to almost 100% mortality in domestic pigs, resulting in huge economic losses. Historically, ASF was mainly circulating in African countries. The incursion of ASF to republic of Georgia in 2007 and China in 2018 constitutes a significant threat to global swine industry today. Within a year of the ASF outbreak in China, there was an almost 40% reduction of the national swine herd. Southeast Asia and European countries also suffered a lot from ASF outbreaks. Outbreaks in Caribbean islands (Dominican Republic and Haiti) during 2021 highlights the risk of ASF introduction to the mainland North America. As the third largest pork consumer and producer, ASF introduction into the US could result in 10- 30% swine population reduction. Vaccines are urgently needed for ASF control.

[0004] Tremendous efforts have been dedicated to the development and deployment of an effective ASF vaccine. Among the promising vaccine candidates, live attenuated vaccines (LAVs) are most promising. With CRISPR genome editing technology, new LAV candidates can be readily engineered and tested. Nevertheless, there are significant concerns about LAV vaccines, encompassing critical safety apprehensions such as the potential for recombination with field strains or reverting to virulence, complicating the path to a safe and effective vaccine.

[0005] The key advantage of a subunit vaccine over LAV lies in its enhanced safety profile. Subunit vaccines, which employ viral components rather than replication-competent viruses,overcome the risks associated with reversion to virulence, delayed viraemia, and potential vaccine strain shedding, and limited use in the naive populations. Unfortunately, most historical and contemporary attempts to develop ASFV subunit vaccine candidates have, at best, yielded only partial protection. The primary hurdle in subunit vaccine development for ASFV is the limited knowledge of protective antigens (PAs), primarily due to the virus's complexity. While subunit vaccine candidates have demonstrated their capacity to elicit specific antibodies and T cell responses, these responses have not conferred robust protection.SUMMARY

[0006] Presented herein is an approach to harness capsid proteins for subunit vaccine development by preserving their high-density conformational epitopes and their ability to induce both humoral and cellular immunities without the technical challenges of VLP assembly. The outer shell of ASFV capsid is composed of the major capsid protein P72, which exists as a homotrimer in the capsid, and the minor capsid protein Penton, which exists as a homopentamer in the capsid. Previous attempts to utilize recombinant P72 in subunit vaccine development have been hindered by its tendency to form monomers rather than the more immunogenic native trimers and the requirement for chaperone pB602L for folding. In comparison, the immunogenicity of Penton has not been fully explored. Both P72 and Penton were engineered into membrane-bound and secreted proteins and their immunogenicity was compared to the native intracellular form in mice and pigs through LNP mRNA vaccination. Notably, the membrane-bound and secreted P72 were able to fold into trimers independent of chaperone pB602L and the membrane-bound and secreted Penton also form native pentamer, therefore preserving their conformational epitopes. Quite surprisingly, immunogenicity testing in both mouse and pig models showed that the membrane -bound P72 and Penton elicited significantly stronger antibody and T cell responses than their secreted or intracellular counterparts. Disclosed herein is an approach which enhances folding, formation of native multimeric structures, and immunogenicity of viral capsid proteins, offering a simple strategy for developing safe and effective subunit vaccines for ASFV. This approach is useful for enhancing immunogenicity of any intracellular proteins of pathogen origin or otherwise for preventive and therapeutic vaccination.

[0007] The present invention relates, in part, to the discovery that an mRNA-LNP platform combined with ASFV capsid engineering can enhance B cell and T cell responses. The present invention further relates, in part, to the discovery of a comprehensive approach to dissect the immune response profiles of rationally selected ASFV antigens, and developednew strategies for antigen combination for developing a safe and effective mRNA-based subunit vaccine for ASFV and other viruses. The methodologies developed herein, in some embodiments, may also be applied to develop subunit vaccines for other pathogens with large genomes, such as the monkeypox virus.

[0008] Aspects of the disclosure herein provide a vaccine comprising (a) one or more engineered nucleic acids, wherein the one or more engineered nucleic acids encode African swine fever virus (ASFV) antigens comprised of at least one B cell antigen and at least one T cell antigen; and (b) a lipid nanoparticle (LNP).

[0009] In some embodiments, the ASFV antigens comprise p30, CD2v, C-lectin (EP153R), p22, p54, pE199L, E248R, P72, and / or Penton. In some embodiments, the p72 and / or Penton ASFV antigens comprise a signal peptide of human CD8a on the N-terminus. In some embodiments, the ASFV antigens further comprise a CD8a hinge domain, CD8a transmembrane domain (TMD), and a short cytoplasmic region of CD8a on the C-terminus.

[0010] In some embodiments, the at least one B cell antigen is Penton. In some embodiments, the Penton is mutated to remove a site for glycosylation. In some embodiments, the Penton comprises a N180Q mutation.

[0011] In some embodiments, the at least one T cell antigen comprises multiple T cell epitopes (MTE). In some embodiments, the ASFV antigen, optionally the P30 antigen, comprising the MTE further comprises (a) a GGGS linker; (b) a P2A self-cleavage site; and / or (c) an internal ribosome entry site.

[0012] In some embodiments, the ASFV antigen comprises EP153R, P72, and Penton. In some embodiments, the ASFV antigen further comprises p54. In some embodiments, the ASFV antigen further comprises p30.

[0013] In some embodiments, the ASFV antigen comprises: (a) a T cell antigen comprising multiple T cell epitopes (MTE), CD2v, p30, and P72; (b) a T cell antigen comprising multiple T cell epitopes (MTE), CD2v, E199L, and P72; (c) a T cell antigen comprising multiple T cell epitopes (MTE), CD2v, E199L, P30, and P72; (d) a T cell antigen comprising multiple T cell epitopes (MTE), CD2v, E199L, P30, P72, and Penton; (e) a T cell antigen comprising multiple T cell epitopes (MTE), P72, Penton, EP153R, E199L, and P54; or (f) a T cell antigen comprising multiple T cell epitopes (MTE), EP153R, E199L, P54, CD2v, and P22.

[0014] In some embodiments, the one or more engineered nucleic acids comprise a DNA.

[0015] In some embodiments, the one or more engineered nucleic acids comprises a polyribonucleotide .

[0016] In some embodiments, the LNPs comprise cholesterol, an ionizable lipid, a lipid comprising a polyethylene glycol (PEG) moiety, and a lipid comprising a phosphatidylcholine or phosphatidylethanolamine moiety.

[0017] Further aspects of the disclosure herein provide a method of inducing an immune response to African swine fever virus (ASFV) in a subject, comprising administering to the subject a vaccine comprising: (a) one or more engineered nucleic acids, wherein the one or more engineered nucleic acids encode African swine fever virus (ASFV) antigens comprised of at least one B cell antigen and at least one T cell antigen; and (b) a lipid nanoparticle (ENP), wherein the vaccine is in an amount effective to induce an immune response.

[0018] In some embodiments, the ASFV antigens comprise p30, CD2v, C-lectin (EP153R), p22, p54, pE199E, E248R, P72, and / or Penton. In some embodiments, the ASFV antigens comprise a signal peptide of human CD8a on the N-terminus. In some embodiments, the ASFV antigens further comprise a CD8a hinge domain, CD8a transmembrane domain (TMD), and a short cytoplasmic region CD8a on the C-terminus.

[0019] In some embodiments, the subject is a pig.

[0020] In some embodiments, the administering comprises a first intramuscular injection. In some embodiments, the administering further comprises a second intramuscular injection after the first intramuscular injection.

[0021] In some embodiments, the immune response is a protective immune response.

[0022] Further aspects of the disclosure herein provide a method of engineering a subunit vaccine, the method comprising: (a) identifying one or more intracellular viral antigens from a target virus; (b) engineering the one or more intracellular viral antigens as membrane-bound proteins; (c) engineering multiple T cell epitopes (MTE); (d) determining the effector function profiles of antigen- specific antibodies and the levels of B and T cell responses induced by different intracellular viral antigens and MTE; and (e) determining the antigen combination for subunit vaccine with specific immune response profile; and optionally (f) producing the subunit vaccine.

[0023] In some embodiments, the engineering one or more intracellular viral antigens as membrane-bound proteins comprises adding a signal peptide of human CD8a on the N- terminus of the one or more intracellular viral antigens, and a CD8a hinge domain, CD8a transmembrane domain (TMD), and a short cytoplasmic region CD8a on the C-terminus of the one or more intracellular viral antigens. In some embodiments, the engineering multiple Tcell epitopes (MTE) wherein the MTE comprise a GGGS linker; a P2A self-cleavage site; and / or an internal ribosome entry site.

[0024] In some embodiments, determining the effector function profiles comprise measuring antibody-dependent complement deposition (ADCD); antibody dependent cellular cytotoxicity (ADCC), and / or antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the determining B cell responses comprise measuring antibody secreting B cells (ASC). In some embodiments, the determining T cell responses comprise measuring secretion of cytokine IFN-y and / or TFN-OC.

[0025] Further aspects of the disclosure herein provide a composition comprising an mRNA encoding one or more peptides, wherein the one or more peptides comprise an N-terminal human CD8a signal peptide linked to an intracellular viral protein from a target virus, optionally wherein the intracellular viral protein is linked to a CD8a hinge domain, a CD8a transmembrane domain (TMD), and a cytoplasmic region of CD8a on the C-terminus.

[0026] Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having," “containing”, “involving”, and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. The figures are illustrative only and are not required for enablement of the invention disclosed herein. In the drawings:

[0028] FIGs. 1A-1C. Engineering of ASFV capsid proteins. FIG. 1A shows schematics of vectors expressing three forms of ASFV immunogen. Secreted (S) form contains a signal peptide (SP) in the N-terminus of P72 or Penton. In addition to SP, the membrane-bound(MB) form contains the CD8a hinge, transmembrane domain (TMD), and a truncated cytoplasmic tail. FIGs. 1B-1C show predicted cellular localizations of three forms of Penton (FIG. IB) and P72 (FIG. 1C). The figure was created with BioRender.com.

[0029] FIGs. 2A-2D. Characterization of three forms of P72 and trimer formation. FIG. 2 A (top) shows a representative WB image showing the protein size was denoted as P72(M) to represent the P72 monomer, a-actin was included as a reference for protein size calibration and loading control. FIG. 2A (bottom) shows relative expression levels of all three forms of P72 normalized to the co-expression of IC-P72 and pB602L. Results presented as mean ± standard deviation based on three independent experiments. FIG. 2B shows detection of S- P72 in culture supernatants by ELISA. Culture supernatants were collected from untransfected, empty vector transfected, and S-P72 (w / o P17 or pB602L)-transfected HEK293T cells, followed by ELISA. P72 concentrations were determined through a standard curve and presented as the mean ± SD based on three technical replicates. Control was without culture supernatant. Statistical analysis was conducted using one-way analysis of variance (ANOVA) against empty vector transfected cells. * p<0.05 and ****p<0.0001. FIG. 2C Pearson correlation coefficient of IC-, S- and MB-P72 and cell membrane as measured by ImageJ in two separate experiments. FIG. 2D shows size exclusion chromatography elution profiles of HA-tagged S-P72 (top) and MB-P72 (bottom) protein samples. Black arrows indicate the elution positions of the P72 trimer and monomer.

[0030] FIGs. 3A-3E. Characterization and de-glycosylation of engineered Penton. FIG. 3A shows Western blot analysis of glycosylation and de-glycosylation of engineered Penton. HEK 293T cells were transfected with different Penton plasmids and cell lysates were collected at 48 h post transfection. PNGase treated and untreated clear lysates were subject to SDS-PAGE followed by Western blotting using anti-HA tag mouse monoclonal antibody and goat anti-mouse IgG secondary antibody (IRDYE® 800CW). Pwt, wildtype Penton, PNISOQ: Penton with N to Q mutation at position 180. FIG. 3B shows a comparison of mean fluorescent intensity (MFI) of FITC-HA positive cells transfected with different Penton constructs. FIG. 3C shows a comparison of mean fluorescence intensity (MFI) from IFA images from three independent experiments of IC-, S- and MB-PWT transfected HEK293T cells. FIG. 3D shows Penton and plasma membrane colocalization was calculated by Pearson Coefficients. HEK 293T cells were transfected with plasmids expressing HA-tagged Penton proteins. In 48 hours post transfection, cells were fixed, permeabilized, and stained with FITC-conjugated anti-HA antibody for different assays. Cell plasma membrane was stainedusing anti-ZO-1 rabbit polyclonal antibody followed by Alexa Fluor™ 594-labeled goat antirabbit IgG secondary antibody. Experiments were performed in triplicate technically and repeated at least two times. Representative FACS plot from technical triplicates are shown. Error bars display mean + / - standard derivation (SD), and statistical analysis was performed using one-way ANOVA. ***, P<0.001; ****, P<0.0001. FIG. 3E shows flow cytometry analysis of Penton expression following transfection of HEK293T cells with LNP-mRNA encoding IC-PWT, S-PN180Q and MB-PN180Q. Cells were permeabilized and stained with AF488 conjugated mouse anti-HA. Control (Ctl) was untransfected cells stained the same way.

[0031] FIG. 4. In vitro validation of mRNA-LNP encoding three forms of P72 and Penton. Transfection of HEK293T cells with MB-P72, S-P72, and IC-P72 / pB602L. Cells were lysed 48 hours after transfection and cell lysates were used for Western blotting analysis using mouse anti-P72 followed by HRP-conjugated goat anti-mouse antibodies. Quantification of P72 protein levels using Western blot analysis. P72 monomer size denoted as P72(M), with a-actin as a reference. Histogram shows relative expression levels of P72. Determination of P72 expression levels in HEK293T and Vero cells by normalizing to IC-P72* (IC- P72 / pB602L). Results presented as mean ± standard deviation based on two independent experiments.

[0032] FIGs. 5A-5F. MB-capsid antigens elicited higher levels of humoral responses than the corresponding S- or IC- forms of antigens. FIG. 5 A shows scheme of immunization. BALB / c mice (n=5 mice per group) were intramuscularly (i.m.) injected with LNP mRNA encoding IC-P72 plus pB602L (IC-P72*), S-P72 and MB-P72 and boosted at day 21. Sera were collected before and 14 days after each immunization. Spleen and bone marrow were harvested at day 35. FIG. 5B shows total P72-specific IgM titers at day 14 and day 35. Serum from different groups were 3-fold serially diluted and subjected to antigen- specific ELISA. The titer was determined by interpolating cutoff value (OD450 = Average of placebo + 3 STDEV) from the standard curve of serially diluted sample. FIG. 5C shows total P72-specific IgG titers at day 14 and day 35. Placebo (blue), IC-P72* (purple), S-P72 (magenta), and MB- P72 (orange). Sera from different groups were 3-fold serially diluted and assayed for P72- specific IgG by ELISA. The titer was determined by interpolating cutoff value (OD450 = Average of placebo + 3 SD) from the standard curve of serially diluted samples. FIG. 5D shows the frequencies of IgG secreting cells per million BMPCs (n=5). per million bone marrow cells by ELISPOT. 105bone marrow cells were incubated in wells precoated withanti-IgG for 36 hours and IgG was detected with anti-mouse IgG detecting antibody. Antibody secreting B cells (ASC) were detected by ELISPOT reader. FIG. 5E shows total Penton- specific IgG titers at day 14 and day 35. Serum from different groups were 2-fold serially diluted and subjected to antigen- specific ELISA. End point IgG titer was determined by interpolating cutoff value (OD405 = Average of placebo + 3 STDEV) from the standard curve of serially diluted sample. Placebo (blue), IC-Pwt (purple), S-PN180Q (magenta), and MB-PN180Q (orange) (n=5 mice per group). FIG. 5F shows histogram showing the proportion of antigen- specific IgG spots in total IgG- secreting BMPCs. X-axis represents different groups of mice (n=5 per group). Error bars display mean + / - standard derivation (SD). NS, no significance; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

[0033] FIGs. 6A-6H. MB-capsid antigens elicited higher levels of T cell responses than the corresponding S- or IC- forms of antigens. T cell response for P72 were assayed using single color IFN-y / TNF-a ELISpot kit by measuring the number of cytokine- secreting lymphocytes in mouse spleen (n=5) after stimulation with P72 peptide pool for 36 h. FIG. 6A shows the number of IFN-y secreting cells (SFC) per 106splenocytes from different treatment groups. FIG. 6B shows the number of TNF-a secreting cells (SFC) per 106splenocytes from different treatment groups. T cell response for Penton were assayed using IFN-y / TNF-a dual color ELISpot by measuring the number of cytokine- secreting lymphocytes in mouse spleen (n=5) after stimulation with Penton for 36 h. FIGs. 6C-6E show the number of IFN-y (FIG. 6C), TNF-a (FIG. 6D), and IL-4 (FIG. 6E) secreting cells (SFC) per 106 splenocytes from different groups (n=5). Splenocytes were stimulated with recombinant Penton protein for 36 hours and cytokine-secreting cells were quantified by an IFN-y / TNF-a dual color ELISpot or IL-4 ELISpot assay. FIG. 6F shows a comparison of percentages of CD4+B220'CD44+PD- 1+CXCR5+Tfh cells in the spleen of immunized mice. Splenocytes were stained with antibodies specific for CD4, B220, CD44, PD-1, and CXCR5 followed by flow cytometry. Tfh cells were gated on CXCR5+and PD-1+population among the CD44+CD4+B220‘ T cells. FIG. 6G shows quantification of the numbers of germinal centers from spleen sections of immunized mice. Spleen sections were stained for IgD, CD35, CD3s, and Ki-67, imaged using TissueFAXS fluorescent slide scanner and analyzed using StrataQuest software. Colors denote different cellular markers: blue for Ki67+active proliferating cells, grey for CD35+follicular dendritic cells, yellow for CD3+T cells, and red for IgD+B cell follicles. Scale bar is 500 pm. Error bars display mean ± SD. FIG. 6H shows the frequencies of Penton- specific IgG secreting cells among total IgG-secreting bone marrow cells by ELISPOT (n=5). Plasmacells were enriched from bone marrow using anti-CD138 magnetic beads. 104enriched cells were incubated with Penton coated wells to assay for Penton- specific IgG-secreting cells and 103enriched cells were incubated in wells coated with anti-IgG to assay for total IgG- secreting cells. Data are shown as mean ± SD. One-way ANOVA was used for statistical analysis between different groups. NS, no significance; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

[0034] FIGs. 7A-7B. T follicular helper (TFH) cell and Germinal center (GC) responses from immunized mice. FIG. 7A shows a histogram showing the percentage of CXCR5+PD-1+TFH cells from different treatment groups (n=5 per group). Quantification data of splenic germinal center are shown in FIG. 7B for number of GCs per section, and show part of Spleen embedded in O.C.T. was snap-frozen in liquid nitrogen, followed by cryo-sectioning. Slides were air-dried, blocked, and stained with abovementioned antibodies for different cellular markers. Stained slides were subject to autofluorescence quenching treatment and coverslips were mounted using antifade mountant, followed by imaging using TissueFAXS fluorescent slide scanner and analyzed using StrataQuest software. X-axis represents different groups of mice (n=5 per group). Error bars display mean + / - standard derivation (SD). NS, no significance; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

[0035] FIGs. 8A-8E. Immunogenicity evaluation of MB-capsid antigen mRNA vaccines in pigs. FIG. 8A shows two groups of pigs (n=4 per group) were immunized with LNP-mRNAs expressing MB-P72 or MB-PN180Q, and one control group of pigs (n=2) were injected with sterile PBS at day 0 and 21. Sera were collected before immunization and weekly after immunization for assaying antigen- specific IgG responses by ELISA. Spleen was harvested at day 35 and single cell suspension were stimulated with a P72 peptide pool or recombinant Penton protein to assay IFN-y secreting cells by ELISPOT FIG. 8B shows IgG antibody response induced by MB-P72, MB-PNisoQOver time. Weekly collected pig serum was assayed by indirect ELISA to test antigen- specific IgG response. Bars of different colors represent each individual LNP-mRNA as shown. FIG. 8C shows IFN-y ELISpot showing the frequencies of IFN- y secreting cells (SFC) per 106splenocytes from MB-P72 or MB- PN180Q immunized pigs. FIG. 8D shows comparison of antigen- specific IgG titers among MB-P72 or MB-PN180Q immunized mice and pigs. FIG. 8E shows comparison of IFN-y secreting cells among MB-P72 or MB-PN180Q immunized mice and pigs.

[0036] FIGs. 9A-9C. Rational selection, design, and in vitro validation of ASFV mRNA vaccine candidate antigens. FIG. 9A shows a schematic diagram for ASFV of extracellularenveloped form. Each layer of viral structure was illustrated with distinctive colors including outer membrane, capsid layer, inner membrane, core shell, and viral DNA genome. Antigens selected under each layer are depicted under each layer. FIG. 9B shows the three different designs for expressing T cell-directed antigens. Top shows P30-MTE that have P30 fused with multi-T cell epitope (MTE) antigen by a GGGS (SEQ ID NO: 113) linker. Middle shows P30-P2A-MTE in which P2A self-cleavage site links P30 and MTE. Bottom shows an internal ribosome entry site (IRES)-linked construct (P30-IRES-MTE). All three constructs contain an HA tag in C terminus for easy detection. FIG. 9C shows flow cytometry analysis for expression of ASFV antigens following lipid nanoparticle (LNP)-mRNA transfection of HEK 293T cells. Monolayer HEK 293T cells were transfected with DNA plasmids by polyethylenimine or LNP-mRNAs by direct adding to cells. Cells were harvested and fixed at 48 hours post transfection and stained with mouse mAbs specific for P72, P54, P30, and mouse anti-HA tag mAb for CD2v, C-lectin, P22, pE199L, pE248R).

