Conjugated polypeptides and vaccines for inducing immune responses

Abstract

To provide new, safe, effective and scalable vaccines and vaccination methods that provide robust antibody responses against pathogens such as SARS-CoV-2 and potentially enhance T-cell responses.SOLUTION: A vaccine for inducing an immune response against a pathogen in a mammal is provided, the vaccine comprising a conjugate polypeptide comprising an antigen from the pathogen linked to a ligand or antibody fragment that specifically binds to a surface protein present on an immune cell.SELECTED DRAWING: Figure 11

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 027,250, filed May 19, 2020, and U.S. Provisional Application No. 63 / 058,362, filed July 29, 2020, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0002] STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with government support under Grant No. R01 AI118451 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] background While traditional vaccine development for previously unknown pathogens takes years, protecting human health may require a more rapid response, measured in months (1). However, rapid development is hampered by several factors, including the lack of appropriate animal models for newly emerging pathogens; the risk of antibody-dependent enhancement of infection (ADEI), which can occur whenever a suboptimal antibody response is induced (2); and the difficulty of developing new manufacturing processes for subunit, attenuated, or vectored vaccines. Additionally, while the induction of high-titer neutralizing antibodies (nAbs) seems an obvious approach, it is unclear what titers of nAbs are actually protective, and how this threshold varies across age extremes and comorbidities.

[0004] With regard to SARS-CoV-2, the immune correlates of successful vaccination remain largely unknown. Most coronavirus vaccines currently under development target the most variable portion of the spike glycoprotein and induce antibody responses only against the virus present in the vaccine. For SARS-CoV-1, escape mutants arise both in vitro and in mice in the presence of a single anti-receptor binding domain (RBD) nAb or a combination of two nAbs (2,3). Furthermore, as noted above, vaccines that exclusively elicit antibodies must be approached with caution due to the potential for ADEI, especially when antibody levels are low (4). Indeed, high concentrations of antisera against SARS-CoV-1 have been shown to neutralize viral infectivity, whereas diluted antibodies caused ADEI in human promonocyte cultures, resulting in cytopathic effects and increased levels of TNF-α, IL-4, and IL-6 (5-7). Furthermore, vaccine candidates based on the full-length SARS-CoV-1 spike have been demonstrated to induce non-neutralizing antibodies, and immunized animals were not protected. Instead, immunized animals experienced adverse effects such as enhanced hepatitis, increased morbidity, and a stronger inflammatory response (8,9).

[0005] T cell responses induced by CoV vaccines also play an important role in protection and clearance. Clearance of MERS-CoV infection was not possible in T cell-deficient mice, but was achieved in mice lacking B cells (10). Furthermore, airway memory CD4+ T cells have been shown to mediate protective immunity against SARS-CoV-1 and MERS-CoV (11). However, most vaccine types do not induce large numbers of memory CD4+ T cells.

[0006] CMV-vectored vaccines can induce robust antibody responses. While CMV vaccines induce weak antibody responses to some transgenes driven by heterologous promoters, CMV infection and vaccination induce robust antibody responses to proteins expressed under the control of the endogenous pp65b promoter. For example, it was found that rhesus macaques vaccinated with a CMV vaccine carrying the Ebola virus glycoprotein (GP) under the control of the pp65b promoter were able to produce GP-specific antibodies (21).

[0007] Another important feature of CMV vectors for use against emerging pathogens is their ability to be re-administered to previously exposed individuals, allowing for the repeated use of CMV-vectored vaccines to protect against a range of emerging threats over time.

[0008] However, despite the immunological advantages of CMV-vectored vaccines, when these vaccines are delivered as live viruses, practical obstacles hinder the rapid development of CMV vaccines for human clinical use. One obstacle is the extreme difficulty of producing a homogeneous test article on a large scale from a slow-growing, mutable betaherpesvirus (29).

[0009] Thus, there is a need for novel, safe, effective, and scalable vaccines and vaccination methods that provide robust antibody responses against pathogens such as SARS-CoV-2 and potentially also enhance T cell responses. The present disclosure addresses this need and offers other advantages. Summary of the Invention

[0010] Quick Overview In one aspect, the present disclosure provides a vaccine for inducing an immune response to a pathogen in a mammal, the vaccine comprising a conjugated polypeptide comprising an antigen from the pathogen linked to a ligand or antibody fragment that specifically binds to a surface protein present on an immune cell.

[0011] In some embodiments, the surface protein is an abundant T cell surface protein involved in signal transduction and / or adhesion. In some embodiments, the abundant T cell surface protein is CD2, CD3, CD4, or CD5. In some embodiments, the abundant T cell surface protein is CD2 or CD3. In some embodiments, the immune cell is a T cell or an antigen-presenting cell (APC). In some embodiments, the ligand is the ectodomain of a cell adhesion molecule. In some embodiments, the cell adhesion molecule is CD58. In some embodiments, the surface protein is preferentially or exclusively expressed by T cells. In some embodiments, the antibody fragment is an scFv chain derived from an antibody.