[0037] FIGs. 10A-10D. In vivo evaluation of candidate antigens in mice. FIG. 10A shows IgG antibody response from a mouse study. 12 groups of mice treated with different LNP- mRNAs or PBS as placebo control. Each group of mice (n=5) were immunized with 5 pg of LNP-mRNA and boosted in three weeks. Blood were collected before priming and two weeks after each injection. Mice were euthanized in two weeks at week 5 after boost and spleen tissue was collected for immune analysis. Mouse serum (n=5 per group) collected from 14 days post immunization (DPI) and 35 DPI were subject to antigen- specific total IgG analysis by ELISA. For P30-MTE groups, antibodies against P30 protein were measured. The endpoint titer was defined as the reciprocal of the highest serum dilution that gives a reading above the cutoff which was determined by average OD405 readings of placebo mice + 3 standard deviation. FIGs. 10A-10B show T cell immunity was assayed by IFN-y and TNF-a dual ELISpot using isolated splenocytes for CD2v, C-Lectin, Penton, P22, P54 (FIG. 10B), and P30, MTE (FIG. 10B). 105splenocytes from each mouse were seeded on ELISpot plate and stimulated with 5 pg / mL of either purified proteins or MTE peptide cocktail.PMA / ionomycin cocktail and medium were included as positive, negative stimulation control, respectively. Dual-cytokine ELISpot was performed in 36 hours post stimulation by referring to manufactural instructions. The number of spot-forming cells (SFC) per 105splenocytes was calculated and plotted. FIG. 10D shows mean fluorescent intensity and calculated inhibiting percentage for hemoadsorption inhibition assay (HADIA) were assessed for CD2v antibodies. Mouse serum (n=5) from LNP-mRNA expressing CD2v immunizedgroup was treated with receptor destroying enzyme (RDE) to remove unspecific factors inhibiting hemoadsorption, followed by mixing with HEK 293T cells expressing HA-tagged CD2v protein. After incubation to form immune-complex, 2% porcine red blood cells (RBC) were added and kept for 24 hours in cell incubator. Cells were fixed, permeabilized, and stained with mouse anti-HA mAb and rabbit anti-RBC pAb. AF594-labled anti-rabbit and AF488-labled anti-mouse IgG were added for secondary antibody staining. Cells were counterstained with DAPI before imaging using confocal microscope. For EEISpot assay, one-way ANOVA for EEISpot assay and student’s t-test for HADIA were used for statistical analysis. Each circle represents one mouse in FIGs. 10A-10D. NS, no significance; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

[0038] FIGs. 11A-11D. Immunogenicity evaluation of candidate antigens in pigs. Ten groups of pigs (n=4 per group) were injected with LNP-mRNAs expressing P72, Penton, P22, pE199L, P54, CD2v, C-Lectin, P30-IRES-MTE, P30-P2A-MTE, PBS and boosted in three weeks. Two weeks after boost, pig study was terminated for tissue collection. Blood samples were collected before immunization and weekly after each injection. FIG. 11 A shows IgG antibody response. Weekly collected pig serum were assayed by indirect ELISA to test antigen- specific IgG response. X-axis represents time points for blood collection. Histograms of different colors represent each individual LNP-mRNAs. FIG. 11B shows IFN-y ELISpot showing cellular response in 105splenocytes after stimulation by different antigens. Y-axis shows the number of spot-forming cells (SFC). FIGs. 11C-11D show Pearson correlations for antibody response (FIG. 11C) and cellular response (FIG. 1 ID) evaluated in pigs (X-axis) and mice (Y-axis). Opened circles represents individual antigens for CD2v, C-lectin, P72, Penton, P54, P22, pE199L, P30 (FIG. 11C) and an additional MTE (FIG. 1 ID). Arrows in (FIG. 1 ID) point to MTE. Equations for the linear regression and Pearson correlation coefficients along with P values were shown. Shading area shows the filled error bars.

[0039] FIGs. 12A-12D. Evaluation of the candidate cocktail mRNA vaccine in mice. FIGs. 12A shows design of four candidate cocktail vaccines. 5 pg per LNP-mRNA was used to prepare the cocktail vaccine. Four groups of mice (n=5 per group) were injected with respective cocktail vaccine and boosted in three weeks and terminated in two weeks after boost. FIG. 12B shows a comparison of antigen-specific IgG titers at day 14 and 35 after immunization for the indicated antigens. FIG. 12C shows a comparison of IFN-y ELISpots in splenocytes following stimulation with MTE peptide cocktail, P72 peptides, and CD2v protein. FIG. 12D shows Pearson correlations for IgG (top) and T cell (bottom) responsesinduced by cocktail vaccination (X-axis) and individual antigen vaccination (Y-axis). Each circle represents one antigen: P30, P72, CD2v, Penton, E199L, P22 (top), and MTE, P72, CD2v (bottom). Equations and correlation coefficients are shown.Cl, C2, C3 and C4 represents cocktail 1, cocktail 2, cocktail 3, and cocktail 4, respectively.

[0040] FIGs. 13A-13G. Correlations of B and T cell responses. FIG. 13A shows the correlation coefficient was calculated for IgG and IgM titers induced by P72. The correlation coefficient was calculated for ASC and IgG titers and ASC and IgM titers, shown in (FIG. 13B) and (FIG. 13C) respectively. The correlation coefficient was calculated for IFNy / IgG, TNF-a / IgG, IFNy / IgM, and TNF-a / IgM titers, shown in (FIGs. 13D-13G), respectively. The plots were generated, and correlation coefficients were calculated using GraphPad Prism 8.0.

[0041] FIGs. 14A-14B: Schematic diagrams for DNA and mRNA construct design. FIG. 14A shows DNA constructs encoding membrane-bound (MB) P72, MB-Penton with a single mutation for de-glycosylation, and other genes including CD2v, C-lectin, P22, pE199E, pE248R, and P54. The membrane- anchoring strategy was employed by addition of signal peptide (SP) in N terminus, a hinge, transmembrane domain (TMD), and short cytoplasmic tail in C terminus and P72 and Penton. A HA tag was added in the C terminus of all constructs except MB-P72 of which a specific mAb was used for detection. Kozak sequence was added in N terminus of all constructs in aid of protein expression. The vertical line represents N180Q mutation to remove the glycan site on Penton. SP, signal peptide. FIG. 14B shows mRNA construct design. To produce mRNA for ENP encapsulation, all designed genes were inserted into a pUC vector containing 5’ untranslational region (5’UTR) in N terminus, 3’UTR and PolyA in C terminus. The in vitro transcribed mRNA undergoes post- transcriptional capping using Vaccinia Capping Enzyme. For testing in mice, mRNA constructs for all but P72 and MTE contain a HA tag in C terminus of each gene, while the HA tag was removed for testing in mice and pigs.

[0042] FIG. 15: Total IgG response in mice for individual antigens on the indicated days. Antigen- specific IgG responses normalized to day 0 (before immunization). The endpoint titer was defined as the reciprocal of the highest serum dilution that gives a reading above the cutoff which was determined by average OD405 readings of placebo mice + 3 standard deviation

[0043] FIG. 16. Total IgG response for individual antigens in pigs. Serum samples from pigs immunized with ENP-mRNAs expressing individual antigens were weekly collected till termination at day 35. Serum from all time points were 2-fold serially diluted and subjectedto antigen- specific ELISA for CD2v, C-lectin, P72, Penton, P22, P54, pE199L, and p30 (P30- IRES-MTE, P30-P2A-MTE). The End point IgG titer was determined by interpolating cutoff value (OD405 = Average of placebo + 3 STDEV) from the standard curve of serially diluted sample. Antigen- specific IgG responses normalized to day 0 (before immunization). For MTE groups, IgG titers specific for P30 were measured. Data was analyzed by using GraphPad, PRISM. X-axis represents sampling time point and the top shows the name of each immunization group.

[0044] FIGs. 17A-17B. Comparison of memory T cell responses induced by native and engineered Penton. Splenocytes collected at week 2 after second immunization were subject to multi-color staining (CD3, CD4, CD8, CD44, CD62L) for effector memory CD8 T cell response. Single live cells were gated on CD3+CD8+CD44hlghCD62L'to identify effector memory CD8 T cells. FIG. 17A shows fluorophore minus one (FMO) were used to identify CD44 or CD62L positive populations. FIG 17B shows a comparison of percentages of effector memory CD8 T cells in four groups of mice.

[0045] FIGs. 18A-18F. Evaluation of antibody effector functions. FIG. 18A shows a schematic diagram of ADCD assay. FIG. 18B shows a comparison of percentages of lysis of CHO cells among sera from pigs immunized with the indicated ASFV antigens. The antigenspecific ADCD was calculated by normalizing to no- serum control first and then the control group serum. FIG. 18C shows a schematic diagram of ADCC assay. FIG. 18D shows a comparison of percentages of lysis of CHO cells among sera from pigs immunized with the indicated ASFV antigens. The antigen- specific ADCC was calculated by normalizing to noserum control first, then the Triton X-100 positive control well, and finally the control group serum. FIG. 18E shows a schematic diagram of ADCP assay. FIG. 18F shows a comparison of phagocytic scores among sera from pigs immunized with the indicated ASFV antigens. P30-IRES and P30-P2A refer to P30-specific antibodies following P30-IRES-MTE and P30- P2A-MTE immunization, respectively. Phagocytic score was determined by multiplying the percentage of fluorescent-bead-positive cells by the mean fluorescent intensity (MFI) of the same population. The antigen- specific phagocytosis was computed by normalizing the phagocytic score to control group pigs. Each circle represents one pig. *, P<0.05; **, P<0.01;***, P<0.001; ****, P<0.0001.

[0046] FIG. 19. shows computational analysis of immune response profiles of specific antigens. A polar bar plot of rank scores (1 to 8) of the indicated antigens in fiveimmunological categories is shown. The ggplot2 library in R is used to generate the Polar bar plot of rank scores for each antigen.

[0047] FIGs. 20A-20C. Comparison of rank scores of antigen combinations for cocktail vaccines. 3-way (FIG. 20A), 4-way (FIG. 20B), and 5-way (FIG. 20C) antigen combinations based on rank sums of 5 immune parameters ("IgG","IFN-y","ADCD","ADCC","ADCP"), 4 parameters (,TgG",,TFN-Y","ADCC","ADCD"), 3 parameters (,TgG",,TFN-Y","ADCC"), and 2 parameters ("IgG","IFN-Y") colored in black, blue, green, and red, respectively. Arrows point to the combinations with highest sum scores in different categories.

[0048] FIGs. 21A-21B. Representative plasmid maps for CD2v protein expression (FIG. 21A) and mRNA synthesis (FIG. 21B). DNA constructs encoding ASFV genes were shown as an example with detailed elements.DETAILED DESCRIPTION

[0049] The disclosure herein discloses an mRNA-LNP platform combined with ASFV capsid engineering to enhance B cell and T cell responses. It is shown in the examples that ASFV capsid proteins P72 and Penton can be engineered as membrane -bound and secreted forms while preserving their multimeric structure without the use of chaperon pB602L. Notably, the membrane-bound and secreted P72 were able to fold into trimers independent of chaperone pB602L and the membrane-bound and secreted Penton also form native pentamer, therefore preserving their conformational epitopes. Surprisingly, immunogenicity testing in both mouse and pig models showed that the membrane -bound P72 and Penton elicited significantly stronger antibody and T cell responses than their secreted or intracellular counterparts. Disclosed herein, in part, is an approach which enhances folding, formation of native multimeric structures, and immunogenicity of viral capsid proteins, offering a simple strategy for developing safe and effective subunit vaccines for viruses with large genomes, such as ASFV. More broadly, the same approach should improve immunogenicity of any intracellular proteins of pathogen origin or otherwise for preventive and therapeutic vaccination. The inventors further discovered a comprehensive approach to dissect the immune response profiles of rationally selected viral antigens and developed new strategies for antigen combination for developing a safe and effective mRNA-based subunit vaccine for viruses such as ASFV. The methodologies developed herein, in some embodiments, may be applied to develop subunit vaccines for pathogens with large genomes, such as the monkeypox virus.

[0050] Reference numbers in brackets “()” herein refer to the corresponding literature listed in the Reference list, which forms a part of this Specification, and the literature is incorporated by reference herein.

[0051] There are four main hurdles to develop effective viral vaccines based on large genome viruses, such as ASFV (ASFV is provided as a non-limiting example): 1) Viral antigens to elicit protective immunity against ASFV infection have not been identified. ASFV encodes more than 170 proteins. Selecting candidate antigens for eliciting host immunities is a challenge and researchers have tested many candidate proteins in animals with no success resulting in full protection from ASFV infection; 2) ASFV contains a complex multi-layer structure. During the viral replication cycle, both newly released extracellular virion containing all layers and intracellular particle without the outer membrane are infectious. 3) ASFV employs multiple entry mechanisms to infect target cells, including receptor-mediated endocytosis, clathrin-mediated endocytosis, macropiniocytosis, and phagocytosis. 4) Immunological correlates of protective immunity against ASFV are unknown. ASFV protective immunity remains poorly defined. Neutralizing antibody responses elicited by an inactivated ASFV vaccine was not sufficient to protect pigs from infection (14) .Immune responses by attenuated virus confer protection (15). T cell immunity likely also plays a pivotal role in protection. Lymphocyte depletion studies showed that cytotoxic CD8+lymphocytes are important for ASFV clearance IFN-gamma responsescorrelate with the degree of cross-protection against heterologous ASFV challenge (17). Therefore, an effective ASFV vaccine should induce both humoral and cellular immunities.

[0052] ASFV is the only member in Asfarviridae, which is a family of Nucleocytoplasmic large DNA viruses (NCLDV) containing a group of large dsDNA viruses or giant viruses characterized by the most complex virion structure, large genomes, and protein-coding capacity Poxviridae, another well-characterized family in NCLDV, is close to Asfarviridae phylogenetically (FIG. 15). As a key member of Poxviridae, the use of Vaccinia virus (VACV) as a vaccine contributes tremendously to the eradication of smallpox disease caused by Variola virus VACV can form two infectious virions: ExtracellularEnveloped Virion (EEV) and Intracellular Mature Virion (IMV). Multiple studies have demonstrated that subunit vaccines encompassing two EEV antigens (B5R and A33R) and two IMV antigens (L1R and A27L) provide complete protection against lethal VACV challenge (20-23). ASFV has homologous genes to B5R, A33R, L1R and A27L in VACV. It is possible their use as immunogens may stimulate protection against ASFV.

[0053] There are several reports of ASFV proteins for immune protection. P72, P54, and P30 are reported to inhibit both virus attachment and internalization (24). Immunization with a combination of P54 and P30 provides partial protection against lethal virus challenge while individual immunizations of either P54 or P30 did not affect the disease course (25). ASFV EP402R (CD2v) and EP153R (C-lectin) contains many T cell epitopes and deletion of both genes reduced viral persistence in blood while deletion of EP153R (C-lectin) alone has no effect, suggesting a synergistic role of these two proteins in disease protection (26, 27). CD2v also mediates hemadsorption (28, 29). In addition to T cell responses, both P72 and CD2v homologous to VACV protective antigens were also reported to induce strong neutralizing antibody and cellular responses which ultimately provides partial protection against lethal viral challenge (24, 29-34). KP177R (P22) is a high immunogenic protein with application for serological diagnostics and potential role in maintaining virion structure (35, 36). Viral proteins responsible for membrane fusion are also potential targets of neutralizing antibodies. ASFV pE248R is required for virus infectivity and early post entry events (37). pE199L was shown to mediates virus entry by enabling membrane fusion and core penetration.

[0054] Aspects of the disclosure herein comprise an mRNA-LNP platform combined with ASFV capsid engineering to enhance B cell and T cell responses. The inventors demonstrated that ASFV capsid proteins P72 and Penton can be engineered as membrane-bound and secreted forms while preserving their multimeric structure without the use of chaperon pB602L. A single amino acid mutation successfully eliminates glycosylation on the engineered Penton protein. The immunogenicity of the mRNA vaccines in both mouse and pig models were tested, and unexpectedly, the results showed that MB-P72 and MB-PNISOQ elicited significantly stronger B cell and T cell responses. This proof-of-concept study demonstrates a novel approach for engineering viral capsid proteins as immunogens, highlighting its potential for developing highly effective vaccines and its applicability to diverse uses.

[0055] In some aspects, provided herein are technologies (e.g., compositions and methods) for augmenting, inducing, promoting, enhancing and / or improving an immune response against viral antigens (e.g., ASFV antigens) or a component thereof (e.g., a protein or portion thereof). In some embodiments, technologies provided herein are designed to augment, induce, promote, enhance and / or improve immunological memory against virus (e.g., ASFV virus) or a component thereof (e.g., a protein or portion thereof). In some embodiments, technologies described herein are designed to act as an immunological boost to a primaryvaccine, such as a vaccine directed to an epitope and / or epitopes of a virus (e.g., ASFV). In some embodiments, compositions of the present disclosure comprise one or more engineered nucleic acids (e.g., one or more polynucleotides) that encode one or more antigens (e.g,, epitopes) from a virus (e.g., ASFV). In some embodiments, the present disclosure provides vaccines or other compositions comprising nucleic acids encoding such viral (e.g., ASFV) epitopes; those skilled in the art will appreciate from context when reference to a particular polynucleotide (e.g., a DNA or RNA) as “encoding” such epitopes in fact is referencing a coding strand or its complement. In some embodiments, a polynucleotide is a polyribonucleotide .Antigens

[0056] In some aspects, the present disclosure provides pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) that deliver particular viral antigen constructs (e.g. large genome viral antigens, such as, ASFV antigens) to a subject (e.g., a patient) and related technologies (e.g., methods). In some embodiments a subject is a non-human mammal, such as a pig, cow or rodent. In some embodiments the subject is a human.

[0057] In some embodiments, the present disclosure provides certain viral antigen constructs (e.g., ASFV antigens) particularly useful in effective vaccination.

[0058] Antigens utilized in accordance with the present disclosure are or include viral components (e.g., proteins or fragments or epitopes thereof, including epitopes that may comprise non-naturally occurring amino acids), which components induce immune responses when administered to humans (or other animals such as rodents and non-human primates susceptible to viral infection).

[0059] In some embodiments, viral antigens utilized in provided pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) include at least one B cell antigen and at least one T cell antigen, as described herein. In some embodiments, delivered antigens include both a B-cell antigen and a T cell antigen encoded by one or more engineered nucleic acids. In some embodiments, antigens utilized in provided pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) include multiple T cell epitopes (MTEs). In some embodiments, antigens utilized in provided pharmaceutical compositions (e.g., immunogenic composition, e.g., vaccine), together, include B cell, CD4 T cell and CD8 T cell epitopes. Indeed, in some embodiments, the present disclosure defines particularly useful epitopes for inclusion in viral vaccines, and / or provides antigens thatinclude them. Exemplary viral antigens can be antigens, and the present disclosure provides pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) that deliver particular ASFV antigen constructs to a subject (e.g., a patient) and related technologies (e.g., methods).

[0060] In some embodiments, the present disclosure provides certain engineered nucleic acids encoding antigens derived from large genome viruses particularly useful in effective vaccination. Viruses having large genomes are known in the art and include, but are not limited to ASFV, monkeypox virus, smallpox or variola virus, vaccinia virus.

[0061] In some embodiments, the present disclosure provides certain engineered nucleic acids encoding ASFV antigens particularly useful in effective vaccination.

[0062] Antigens utilized in accordance with the present disclosure are or include ASFV components (e.g., proteins or fragments or epitopes thereof, including epitopes that may comprise non-amino acid, e.g., carbohydrate moieties), which components induce immune responses when administered to humans (or other animals such as rodents and non-human primates susceptible to ASFV infection).

[0063] In some embodiments, antigens utilized in provided pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) include at least one B cell antigen and at least one T cell antigen (e.g., wherein the at least one T cell antigen comprises multiple T cell epitopes), as described herein. Exemplary ASFV antigens and / or epitopes for use in compositions described herein can be found in, e.g., Table 1, Table 5, and / or Table 10 herein. Other ASFV antigens and / or epitopes known in the art can be useful for compositions described herein are also contemplated for use.

[0064] Some aspects of the disclosure provide a vaccine comprising one or more engineered nucleic acids, also referred to herein as polynucleotides (e.g., a polyribonucleotide), wherein the one or more engineered nucleic acids encode viral antigens. For example, in some embodiments, a vaccine comprises one or more engineered nucleic acids, wherein the one or more engineered nucleic acids encode African swine fever virus (ASFV) antigens. In some embodiments, an ASFV antigen is a B cell antigen. In some embodiments, an ASFV antigen is a T cell antigen. In some embodiments, a vaccine comprises one or more engineered nucleic acids encode ASFV antigens comprised of at least one B cell antigen and at least one T cell antigen. In some embodiments, a B cell antigen is Penton. In some embodiments, a T cell antigen comprises multiple T cell epitopes (MTE).

[0065] In some embodiments, a viral antigen (e.g., an ASFV antigen) comprising the MTE further comprises a GGGS linker; a P2A self-cleavage site; and / or an internal ribosome entry site.

[0066] The P72 protein, also known as the major capsid protein, plays a pivotal role in capsid assembly and structural integrity. Its high immunogenicity, driving both B cell and T cell responses, makes it an appealing target for vaccine research. Various vaccine strategies have revolved around recombinant P72 protein, plasmid DNA or viral vectors engineered for P72 expression, aiming to induce a protective immune response in swine. Although P72-based vaccines have demonstrated potential in stimulating antibody production, their capacity to contribute protection against ASFV remains suboptimal. This limitation is likely partly attributed to that the native P72 forms trimer whereas recombinant P72 is usually monomeric. It has been revealed that P72 initially presents as monomers but must undergo correct trimeric formation to prevent aggregation. Additionally, the viral protein pB602L is known to function as a critical chaperone for P72 folding, trimer formation and subsequent capsid assembly. As conformational epitopes are pivotal for inducing neutralizing antibodies, the use of P72 trimers as immunogen would be critical for inducing optimal neutralizing antibody responses. Moreover, P72 can also induce T cell responses, which are essential for protective immunity. In some embodiments, a provided vaccine comprises an engineered nucleic acid (e.g., a polynucleotide) encoding a P72 protein, or fragment or epitope thereof. In some embodiments, a polynucleotide as described herein encodes a polypeptide, where the polypeptide comprises one or more antigens, and where the one or more antigens comprises a P72 protein, or fragment or epitope thereof. A non-limiting example of a nucleic acid encoding P72 (B646L) gene is from Georgia 2007 / 1 strain (NCBI Reference Sequence: NC_044959.2).

[0067] In some embodiments, a provided vaccine comprises an engineered nucleic acid (e.g., a polynucleotide) encoding a p30 protein, or fragment or epitope thereof. In some embodiments, a polynucleotide as described herein encodes a polypeptide, where the polypeptide comprises one or more antigens, and where the one or more antigens comprises a p30 protein, or fragment or epitope thereof. A non-limiting example of a nucleic acid encoding p30 (CP204L) gene is from Georgia 2007 / 1 strain (NCBI Reference Sequence: NC_044959.2).

[0068] In some embodiments, a provided vaccine comprises an engineered nucleic acid (e.g., a polynucleotide) encoding a CD2v protein, or fragment or epitope thereof. In someembodiments, a polynucleotide as described herein encodes a polypeptide, where the polypeptide comprises one or more antigens, and where the one or more antigens comprises a CD2v protein, or fragment or epitope thereof. A non-limiting example of a nucleic acid encoding CD2v (EP402R) gene is from Georgia 2007 / 1 strain (NCBI Reference Sequence: NC-044959.2).

[0069] In some embodiments, a provided vaccine comprises an engineered nucleic acid (e.g., a polynucleotide) encoding a C-lectin (EP153R) protein, or fragment or epitope thereof. In some embodiments, a polynucleotide as described herein encodes a polypeptide, where the polypeptide comprises one or more antigens, and where the one or more antigens comprises a C-lectin (EP153R) protein, or fragment or epitope thereof. A non-limiting example of a nucleic acid encoding C-type lectin (EP153R) gene is from Georgia 2007 / 1 strain (NCBI Reference Sequence: NC_044959.2).

[0070] In some embodiments, a provided vaccine comprises an engineered nucleic acid (e.g., a polynucleotide) encoding a p22 protein, or fragment or epitope thereof. In some embodiments, a polynucleotide as described herein encodes a polypeptide, where the polypeptide comprises one or more antigens, and where the one or more antigens comprises a p22 protein, or fragment or epitope thereof. A non-limiting example of a nucleic acid encoding P22 (KP177R) gene is from Georgia 2007 / 1 strain (NCBI Reference Sequence: NC_044959.2).

[0071] In some embodiments, a provided vaccine comprises an engineered nucleic acid (e.g., a polynucleotide) encoding a p54 protein, or fragment or epitope thereof. In some embodiments, a polynucleotide as described herein encodes a polypeptide, where the polypeptide comprises one or more antigens, and where the one or more antigens comprises a p54 protein, or fragment or epitope thereof. A non-limiting example of a nucleic acid encoding P54 (E183L) gene is from Georgia 2007 / 1 strain (NCBI Reference Sequence: NC_044959.2).

[0072] In some embodiments, a provided vaccine comprises an engineered nucleic acid (e.g., a polynucleotide) encoding a pE199L protein, or fragment or epitope thereof. In some embodiments, a polynucleotide as described herein encodes a polypeptide, where the polypeptide comprises one or more antigens, and where the one or more antigens comprises a pE199L protein, or fragment or epitope thereof. A non-limiting example of a nucleic acid encoding pE199L gene is from Georgia 2007 / 1 strain (NCBI Reference Sequence: NC_044959.2).

[0073] In some embodiments, a provided vaccine comprises an engineered nucleic acid (e.g., a polynucleotide) encoding a E248R protein, or fragment or epitope thereof. In some embodiments, a polynucleotide as described herein encodes a polypeptide, where the polypeptide comprises one or more antigens, and where the one or more antigens comprises a E248R protein, or fragment or epitope thereof. A non-limiting example of a nucleic acid encoding pE248R gene is from Georgia 2007 / 1 strain (NCBI Reference Sequence: NC-044959.2).

[0074] In some embodiments, a provided vaccine comprises an engineered nucleic acid (e.g., a polynucleotide) encoding a Penton protein, or fragment or epitope thereof. In some embodiments, a polynucleotide as described herein encodes a polypeptide, where the polypeptide comprises one or more antigens, and where the one or more antigens comprises a Penton protein, or fragment or epitope thereof. A non-limiting example of a nucleic acid encoding Penton (H240R) gene is from Georgia 2007 / 1 strain (NCBI Reference Sequence: NC_044959.2). In some embodiments, a Penton is mutated to remove a site for glycosylation. In some embodiments, the Penton comprises a N180Q mutation.

[0075] In some embodiments, a provided vaccine comprises one or more engineered nucleic acids (e.g., a polynucleotide) encoding p30, CD2v, C-lectin (EP153R), p22, p54, pE199L, E248R, P72, and / or Penton.

[0076] In some embodiments, one or more engineered nucleic acids encoding viral antigens (e.g., ASFV antigens) are engineered to encode a secreted form of the viral antigen (e.g., ASFV antigen). For example, to express a viral antigen as a secreted protein, in some embodiments, a signal peptide is added to the N-terminus of the sequence encoding the antigen. In some embodiments, the signal peptide of human CD8a is added to the N- terminus. In some embodiments, the signal peptide of human CD8a is added to the N- terminus of a viral antigen. In some embodiments, the signal peptide of human CD8a is added to the N-terminus of p30, CD2v, C-lectin (EP153R), p22, p54, pE199E, E248R, P72, and / or Penton.

[0077] In some embodiments, one or more engineered nucleic acids encoding viral antigens (e.g., ASFV antigens) are engineered to encode a membrane-bound form of the viral antigen (e.g., ASFV antigen). For example, to express a viral antigen as a membrane-bound protein, a signal peptide is added to the N-terminus of the sequence encoding the antigen, and a hinge domain, a transmembrane domain (TMD), and a short cytoplasmic region from the same signal peptide is added to the C-terminus of the viral antigen. In some embodiments, toexpress a viral antigen as a membrane-bound protein, the signal peptide of human CD8a is added to the N-terminus of the sequence encoding the antigen, and a hinge domain, a transmembrane domain (TMD), and a short cytoplasmic region from the human CD8a signal peptide is added to the C-terminus of the viral antigen. In some embodiments, the addition of the CD8a hinge domain does not induce significant structural alterations and provides sufficient flexibility and space to accommodate formation of a viral antigen multimer. In some embodiments, to express a viral antigen as a membrane-bound protein, the signal peptide of human CD8a is added to the N-terminus of the sequence encoding the antigen (e.g., p30, CD2v, C-lectin (EP153R), p22, p54, pE199L, E248R, P72, and / or Penton), and a hinge domain, a transmembrane domain (TMD), and a short cytoplasmic region from the human CD8a signal peptide is added to the C-terminus of the viral antigen (e.g., p30, CD2v, C-lectin (EP153R), p22, p54, pE199L, E248R, P72, and / or Penton).

[0078] In some embodiments, a provided vaccine comprises one or more engineered nucleic acids encoding EP153R, P72, and Penton.

[0079] In some embodiments, a provided vaccine comprises one or more engineered nucleic acids encoding EP153R, P72, Penton, and p54.

[0080] In some embodiments, a provided vaccine comprises one or more engineered nucleic acids encoding EP153R, P72, Penton, and p30.

[0081] In some embodiments, a provided vaccine comprises one or more engineered nucleic acids encoding EP153R, P72, Penton, p54, and p30.

[0082] In some embodiments, a provided vaccine comprises one or more engineered nucleic acids encoding a T cell antigen comprising multiple T cell epitopes (MTE), CD2v, p30, and P72.

[0083] In some embodiments, a provided vaccine comprises one or more engineered nucleic acids encoding a T cell antigen comprising multiple T cell epitopes (MTE), CD2v, E199L, and P72.

[0084] In some embodiments, a provided vaccine comprises one or more engineered nucleic acids encoding a T cell antigen comprising multiple T cell epitopes (MTE), CD2v, E199L, P30, and P72.

[0085] In some embodiments, a provided vaccine comprises one or more engineered nucleic acids encoding a T cell antigen comprising multiple T cell epitopes (MTE), CD2v, E199L, P30, P72, and Penton.

[0086] In some embodiments, a provided vaccine comprises one or more engineered nucleic acids encoding a T cell antigen comprising multiple T cell epitopes (MTE), P72, Penton, EP153R, E199L, and P54.

[0087] In some embodiments, a provided vaccine comprises one or more engineered nucleic acids encoding a T cell antigen comprising multiple T cell epitopes (MTE), EP153R, E199L, P54, CD2v, and P22.

[0088] In some embodiments, a provided vaccine comprises one or more engineered nucleic acids comprising DNA. In some embodiments, a provided vaccine comprises one or more engineered nucleic acids comprising RNA (e.g., a polyribonucleotide). In some embodiments, a provided vaccine comprises one or more engineered nucleic acids comprising mRNA.

[0089] In some embodiments, a viral antigen (e.g., AFSV antigen) described herein may be a fragment or epitope thereof. In some embodiments, a viral antigen (e.g., ASFV antigen) may include one or more sequence alterations relative to a particular reference viral protein, or fragment or epitope thereof. For example, in some embodiments, a viral antigen (e.g., AFSV antigen) may include one or more sequence variations found in circulating strains or predicted to arise, e.g., in light of assessments of sequence conservation and / or evolution of viral proteins over time and / or across strains. Alternatively or additionally, in some embodiments, a viral antigen (e.g., AFSV antigen) may include one or more sequence variations selected, for example, to impact stability, folding, processing and / or display of the antigen or any epitope thereof.

[0090] In some embodiments, a viral antigen (e.g., AFSV antigen), or fragment or epitope thereof, as described herein shows at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with a relevant corresponding reference protein, fragment or epitope. In some embodiments, a viral antigen (e.g., AFSV antigen), or fragment or epitope thereof, as described herein shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology (i.e., identity or conservative substitution as is understood in the art) amino acid sequence identity with a relevant corresponding reference protein, fragment or epitope. Moreover, in some embodiments, a viral antigen (e.g., AFSV antigen), or fragment or epitope thereof, as described herein shares conserved amino acid residues (e.g., at corresponding positions) with a relevant corresponding reference protein,fragment or epitope. In some embodiments, a viral antigen (e.g., AFSV antigen), or fragment or epitope thereof, as described herein shows at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32. Those skilled in the art will appreciate that, in general, lower percent identity or homology may be tolerated for shorter peptides, as a single change will by definition have a larger impact on percent identity or homology when considered relative to a smaller number of residues. For example, those skilled in the art will appreciate that, for sequences longer than about 20 amino acids, percent identity or homology is typically greater than about 80%; for sequences longer than about 50 amino acids, percent identity or homology is typically greater than about 90%.

[0091] In some embodiments, a nucleic acid sequence encoding a viral antigen (e.g., AFSV antigen), or fragment or epitope thereof as described herein shows at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a relevant corresponding reference nucleic acid sequence. In some embodiments, a nucleic acid sequence encoding a viral antigen (e.g., AFSV antigen), or fragment or epitope thereof, as described herein shows at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31.

[0092] In some embodiments, a vaccine described herein comprises (a) one or more engineered nucleic acids, wherein the one or more engineered nucleic acids encode African swine fever virus (ASFV) antigens comprised of at least one B cell antigen and at least one T cell antigen; and (b) a lipid nanoparticle (ENP). In some embodiments, a vaccine described herein comprises (a) one or more engineered nucleic acids, wherein the one or more engineered nucleic acids encode African swine fever virus (ASFV) antigens comprised of at least one B cell antigen and at least one T cell antigen; and (b) a lipid nanoparticle (ENP), wherein the one or more engineered nucleic acids comprise a sequence comprising at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32. In some embodiments, the one or more engineered nucleic acids comprise a sequence comprising atleast 70% identity with SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32. In some embodiments, the one or more engineered nucleic acids comprise a sequence comprising at least 80% identity with SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, or 32. In some embodiments, the one or more engineered nucleic acids comprise a sequence comprising at least 90% identity with SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32. In some embodiments, the one or more engineered nucleic acids comprise a sequence comprising at least 95% identity with SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32. In some embodiments, the one or more engineered nucleic acids comprise a sequence comprising at least 99% identity with SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32. In some embodiments, the one or more engineered nucleic acids comprise a sequence comprising 100% identity with SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32.

[0093] In some embodiments, a vaccine described herein comprises (a) one or more engineered nucleic acids, wherein the one or more engineered nucleic acids encode African swine fever virus (ASFV) antigens comprised of at least one B cell antigen and at least one T cell antigen; and (b) a lipid nanoparticle (LNP), wherein the one or more engineered nucleic acids encode African swine fever virus (ASFV) antigens comprising an amino acid sequence comprising at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, or 31. In some embodiments, the one or more engineered nucleic acids encode African swine fever virus (ASFV) antigens comprising an amino acid sequence comprising at least 70% identity with SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31. In some embodiments, the one or more engineered nucleic acids encode African swine fever virus (ASFV) antigens comprising an amino acid sequence comprising at least 80% identity with SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31. In some embodiments, the one or more engineered nucleic acids encode African swine fever virus (ASFV) antigens comprising an amino acid sequence comprising at least 90% identity with SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31. In some embodiments, the one or more engineered nucleic acids encode African swine fever virus (ASFV) antigens comprising an amino acid sequence comprising at least 95% identity with SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31. In some embodiments, the one or more engineered nucleic acids encode African swine fever virus (ASFV) antigens comprising anamino acid sequence comprising at least 99% identity with SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31. In some embodiments, the one or more engineered nucleic acids encode African swine fever virus (ASFV) antigens comprising an amino acid sequence comprising 100% identity with SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31.

[0094] In some embodiments, a vaccine described herein comprises (a) one or more engineered nucleic acids, wherein the one or more engineered nucleic acids encode African swine fever virus (ASFV) antigens comprised of at least one B cell antigen and at least one T cell antigen; and (b) a lipid nanoparticle (LNP), wherein the ASFV antigen comprise: CD2v- E199E-P30-P72-EP153R; CD2v-E199E-P30-P72-p22; CD2v-E199E-P30-P72-p54; CD2v- E 199E-P30-P72-E248R; CD2v-E 199E-P30-P72-penton; CD2v-E 199E-P30-EP 153R-p22; CD2v-E199E-P30-EP153R-p54; CD2v-E199E-P30-EP153R-E248R; CD2v-E199E-P30- EP153R-penton; CD2v-E199E-P30-p22-p54; CD2v-E199E-P30-p22-E248R; CD2v-E199E- P30-p22-penton; CD2v-E199E-P30-p54-E248R; CD2v-E199E-P30-p54-penton; CD2v- E 199L-P30-E248R-penton; CD2v-E 199L-P72-EP 153R-p22; CD2v-E 199L-P72-EP 153R- p54; CD2v-E199L-P72-EP153R-E248R; CD2v-E199L-P72-EP153R-penton; CD2v-E199L- P72-p22-p54; CD2v-E199L-P72-p22-E248R; CD2v-E199L-P72-p22-penton; CD2v-E199L- P72-p54-E248R; CD2v-E199L-P72-p54-penton; CD2v-E199L-P72-E248R-penton; CD2v- E199L-EP153R-p22-p54; CD2v-E199L-EP153R-p22-E248R; CD2v-E199L-EP153R-p22- penton; CD2v-E199L-EP153R-p54-E248R; CD2v-E199L-EP153R-p54-penton; CD2v- E199L-EP 153R-E248R-penton; CD2v-El 99L-p22-p54-E248R; CD2v-E 199L-p22-p54- penton; CD2v-E199L-p22-E248R-penton; CD2v-E199L-p54-E248R-penton; CD2v-P30- P72-EP153R-p22; CD2v-P30-P72-EP153R-p54; CD2v-P30-P72-EP153R-E248R; CD2v- P30-P72-EP153R-penton; CD2v-P30-P72-p22-p54; CD2v-P30-P72-p22-E248R; CD2v-P30- P72-p22-penton; CD2v-P30-P72-p54-E248R; CD2v-P30-P72-p54-penton; CD2v-P30-P72- E248R-penton; CD2v-P30-EP153R-p22-p54; CD2v-P30-EP153R-p22-E248R; CD2v-P30- EP 153R-p22-penton; CD2v-P30-EP 153R-p54-E248R; CD2v-P30-EP 153R-p54-penton;CD2v-P30-EP153R-E248R-penton; CD2v-P30-p22-p54-E248R; CD2v-P30-p22-p54-penton; CD2v-P30-p22-E248R-penton; CD2v-P30-p54-E248R-penton; CD2v-P72-EP153R-p22-p54; CD2v-P72-EP 153R-p22-E248R; CD2v-P72-EP 153R-p22-penton; CD2v-P72-EP 153R-p54- E248R; CD2v-P72-EP153R-p54-penton; CD2v-P72-EP153R-E248R-penton; CD2v-P72- p22-p54-E248R; CD2v-P72-p22-p54-penton; CD2v-P72-p22-E248R-penton; CD2v-P72- p54-E248R-penton; CD2v-EP153R-p22-p54-E248R; CD2v-EP153R-p22-p54-penton; CD2v-EP 153R-p22-E248R-penton; CD2v-EP 153R-p54-E248R-penton; CD2v-p22-p54-E248R- penton; E199L-P30-P72-EP153R-p22; E199L-P30-P72-EP153R-p54; E199L-P30-P72- EP153R-E248R; E199L-P30-P72-EP153R-penton; E199L-P30-P72-p22-p54; E199L-P30- P72-p22-E248R; E199L-P30-P72-p22-penton; E199L-P30-P72-p54-E248R; E199L-P30- P72-p54-penton; E199L-P30-P72-E248R-penton; E199L-P30-EP153R-p22-p54; E199L-P30- EP 153R-p22-E248R; E 199L-P30-EP 153R-p22-penton; E 199L-P30-EP 153R-p54-E248R; E 199L-P30-EP 153R-p54-penton; E 199L-P30-EP 153R-E248R-penton; E 199L-P30-p22-p54- E248R; E199L-P30-p22-p54-penton; E199L-P30-p22-E248R-penton; E199L-P30-p54- E248R-penton; E199L-P72-EP153R-p22-p54; E199L-P72-EP153R-p22-E248R; E199L-P72- EP 153R-p22-penton; E 199L-P72-EP 153R-p54-E248R; E 199L-P72-EP 153R-p54-penton; E 199L-P72-EP 153R-E248R-penton; E 199L-P72-p22-p54-E248R; E 199L-P72-p22-p54- penton; E199L-P72-p22-E248R-penton; E199L-P72-p54-E248R-penton; E199L-EP153R- p22-p54-E248R; E 199L-EP 153R-p22-p54-penton; El 99L-EP 153R-p22-E248R-penton;E 199L-EP 153R-p54-E248R-penton; E 199L-p22-p54-E248R-penton; P30-P72-EP 153R-p22- p54; P30-P72-EP153R-p22-E248R; P30-P72-EP153R-p22-penton; P30-P72-EP153R-p54- E248R; P30-P72-EP153R-p54-penton; P30-P72-EP153R-E248R-penton; P30-P72-p22-p54- E248R; P30-P72-p22-p54-penton; P30-P72-p22-E248R-penton; P30-P72-p54-E248R- penton; P30-EP153R-p22-p54-E248R; P30-EP153R-p22-p54-penton; P30-EP153R-p22- E248R-penton; P30-EP153R-p54-E248R-penton; P30-p22-p54-E248R-penton; P72-EP153R- p22-p54-E248R; P72-EP153R-p22-p54-penton; P72-EP153R-p22-E248R-penton; P72- EP153R-p54-E248R-penton; P72-p22-p54-E248R-penton; EP153R-p22-p54-E248R-penton; CD2v-E 199L-P30-P72-EP 153R-p22; CD2v-E 199L-P30-P72-EP 153R-p54; CD2v-E 199L- P30-P72-EP 153R-E248R; CD2v-E 199L-P30-P72-EP 153R-penton; CD2v-E 199L-P30-P72- p22-p54; CD2v-E199L-P30-P72-p22-E248R; CD2v-E199L-P30-P72-p22-penton; CD2v- E199L-P30-P72-p54-E248R; CD2v-El 99L-P30-P72-p54-penton; CD2v-E 199L-P30-P72- E248R-penton; CD2v-E 199L-P30-EP 153R-p22-p54; CD2v-E 199L-P30-EP 153R-p22- E248R; CD2v-E 199L-P30-EP 153R-p22-penton; CD2v-El 99L-P30-EP 153R-p54-E248R; CD2v-E 199L-P30-EP 153R-p54-penton; CD2v-E 199L-P30-EP 153R-E248R-penton; CD2v- E199L-P30-p22-p54-E248R; CD2v-El 99L-P30-p22-p54-penton; CD2v-E 199L-P30-p22- E248R-penton; CD2v-E199L-P30-p54-E248R-penton; CD2v-E199L-P72-EP153R-p22-p54; CD2v-E 199L-P72-EP 153R-p22-E248R; CD2v-E 199L-P72-EP 153R-p22-penton; CD2v- E 199L-P72-EP 153R-p54-E248R; CD2v-E 199L-P72-EP 153R-p54-penton; CD2v-E 199L- P72-EP 153R-E248R-penton; CD2v-El 99L-P72-p22-p54-E248R; CD2v-E 199L-P72-p22- 1p54-penton; CD2v-E199L-P72-p22-E248R-penton; CD2v-E199L-P72-p54-E248R-penton;CD2v-El 99L-EP 153R-p22-p54-E248R; CD2v-E 199L-EP 153R-p22-p54-penton; CD2v- E 199L-EP 153R-p22-E248R-penton; CD2v-E 199L-EP 153R-p54-E248R-penton; CD2v- E199L-p22-p54-E248R-penton; CD2v-P30-P72-EP 153R-p22-p54; CD2v-P30-P72-EP 153R- p22-E248R; CD2v-P30-P72-EP 153R-p22-penton; CD2v-P30-P72-EP 153R-p54-E248R; CD2v-P30-P72-EP 153R-p54-penton; CD2v-P30-P72-EP 153R-E248R-penton; CD2v-P30- P72-p22-p54-E248R; CD2v-P30-P72-p22-p54-penton; CD2v-P30-P72-p22-E248R-penton; CD2v-P30-P72-p54-E248R-penton; CD2v-P30-EP153R-p22-p54-E248R; CD2v-P30- EP 153R-p22-p54-penton ; CD2v-P30-EP 153R-p22-E248R-penton; CD2v-P30-EP 153R-p54- E248R-penton; CD2v-P30-p22-p54-E248R-penton; CD2v-P72-EP153R-p22-p54-E248R; CD2v-P72-EP 153R-p22-p54-penton; CD2v-P72-EP 153R-p22-E248R-penton; CD2v-P72- EP 153R-p54-E248R-penton; CD2v-P72-p22-p54-E248R-penton; CD2v-EP 153R-p22-p54- E248R-penton; E 199L-P30-P72-EP 153R-p22-p54; E 199L-P30-P72-EP 153R-p22-E248R; E 199L-P30-P72-EP 153R-p22-penton; E 199L-P30-P72-EP 153R-p54-E248R; E 199L-P30- P72-EP 153R-p54-penton; E 199L-P30-P72-EP 153R-E248R-penton; E 199L-P30-P72-p22- p54-E248R; E199L-P30-P72-p22-p54-penton; E199L-P30-P72-p22-E248R-penton; E199L- P30-P72-p54-E248R-penton; El 99L-P30-EP 153R-p22-p54-E248R; E 199L-P30-EP 153R- p22-p54-penton; E 199L-P30-EP 153R-p22-E248R-penton; El 99L-P30-EP 153R-p54-E248R- penton; E199L-P30-p22-p54-E248R-penton; E199L-P72-EP153R-p22-p54-E248R; E199L- P72-EP 153R-p22-p54-penton; E 199L-P72-EP 153R-p22-E248R-penton; E 199L-P72-EP 153R-p54-E248R-penton; E 199L-P72-p22-p54-E248R-penton; E 199L-EP 153R-p22-p54- E248R-penton; P30-P72-EP153R-p22-p54-E248R; P30-P72-EP153R-p22-p54-penton; P30- P72-EP 153R-p22-E248R-penton; P30-P72-EP 153R-p54-E248R-penton; P30-P72-p22-p54- E248R-penton; P30-EP153R-p22-p54-E248R-penton; P72-EP153R-p22-p54-E248R-penton; CD2v-E 199L-P30-P72-EP 153R-p22-p54; CD2v-E 199L-P30-P72-EP 153R-p22-E248R; CD2v-E 199L-P30-P72-EP 153R-p22-penton; CD2v-E 199L-P30-P72-EP 153R-p54-E248R; CD2v-E 199L-P30-P72-EP 153R-p54-penton; CD2v-E 199L-P30-P72-EP 153R-E248R- penton; CD2v-E 199L-P30-P72-p22-p54-E248R; CD2v-E 199L-P30-P72-p22-p54-penton; CD2v-E199L-P30-P72-p22-E248R-penton; CD2v-E199L-P30-P72-p54-E248R-penton; CD2v-E 199L-P30-EP 153R-p22-p54-E248R; CD2v-E 199L-P30-EP 153R-p22-p54-penton; CD2v-E 199L-P30-EP 153R-p22-E248R-penton; CD2v-E 199L-P30-EP 153R-p54-E248R- penton; CD2v-E 199L-P30-p22-p54-E248R-penton; CD2v-El 99L-P72-EP 153R-p22-p54- E248R; CD2v-E 199L-P72-EP 153R-p22-p54-penton; CD2v-E 199L-P72-EP 153R-p22-E248R-penton; CD2v-E199L-P72-EP153R-p54-E248R-penton; CD2v-E199L-P72-p22-p54- E248R-penton; CD2v-E 199L-EP 153R-p22-p54-E248R-penton; CD2v-P30-P72-EP 153R- p22-p54-E248R; CD2v-P30-P72-EP 153R-p22-p54-penton; CD2v-P30-P72-EP 153R-p22- E248R-penton; CD2v-P30-P72-EP153R-p54-E248R-penton; CD2v-P30-P72-p22-p54- E248R-penton; CD2v-P30-EP 153R-p22-p54-E248R-penton; CD2v-P72-EP 153R-p22-p54- E248R-penton; E 199L-P30-P72-EP 153R-p22-p54-E248R; E 199L-P30-P72-EP 153R-p22- p54-penton; E 199L-P30-P72-EP 153R-p22-E248R-penton; E 199L-P30-P72-EP 153R-p54- E248R-penton; E199L-P30-P72-p22-p54-E248R-penton; E199L-P30-EP153R-p22-p54- E248R-penton; El 99L-P72-EP 153R-p22-p54-E248R-penton; P30-P72-EP 153R-p22-p54- E248R-penton; CD2v-El 99L-P30-P72-EP 153R-p22-p54-E248R; CD2v-E 199L-P30-P72- EP 153R-p22-p54-penton ; CD2v-E 199L-P30-P72-EP 153R-p22-E248R-penton; CD2v- E199L-P30-P72-EP153R-p54-E248R-penton; CD2v-E199L-P30-P72-p22-p54-E248R- penton; CD2v-E 199L-P30-EP 153R-p22-p54-E248R-penton; CD2v-E 199L-P72-EP 153R- p22-p54-E248R-penton; CD2v-P30-P72-EP 153R-p22-p54-E248R-penton; E 199L-P30-P72- EP 153R-p22-p54-E248R-penton; or CD2v-E 199L-P30-P72-EP 153R-p22-p54-E248R- penton, and optionally a T cell antigen comprising multiple T cell epitopes (MTE) in combination with each of the antigens.Lipid Nanoparticles (LNPs)