[0012] In some embodiments, the conjugated polypeptide further comprises a lipid anchor, a transmembrane segment, a multimerization domain, or any combination of these elements. In some embodiments, the lipid anchor is a glycosylphosphatidylinositol anchor. In some embodiments, the addition of the lipid anchor is directed by a signal sequence. In some embodiments, the signal sequence is derived from CD55. In some embodiments, the transmembrane segment is derived from a PDGF receptor, glycophorin A, or SARS-CoV-2 spike protein. In some embodiments, the multimerization domain is derived from T4 fibritin. In some embodiments, the multimerization domain is an Fc domain. In some embodiments, the Fc domain is located at the C-terminus of the conjugated polypeptide. In some embodiments, the Fc domain is a human IgG1 Fc domain. In some embodiments, the conjugated polypeptide is a fusion protein comprising an antigen and a ligand or an antibody fragment within a single polypeptide chain.

[0013] In some embodiments, the antibody fragment is an scFv chain derived from an antibody, and the VH and VL regions of the scFv are separated by a flexible linker. In some embodiments, the flexible linker is 12 amino acids or more in length, and the conjugated polypeptide preferentially binds to the surface protein as a monomer. In some embodiments, the flexible linker is less than 12 amino acids in length, and the conjugated polypeptide preferentially binds to the surface protein as a multimer. In some embodiments, the multimer is stabilized by disulfide bonds between the monomer units. In some embodiments, the flexible linker is 5 amino acids in length. In some embodiments, the conjugated polypeptide further comprises a tPA leader sequence. In some embodiments, the tPA leader sequence is 23 amino acids in length.

[0014] In some embodiments, the vaccine further comprises a second antigen derived from a pathogen. In some embodiments, the pathogen is a virus. In some embodiments, the virus is SARS-CoV-2. In some embodiments, the antigen present in the conjugated polypeptide comprises the SARS-CoV-2 spike glycoprotein or a fragment thereof. In some embodiments, the fragment of the SARS-CoV-2 spike glycoprotein comprises the S1 domain or the receptor binding domain (RBD). In some embodiments, the second antigen comprises the SARS-CoV-2E, M, N, nsp3, nsp4, or nsp6 protein, or a fragment of one of these proteins. In some embodiments, the second antigen comprises a fusion protein comprising the SARS-CoV-2E and M proteins, or a fragment thereof. In some embodiments, the mammal is a human. In some embodiments, the vaccine is formulated for subcutaneous injection. In some embodiments, the conjugated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 and SEQ ID NO:27.

[0015] In another aspect, the present disclosure provides a vaccine for inducing an immune response to a pathogen in a mammal, the vaccine comprising a polynucleotide encoding a conjugate polypeptide comprising an antigen from the pathogen fused to a ligand or antibody fragment that specifically binds to a surface protein present on an immune cell.

[0016] In some embodiments of the vaccine, the surface protein is an abundant T cell surface protein involved in signal transduction and / or adhesion. In some embodiments, the abundant T cell surface protein is CD2, CD3, CD4, or CD5. In some embodiments, the abundant T cell surface protein is CD2 or CD3. In some embodiments, the immune cell is a T cell or an antigen-presenting cell (APC). In some embodiments, the ligand is an ectodomain of a cell adhesion molecule. In some embodiments, the cell adhesion molecule is CD58. In some embodiments, the surface protein is preferentially expressed by T cells. In some embodiments, the antibody fragment is an scFv chain derived from an antibody. In some embodiments, the conjugated polypeptide further comprises a lipid anchor, a transmembrane segment, a multimerization domain, or any combination of these elements. In some embodiments, the lipid anchor is a glycosylphosphatidylinositol anchor. In some embodiments, addition of the lipid anchor is directed by a signal sequence. In some embodiments, the signal sequence is derived from CD55. In some embodiments, the transmembrane segment is derived from a PDGF receptor, glycophorin A, or SARS-CoV-2 spike protein. In some embodiments, the multimerization domain is derived from T4 fibritin. In some embodiments, the multimerization domain is an Fc domain. In some embodiments, the Fc domain is located at the C-terminus of the conjugated polypeptide. In some embodiments, the Fc domain is a human IgG1 Fc domain.

[0017] In some embodiments, the VH and VL regions of the scFv are separated by a flexible linker within the conjugated polypeptide. In some embodiments, the flexible linker is 12 amino acids or more in length, and the conjugated polypeptide preferentially binds to the surface protein as a monomer. In some embodiments, the flexible linker is less than 12 amino acids in length, and the conjugated polypeptide preferentially binds to the surface protein as a multimer. In some embodiments, the multimer is stabilized by a disulfide bond. In some embodiments, the flexible linker is 5 amino acids in length. In some embodiments, the conjugated polypeptide comprises a tPA leader sequence. In some embodiments, the tPA leader sequence is 23 amino acids in length.