[0095] As used herein, the term “nanoparticle” refers to a particle having an average size suitable for parenteral administration. In some embodiments, a nanoparticle has a longest dimension (e.g. , a diameter) of less than 1 ,000 nanometers (nm). In some embodiments, a nanoparticle may be characterized by a longest dimension (e.g., a diameter) of less than 300 nm. In some embodiments, a nanoparticle may be characterized by a longest dimension (e.g., a diameter) of less than 100 nm. In many embodiments, a nanoparticle may be characterized by a longest dimension between about 1 nm and about 100 nm, or between about 1 pm and about 500 nm, or between about 1 nm and 1 ,000 nm. In many embodiments, a population of nanoparticles is characterized by an average size (e.g., longest dimension) that is below about 1,000 nm, about 500 nm, about 100 nm, about 50 nm, about 40 nm, about 30 nm, about 20 nm, or about 10 nm and often above about 1 nm. In many embodiments, a nanoparticle may be substantially spherical so that its longest dimension may be its diameter. In some embodiments, a nanoparticle has a diameter of less than 100 nm as defined by the National Institutes of Health.

[0096] In some embodiments, a nanoparticle is a lipid nanoparticle.

[0097] In some embodiments, one or more engineered nucleic acids of the disclosure is administered in the form of lipid nanoparticles (LNPs). The LNPs may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated.

[0098] In some embodiments, the LNPs comprise one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids.

[0099] In some embodiments, the LNPs comprises a cationic lipid, a neutral lipid, a steroid, a polymer conjugated lipid; and the RNA, encapsulated within or associated with the lipid nanoparticle.

[0100] In some embodiments, the LNPs comprise cholesterol, an ionizable lipid, a lipid comprising a polyethylene glycol (PEG) moiety, and a lipid comprising a phosphatidylcholine or phosphatidylethanolamine moiety.

[0101] Lipid nanoparticles contemplated for use with the compositions (e.g., vaccines) and methods described herein are described in International Publication No. WO2023 / 064612, WO / 2024Z 184500, WO / 2023 / 031392, WO / 2023 / 031394, and WO / 2021 / 123332, which is incorporated by reference in its entirety.Methods of Administration

[0102] Vaccines and / or compositions comprising vaccines (e.g., pharmaceutical compositions) described herein may be administered to a subject. As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active agent is present in unit dose amount appropriate for administration in a prophylactic or therapeutic regimen that shows a statistically significant probability of achieving a predetermined prophylactic or prophylactic or therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for parenteral administration, for example, by subcutaneous, intramuscular, or intravenous injection as, for example, a sterile solution or suspension formulation. In some embodiments, a vaccine described herein is administered to a subject via a first intramuscular injection. In some embodiments, a vaccine described herein is administered to a subject the via a second intramuscular injection after the first intramuscular injection. In some embodiments, the time between the first intramuscular injection and the second intramuscular injection is about 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days (e.g., one week). In some embodiments, the time between the first intramuscular injection and the second intramuscular injection is about one week, about two weeks, about three weeks, about four weeks, about five weeks, or about six weeks after the first intramuscular injection. In some embodiments, the time between the first intramuscular injection and the second intramuscular injection is about three weeks.

[0103] The term "pharmaceutically effective amount" or "prophylactic or therapeutically effective amount" or “amount effective” refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of the treatment of a particular disease, a desired reaction in some embodiments relates to inhibition of the course of the disease. In some embodiments, such inhibition may comprise slowing down the progress of a disease and / or interrupting or reversing the progress of the disease. In some embodiments, a desired reaction in a treatment of a disease may be or comprise delay or prevention of the onset of a disease or a condition. An effective amount of pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) described herein will depend, for example, on a disease or condition to be treated, the severity of such a disease or condition, individual parameters of the patient, including, e.g., age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, doses of pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[0104] As used herein, the term “effective amount to induce an immune response” refers to the amount which achieves a desired reaction such as a protective immune response. As used herein, the term “protective immune response” refers to both the humoral immune response and the cell-mediated immune response. The humoral immune response involves the stimulation of the production of antibodies by B lymphocytes that, for example, neutralize infectious agents, block infectious agents from entering cells, block replication of said infectious agents, and / or protect host cells from infection and destruction. The cell-mediated immune response refers to an immune response that is mediated by T-lymphocytes and / or other cells, such as macrophages, against an infectious agent, exhibited by a vertebrate (e.g., a human), that prevents or ameliorates infection or reduces at least one symptom thereof. Thus,a protective immune response, as described herein, refers to immunity or eliciting an immune response against an infectious agent, which is exhibited by a vertebrate that prevents or ameliorates an infection or reduces at least one symptom thereof. Specifically, induction of a protective immune response from administration of the vaccine is evident by elimination or reduction of the presence of one or more symptoms of viral infection, e.g., ASF infection, or a reduction in the duration or severity of such symptoms. In some embodiments, an immune response provides a subject with some degree of protection from viral disease, but not complete resistance. For example, in some embodiments, an immune response may be the induction of neutralizing antibodies and cellular responses which provide partial protection against a lethal viral challenge. In some embodiments, antigens described herein have demonstrated capacity to elicit specific antibodies and T cell responses. In some embodiments the protective immune response, through induction of an antibody and / or T-cell response in a subject, is sufficient to reduce development of one or more symptoms or the severity of one or more symptoms, to reduce the likelihood of viral infection or reduce the severity of the viral infection relative to a subject that has not received a vaccine against the virus. In some embodiments, a protective immune response refers to any immune response that may completely prevent detectable viral infection, significantly limit symptoms associated with viral infection, or eradication of viral infection. In some embodiments, a protective immune response may result from neutralizing antibodies, B cell immunity, and / or broad cellular immunity that provides complete protection against a lethal viral challenge. In some embodiments, antigens described herein have demonstrated capacity to elicit a protective immune response. A reduction in a symptom may be determined subjectively or objectively, e.g., self-assessment by a subject, by a clinician's assessment or by conducting an appropriate assay or measurement, including, e.g., a quality of life assessment, a slowed progression of a viral infection or additional symptoms, or suitable assays (e.g. antibody titer, RT-PCR antigen detection, and / or B-cell or T-cell activation assay). An effective response may also be determined by directly measuring (e.g., RT-PCR) virus load in biological samples, which reflects the amount of virus shedding).

[0105] In some embodiments, a subject is in need of a vaccine described herein. For example, in some embodiments, a subject in need of a vaccine comprising one or more engineered nucleic acids encoding African swine fever virus (ASFV) antigens comprised of at least one B cell antigen and at least one T cell antigen is a pig.Methods of engineering a subunit vaccine

[0106] Some aspects of the disclosure method of engineering a subunit vaccine, the method comprising: (a) identifying one or more intracellular viral antigens from a target virus; (b) engineering the one or more intracellular viral antigens as membrane-bound proteins; (c) engineering multiple T cell epitopes (MTE); (d) determining the effector function profiles of antigen- specific antibodies and the levels of B and T cell responses induced by different intracellular viral antigens and MTE; and (e) determining the antigen combination for subunit vaccine with specific immune response profile; and optionally (f) producing the subunit vaccine.

[0107] As described herein, a viral antigen may be engineered as a membrane-bound protein. In some embodiments, engineering one or more intracellular viral antigens as membrane-bound proteins comprises adding a signal peptide of human CD8a to the N- terminus of the one or more intracellular viral antigens, and a CD8a hinge domain, CD8a transmembrane domain (TMD), and a short cytoplasmic region CD8a to the C-terminus of the one or more intracellular viral antigens.

[0108] In some embodiments, determining the effector function profiles of antigenspecific antibodies comprise measuring one or more effector function. In some embodiments, the effector function is antibody-dependent complement deposition (ADCD). In some embodiments, the effector function is antibody dependent cellular cytotoxicity (ADCC). In some embodiments, the effector function is antibody-dependent cellular phagocytosis (ADCP). In some embodiments, an effector function profile is determined by measuring one or more of antibody-dependent complement deposition (ADCD), antibody dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP).

[0109] In some embodiments, ADCD is measured with a CHO cell assay, wherein the CHO cells stably express individual viral antigens (e.g., ASFV antigens) incubated with heatinactivating pig serum, followed by addition of non-heat inactivated serum from placebo pics as a source of complement, followed by cell lysis quantified by flow cytometry. Other assays known in the art for measuring ADCD are also contemplated for use with the methods described herein.

[0110] In some embodiments, ADCC is measured with a CHO cells assay, wherein the CHO cells stably express individual viral antigens (e.g., ASFV antigens) are incubated with heat- inactivated pig serum, followed by the addition of pig peripheral blood mononuclear cells (PBMCs) as a source of natural killer (NK) cells, and lysis of CHO cells isquantified by a luminescent assay. Other assays known in the art for measuring ADCC are also contemplated for use with the methods described herein.

[0111] In some embodiments, ADCP is measured with fluorescent beads that are coated with specific viral antigens (e.g., ASFV antigens) incubated with immune sera and a pig macrophage cell line 3D4 / 31, and phagocytosis of labeled beads is quantified by flow cytometry. Other assays known in the art for measuring DCP are also contemplated for use with the methods described herein.

[0112] In some embodiments, determining B cell responses comprise measuring antibody secreting B cells (ASC). Antibody secreting B cells can be measured, for example, by using ELISpot assays and / or flow cytometry. Other methods known in the art for measuring ASCs are also contemplated for use with the methods described herein.

[0113] In some embodiments, the determining T cell responses comprise measuring secretion of cytokine IFN-y and / or TFN-OC. In some embodiments, cytokine levels (e.g., IFN- y and / or TFN-OC) may be measured using enzyme-linked immunosorbent assay (ELISA), which enables the specific and quantitative detection of individual cytokines via antibodyantigen binding interactions. For multiplexed analysis, bead-based immunoassays, including those utilizing Luminex xMAP technology, allow simultaneous quantification of multiple cytokines in a single sample through the use of distinguishably labeled capture beads. Similarly, cytometric bead array (CBA) assays, which leverage flow cytometry for beadbased multiplex detection, permit the concurrent measurement of multiple cytokines with cell-type resolution. Cytokine secretion (e.g., IFN-y and / or TFN-OC) may also be measured via the enzyme-linked immunospot (ELISpot) assay, which can detect cytokine secretion at the single-cell level, thereby enabling sensitive assessment of immune cell activity, particularly in response to antigenic stimulation. Alternatively, intracellular cytokine staining (ICS) followed by flow cytometric analysis may be used to detect intracellular cytokine accumulation within individual immune cells after pharmacological stimulation and fixation / permeabilization. In some embodiments, quantitative polymerase chain reaction (qPCR) or reverse transcription qPCR (RT-qPCR) may be utilized to measure cytokine mRNA transcript levels, thereby providing an indirect but highly sensitive assessment of cytokine expression. Where protein confirmation is desired, Western blot analysis may be used to detect specific cytokines in cell lysates or supernatants, though it is typically limited to semi-quantitative analysis. Other assays known in the art for measuring cytokine secretion are contemplated for use with the methods described herein.

[0114] It should be understood that the subject matter defined in the appended claims is not necessarily limited to the specific implementations described above. The specific implementations described above are disclosed as examples only.EXAMPLES

[0115] As described herein, an mRNA-LNP platform was combined with ASFV capsid engineering to enhance B cell and T cell responses. It was shown that ASFV capsid proteins P72 and Penton can be engineered as membrane-bound and secreted forms while preserving their multimeric structure without the use of chaperon pB602L. A single amino acid mutation successfully eliminates glycosylation on the engineered Penton protein. The immunogenicity of the mRNA vaccines disclosed herein were tested in both mouse and pig models. Surprisingly, the results showed that MB-P72 and MB-PNISOQ elicited significantly stronger B cell and T cell responses. This data demonstrate a novel approach for engineering viral capsid proteins as immunogens, highlighting its use for developing highly effective vaccines and its applicability to diverse uses.

[0116] Further as disclosed herein, immunogenicity of ASFV antigens were rationally selected and comprehensively evaluated, including i) ASFV homologs corresponding to the subunits of the protective VACV vaccine; ii) promising immunogens reported in previous studies; iii) viral capsid proteins in membrane-bound form for more efficient induction of antibody responses; and iv) multiple T cell epitopes (MTE) for inducing broad and robust cellular immunity. In mRNA-ENP formulation, the selected ASFV antigens stimulated concordant antigen- specific antibody and T cell responses in both mice and pigs, with the MTE inducing the most potent cellular immunity. By combining the effector function profiles of antigen- specific antibodies and the levels of B and T cell responses induced by different ASFV antigens, optimal antigen combinations for cocktail vaccines with specific immune response profiles, were developed and further validated in mice. The approach disclosed herein represents a comprehensive investigation of ASF mRNA subunit vaccines incorporating rational antigen selection, protein engineering, T cell-directed antigen design, and profiling of immune responses. These results provide a basis for further evaluating the subunit mRNA vaccine candidates in challenge studies. The innovative strategies reported herein should be applicable to design vaccines for other large complex DNA viruses such as monkeypox.Example 1. Membrane Expression Enhances Folding, Multimeric Structure Formation, and Immunogenicity of Viral Capsid Proteins

[0117] Vaccination remains a cornerstone of infectious disease control. Traditional vaccines based on inactivated and live- attenuated infectious agents have provided protection against many pathogens, but they are either ineffective or have unacceptable safety concerns in protecting against certain pathogens. For example, African swine fever virus (ASFV) causes a highly contagious and deadly disease in both domestic and wild swine, with devastating economic consequences for the global pork industry (67-69). Inactivated ASFV vaccines do not confer protection while the live-attenuated vaccines are effective but with high risk of reversion to virulence (70,71). Subunit vaccines based on recombinant proteins and nucleic acids offer greater precision and safety (72). Central to the success of the subunit vaccine development is the design of antigens capable of eliciting strong and protective immune responses.

[0118] Viral envelope and capsid proteins are among the most explored antigens for subunit vaccine development. Naturally, envelope proteins are displayed on the surface of virion (or infected host cells) in large numbers. The resulting high epitope density and accessibility to B cell recognition induces potent antibody responses. In contrast, viral capsid proteins are synthesized in the cytosol and often require chaperone for folding and assembly into capsid for packaging viral genome. The use of capsid proteins in subunit vaccine is often hampered by the complexity in their production due to susceptibility to misfolding and dependence on specific post-translational modifications, and poor immunogenicity as recombinant capsid proteins may not fold into their natural multimeric structures for inducing antibody responses against conformational epitopes that many neutralizing antibodies target (73). A more successful approach has been to express and assemble viral capsid proteins into virus-like particle (VLP), as demonstrated by VLP-based vaccines for hepatitis B (HBV)8, human papillomavirus (HPV)(74), and hepatitis E (HEV)(75). Although VLP preserves conformational epitopes and the high epitope density of the natural viral capsids and therefore induces potent antibody responses, it does not induce any significant CD8+T cell response. In addition, VLP production in scale has not been possible for many pathogenic viruses due to the complexity of capsid assembly.ResultsFolding and trimer formation of the membrane-bound and secreted P72 do not require chaperone

[0119] Recent structural studies revealed that the outer capsid shell of ASFV is composed of a major capsid protein P72, which forms homotrimer, and a minor capsid protein Penton, which forms a homopentamer (5,6). The outer capsid shell is stabilized by three minor proteins underneath, including P17 that glues together P72 trimers (5,6). To enhance immunogenicity of P72 and Penton by maintaining their native multimeric structures and therefore conformational epitopes, P72 and Penton were expressed as the membranebound (MB) and secreted (S) proteins, in addition to their native intracellular (IC) form. Specifically, to express P72 as a secreted protein (S-P72) the signal peptide of human CD8a was added to the N-terminus of P72. To express P72 as a membrane-bound protein (MB- P72), in addition to the signal peptide, the hinge domain, transmembrane domain (TMD), and a short cytoplasmic region from the same CD8a were added to the C-terminus of P72. CD8a signal peptide, hinge and transmembrane domains were chosen because they are widely used to express chimeric antigen receptors on the cell surface (84). AlphaFold2(85) and PyMOL modeling suggested that the addition of the CD8a hinge domain does not induce significant structural alterations of P72 nor affects the formation of P72 trimer probably because the long CD8a hinge domain provides sufficient flexibility and space to accommodate P72 trimer formation (FIGs. 1A, IE), consistent with a previous report24.

[0120] The requirement of chaperone pB602L, a viral protein known to facilitate P72 folding and trimer formation (82,83), and P17 was determined in P72 expression. HEK293T and Vero cells were transiently transfected with vectors expressing IC-, S- and MB-P72 with or without co-transfection of P17 or pB602L, followed by Western blotting analysis of cell lysates. The level of IC-P72 expression was minimal by itself or in the presence of P17 but greatly elevated in the presence of pB602L. In contrast, the expression of S-P72 and MB-P72 was readily detected, and their level of expression was not significantly affected by coexpression of P17 or pB602L. Approximately 2 ng / mL of S-P72 was detected by ELISA in the supernatants of S-P72-transfected HEK293T cells. Notably, this level was not affected by the co-expression of P17 or pB602L. Immunofluorescence assay (IFA) showed colocalization of MB-P72 with the cell membrane. Based on Pearson correlation, colocalization of P72 and cell membrane was 97.5% for MB-P72, 40.5% for S-P72, and 8.3% for IC- P72 / pB602L.

[0121] To assay P72 trimer formation, four approaches were used. First, cell lysates were subjected to SDS-PAGE under non-reducing conditions followed by Western blotting. A dominant P72-reactive band was detected at approximately 240 kDa, suggesting trimerformation. Consistently, formation of P72 trimer from IC-P72 was only detected in the presence of pB602L, while formation of P72 trimer from S-P72 and MB-P72 did not require pB602L. Second, HA-tagged S-P72 and MB-P72 were expressed and purified and P72 trimers and monomers were quantified by size exclusion chromatography. As shown by Table 7, 50.8% of S-P72 and 36.8% MB-P72 were trimers. Third, the structural similarity of trimers formed by HA-tagged S-P72 and MB-P72 was confirmed by transmission electron microscopy (TEM) to those of native ASFV virions (PDB ID: 6L2T, EMDB ID: 0814) (5) and previously reported P72 structures (PDB ID: 6KU9, EMDB ID: 0776) (87). Finally, monoclonal antibody 4H3, which is specific for a confirmational epitope of the native P72 trimer(88,89), was able to detect both MB-P72 and S-P72 by IP-Western (FIG. 20A), by flow cytometry (FIG. 20B), and by confocal microscopy, suggesting both MB-P72 and S-P72 can form native P72 trimer. Together, these results show that while folding and trimer formation of native P72 (IC-P72) requires chaperone pB602E, folding and trimer formation of S-P72 and MB-P72 do not require pB602E, probably due to re-direction of P72 synthesis into the endoplasmic reticulum (ER) by the signal peptide.

[0122] To develop P72 mRNA vaccines, MB-P72, S-P72, IC-P72, and pB602E were cloned into the pUC57 vector, which includes a T7 promoter, 5’UTR, 3’UTR, and polyA tail. The mRNAs were synthesized via in vitro transcription using T7 polymerase28and transfected into HEK293T or Vero cells using MessengerMax™. P72 expression was confirmed by Western blotting analysis with relative expression of IC-P72:S-P72:MB-P72 at 1:2:8 (FIG. 4). The MB-P72 and S-P72 mRNAs were formulated into lipid nanoparticles (ENP) individually and IC-P72 and pB602E mRNAs were formulated together into ENP. The resulting LNP-mRNAs exhibited a narrow size distribution of approximately 115 nm and an encapsulation efficiency exceeding 91% (Table 8). Transfection of LNP-mRNAs into HEK293T cells led to robust expression of MB-P72, S-P72, and IC-P72 as detected by flow cytometry, suggesting successful expression of the three forms of P72 via mRNAs.Penton forms pentameric structure without viral chaperone

[0123] Penton, referred to as PWT, was also expressed in membrane-bound (MB), secreted (S), and native intracellular (IC) forms. Because of a lack of antibodies specific for Penton, a HA-tag was added at the C-terminal of Penton for easy detection. Structural modeling with PyMOL indicated that the hinge domain provides sufficient flexibility to accommodate pentamer formation, consistent with a previous report24. HEK293T cells were transiently transfected with vectors expressing IC-, S- and MB-PWT followed by Westernblotting analysis of cell lysates. The IC-PWT migrated at the predicted size of 28.9 kDa. S-PWT produced an additional band slightly larger than that of IC-PWT; and MB-PWT migrated slightly larger than the predicted 36.9 kDa (FIG. 3A). Treatment of cell lysates with PNGase F, which removes N-linked oligosaccharides, did not affect the size of IC-PWT, but reduced the larger S-PWT to the same size as IC-PWT and the size of MB-PWT slightly, indicating glycosylation of both S-PWT and MB-PWT. Because the N-linked glycosylation may shield B cell epitopes and hinder immune responses to the native protein, a single NLTT glycosylation motif was identified in the wildtype Penton that is exposed and mutated N at residue 180 to Q (N180Q). S-PNISOQ and MB -PNISOQ produced a single band at the same size as the nonglycosylated counterparts (FIG. 3A), suggesting successful abolition of N-linked glycosylation. Further characterization using confocal microscopy revealed that IC-PWT and MB-PWT were expressed at similar levels, significantly higher than S-PWT (FIG. 3C). The N180Q mutation did not significantly alter their levels of expression.

[0124] To detect the secretion of Penton, culture supernatants from transfected HEK293F cells were concentrated and analyzed by Western blotting. Both non-glycosylated and glycosylated S-PWT were detected in the supernatants of S-Pwr-transfected cells, while only non-glycosylated S-PNISOQ was detected in the supernatants of S-PNi80Q-transfected cells. IC-PWT and MB-PNISOQ were not detected in the culture supernatants. To investigate subcellular localization, HEK293T cells transfected with HA-tagged Penton were permeabilized and stained with anti-HA and Zonula Occludens (ZO)-l, a cell membrane marker, followed by confocal imaging. MB-PNISOQ predominantly colocalized with ZO-1 on the cell membrane, whereas IC-PWT and S-PNISOQ primarily localized within the cytosol. Quantification of the Pearson colocalization coefficient showed that more than 80% of MB- PNISOQ colocalized with ZO-1, compared to only 35% for S-PNISOQ and 10% for IC-PWT (FIG. 3E). Furthermore, pentameric Penton formation was directly assessed by Western blotting analysis using SDS-PAGE under non-reducing conditions. Cell lysates from IC-PWT and S- PNISOQ transfected HEK293T cells gave rise to two predominant bands corresponding to monomeric and pentameric forms of Penton, whereas only pentamer was detected in MB- PNisoQ-transfected cells. Additionally, IC-PWT, S-PNISOQ, and MB-PNISOQ mRNAs were prepared and formulated into LNP. Physicochemical analysis confirmed a narrow particle size distribution (-120 nm) with over 94% encapsulation efficiency (Table 8). Expression of IC-PWT, S-PNISOQ, and MB-PNISOQ was validated by flow cytometry analysis of LNP-mRNA transfected HEK293T cells (FIG. 3E.

[0125] Together, these results show that Penton can form pentamer without viral chaperone and its expression in membrane-bound form further promotes pentamer formation. Redirection of Penton synthesis in the ER and Golgi leads to N-glycosylation, which is abolished by the N180Q mutation.MB-P72 is more immunogenic than S-P72 and. IC-P72 in mice

[0126] To compare the immunogenicity of MB-, S- and IC-P72, BALB / c mice were immunized intramuscularly with 5 pg of mRNA in LNP formulation (using ARV L002 ionizable lipid)28at day 0 and 21 (FIG. 5A). Sera were collected before immunization and two weeks after each immunization (days 14 and 35) and used to assay P72-specific IgG by ELISA. Spleen and bone marrow were harvested at day 35 and used for assaying IFN-y- and TNF- ex- secreting T cells and antibody secreting B cells (ASC) by ELISPOT, respectively. P72-specific IgG titers 14 days following the first immunization were 62.4 for IC-P72 plus pB602L (shown as IC-P72* on Figs), 129 for S-P72 and 330 for MB-P72 (FIG. 5C). Following the boost, the titers increased to 93 for IC-P72*, 4576 for S-P72 and 50820 for MB-P72. Consistently, the frequency of ASCs in the bone marrow at day 35 tended to be higher in MB- and S-P72 immunized mice (FIG. 5D). Furthermore, the frequencies of IFN-y- or TNF- a- secreting cells following stimulation of splenocytes with a P72 peptide pool (Table 6) tended to be higher in MB-P72 immunized mice (FIGs. 6A-6B). In a separate experiment where P72 mRNAs were formulated in LNP using ionizable lipid SM-102, MB-P72 also induced higher antigen-specific IgG responses than IC-P72* (FIG. 13C). These results show that MB-P72 is more immunogenic than S-P72 and IC-P72 in inducing antibody and T cell responses in mice.

[0127] To investigate the mechanisms underlying the enhanced antibody response induced by MB-P72, the follicular T helper (Tfh) cell response was assessed in the spleen at day 35. Tfh cells were identified as CD4+B220'CD44+PD-l+CXCR5+(FIG. 21). The frequencies of Tfh cells were significantly higher in the spleen of MB-P72 immunized mice (6.6%) than S-P72 immunized mice (4.4%), IC-P72* immunized mice (4.8%) and placebo mice (1.6%) (FIG. 6F). Spleen sections were stained for IgD to identify B cell follicles, CD35 for follicular dendritic cells, CD3s for T cells, and Ki-67 for actively proliferating cells. MB- P72 immunization induced significantly more germinal centers (21 per spleen section) compared to immunization with IC-P72* (13 per spleen section) and S-P72 (15 per spleen section) (FIG. 6G). These findings suggest that the enhanced antibody response observedwith MB-P72 immunization is associated with increased germinal center formation and a stronger Tfh cell response.MB-Penton is more immunogenic than S-Penton and IC-Penton in mice

[0128] The immunogenicity of IC-, S- and MB-Penton was also compared by immunizing BALB / c mice with 5 pg of IC-PWT, S-PNISOQ, and MB-PNISOQ mRNA in LNP formulation (using ARV L002 ionizable lipid) at day 0 and 21. Sera was collected before immunization and two weeks after each immunization (days 14 and 35) and used to assay Penton- specific IgG by ELISA. The IgG titers were 159 for IC-PWT, 95 for S-PNISOQ, and 427 for MB-PNISOQ 14 days after the first immunization (FIG. 5E). 14 days after the second immunization, the titers rose to 494 for IC-PWT, 177 for S-PNISOQ, and 3056 for MB-PNISOQ. Consistently, the frequency of Penton- specific ASCs in the bone marrow at day 35 was significantly higher in MB-PNISOQ immunized mice than in IC-PWT and S-PNISOQ immunized mice (FIG. 6H). Similarly, the frequencies of IFN-y-,TNF-a, and IL4-secreting T cells following stimulation of splenocytes with recombinant Penton protein were significantly higher in MB-PNISOQ immunized mice than in IC-PWT and S-PNISOQ immunized mice (FIGs. 5D, 6A-6B). In a separate experiment where Penton mRNAs were formulated in ENP using ionizable lipid SM-102, MB-PNISOQ also stimulated higher antigen- specific IgG responses than IC-PWT and S-PNISOQ (FIGS. 13D-13G). These results show that MB-PNISOQ is more immunogenic than IC-PWT and S-PNISOQ in inducing both antibody and T cell responses in mice.

[0129] The mechanisms underlying enhanced immune responses induced by MB- PNISOQ were investigated. The frequencies of CD4+B220'CD44+PD-l+CXCR5+Tfh cells were significantly higher in the spleens of MB-PNISOQ immunized mice (7.4%) than IC-PWT immunized mice (5.1%), S-PNISOQ immunized mice (4.1%) and placebo mice (1%) (FIG. 7A and FIG. 21). Germinal center analysis revealed that MB-PNISOQ induced significantly more germinal center formations than IC-PWT and S-PNISOQ. Mice immunized with MB-PNISOQ had an average of 26 germinal centers per section, which was 2.2 times higher than in S-PNISOQ- immunized mice (12 per section, p=0.019) and 2.5 times higher than in IC-Pwr-immunized mice (10 per section, p=0.008) (FIG. 7B). The memory responses induced by IC-PWT, S- PNISOQ, and MB-PNISOQ mRNA vaccination were also investigated by quantifying percentages of CD3+CD8+CD44+CD62E“ effector memory T cells. The frequency of effector memory CD8+T cells tended to be higher in the spleen of MB-NPISOQ immunized mice than IC-PWT or S-NPI8OQ immunized mice (FIGs. 17A-17B). These results show that the enhanced antibodyresponse observed with MB-PNISOQ immunization is associated with increased germinal center formation and a stronger Tfh cell response.Membrane-bound P72 and Penton induce robust antibody and T cell responses in pigs

[0130] The immunogenicity of the MB-P72 and MB-PNISOQ mRNA vaccines was further assessed in pigs. Five-week-old piglets were injected intramuscularly with 30 pg of mRNA in LNP formulation at day 0 and 21 (FIG. 8A). Sera were collected before immunization and weekly following immunization for assaying antigen- specific IgG titers. Spleen were harvested at day 35 for assaying T cell responses. Antigen- specific IgG responses became detectable 14 days after the first immunization, increased steadily afterwards, and reached the highest level at day 35, i.e., 14 days after boost (FIG. 8B). Specifically, P72-specific IgG titers were 174 at day 14 and increased to 81359 at day 35; and Penton- specific IgG titers were 332 at day 14 and increased to 8086 at day 35. Antigenspecific T cell responses were assessed by ELISPOT following stimulation of splenocytes with a P72 peptide pool or recombinant Penton protein. The frequencies of IFN-y-secreting cells were 438 and 400 per 106splenocytes from MB-P72 and MB-PNISOQ immunized pigs, respectively (FIG. 8C). These results show that both MB-P72 and MB-PNISOQ induce robust antibody and T responses in pigs.

[0131] A comparative analysis of MB-P72- and MB-PNisoQ-induced antibody and T cell responses was conducted between mice and pigs. The antigen- specific IgG responses were nearly identical between the two species for both MB-P72 and MB-PNISOQ (FIG. 8D). For MB-P72-induced T cell responses, IFN-y levels were lower in mice compared to pigs, whereas for MB-PNisoQ-induced T cell responses, IFN-y levels were higher in mice than in pigs (FIG. 8E). However, none of these differences were statistically significant. These findings indicate that MB-P72 and MB-PNISOQ elicit similar antibody and T cell responses in both mice and pigs, cross-validating the experimental outcomes.Discussion associated with Example 1

[0132] As presented herein, mRNA vaccine technology was combined with protein engineering to tackle the longstanding challenge of low immunogenicity associated with intracellular viral proteins. Using ASFV capsid proteins P72 and Penton as models, it was shown that when these proteins are expressed as membrane-bound and secreted forms, they fold and assemble into native multimeric structures without the help of viral chaperone. Importantly, both the membrane-bound P72 and Penton induced significantly strongerhumoral and cellular immune responses in mice than their secreted and intracellular counterparts. The approach disclosed herein provides a simple approach to enhance antigen presentation and immunogenicity of intracellular proteins, opening the possibility for developing safe and effective subunit vaccines.

[0133] ASFV capsid proteins P72 and Penton were engineered as membrane-bound and secreted proteins, which facilitated their folding and assembly into native multimeric structures. Naturally, viral capsid proteins are synthesized in the cytosol of infected host cells. With the help of chaperones, the newly synthesized capsid proteins are folded and assembled into intermediate multimeric structures and finally into much larger and more complex capsid, into which viral genome is packaged. The intermediate for P72 is a homotrimer and for Penton a homopentamer11,12. Studies have shown that folding and trimerization of P72 requires an ASFV-encoded chaperone pB6O2L20,21,25. Consistently, it was found that expression, folding and trimerization of the native intracellular P72 requires chaperone pB602L. In contrast, the membrane-bound and secreted P72 were readily detected without pB602L. Furthermore, -50% of the membrane-bound and secreted P72 form trimers based on non-reducing gel electrophoresis followed by Western blotting, size exclusion chromatography and transmission electron microscopy of purified P72. Moreover, monoclonal antibody 4H3, which specifically recognizes a conformational epitope of the native P72 trimer26,27, readily detected S-P72 and MB-P72 proteins as well as intracellular P72 (IC-P72) co-expressed with chaperone pB602L. As newly synthesized membrane-bound and secreted proteins are directed into the ER and Golgi apparatus, the oxidative environment of the ER likely facilitated the proper folding, disulfide bond formation and trimerization of P72 without the chaperone pB602L29 33. In comparison, approximately 30% of both intracellular and secreted Penton formed pentamer, but almost 100% of the membrane-bound Penton was detected as pentamer. It is possible that the long and flexible hinge used to present Penton (and P72) on the cell surface may help to stabilize the multimeric complexes. It is notable that the membrane-bound and secreted Penton became glycosylated likely during their transition through the Golgi apparatus. A single amino acid mutation of N180Q was introduced to abolish the glycosylation and therefore to preserve immunogenic potential without masking antigenic epitopes by glycans34. The ability to express P72 and Penton in their native multimeric structures through a simple engineering of them as the membranebound or secreted proteins should help to preserve their conformational epitopes, providing a structural basis for inducing the appropriate antibody responses.

[0134] It was shown that the membrane-bound P72 and Penton induced significantly stronger immune responses than their secreted and intracellular counterparts in mice when delivered through mRNA in LNP formulation. Antigen- specific IgG titer induced by MB-P72 was 540-fold higher than that induced by IC-P72* (50,820 vs. 93) 14 days after boost. Consistently, the frequencies of antibody secreting cells in the bone marrow and the frequencies of T cells that were stimulated by P72 peptides to secrete IFN-y and TNF-oc tended to be higher although not statistically significant. MB-P72 also induced potent IgG responses in pigs, reaching a titer over 80,000 14 days after second immunization. Similarly, antigen- specific IgG titer induced by MB-PNISOQ was 19-fold higher than that induced by IC- PWT (3056 vs. 494) 14 days after boost. Consistently, the frequencies of antibody secreting cells in the bone marrow and frequencies of T cells that were stimulated by recombinant Penton to secrete IFN-y, TNF-oc, and IL-4were all significantly higher following immunization with MB-PNISOQ than IC-PWT. MB-PNISOQ also induced potent IgG responses in pigs, reaching a titer over 8,000 14 days after second immunization. Enhanced antibody responses are associated with elevated Tfh response and germinal center formation in the spleen. These results are consistent with previous observation showing that the membranebound Spike protein induced stronger antibody response than the secreted form of Spike protein35,36. Multiple factors may have contributed to the enhanced immunogenicity of the membrane-bound P72 and Penton. First, a large number of the membrane-bound P72 and Penton are displayed on the cell surface. The increased epitope density and their readily accessible to B cell recognition likely help to stimulate antibody responses. Second, the formation of trimeric P72 and pentameric Penton when expressed in the membrane -bound form preserves conformational epitopes, which could stimulate additional antibody responses. Third, a higher level of the membrane-bound P72 or Penton may be expressed following LNP mRNA vaccination. The induction of significantly stronger antibody and T cell responses by both the membrane -bound P72 and Penton, which differ in size and multimeric configuration, suggests this finding could be a general phenomenon.

[0135] The success of the approach disclosed herein with ASFV capsid proteins highlights its potential for developing safe and effective subunit vaccine for ASFV as well as application to other intracellular viral antigens. Many viral capsid proteins face similar challenges in antigen accessibility and presentation. By applying protein engineering to enhance extracellular presentation on the membrane, coupled with the flexibility of mRNA vaccination, this simple approach could be a powerful tool for developing subunit vaccinesagainst a broad spectrum of intracellular pathogens, without resorting the use of artificial multimeric structures such as foldon37. An approach disclosed herein enables the targeting of intracellular viral proteins that have historically been difficult to use as immunogens due to their reliance on specific intracellular pathways and post-translational modifications for folding and multimeric formation.Methods and Materials associated with the DisclosureAnimal Experiments

[0136] All procedures were conducted in accordance with the approved animal protocol 0322-021-25, which had been reviewed and approved by the Massachusetts Institute of Technology (MIT) Committee on Animal Care. The experiments adhered to the established guidelines for animal care. Female BALB / c mice, 7 weeks old, were procured from Charles River Laboratories and were housed within an MIT animal facility. LNP- mRNA vaccination of pigs was performed according to the protocols approved by Committee on Animal care (protocol number 2308000566) and Midwest Veterinary Service (MVS), Inc. (protocol number 24005).Cells and Plasmids

[0137] HEK293T (ATCC CRL-3216) cells were purchased from the American Type Culture Collection (ATCC) and grown in DMEM supplemented with 2 mM L-glutamine, non-essential amino acids, 100 U / mL gentamicin, and 10 % fetal bovine serum (FBS) (Invitrogen Life Technologies). Vero (ATCC CCL-81) and MDCK (ATCC CCL-34) cells were purchased from the American Type Culture Collection (ATCC) and grown in Eagle's Minimum Essential Medium (EMEM), 10 % fetal bovine serum (FBS) (Invitrogen Life Technologies) and 1 X pen / strep. 3D4 / 31 (ATCC CRL-2844) cells were purchased from ATCC and grown in RPML1640, 10 % filter sterilized FBS (ATCC), and 1 X pen / strep.Gene information of ASFV viral protein P72, Penton, P17, and pB602L was obtained from ASFV Georgia 2007 / 1 (Gene bank # FR682468.2). ASFV DNA encoding for three forms (membrane-bound, secreted, and intracellular) of P72 and Penton, pB602L, and P17 were codon optimized to swine and synthesized by GenScript (Piscataway NJ) and cloned into phCMVl and / or phCMV3 (HA-tagged) (Promega) via the NEBuilder® HiFi DNA Assembly (NEB, #M5520AVIAL), and the corresponding ASFV plasmids were constructed for the following characterization. Secreted form of capsid proteins was engineered by addition of signal peptide from a human CD8a (GenBank ID: NP_001139345.1) to the N terminus, whilethe membrane-bound form encompasses an additional CD8a stalk region or hinge, transmembrane region, and short cytoplasmic tail to the C terminus.Structural Modeling

[0138] The three-dimensional (3D) structures of both capsid protein subunit and multimeric forms (trimer for P72, pentamer for Penton) were predicted by AlphaFold223, followed by structural visualization using the open-source PyMOL system (PyMOL Molecular Graphics System, version 1.7, Schrodinger, LLC). Predicted structures of engineered antigens with top-ranked models were aligned with the intracellular or native form of each capsid protein for similarity analysis by calculation of the root-mean-square deviation (RMSD) metric score (the lower the score the more similar the structures).Transfection of Plasmid Expressing ASFV Viral Protein in Mammalian Cells

[0139] Confluent monolayers of HEK293T, Vero, and 3D4 / 31 cells were transfected with constructed plasmids using Lipofectamine 3000 transfection reagent (Invitrogen, #L3000008) according to manufacturer’s protocol. Forty-eight hours after transfection, cells or the whole cell lysate were used for further analysis.Confocal microscopy

[0140] Intracellular staining: Preconfluent monolayers were rinsed with phosphate- buffered saline (PBS), fixed with 4% paraformaldehyde at room temperature for 20 min, washed three times with PBS and rinsed for 5 min after each wash, permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS-T) at 4 °C for 10 min, washed with PBS for 5 min at room temperature, blocked with PBS-B solution (PBS containing 4% BSA) at 37 °C for 30 min and incubated with mouse anti-P72 antibody (MyBioSource's, clone 10G5 or clone 4H3 (obtained from The Pirbright Institute, ITB01736)anti-HA antibody (clone 16B12), or anti-His antibody (clone ADI.1.10) at room temperature for 1 h, followed by three washes with PBS. Then, the cells were incubated with fluorescein conjugated (AF488, CF568) goat anti-mouse antibody at room temperature in dark for 1 h, followed by 1 pg / mL 4 ' ,6-diamidino-2-phenylindole dihydrochloride (DAPI) solution incubation for 15 min. Cells were then washed three times as described above and observed using a fluorescence microscope (Olympus FV1200 Laser Scanning Confocal Microscope).

[0141] Surface Staining: Preconfluent monolayers were rinsed with phosphate- buffered saline (PBS), blocked with PBS-B solution (PBS containing 4% BSA) at 37 °C for 30 min and incubated with mouse anti-P72 antibody (MyBioSource's), anti-HA antibody, oranti-His antibody on ice for 1 h, followed by three washes with PBS. Then, the cells were incubated with fluorescein conjugated (CF568) goat anti-mouse antibody on ice in dark for 1 h, followed by lx CellMask™ Green (Invitrogen, # C37608) and 1 pg / mL DAPI solution incubation for 15 min. Cells were then washed three times as described above and observed using a fluorescence microscope (Olympus FV1200 Laser Scanning Confocal Microscope).SDS-PAGE and Western Blotting Analysis

[0142] The transfected cells were harvested and lysed using RIPA lysis buffer (Thermo Fisher, #89900) following the manufacturer's instructions. Subsequently, the lysed samples were combined with one-third of the total volume of 4 x sample loading buffer, with or without SDS or P-mercaptoethanol, and boiled for 10 min. To detect native proteins under non-reducing conditions, cleared cell lysate was incubated with 4x NuPAGE LDS sample buffer (Thermo Fisher Scientific) without heat-denaturing and P-mercaptoethanol reducing. To detect N-linked glycosylation of Penton, transfected cell lysates were denatured first, then incubated with PNGase F (New England Biolabs) at 37 °C for 1 h. The protein samples were resolved by SDS-PAGE using gels of varying percentages (8%, 10%, or 12%). After electrophoresis, the separated proteins were transferred onto a nitrocellulose membrane, which was subsequently blocked using 5% skimmed milk at 4 °C overnight. Next, the membranes were subjected to immunoblotting using specific primary antibodies, including anti-P72, anti-HA, anti-His, and anti-actin or anti-tubulin antibodies, at room temperature for 1 h. The membranes were washed three times with PBST (Phosphate Buffered Saline with 0.05% Tween-20) and then incubated with anti-mouse HRP-conjugated secondary antibodies (Thermo Fisher) for 1 h at room temperature. Subsequently, the membranes were washed three times with PBST. The protein bands were visualized using the Bio-Rad system (BioRad, USA), and the intensities of the bands were quantified using Image Lab imaging software (Bio-Rad).Flow Cytometry Analysis

[0143] Transfected cells expressing ASFV viral proteins were detached using trypsin, mixed with an excess of PBS, and centrifuged at 1200 rpm for 10 min. The cells were washed once with FACS buffer (PBS, 2% BSA) and resuspended in 200 pL of FACS buffer. To minimize nonspecific binding of mouse monoclonal antibodies, the Rhesus Fc Receptor Binding Inhibitor was added to the samples. The samples were then incubated on ice for 20 min without additional washing. Subsequently, without washing the samples, primary antibody staining was performed using a 1:1000 dilution of mouse anti-P72 or anti-HAantibody for 30 min on ice. After staining, the cells were washed three times with FACS buffer. Next, a 1:500 dilution of anti-mouse- AF647 secondary antibody was applied, and the cells were incubated for 30 min on ice, followed by three washes with FACS buffer. Finally, just before analysis, 5-10 pL of propidium iodide (PI) staining solution was added to each sample. The stop count for analysis was set based on viable cells as determined from a dotplot of forward scatter versus PI.Recombinant Protein Production and Sample Preparation for P72 Primer Characterization

[0144] The synthesized genes were cloned into the phCMV3 vector separately. A C- terminal HA tag was included in this vector. HEK293T cells were cultured with DMEM plus 10 % FBS at 37 °C and 5 % CO2. For a 60 mm cell culture dish, the cells were transfected with plasmids containing 10 pg MB-P72 or S-P72, 10 pg IC-P72 and 5 pg pB602L plasmid mixture and 30 or 45 pg PEI well mixed in Opti-MEM (Gibco, #31985062). The transfected cells were harvested 48 h post-transfection with RIPA buffer and cocktail protease inhibitors (Thermo scientific, # A32965) for 30 min on ice then scraped off the dish. The whole cell lysate was sonicated for 2 min and the cell lysate was centrifuged for 60 min at 20,000 rpm (JA 25.50 rotor, Beckman). The recombinant P72 protein in the supernatant were collected and applied to the anti-HA Magnetic Beads (MCE, #HY-K0201). The bound protein was washed with the resuspended buffer five times and then was eluted from the beads with a buffer containing 2 mg / mL HA peptide. The eluted sample was concentrated and further purified by Superdex 200 increase 10 / 300 GL size exclusion column (Cytiva 28990944) via capillary loop running in a buffer containing 20 mM HEPES at pH 7.4 and 300 mM NaCl. The P72 protein from size exclusion column peak was concentrated and kept at -80 °C for future use.

[0145] In sample preparation for negative stained-electron microscopy (TEM), 10 pL of protein sample and buffer containing solution was dropped on a 200 meshes copper grid coated with a continuous carbon film and waited for 60 sec and removed excess solution by touching the grid with a kimwipes and then 10 pL of negative staining solution, phosphotungstic acid, 1% aqueous solution was dropped on the TEM grid and immediately removed it by kimwipes and 10 pL of the stain was then applied to the grid and after 30 sec, the excess stain was removed by touching the edge with kimwipes. Finally, dried the grid at room temperature. After that, the grid was mounted on a JEOL single tilt holder equipped in the TEM column. The specimen was cooled down by liquid-nitrogen and imaging on a JEOL 2100 FEG microscope was done using minimum dose method that were essential to avoidsample damage under the electron beam. The microscope was operated at 200 kV and with a magnification in the ranges of 10,000-60,000 for assessing particle size and distribution. All images were recorded on a Gatan 2k x 2k UltraScan CCD camera.Production of mRNA and LNP Formulation

[0146] Genes encoding IC-P72, S-P72, MB-P72, pB602L, IC-Pwr, S-PNISOQ and MB- PNISOQ were inserted into pUC57 (primers are listed in Table 6), which contains a T7 promoter, 5UTR, 3’UTR, and polyA tail. The linearized plasmids were subject to in vitro transcription using pseudo-UTP. After mRNA purification and capping reaction, an aqueous phase of mRNA was prepared by diluting mRNA stock in 10 mM citrate buffer. The organic phase of lipid nanoparticles was prepared by adding 200 proof ethanol with lipid stock solutions which contained ionizable lipid SM102 or L002 (Advanced RNA Vaccine Technologies, ARV), DSPC helper lipid (Avanti Polar Lipids), cholesterol (Avanti Polar Lipids), and DMG-PEG-2000 (Avanti Polar Lipids). LNP-mRNA formulations were prepared by mixing organic and aqueous phases at a ratio of 1:3. RiboGreen assay (Thermo Fisher) was performed by following manufacture’s instruction to quantify mRNA after formulation. Encapsulation efficiency was measured by Picogreen assay. To validate LNP delivery of mRNAs, HEK293T and Vero cells were seeded onto 24-well tissue culture plate and 500 ng of LNP-mRNA diluted in Opti-MEM was added in individual well. Cells were collected in 48 h and protein expression was analyzed by flow cytometry and Western blotting as described below.Expression ofASFV mRNA in vitro

[0147] ASFV mRNAs were transfected into HEK293T and Vero cells with MessengerMax (Invitrogen) for naked mRNA or formulated mRNA-LNP directly. After 48h, the expression was evaluated with flow cytometry (FACS), immunofluorescence assay (IFA), and western blot (WB). For IFA, cells were incubated with mouse anti-P72 antibody or mouse anti-HA antibody and AF488 conjugated anti-mouse secondary antibody (Abeam), and images were taken with a fluorescence microscope (Olympus FV1200 Laser Scanning Confocal Microscope). For FACS, cells were trypsinized and incubated with mouse anti-P72 antibody or mouse anti-HA antibody and AF488 conjugated anti-mouse secondary antibody (Abeam). Data was acquired with LSR Fortessa HTS-2 (BD Biosciences) and analyzed with FlowJo (BD Biosciences). For WB, cells were harvested and denatured in lysis buffer. Samples were loaded and run in 8 or 12 % SDS-PAGE gel and transferred to nitrocellulose membrane. The membrane was incubated with mouse anti-P72 antibody or mouse anti-HAantibody and HRP conjugated anti-mouse secondary antibody (Invitrogen, Cat#62-6520).Anti a-actin HRP Antibody for protein loading control was purchased from Santa Cruz Biotechnology (sc-47778 HRP).Immunization of Mice

[0148] To assess immunogenicity of individual antigens in mice, six to eight-week- old female BALB / c mice were purchased from Charles River and housed in animal facility at MIT. Briefly, 5 mice were assigned to each group and immunized intramuscularly with 50 pL 5 pg LNP-mRNA (SM102 or ARV-L002) diluted in PBS. All mice were boosted three weeks later. Serum samples were collected from submandibular vein prior to immunization and two weeks after each injection. Mice were euthanized at day 35 and spleen tissue and bone marrow were collected for analysis of cellular immunity.Immunization of Pigs

[0149] A total of ten four-week-old piglets were randomly assigned to three groups: four piglets each in the MB-P72 and MB-PNISOQ groups, and two piglets in the control group, which received sterile PBS. 30 pg of LNP-mRNA expressing each individual antigen was diluted to 1 mF in sterile PBS and injected to the back of ear intramuscularly and boosted three weeks later. Serum samples were collected before vaccination and weekly after each injection. Body weight of each pig was measured before the study and at the termination. All pigs were euthanized two weeks after boost and spleens were collected for testing T cell responses.Antibody Measurement with ELISA

[0150] Each well of a flat-bottomed, high-binding 96-well plate (Santa Cruz Biotechnology) was coated with approximately 50 pL of antigen solution, prepared at a concentration of 5 pg / mL of purified recombinant ASFV viral protein in PBS (pH 7.4). The plates were then incubated at 4 °C overnight. Following the incubation, the antigen solutions were carefully aspirated, and the plates underwent five washes using PBS Tween (PBST, containing 0.05% v / v Tween-20; pH 7.4). Subsequently, the wells were filled with blocking buffer (5% skimmed milk in PBS) and incubated at 37 °C. After a 1 h incubation at room temperature, the blocking buffer was aspirated, and all wells received an additional five washes with PBST. For the subsequent step, mouse sera were subjected to three-fold serial dilutions, starting at a 1:100 dilution, in PBS containing 5% skimmed milk. These diluted sera were added to the plates at a volume of 100 pF per well and incubated for 1 h at 37 °C.Following this incubation, the plates were washed five times with PBST. To detect IgG, goat anti-mouse IgG conjugated to HRP (Cell Signaling Technology, diluted 1:2,000 in PBS with 5% skimmed milk) was used as the secondary antibody. The secondary antibody solution was added to the plates, and the plates were incubated at 37 °C for 1 h. Afterward, the plates underwent five additional washes using PBST. To develop the assay, 100 pL of HRP substrate 3,3',5,5'-tetramethylbenzidine (TMB) (Cell Signaling Technology) was added to each well. The plates were gently shaken for 15 min, and the reaction was stopped by adding 100 pL of a stop solution (0.2 M H2SO4) to each well. The absorbance was measured at two wavelengths, 450 nm (signal) and 570 nm (background), using a Tecan microplate reader. End-point titers were determined as the highest serum dilution at which the optical density difference, compared to serum from sham- vaccinated mice at the same dilution, exceeded 0.005.Enzyme-linked. Immunospot (ELISPOT) assay

[0151] Splenocytes from vaccinated mice were evaluated for antigen specific IFN-y by Enzyme-linked immunospot (ELISPOT). ELISPOT assays were performed using a Mouse IFN-y, TNF-a, and IL-4 Single-Color ELISPOT kit from ImmunoSpot followed the manufacturer’s instruction. Briefly, splenocytes were plated at 4 x 105cells / well and cocultured with either 5 pg / mL P72 peptide pool (Core facility, Koch Institute), 2 pg / mL recombinant Penton protein, Cell Stimulation Cocktail (500X) containing phorbol 12- myristate 13-acetate (PMA) and ionomycin (eBioscience, # 00-4970-93) or medium alone in a total volume of 200 pL / well T cell media for 36 h at 37 °C in 5 % CO2. The plates were incubated with detection antibodies, Biotin-IFN-y and Streptavidin-HRP at RT for 1-2 h, respectively. The plates were developed with 50 pL / well the development solution for up to 30 min. Color development was stopped by washing under running tap water. After air-dried, colored spots were counted using a CTL ELISPOT Reader System and CTL ELISPOT Software.

[0152] For B cell ELISpot, bone marrow from mouse femur and tibia of both hind limbs were collected into PBS supplemented with 1 mM EDTA and 2% FBS. Bone marrow plasma cells (BMPCs) were further enriched using EasySep™ Release Mouse CD138 Positive Selection Kit (StemCell Technologies) and cryopreserved in 10% DMSO in FBS. A mouse IgG single-color ELISpot kit (ImmunoSpot) was used to determine total IgG-secreting cells in enriched BMPCs according to the manufacturer’s instructions. ASFV proteins (300 ng / well) were coated onto the ELISpot plate to determine the antigen- specific -binding IgG-secreting cells in BMPCs. ELISpot plates were further analyzed using ELISpot plate reader (Cellular Technology).Germinal Center Reactions

[0153] To image the germinal centers, one fifth of the spleen was embedded in Optimal Cutting Temperature (O.C.T.) solution (VWR) solution for 5 min at room temperature followed by snap-freezing in liquid nitrogen. Tissue blocks were stored in -80 °C until cryostat sectioning into 6 pm sections using a microtome. Sectioned tissue slides were first air dried for 30 min and an unbroken circle was drawn with a PAP pen (Vector Laboratories) to create a hydrophobic barrier. The slides were blocked in room temperature (RT) for 2 h by adding PBS containing 2% BSA, 10% normal goat serum, and TruStain FcX™ (anti-mouse CD16 / 32) Antibody (Biolegend). Slides were washed three times with 0.5% Tween-20 in PBS and permeabilized using 2% Trion X-100 (Sigma) followed by staining according to a modified procedure published previously43. Briefly, slides were stained with a primary antibody cocktail containing AF647-labled rat anti-mouse IgD antibody (clone l l-26c.2a, Biolegend), Hamster anti-Mouse CD3e (Clone 500A2, BD Biosciences), biotin-conjugated CD21 / CD35 antibody (Clone 8D9, Invitrogen), AF488- conjugated Ki-67 antibody (Clone SolA15, Invitrogen). After overnight incubation at 4°C, slides were washed with PBST for three times followed by adding secondary antibody cocktail containing AF594-conjugated goat anti-hamster IgG (Jackson ImmunoResearch) and AF546-conjugated Streptavidin (Invitrogen). Slides were washed three times and treated with a ReadyProbes™ Tissue Autofluorescence Quenching Kit (Invitrogen) and coverslips were mounted using SlowFade™ Diamond Antifade Mountant (Invitrogen). Mounted slides were protected from light and kept at 4 °C until imaging using TissueFAXS SL Fluorescent Slide Scanner with 20x objective and images were analyzed using StrataQuest software (TissueGnostics).Follicular T helper Cell Response

[0154] Isolated splenocytes were treated with ACK red blood cell lysis buffer (Gibco) and blocked for Fey receptor blockade using TruStain FcX™ (anti-mouse CD 16 / 32) Antibody (Biolegend). Cells were stained by an antibody cocktail containing PE Rat AntiMouse CD279 (PD-1) (Clone RMP1-30, BD bioscience), FITC anti-mouse CD4 (Clone GK1.5, Biolegend), PE / Cy7 anti-mouse / human CD44 (Clone IM7, BioLegend), BUV395 Rat anti-mouse CXCR5 (Clone 2G8, BD Bioscience). Stained cells were washed three times andstained for viability by DAPI. Samples were analyzed by flow cytometry on a BD Symphony A3 and analyzed on FlowJo software.Statistical Analysis

[0155] GraphPad Prism 8.0 was used for conducting statistical analyses and generating data plots. Two-tailed Student's t-tests were used to assess distinctions between two independent sample groups. One-way analysis of variance (ANOVA) was used for comparisons involving multiple sample groups. The results are presented in the form of mean values along with their standard deviations (mean ± SD). A threshold of P < 0.05 was considered statistically significant. Significance levels were defined as follows: ns (not significant) for P > 0.05; **** for P < 0.0001; *** for P < 0.001; ** for P < 0.01; and * for P < 0.05.Example 2. Selection, Design and Immunogenicity Studies of ASFV Antigens for Subunit mRNA Cocktail Vaccines with Specific Immune Response Profiles

[0156] African swine fever (ASF) is a highly contagious swine viral disease that causes almost 100% mortality in domestic pigs. Currently the United States is free of ASF and incursion to the US swine population could result in approximately $50 billion losses (J). The causative agent, African swine fever virus (ASFV), is a large DNA virus with a doublestranded DNA genome of 170 to 190 kb, encoding at least 150 proteins. Although a live attenuated vaccine was licensed in Vietnam in 2023 and in the Philippines in 2024, it has major safety concerns (2). A safe and effective subunit ASF vaccine is urgently needed.

[0157] ASF vaccine development faces several major hurdles: i) ASFV is classified as a biosafety level 3 (BSL-3) agent. The requirement for BSL-3 containment for any virus work limits the research on the virus and vaccine development, ii) Viral antigens to elicit protective immunity against ASFV infection have not been identified, impeding the development of effective subunit vaccines, iii) ASFV exhibits complex multi-layer structures. Both the extracellular enveloped virion (EEV), i.e., with the outer membrane, and intracellular mature virion (IMV), i.e., without the outer membrane, are infectious (3). iv) ASFV can infect host macrophages through multiple entry mechanisms, including receptor- mediated endocytosis, clathrin-mediated endocytosis, macropiniocytosis, and phagocytosis. As a result, there is no reliable viral neutralization assay for measuring anti-viral antibody responses induced by any single antigen, v) Immunological correlates of protective immunity against ASFV are not fully defined. Studies have shown that antibody responses elicited byinactivated virus were not sufficient to protect pigs from infection (4), whereas immune responses by live attenuated virus confer protection (5). Therefore, T cell immunity likely play a pivotal role in the protection. Lymphocyte depletion studies showed that cytotoxic CD8+lymphocytes are important for ASFV clearance (6). IFN-y responses correlate with the degree of cross-protection against heterologous ASFV challenge (7). Therefore, an effective ASFV vaccine must induce both humoral and cellular immunities.

[0158] ASFV is the only member in Asfarviridae, which belongs to the phylum of nucleocytoplasmic large DNA viruses (NCLDV), characterized by the complex virion structures, large genomes and protein-coding capacity. Poxviridae, a well-characterized family in NCLDV, is the most closely related to Asfarviridae phylogenetically (FIG. 15). As a key member of Poxviridae, the use of vaccinia virus (VACV) as a vaccine has contributed tremendously to the eradication of smallpox disease caused by Variola virus. Like ASFV, VACV also produces two infectious virions: EEV and IMV. Studies have shown that subunit vaccines, composing two EEV antigens (B5R and A33R) and two IMV antigens (L1R and A27L), provide complete protection against lethal VACV challenge in mice (S-77), suggesting the feasibility of developing safe and effective subunit vaccines for NCLDV with appropriate viral immunogens.

[0159] The success of lipid nanoparticle (LNP) -delivered mRNA encoding the spike protein as COVID-19 vaccines motivates broad applications of mRNA-based vaccines. In mRNA vaccines, proteins are synthesized by the host cells and likely maintain native structural conformation, and therefore could induce both humoral and cellular immunities. Antibodies produced by B cells could block viral infection of host cells through neutralization and help to inactivate or eliminate viruses through other effector mechanisms, such as antibody-dependent complement deposition (ADCP), antibody-dependent cellular phagocytosis (ADCP), and antibody-dependent cellular cytotoxicity (ADCC). T cells could recognize virus-infected cells and help to clear the virus through cytokines or directly killing of the infected cells. In addition, mRNA vaccines are especially suited for developing subunit vaccines as multiple mRNAs can be formulated into the same LNP, greatly simplifying the manufacturing process.ResultsSelection and design of ASFV candidate antigens

[0160] To identify viral antigens for a safe and effective subunit mRNA vaccine for ASF, ASFV antigens were selected using the following approaches. First, we selected ASFVhomologs corresponding to the subunits of the protective VACV vaccine because ASFV is most closely related to VACV (FIG. 15) and both viruses produce two types of infectious virions (EEV and IMV). Four VACV antigens were used in the subunit vaccine, including A33R and B5R from EEV and L1R and A27L from IMV (Table 9). Thus, ASFV EP153R was selected, encoding C-type lectin, which is localized on the outer membrane and shares 18% amino acid identity with VACV A33R and ASFV CD2-like protein (CD2v or EP402R), a major outer membrane protein with a size around 41 kDa, which shares 12% amino acid identity with VACV B5R (42 kDa) (FIG. 9A). L1R, localized on IMV of VACV, is responsible for membrane fusion. In ASFV, both E248R and E199L have been reported to play a similar role (72, 73) and share 14% and 8% amino acid identities with L1R, respectively. VACV A27L exists as a trimer in the capsid on IMV and contributes to attachment to cell heparan sulfate receptor (74, 75). In ASFV, the viral capsid is formed by two surface-exposed proteins, the trimeric P72 (B646L) and pentameric Penton (H240R), and several unexposed proteins, M1249L, P17, and P49, which form a network beneath P72 and Penton (76). Because both P72 and Penton are viral proteins expressed in the cytoplasm of infected cells and exist in multimeric forms in the capsid, they were engineered as membrane-bound (MB) form to preserve the multimeric structures and to more effectively induce antibody responses (77). It was shown that the monoclonal antibody clone #4H3 that binds to the native P72 (7S) also recognizes the membrane-bound form of P72 (MB-P72) (77). When Penton was expressed as the membrane-bound form (MB-Penton), it was found that it became glycosylated probably due to its synthesis in the endoplasmic reticulum (ER) and transitioning through Golgi apparatus. To prevent the unnatural glycosylation, which may interfere with induction of proper antibody response, the N-glycosylation site (asparagine at amino acid residual 180) was identified and the asparagine was mutated to glutamine (N180Q) (MB-PNISOQ) (77). Therefore, MB-P72 and MB-PNISOQ are tested here as candidate vaccine antigens. For simplicity, MB-P72 and MB-PNISOQ are referred to as P72 and Penton herein. All other candidate antigens were expressed in their native forms.

[0161] Second, ASFV antigens that have shown to be promising were selected. ASFV P72 and CD2v were reported to induce neutralizing antibodies, which provide partial protection against lethal viral challenge (79-25). ASFV EP153R was shown to contain several T cell epitopes and deletion of both EP153R and CD2v genes from ASFV resulted in reduced viremia and disease symptoms (26, 27). ASFV P54 was shown to induce neutralizing antibody response or partial protection against viral challenge (4, 28). E199L and E248Rwere reported to play a critical role in membrane fusion (12, 29). ASFV P22 is a highly immunogenic protein with application for serological diagnostics and potential role in maintaining virion structure (30, 31). Hence, besides P72 and Penton, CD2v, EP153R, P54, P22, E199L and E248R, were also selected, which are all localized on the viral envelope, as candidate antigens for evaluation (FIG. 9A).

[0162] Third, MTE was developed to stimulate strong cellular immunity against ASFV as both antibody and T cell responses contribute to the protection against ASFV. T cell epitopes were selected based on i) searching IEDB database and literature for experimentally identified epitopes by IFN-y EEISpot and / or MHC / mass spectrometry using recovered pig lymphocytes, and ii) NetMHCpan prediction of epitopes that bind to the most prevalent swine leukocyte antigen (SEA) alleles (SEA-E0101, SEA-E0401 and SEA-E0801) and those with highest binding affinity from the abundantly expressed viral proteins (32, 33) were selected. A total of 27 epitopes were selected (Table 10, including 22 experimentally identified epitopes (6 from EP153R, 4 from PP62, 5 from MGF100-1E, 1 each from A238E, K145R, MGF505-7R, P34, and P37, 2 from P150) (6, 27, 34-38), and 5 predicted epitopes (3 from M448R and 2 from MGF505-7R). The 6 overlapped epitopes from EP153R were covered by a 56 amino acid fragment and the 5 overlapped epitopes from MGF100-1E were covered by a 29 amino acid fragment. Five additional amino acid residues from the native proteins were added at both ends of the two fragments. Five additional amino acid residues from the native proteins were also added to each of the rest of 16 epitopes for facilitating proteasomal degradation. All epitopes were fused together in the order of M448R, MGF505-7R, PP62, MGF100-1E, A238E, K145R, P34, P150, P37, and EP153R, which avoid disordered polypeptide structure based on Alphafold 2 prediction (39). Notably, the 6 known T cell epitopes from CD2v and 1 from P72 were not included in the MTE design as these two antigens were tested separately. In addition, to facilitate the expression of the MTE antigen, the highly expressed ASFV P30 is linked to MTE by a direct GGGS linker (P30-MTE), or a P2A self-cleavage site (P30-P2A-MTE), or an internal ribosome entry site (40) signal (P30- IRES-MTE) for direct translation of MTE (FIG. 9B). Notably, P30 is highly immunogenic and has also been reported to induce neutralizing antibodies (4, 28, 41). Similar strategies to enhance protein expression by employing fusion partners have been reported before (42, 43).

[0163] In total, 11 vectors expressing CD2v, EP153R, P72, Penton, P22, E199L, E248R, P54, P30-MTE, P30-P2A-MTE, and P30-IRES-MTE were constructed and tested based on sequences from the genotype II Georgia 2007 / 1 strain (FIGs. 9A-9B, 16A and FIG.21A). Because specific antibodies are not available for all selected ASFV proteins, except P72, P54 and P30, a HA tag was added at the C-terminus of the most proteins for easy monitoring of their expression. The HA tag was removed for immunogenicity studies in pigs.Validation of expression of candidate antigens in vitro

[0164] Expression of the selected ASFV candidate antigens was validated in cell lines. Abundant fluorescence signals were detected for all vectors in the transfected but not untransfected cells (FIG. 9C). Expressions of CD2v, P22, P54, EP153R, E199E, and E248R were also detected by Western blotting. Notably, CD2v and EP153R were highly glycosylated, consistent with previous reports (44, 45). Expression of P72 and Penton were validated previously (77). Both P30 and MTE from all three different designs were readily detected in the cytosol of transfected cells (FIG. 9D). Thus, the selected ASFV antigens can be expressed in cell lines.

[0165] Next, mRNA for each candidate antigen was prepared by in vitro transcription and formulated the mRNA in ENP. Briefly, DNA fragments encoding P72, Penton, CD2v, EP153R, E199E, E248R, P22, P54, P30-MTE, P30-P2A-MTE, and P30-IRES-MTE were inserted into a pUC plasmid containing the T7 promoter, 5’UTR, 3’UTR, and polyA (FIG. 14B and FIG. 21B). The ENP-mRNA formulations were further subject to physicochemical analysis, including encapsulation efficiency, poly dispersity index, and hydrodynamic size (77, 46). Results showed an average particle size of 80 - 120 nm in diameter and a low poly dispersity index (PDI) of 0.1 to 0.2 (Table 11), suggesting that the ENP-mRNA particles have a uniform particle size distribution, optimal for particle internalization and biodistribution. ENP-mRNAs were transfected into HEK 293T cells and expression of all candidate antigens was readily detected by flow cytometry. These results suggest that formulated ENP-mRNAs are efficiently translated.Induction of antibody and T cell responses in mice by LNP-mRNA vaccination

[0166] The immunogenicity of the candidate antigens was first evaluated in mice. Mice were immunized separately with each of the LNP-mRNAs at day 0 and 21 and sera were collected at day 14 and 35 for measuring antigen- specific IgG titers by ELISA. Compared to before immunization, antigen- specific IgG responses, as indicated by the endpoint titers, were significantly induced in all immunized mice 14 days after the first immunization and further boosted at day 35 (FIG. 15). However, the levels of IgG titers varied significantly among the different antigens. For example, 14 days after the first immunization antigen- specific IgG titer was only 1.2-fold over the background for E248Rwhereas the titer was 125.2-fold for P22. Fourteen days following the boost, the titer was increased to 126 (2.2-fold) for E248R and to 521,675 (83-fold) for P22. Among the 11 different antigens, P22, P30-IRES-MTE and P30-P2A-MTE induced the highest titers of antigen- specific IgG responses after boost, followed by E199L, P72, Penton, P54 and CD2v, with the lowest for EP153R, E248R and P30-MTE (FIG. 15). Similarly, LNP-mRNA immunization induced significant antigen- specific T cell responses as indicated by ELISPOT assay for cytokines IFN-y and TFN-OC following re- stimulation of splenocytes with either specific recombinant proteins or P72 peptides (FIG. 10B). T cell responses also varied significantly among different antigens. For example, CD2v stimulated highest level of cytokine-secreting spots, followed by EP153R, P54, Penton, TNF-oc, P72 , and P22. These results show that mRNA expressing the selected ASFV antigens induce both antibody and T cell responses, but the magnitudes of the immune responses vary considerably among the different antigens.

[0167] The three MTE constructs were also used to immunized mice separately. P30- IRES-MTE induced the highest titer of anti-P30 IgG (FIG. 15) and highest numbers of MTE- specific IFN-y and TNF-a spots (FIG. 10C), followed by P30-P2A-MTE. P30-MTE induced the lowest antibody and T cell responses. Notably, the MTE-specific T cell responses, as indicated by the numbers of IFN-y and TNF-a ELISpots, were significantly higher than P30- specific responses, indicating that the T cell-directed antigen (MTE) is potent. Compared to P30-MTE, where P30 is fused with MTE via a GGGS linker, in P30-P2A-MTE, P30 and MTE are synthesized as a single polypeptide that is cleaved at P2A site, and in P30-IRES- MTE, P30 and MTE are synthesized separately through the internal ribosomal entry site (40). These results show that a complete separation of P30 and MTE translation through IRES is the most effective for inducing both P30-specific antibody responses and MTE-specific T cell responses.

[0168] ASFV induced hemadsorption is characterized by the adherence of red blood cells to the surface of infected cells (47). This phenomenon is primarily mediated by viral protein CD2v expressed on the surface of infected cells. Sera from unimmunized mice did not show significant inhibition of hemadsorption or “rosette” formation surrounding CD2v- expressing cells, while the sera from CD2v LNP-mRNA immunized mice inhibited “rosette” formation. Quantification of percentages of inhibition showed that CD2v immunized sera yielded 64% inhibition of hemadsorption as compared to 13% with control sera (FIG. 10E). These results show that although CD2v-specific IgG titer is relatively low compared to thoseinduced by other ASFV antigens (FIG. 15), the antibodies can specifically block CD2v- mediated hemadsorption.Induction of antibody and T cell responses in pigs by LNP-mRNA vaccination

[0169] Immunogenicity of the following ASFV antigens was next evaluated in pigs based on their induction of relatively higher levels of antibody and T cell responses in mice, including P72, Penton, P22, E199L, P54, CD2v, EP153R, P30-IRES-MTE, P30-P2A-MTE. Nine groups of commercial piglets at 6 weeks of age were immunized separately with 30 pg mRNA in LNP formulation for each antigen (4 pigs per group) twice at day 0 and day 21 (FIG. 11). No clinical signs were observed throughout the study and immunized pigs gained as much weight as control pigs, suggesting that LNP-mRNA is safe in pigs. Sera were collected every 7 days for measuring antigen-specific IgG titers.

[0170] Antigen- specific IgG responses, as indicated by the endpoint titers, became detectable in all immunized pigs 14 days after the first immunization, increased steadily afterwards, and reached the highest level at day 35, i.e., 14 days after boost (FIG. 16). As observed in mice, the levels of antigen- specific IgG titers varied significantly among the different antigens. Among the 9 antigens, P72, P22, E199L and P30 of P30-IRES-MTE induced the highest titers of antigen- specific IgG responses at day 35, followed by Penton and P30 of P30-P2A-MTE, with the lowest for CD2v, EP153R, and P54. Similarly, LNP- mRNA immunization induced significant antigen- specific T cell responses as indicated by ELISPOT assay for IFN-y-secreting cells following re- stimulation of splenocytes with either recombinant proteins (CD2v, EP153R, Penton, E199L, P54, P22, P30) or P72 peptides or MTE peptides (FIG. 1 IB). P30 and MTE peptides were used separately to stimulate splenocytes from P30-IRES-MITE or P30-P2A-MTE-immunized pigs to measure P30- and MTE-T cell responses. T cell responses also varied significantly among different antigens. For example, MTE of P30-IRES-MTE stimulated highest numbers of antigen- specific IFN-y- secreting spots (103), followed by CD2v (74) and MTE of P30-P2A-MTE (73). These results show that mRNA expressing the selected ASFV antigens induces both antibody and T cell responses in pigs, but the magnitudes of the immune responses vary considerably among the different antigens.

[0171] The correlations of antibody and T cell responses were analyzed for different antigens between pigs and mice. IgG responses induced by the same antigens in pigs and mice were highly correlated with Pearson correlation coefficient of 0.93 (p<0.005) (FIG. 11C). Similar, T cell-mediated IFN-y responses induced by the same antigens in pigs andmice were also highly correlated with Pearson correlation coefficient of 0.87 (p<0.005) (FIG. 1 ID). The similar results between mice and pigs cross-validate the two studies and also suggest that mice can be used to replace pigs for assessing immunogenicity of ASF mRNA vaccines in most cases.Distinct effector functions by antibodies specific for different ASFV antigens

[0172] Due to the large size and structural complexity of virion, ASFV can infect host macrophages through multiple mechanisms, including receptor-mediated endocytosis, clathrin-mediated endocytosis, macropiniocytosis, and phagocytosis. As a result, viral neutralization assay with sera from immunization with a single ASFV antigen is not reliable (49) and antibody-mediated neutralization is not sufficient to confer protection (4). To identify potential host-dependent antiviral functions of antibodies induced by different ASFV antigens, their effector functions were determined, including ADCD, ADCC and ADCP.

[0173] To measure ADCD, CHO cells stably expressing individual ASFV antigens were incubated with heat-inactivated pig serum, followed by addition of non-heat-inactivated serum from placebo pigs as a source of complement, and cell lysis was quantified by flow cytometry (FIG. 18A). ADCD activities of sera varied depending on the immunizing antigens, but overall, the sera from EP153R, P72, Penton, and P30-IRES-MTE-immunized pigs had significantly higher ADCD activities than sera from CD2v, P22, P54, E199L, and P30-P2A-MTE- immunized pigs (25-31% vs. 9-17%, FIG. 18B).

[0174] To measure ADCC, CHO cells stably expressing individual ASFV antigens were incubated with heat-inactivated pig serum, followed by addition of pig peripheral blood mononuclear cells (PBMCs) as a source of natural killer (NK) cells, and lysis of CHO cells was quantified by a luminescent assay (FIG. 18C). Sera from P54-immunized pigs induced highest CHO cell lysis (mean: 76%), followed by sera from EP153R (mean: 64%), Penton (mean: 61.2%), P72 (mean: 56%), CD2v (mean: 51.2%), and P22 (mean: 48.6%) immunized pigs, and sera from E199L, P30-IRES-MTE and P30-P2A-MTE had lowest cell lysis (mean: 19-30%) (FIG. 18D).

[0175] To measure ADCP, fluorescent beads were coated with specific ASFV proteins and incubated with immune sera and a pig macrophage cell line 3D4 / 31, and phagocytosis of labeled beads was quantified by flow cytometry. Sera from P72 and Penton- immunized pigs induced highest levels of ADCP (FIG. 18F), which were 2-3-fold higher than the ADCP induced by sera from pigs immunized with CD2v, EP153R, P22, P54 or E199L.Notably, ADCP activity was lowest for sera from pigs immunized with P30-P2A-MTE, despite robust anti-P30 IgG responses.

[0176] These results show that antibodies induced by different ASFV antigens exhibit different effector functions, which could be exploit for developing cocktail vaccines with desired immune profiles.Identification of antigen combinations for cocktail vaccines by computational analysis

[0177] To identify optimal antigen combinations, computational analysis of five immune parameters was performed: T cell response (IFN-y), antigen- specific IgG level at day 35, ADCD, ADCC, and ADCP. Average values from four pigs per antigen were calculated and normalized by ranking each immune parameter from 1 (lowest) to 8 (highest). P30 of P30-IRES-MTE was used for analysis. The ranking revealed distinct patterns: P22 and P30 induced the highest IgG responses; CD2v and EP153R induced the highest T cell response (IFN-y); Penton and EP153R induced antibodies showing the highest ADCD activities; P54 and EP153R induced antibodies with the highest ADCC activities; and Penton and P72 induced antibodies with the highest ADCP activities (FIG. 19A). When the magnitude of each immune parameters from individual antigen was taken into consideration, similar patten was also found in the chord diagram showing correlations between antigens and immunological categories. Correlation analysis of the five immune parameters across all antigens indicated a positive correlation between Fc-mediated ADCD and ADCP, and between ADCC and T cell response (IFN-y). However, IgG response was negatively correlated with IFN-y and ADCC.

[0178] Optimal antigen combinations were scored by summing the ranks of selected immune parameters. Among the eight antigens tested (CD2v, EP153R, P72, Penton, P22, P54, E199L, P30), all possible 3-way, 4-way, and 5-way combinations were developed based on rank sums of the five immune parameters ("IgG", "IFN-y", "ADCD", "ADCC", "ADCP") (FIGs. 20A-20C). For the highest response across five, four or three parameters, the optimal 3-way combination was EP153R_P72_Penton (FIG. 20A) and the optimal 4-way combination was EP153R_P54_P72_Penton (FIG. 20B). Although the top 5-way combinations across five, four or three parameters were different, but they all included EP153R, P72 and Penton (FIG.20C). When ADCP was excluded because of the potential to enhance ASFV infection of macrophages due to antibody-dependent enhancement (50, 57), the best 3-way cocktail remained EP153R_P72_Penton, with addition of P54 for the 4-way and addition of P54 and P30 for the 5-way combinations. Additionally, when only IgG and Tcell responses were considered, the top 3-way combinations were CD2v_P30_P72 or CD2v_ E199L_P72, the top 4- way combination was CD2v_E199L_P30_P72, and the top 5- way combination was CD2v_E199L_P30_P72_Penton (FIGs. 20A-20C). Although MTE-induced T cell responses and CD2v antibody-mediated hemadsorption inhibition activity were not considered here, optimal combinations for targeted immune profiles provide novel insights for developing cocktail mRNA vaccines.Induction of robust antibody and T cell responses by cocktail mRNA vaccine candidates

[0179] Cocktail vaccines with antigen combinations were evaluated based on computational analysis and the following considerations. All the cocktails contained P30- IRES-MTE and P72 with the former stimulating robust T cell immunity and the latter as a major capsid protein for inducing antibody responses with partial protection (FIG. 12A). P72 was also identified as one of the three antigens in the optimal 3-way combination based on the highest response across five, four or three parameters above (FIGs. 20A-20C). CD2v was included in cocktails 1-3 to provide maximal induction of T cell immunity due to abundant T cell epitopes present in CD2v and induction of antibodies that inhibit hemadsorption, which is critical for controlling viral spread (27, 52). Compared to cocktail 1, Penton was included in cocktail 2 as Penton is a minor but exposed capsid component, was identified as one of the three antigens in the optimal 3-way combination, and no study has examined whether antibodies against Penton is protective. Cocktail 3 contains another highly immunogenic protein, E199E and Cocktail 4 contained P22.

[0180] The immunogenicity of candidate cocktail vaccines was evaluated in mice. Antigen- specific IgG was detected 14 days after the first immunization and the titers were significantly increased (6-528-fold) 14 days following boost (FIG. 12B). At day 35, IgG titers for P30 and P22 were the highest, the same as observed in mice immunized with P30-IRES- MTE or P22 individually (FIG. 15). The IgG titers for the rest of antigens were similar , ranging from 2xl03to 1.6xl04. Similarly, MTE induced the highest level of T cell response as indicated by the numbers of IFN-y EEISPOTs in all cocktails (FIG. 12C), followed by CD2v, with the lowest for P72.

[0181] To further determine whether the immune responses elicited by specific antigens in the cocktail vaccines is comparable to those elicited by individual antigens, Pearson correlation analysis was conducted. The analysis revealed correlation coefficients of 0.95 for IgG antibody responses and 0.80 for T cell responses. Similar levels of antigenspecific IgG and T cell responses induced by specific antigens from immunization withindividual LNP-mRNA versus the cocktail LNP-mRNA suggests that the inclusion of 3 or 4 mRNA in the same LNP formulation does not interfere with induction of immune responses to each antigen in the cocktail. These results also show that the cocktail ASFV vaccines induce potent antibody and T cell responses.Discussion associated with Example 2

[0182] Live ASF vaccines have significant safety concerns, while protein- and DNA- based subunit vaccines showed limited or no protection. Compared to these conventional platforms, the mRNA vaccine platform has demonstrated tremendous advantages, including good safety profiles, ease of manufacturing, expression of antigenic proteins in native conformations, and induction of strong T cell response. As the first step toward developing a safe and effective mRNA subunit vaccine for ASF, as presented herein viral antigens were rationally selected and designed, their immunogenicity was evaluated in mice and pigs, cocktail vaccines with specific immune response profiles were developed, and the induction of robust humoral and cellular immunities of the selected combinations were validated in mice. An mRNA platform was chosen for a potential ASFV subunit vaccine for the following reasons: First, although immunological correlates for a protective ASFV vaccine is not fully defined, studies have shown that antibody responses elicited by inactivated virus were not sufficient to protect pigs from infection (4), whereas immune responses induced by live attenuated virus confer protection (5). Therefore, T cell immunity likely plays a pivotal role in the protection, and an effective ASFV vaccine must induce both humoral and cellular immunities. Second, in mRNA vaccine, immunogens are expressed in native conformation by the host cells and have a higher chance to induce antibodies that recognize the native viral antigens (conformational epitopes), as well as induce CD8+T cell responses that are effective in clearing virus from infected cells. Third, multiple mRNAs can be easily formulated into the same LNP, greatly simplifying the development of a cocktail vaccine.

[0183] A key aspect of subunit vaccine development is the identification of immunogens to include in the vaccine. This is especially challenging for ASFV. In the selection and design of ASFV antigens, the following four novel approaches were used: First, ASFV antigens that are homologs of the subunits of the protective VACV vaccine were selected (S, 9). Asfarviridae is phylogenetically most closely related to Poxviridae. A VACV subunit vaccine with two antigens from EEV and two antigens from IMV confers complete protection against lethal challenge in mice (9, 10, 53), which was correlated with induction of serum-neutralizing antibodies and vaccinia virus- specific CD8+T cells (54, 55). Thus, thecorresponding ASFV antigens were selected based on the same localization on virion, similar functions, and amino acid sequence homology (Table 9). Second, ASFV antigens P72 and Penton were selected because structural study identified them as exposed on the viral capsid and that P72 is the major capsid protein and Penton is a minor capsid protein (76). In particular, Penton has never been investigated as a vaccine antigen. Third, even for promising ASFV antigens identified previously, antigens were designed through protein engineering in order to induce strong immune responses. For example, P72 and Penton were expressed, both are synthesized in the cytoplasm of infected cells, in membrane-bound form, which form multimeric structures without viral chaperone and induced stronger antibody responses (77). Fourth, T cell-directed (MTE) antigen was designed to induce broad and robust cellular immunity. MTE contains 27 experimentally identified and computationally predicted T cell epitopes with majority being CD8 T cell epitopes. To facilitate MTE expression, MTE was linked to the abundantly expressed P30 through three strategies, GGGS linker, P2A, and IRES with the expectation that efficient translation of P30 may facilitates synthesis of MTE downstream. In vivo testing of the three MTE designs showed that LNP-mRNA expressing P30-IRES-MTE stimulated strongest T cell responses (FIGs. 10A, 10B-10D, 11A, 11C-E, 18 and 19), probably due to the direct initiation of MTE expression via IRES element. Based on these four criteria, eleven ASFV antigens were selected: two each from the outer membrane and capsid, five from the inner membrane (FIG. 9A).

[0184] The immunogenicity of the selected ASFV antigens was evaluated in LNP- mRNA formulation in both mice and pigs with concordant results. However, the magnitude of antibody and T cell responses induced by different ASFV antigens was quite different. For example, P30, P22, P72 and E199L induced robust antibody responses, but T cell responses to P30 and P22 were low (FIGs. 10A, 10C-10F, 11A, 11C-11E, 18 and 19). In contrast, CD2v and EP153R induced only modest antibody responses, probably due to their high level of glycosylation , but induced robust T cell responses. These findings are in line with previous reports of abundant T cell epitopes harbored by CD2v and EP153R (56). Consistent with previous observations (44, 57, 58), E199L mRNA vaccine is quite immunogenic and anti-sera induced by CD2v mRNA vaccine are effective in inhibiting hemadsorption, suggesting the functionality of the induced antibodies. Immunogenicity of Penton has not been investigated. These results show that Penton (in membrane-bound form with N180Q mutation to prevent glycosylation) induces both antibody and T cell responses. These results also show that MTE antigen induced the highest level of T cell responses, suggesting thevalidity of a design disclosed herein. Overall, these results with mRNA vaccines are consistent with previous reports, but the strict comparison may not be possible due to difference in experimental details. For example, in the assay for IgG titer by ELISA, recombinant proteins (CD2v, EP153R, Penton, P54, P22, P30) purified from E. coli were used, which lack glycosylation as compared to their counterparts from mammalian expression system. Furthermore, peptide mixtures were used for recall stimulation of splenocytes from P72 and MTE immunized animals whereas proteins were used for other antigen groups. Nevertheless, induction of divergent antibody and T cell responses by different ASFV mRNA vaccine antigens suggest the need to strategically combine antigens for inducing a balanced immune response for optimal protection (see below).

[0185] Another major challenge of ASFV vaccine development is a lack of reliable viral neutralizing assay due to infection of host macrophages by both EEV and IMV through receptor-mediated endocytosis, clathrin-mediated endocytosis, macropiniocytosis, and phagocytosis. This is further compounded by the requirement of BSL3 containment for any live ASFV work. To get around these obstacles, effector functions were determined, including ADCD, ADCC and ADCP, of anti-sera from pigs immunized with different ASFV antigens. For ADCD and ADCC assay development, the P72 and Penton were engineered to anchor on the cell membrane. Antibodies targeting these two proteins may potentially lead to viral lysis through ADCD and ADCC. The same as antibody and T cell responses, anti-sera induced by different antigens exhibited divergent effector functions (FIGs. 18A-19). Nevertheless, Fc-mediated effector functions, ADCD and ADCP, were correlated, so as ADCC and T cell response (IFN-y). Interestingly, IgG response was negatively correlated with IFN-y and ADCC. To further compare the effector functions, sera from convalescent pigs should be tested in further studies. To develop cocktail vaccines with desired immune response profiles, computational analysis was used to rank order antigen combinations based on the five semi-quantitative immune parameters: the levels of IgG responses, the levels of T cell responses (IFN-y), ADCD, ADCC and ADCP. Notably, the top 3-way combination based on 5-, 4-, and 3-parameters was EP153R, P72 and Penton (FIG. 20A). In the 4-parameter analysis, ADCP was removed for possible concern due to antibody-dependent enhancement (ADE), which has been observed for flavivirus infection of macrophages (59, 60). In the 3- parameter analysis, ADCD was further removed due to its unidentified role in disease protection. In addition, MTE- induced T cell responses and inhibition of hemadsorption by CD2v- specific antibodies were further considered. Four cocktail vaccines were tested and itwas found that antibody and T cell responses induced by specific antigen in the cocktails are highly correlated with immune responses induced by single antigen immunization (FIGs. 12A-12D). These results suggest that strategic combination of different antigens in LNP- mRNA vaccines are likely to achieve specific immune responses profiles and provides a rationale for ASF subunit vaccine design. However, previous studies of ASF subunit vaccines based on recombinant proteins or viral vectors have conferred no or low protection against virus challenge (38, 61). Future studies may evaluate the efficacy of mRNA-based subunit vaccines.

[0186] In summary, a comprehensive approach has been disclosed to dissect the immune response profiles of rationally selected ASFV antigens, and developed new strategies for antigen combination for developing a safe and effective mRNA-based subunit vaccine for ASFV. Without wishing to be bound by theory, the methodologies developed herein may also be applied to the development of subunit vaccines for other pathogens with large genomes, including the monkeypox virus.Additional Materials and Methods associated with the Disclosure

[0187] Cells, growth conditions, and cloning information are detailed in the supplemental materials. Details protocols for flow cytometry, confocal microscopy, indirect ELISA, enzyme-linked immunospot (ELISpot) assay, hemadsorption inhibition assay, and antibody effector assays for ADCD, ADCC, ADCP are described in supplemental materials as well.Design ofT cell-directed vaccine

[0188] ASFV-derived CD4+and CD8+T cell epitopes were searched on the Immune Epitope Database (IEDB) (.iedb.org / ) and limited to those with positive experimental results for IFN-y ELISpot and / or cellular MHC / mass spectrometry ligand presentation. A total of 22 T cell epitopes from genotype II ASFV (Georgia 2007 / 1 strain) PP62, MGF100-1L, A238L, K145R, MGF505-7R, P34, P150, P37, and EP153R were selected for their strong induction of cellular immunity according to previous publications (6, 27, 35, 38, 62, 63). P72 and CD2v contains multiple T cell epitopes which were not included in the T cell-directed vaccine design. Additionally, the online server, NetMHCpan 4.1 ( cbs.dtu.dk / services / NetMHCpan / ) was used to predict binding affinities of all 8-1 Imer peptides in the most abundantly expressed pp220 polyprotein. The most frequent Swine Leukocyte Antigens (SLA-l:0401, SLA-l:0101, SLA-l:0801) were selected for peptide binding. Strong binders with an %Rank- Eluted ligand (EL) <0.1 and affinity <200 mM was selected and included 3 epitopes fromM448R and 2 epitopes from MGF505-7R. The T cell-directed vaccine construct, multiple-T cell epitope (MTE), contained a Kozak sequence followed by P30, Internal Ribosome Entry Site (40) or porcine teschovirus- 1 2A (P2A) or GGGS linker, and individual T cell epitopes with additional 5 amino acid residues (AA) on each side were fused together, plus a HA tag for MTE detection. MTE was synthesized by GenScript and cloned into expression vector with P30, namely P30-MTE, P30-IRES-MTE, and P30-P2A-MTE.Lipid. Nanoparticle (LNP) formulation ofmRNA and validation

[0189] Genes encoding the eleven candidate antigens were inserted into pUC57 vector, which contains essential elements for mRNA in vitro transcription, such as T7 promoter, UTRs, and polyA tail. The linearized plasmids were subject to in vitro transcription and formulated by LNP using a protocol described previously (46). Measurement of the encapsulation efficiency, polydispersity index, and hydrodynamic size of the particles was performed use the same procedure described in previous publications (17, 46). To validate LNP delivery of mRNAs, HEK-293T cells were seeded onto 24-well tissue culture plate and 500 ng of LNP-mRNA diluted in Opti-MEM was added in individual well. Cells were collected in 48 h and protein expression was analyzed by flow cytometry and confocal microcopy as described below.Mouse study

[0190] To assess immunogenicity of individual antigens in mice, six to eight-week- old female BALB / c mice were purchased from Charles River and housed in animal facility at the Massachusetts Institute of Technology (MIT). All animal ethical and welfare standards were met in this study and experiments were approved by the Institutional Animal Care and Use Committee at MIT under protocol number 0322-021-25. Briefly, 5 mice were assigned to each of the 12 treatment groups and immunized intramuscularly with 50 pL 5 pg LNP- mRNAs expressing MB-P72, MB-Penton, CD2v, P22, E199L, E248R, EP153R, P54, P30- MTE, P30-IRES-MTE, and P30-P2A-MTE, separately. The negative control group was immunized with 50 pL PBS. For assessing immunogenicity of candidate vaccine cocktails, a total of 50 pL composed of 5 pg of each selected antigens were injected to the hind limb of a mouse. All mice were boosted three weeks later. Serum samples were collected from submandibular vein prior to immunization and two weeks after each injection. Mice were euthanized at day 35 and spleen tissue was collected for analysis of cellular immunity.Pig study

[0191] LNP-mRNA vaccination of pigs was performed according to the protocols approved by Committee on Animal care (protocol number 2308000566) and Midwest Veterinary Service (MVS), Inc. (protocol number 24005). A total of 36 four-week-old piglets were randomly assigned into nine groups with four pigs for each of the 9 LNP-mRNAs groups (MB-P72, MB-Penton, CD2v, P22, E199L, EP153R, P54, P30-IRES-MTE, and P30- P2A-MTE) and the additional two pigs assigned to control group injected with sterile PBS. 30 ug of LNP-mRNA expressing each individual antigen was diluted to 1 mL in sterile PBS and injected to the back of ear intramuscularly and boosted three weeks later. Serum samples were collected before vaccination and weekly after each injection. Body weight of each pig was measured before the study and at the termination. All pigs were euthanized two weeks after boost.Statistical analysis

[0192] Data was processed using R version 4.4.1 with tidyverse version 2.0.0. The R environment was executed using singularity version 3.5.0 and this docker container docker: / / bumproo / bulk_r441. Four biological replicates for each antigen, except for P30 which had three replicates, were summarized by averaging. Correlation analysis was done using the cor function from the stats package in R with the pearson method and heatmaps were plotted using the Heatmap function from the R package ComplexHeatmap version 2.20.0. These averaged values were then normalized by ranking antigen values for each immunological category from 1 (lowest) to 8 (highest). The ggplot2 library in R is used to generate the Polar bar plot of rank scores of antigens based on the average rank scores. Based on the average Z-scores of antigens in five immunological categories, the pyCirclize module in python3 is used to generate the chord diagram of correlations between antigens and five immunological categories. All possible 5-way, 4-way, and 3-way antigen combinations were then scored by summing the ranks. The sum scores calculated for the following sets of immunological data: (,TgG",,TFN-Y","ADCD","ADCC","ADCP"), ("IgG","IFN- Y'V’ADCC'V’ADCD”), ("IgG","IFN-Y","ADCC"), and (' gG'V’IFN-Y").

[0193] All the histogram charts were expressed as means + / - standard error of mean (SEM). Differences between groups were evaluated by one-way analysis of variance (ANOVA) using Prism software version 6 (GraphPad Software). A P value of <0.05 was regarded as statistically significant difference.Cells and plasmids

[0194] Human embryonic kidney cells (HEK-293T) and Vero E6 cells were maintained in minimum essential medium supplemented with 10% fetal bovine serum, antibiotics (100 units / ml of penicillin and 100 mg / ml of streptomycin) and fungizone (0.25 mg / ml) at 37 °C with 5% CO2. Porcine macrophage 3D4 / 31 cell line was maintained in RPMI with essential supplements including FBS, antibiotics, and fungizone. FreeStyle™ CHO-S cells were obtained from ThermoFisher and cultured in Freestyle™ CHO Expression Medium supplemented with 8 mM E-glutamine at 37 °C , 8% CO2 on an orbital shaker platform rotating at 135 rpm. ASFV P72 (B646E), Penton (H240R), CD2v (EP402R), P22 (KP177R), EP153R (C-type lectin), E248R, E199E, P54 (E183E), P30 (CP204E) genes from Georgia 2007 / 1 strain (NCBI Reference Sequence: NC_044959.2) were synthesized by GenScript and inserted into the phCMV mammalian cell expression vector (MoBiTec). P72 and Penton were engineered for membrane anchoring by addition of secretion signal peptide from human CD8a (GenBank ID: NP_001139345.1) to the N terminus and CD8a stalk region or hinge, transmembrane region, and a short cytoplasmic tail to the C terminus.Flow cytometry

[0195] HEK-293T cells seeded in 6-well tissue culture plate and transfected with 3 pg recombinant phCMV plasmids expressing individual proteins using the linear 25 kDa polyethylenimine (PEI; Santa Cruz Biotechnology) at a 3:1 mass ratio of PEI to DNA. At 48 h post transfection, cells were trypsinized followed by 4% PFA fixation, 2% Triton X-100 permeabilization, and staining using anti- ASFV P72 mAb clone #10G5 (MyBioSource, San Diego, CA), anti-P30 mAb (Aviva Systems Biology, OAEF00154), P54 mAb clone #GT853 (GeneTex, GTX635690), or mAb specific for HA tag (GenScript). Alexa Fluor™ 488- conjugated Goat anti-Mouse IgG (H+E) was used as the secondary antibody and cells nuclei were counterstained with DAPI before data acquisition on a BD ESR Fortessa cytometer.Confocal microscopy

[0196] Vero E6 cells were grown on glass-bottom 35-mm cell culture dishes (MatTek). In 48 hours after transfection of MTE expression vectors, cells were fixed by 4% paraformaldehyde at room temperature (RT) for 15 min followed by permeabilization with 0.5% Triton X-100 for 10 min and then blocked with 2% bovine serum albumin for 30 min. Cells were incubated with mouse anti-P30 mAb (GenScript) and Rabbit anti-HA tag polyclonal antibody (Thermo Fisher) at 37°C for 1 hour. Alexa Fluor™ 488-conjugated goat anti-mouse IgG and Alexa Fluor® 594 AffiniPure™ goat anti-rabbit IgG (H+E) (Jackson ImmunoResearch) were used as secondary antibody. Cells were counterstained with DAPIbefore proceeding with imaging under a confocal microscope (Nikon AIR HD25, Nikon).Images were processed using the program ImageJ (https: / / imagej.net / Fiji).Indirect ELISA

[0197] Recombinant ASFV proteins (full-length Penton, P30, and extracellular domain of P22, P54, CD2v, and C-type lectin) with a HIS tag were inserted into PET28a vector and expressed in a BL21 Escherichia coli system under induction of isopropyl-P-D- thiogalactopyranoside (IPTG), followed by protein purification via Ni-nitrilotriacetic acid (NTA) agarose. ASFV P72, E248R, and E199L with a HA tag in C terminus were expressed in a mammalian expression system and purified using anti-HA beads followed by elution using HA peptides. The assay was conducted by following procedures described previously (7). Cutoff values for each protein were determined by OD value of control group animal samples plus three standard deviations. The endpoint titer for each animal that has the highest dilution giving a reading above cutoff was calculated by interpolating from a sigmoidal standard curve using GraphPad Prism.Enzyme-linked immunospot (ELISPOT) assay

[0198] To evaluate T cell immunity, 105splenocytes from each mouse or pig were cultured in CTL medium and seeded on pre-coated mouse IFN-y / TNF-a Double-Color ELISPOT plate (Cellular Technology Limited) or IFN-y single-color ELISpot plate. P72 peptide cocktail (Table 12) was used for stimulation of splenocytes from P72-immunized pigs, P30 and MTE peptides were used separately to stimulate splenocytes from P30-IRES- MITE and P30-P2A-MTE immunized pigs to measure P30- and MTE-specific T cell response. For all other groups (CD2v, EP153R, Penton, P22, P54, E199L), recombinant proteins were used for stimulation of splenocytes. Cocktail of PMA and ionomycin (Thermo Scientific) was used as positive control and cell culture medium as negative control. Plate was incubated at 37 °C with 5% CO2 for 36 hours followed by staining per manufacture’s protocol (Cellular Technology Limited). Colored spots were counted by an automated immuno spot analyzer (Cellular Technology Limited).Hemadsorption inhibition assay (HADIA)

[0199] CD2v has been reported to contribute to hemadsorption which is a term describing ASFV-infected cells attracting red blood cells to attach on cell surface and form “rosette” pattern. To establish an assay for measuring anti-CD2v antibody-mediated hemadsorption inhibition, HEK 293T cells transfected with CD2v-expression vector wereharvested and mixed with 1:100 diluted serum pretreated with Receptor Destroying Enzyme (RDE) at 37°C for 18h and then 56°C for Ih. After 1 hour incubation of serum with cell, 2% red blood cells (RBC) were added and incubated at 37°C for 24 hours. Monolayer cells were gently washed with PBS for five times and fixed, permeabilized, and blocked for antibody staining. Rabbit anti-RBC polyclonal antibody (LSBio) and mouse anti-HA mAb (Thermo Fisher) were added and incubated for 1 hour at 37°C . Alexa Fluor™ 488-conjugated goat anti-mouse IgG and Alexa Fluor® 594 AffiniPure™ goat anti-rabbit IgG (H+E) (Jackson ImmunoResearch) were used as secondary antibody. Cells were counterstained with DAPI before proceeding with imaging by confocal microscopy (Nikon AIR HD25, Nikon). Images were processed using the program ImageJ (imagej.net / Fiji) and geometric mean fluorescent intensity of AF594 red fluorescence for RBC were calculated and compared between groups.Antibody-dependent complement deposition (ADCD) assay

[0200] The ADCD assay was modified from an established protocol published previously (2). Before testing, pig serum samples were inactivated at 56°C for 30 min. CHO- S cells stably expressing each of the ASFV antigen-P2A-mCherry fusion proteins (CD2v, EP153R, P22, P54, E199E and membrane-bound P72, P30, Penton) were used as target cells and seeded in non-tissue culture treated 96-well plates at a density of 1.0 x 105cells per well. 10-fold diluted inactivated serum were added and mixed briefly with the cells. After incubation for 30 min at 37°C in a humidified 5% CO2 incubator, 50 pF control pig serum was added into each well as the complement source and plates were incubated at 37°C for 18 hours, followed by DAPI staining. Cells were subject to flow cytometry using a BD ESR Fortessa HTS-2 cytometer. Results were analyzed using FlowJo vlO software and the percentage of live CHO cells positive for mCherry (%live) were calculated. mCherry positive represents the population of CHO-S cells with stable ASFV antigen expression. The complement-mediated cytotoxicity was calculated according to the formula: cytotoxicity (%) = ((1 - (%live of sample / %live of no serum Ctrl)) x 100. The antigen- specific cytotoxicity effect was calculated by normalizing to the control group serum.Antibody-dependent cellular cytotoxicity (ADCC) assay

[0201] The ADCC assay was modified from procedures published previously 127,128)'). PBMCs collected from control pigs were used as a source of NK cells and preactivated with IE-2 (final concentration 20ng / mE) and IE- 12 (final concentration 25 ng / mE) at 37°C for 24 hours. CHO cells expressing ASFV proteins were seeded on 96-well plates ata density of 2 x 104cells per well. Inactivated serum was 1:10 diluted and incubated with CHO cells for 30 min at 37°C . 50 pL of 2% Triton was added as positive control for complete cell lysis. 4 x 105Pre-activated PBMCs were added to achieve an effector to target cell ratio of 20:1. Following 24 hours incubation at 37°C in humidified incubator, a CytoTox-Glo™ luciferase based assay was used to measure the luminescence of viable and non-viable cells according to manufacturer’s instructions. Percentage of cell lysis (% lysis) was calculated by dead cell luminescence divided by total cell luminescence. The percentage of antigen- specific cytotoxicity was calculated using the formular: (% lysis of testing sample - % lysis of no serum Ctrl) / (% lysis of triton - % lysis of no serum Ctrl).Antibody-dependent cellular phagocytosis (ADCP) assay

[0202] The ADCP assay was modified from protocols published previously (127- 130). ASFV proteins were biotinylated using EZ-Link™ Micro Sulfo-NHS-LC-Biotinylation Kit and then conjugated to 1 pm red FluoSpheres™ NeutrAvidin™-Labeled Microspheres by overnight incubation at 4°C . Fluorescent microspheres were centrifuged at 13,000 g for 2 min at 4°C and washed twice with 1% BSA in PBS to remove the excess unbound proteins. The antigen-coated microspheres were resuspended in 1% BSA. Effector cells were porcine macrophage 3D4 / 31 cell line labeled with CellTrace™ CFSE Cell Proliferation Kit. To perform the phagocytosis assay, CFSE-labeled 3D4 / 31 cells were seeded on 96-well plate at a density of 1 x 104cells per well and incubated for 24 hours in 5% CO2 incubator. Cell culture media were removed and treated with fucoidan (final concentration 100 pg / mL) for 1 hour at 37 °C to block scavenger receptors. 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(2019). Ictv Report, ICTV Virus Taxonomy Profile: Paramyxoviridae. J Gen Virol 100, 1593-1594.TABLESTable 1: SequencesTable 2. Physicochemical properties of LNP-mRNAsTable 3. SEC analysis ofS-P72 and MB-P72 TrimersTable 4. Comparison of vaccinia virus protective antigens with ASFV homologsTable 5. Epitopes encoded by MTETable 6. Primers and PeptidesTable 7. SEC analysis of purified S-P72 and MB-P72Table 8. Characterization of formulated LNP mRNATable 9. Comparison of vaccinia virus protective antigens with ASFV homologsTable 10. T cell epitope sequence and their viral protein originsTable 11. Physicochemical properties LNP-mRNAsTable 12. Peptide pool used for inducing P72- and MTE-specific T cell responses in vitro

Claims

CLAIMSWhat is claimed is:

1. A vaccine comprising:(a) one or more engineered nucleic acids, wherein the one or more engineered nucleic acids encode African swine fever virus (ASFV) antigens comprised of at least one B cell antigen and at least one T cell antigen; and(b) a lipid nanoparticle (LNP).

2. The vaccine of claim 1, wherein the ASFV antigens comprise p30, CD2v, C-lectin (EP153R), p22, p54, pE199L, E248R, P72, and / or Penton.

3. The vaccine of claim 1 or 2, wherein the p72 and / or Penton ASFV antigens comprise a signal peptide of human CD8a on the N-terminus.

4. The vaccine of claim 3, wherein the ASFV antigens further comprise a CD8a hinge domain, CD8a transmembrane domain (TMD), and a short cytoplasmic region of CD8a on the C-terminus.

5. The vaccine of any one of claims 1 to 4, wherein the at least one B cell antigen is Penton.

6. The vaccine of claim 5, wherein the Penton is mutated to remove a site for glycosylation.

7. The vaccine of claim 6, wherein the Penton comprises a N180Q mutation.

8. The vaccine of any one of claims 1-7, wherein the at least one T cell antigen comprises multiple T cell epitopes (MTE).

9. The vaccine of claim 8, wherein the ASFV antigen, optionally the P30 antigen, comprising the MTE further comprises(a) a GGGS linker;(b) a P2A self-cleavage site; and / or(c) an internal ribosome entry site.

10. The vaccine of claim 2, wherein the ASFV antigen comprises EP153R, P72, and Penton.

11. The vaccine of claim 10, wherein the ASFV antigen further comprises p54.

12. The vaccine of claim 11, wherein the ASFV antigen further comprises p30.

13. The vaccine of claim 2, wherein the ASFV antigen comprises:(a) a T cell antigen comprising multiple T cell epitopes (MTE), CD2v, p30, and P72;(b) a T cell antigen comprising multiple T cell epitopes (MTE), CD2v, E199L, andP72;(c) a T cell antigen comprising multiple T cell epitopes (MTE), CD2v, E199L, P30, and P72;(d) a T cell antigen comprising multiple T cell epitopes (MTE), CD2v, E199L, P30, P72, and Penton;(e) a T cell antigen comprising multiple T cell epitopes (MTE), P72, Penton, EP153R, E199L, and P54; or(f) a T cell antigen comprising multiple T cell epitopes (MTE), EP153R, E199L, P54, CD2v, and P22.

14. The vaccine of any one of claims 1-13, wherein the one or more engineered nucleic acids comprise a DNA.

15. The vaccine of any one of claims 1-13, wherein the one or more engineered nucleic acids comprises a polyribonucleotide.

16. The vaccine of any one of claims 1 to 15, wherein the LNPs comprise cholesterol, an ionizable lipid, a lipid comprising a polyethylene glycol (PEG) moiety, and a lipid comprising a phosphatidylcholine or phosphatidylethanolamine moiety.

17. A method of inducing an immune response to African swine fever virus (ASFV) in a subject, comprising administering to the subject a vaccine comprising:(a) one or more engineered nucleic acids, wherein the one or more engineered nucleic acids encode African swine fever virus (ASFV) antigens comprised of at least one B cell antigen and at least one T cell antigen; and(b) a lipid nanoparticle (LNP), wherein the vaccine is in an amount effective to induce an immune response.

18. The method of claim 17, wherein the ASFV antigens comprise p30, CD2v, C-lectin (EP153R), p22, p54, pE199L, E248R, P72, and / or Penton.

19. The method of claim 17 or 18, wherein the ASFV antigens comprise a signal peptide of human CD8a on the N-terminus.

20. The vaccine of claim 19, wherein the ASFV antigens further comprise a CD8a hinge domain, CD8a transmembrane domain (TMD), and a short cytoplasmic region CD8a on the C-terminus.

21. The method of claim 20, wherein the subject is a pig.

22. The method of any one of claims 17 to 21, wherein the administering comprises a first intramuscular injection.

23. The method of claim 22, wherein the administering further comprises a second intramuscular injection after the first intramuscular injection.

24. The method of any one of claims 17 to 23, wherein the immune response is a protective immune response.

25. A method of engineering a subunit vaccine, the method comprising:(a) identifying one or more intracellular viral antigens from a target virus;(b) engineering the one or more intracellular viral antigens as membrane-bound proteins;(c) engineering multiple T cell epitopes (MTE);(d) determining the effector function profiles of antigen- specific antibodies and the levels of B and T cell responses induced by different intracellular viral antigens and MTE; and(e) determining the antigen combination for subunit vaccine with specific immune response profde; and optionally(f) producing the subunit vaccine.

26. The method of claim 25, wherein the engineering one or more intracellular viral antigens as membrane-bound proteins comprises adding a signal peptide of human CD8a on the N-terminus of the one or more intracellular viral antigens, and a CD8a hinge domain, CD8a transmembrane domain (TMD), and a short cytoplasmic region CD8a on the C- terminus of the one or more intracellular viral antigens.

27. The method of claim 25 or 26, wherein the engineering multiple T cell epitopes (MTE) wherein the MTE comprise a GGGS linker; a P2A self-cleavage site; and / or an internal ribosome entry site.

28. The method of any one of claims 25 to 27, wherein determining the effector function profiles comprise measuring antibody-dependent complement deposition (ADCD); antibody dependent cellular cytotoxicity (ADCC), and / or antibody-dependent cellular phagocytosis (ADCP).

29. The method of any one of claims 25 to 28, wherein the determining B cell responses comprise measuring antibody secreting B cells (ASC).

30. The method of any one of claims 26 to 29, wherein the determining T cell responses comprise measuring secretion of cytokine IFN-y and / or TFN-OC.

31. A composition comprising an mRNA encoding one or more peptides, wherein the one or more peptides comprise an N-terminal human CD8a signal peptide linked to an intracellular viral protein from a target virus, optionally wherein the intracellular viral protein is linked to a CD8a hinge domain, a CD8a transmembrane domain (TMD), and a cytoplasmic region of CD8a on the C-terminus.

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