[0018] In some embodiments, the vaccine further comprises a second polynucleotide encoding a second antigen from a pathogen. In some embodiments, the pathogen is a virus. In some embodiments, the virus is SARS-CoV-2. In some embodiments, the antigen present in the conjugated polypeptide comprises the SARS-CoV-2 spike glycoprotein or a fragment thereof. In some embodiments, the fragment of the SARS-CoV-2 spike glycoprotein comprises the S1 domain or the receptor binding domain (RBD). In some embodiments, the second antigen comprises the SARS-CoV-2E, M, N, nsp3, nsp4, or nsp6 protein, or a fragment of one of these proteins. In some embodiments, the second antigen comprises a fusion protein comprising the SARS-CoV-2E and M proteins, or a fragment thereof. In some embodiments, the mammal is a human. In some embodiments, the vaccine is formulated for electroporation or subcutaneous injection.

[0019] In some embodiments, the polynucleotide encoding the conjugate polypeptide and / or the second polynucleotide encoding the second antigen are codon-optimized. In some embodiments, the polynucleotide encoding the conjugate polypeptide is present in a first expression cassette, the polynucleotide being operably linked to a first promoter, and / or the second polynucleotide encoding the second antigen is present in a second expression cassette, the second polynucleotide being operably linked to a second promoter. In some embodiments, the second promoter is a mammalian promoter. In some embodiments, the mammalian promoter is an EF-1α promoter. In some embodiments, the first and / or second expression cassettes are present in a vector. In some embodiments, the vector is administered as naked DNA. In some embodiments, the vector is a viral vector. In some such embodiments, the viral vector is a cytomegalovirus (CMV), adenovirus, or adeno-associated virus (AAV) vector. In some embodiments, the vaccine further comprises an in vivo transfection reagent. In some such embodiments, the in vivo transfection reagent is in vivo-jetPEI™. In some embodiments, the vaccine is formulated for subcutaneous transfection.

[0020] In some embodiments, the vector is a circular CMV vector comprising: (a) a CMV genome or portion thereof containing a first expression cassette, or a first and a second expression cassette; (b) a bacterial artificial chromosome (BAC) sequence comprising an origin of replication; (c) a first terminase complex recognition locus (TCRL1) comprising at least two viral direct repeat sequences; and (d) a second terminase complex recognition locus (TCRL2) comprising at least two viral direct repeat sequences, wherein the CMV genome or portion thereof is adjacent to TCRL1 and TCRL2, thereby defining a first region of the circular vector extending from TCRL1 to TCRL2 and comprising the CMV genome or portion thereof; and the BAC sequence is located in a second region of the circular vector extending from TCRL1 to TCRL2 and not comprising the CMV genome or portion thereof.

[0021] In some embodiments, the vector is a circular CMV vector comprising: (a) a CMV genome or portion thereof containing a first expression cassette, or a first and a second expression cassette; (b) a sequence comprising an origin of replication that functions in a unicellular organism; and (c) one or more terminase complex recognition loci (TCRLs) comprising recombinantly introduced polynucleotide sequences capable of directing cleavage by an HV terminase complex, wherein the CMV genome or portion thereof is separated from the sequence comprising the origin of replication by the TCRL; the CMV genome or portion thereof is flanked by the TCRL on at least one end; and the sequence comprising the origin of replication is flanked by the TCRL on at least one end.

[0022] In some embodiments, the one or more terminase complex recognition loci comprise a Pac1 site and a Pac2 site. In some embodiments, all of the terminase complex recognition loci comprise a Pac1 site and a Pac2 site. In some embodiments, the first promoter is a viral promoter. In some embodiments, the viral promoter is a pp65b promoter. In some embodiments, the vector is a CMV vector, and the CMV is Towne HCMV. In some embodiments, the polynucleotide encoding the conjugate polypeptide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26 and SEQ ID NO:28.

[0023] In another aspect, the present disclosure provides a conjugated polypeptide comprising an antigen derived from a pathogen linked to a ligand or antibody fragment that specifically binds to a surface protein present on an immune cell.

[0024] In some embodiments of the conjugated polypeptide, the surface protein is CD2, CD3, CD4, or CD5. In some embodiments, the surface protein is CD2 or CD3. In some embodiments, the immune cell is a T cell or an antigen-presenting cell (APC). In some embodiments, the ligand is the ectodomain of a cell adhesion molecule. In some embodiments, the cell adhesion molecule is CD58. In some embodiments, the surface protein is an abundant T cell surface protein involved in signal transduction and / or adhesion. In some embodiments, the ligand or antibody fragment specifically binds to a surface protein that is preferentially or exclusively expressed by T cells. In some embodiments, the antibody fragment is an scFv chain derived from an antibody.

[0025] In some embodiments, the conjugated polypeptide further comprises a lipid anchor, a transmembrane segment, a multimerization domain, or any combination of these elements. In some embodiments, the lipid anchor is a glycosylphosphatidylinositol anchor. In some embodiments, the addition of the lipid anchor is directed by a signal sequence. In some embodiments, the signal sequence is derived from CD55. In some embodiments, the transmembrane segment is derived from a PDGF receptor, glycophorin A, or SARS-CoV-2 spike protein. In some embodiments, the multimerization domain is derived from T4 fibritin. In some embodiments, the multimerization domain is an Fc domain. In some embodiments, the Fc domain is located at the C-terminus of the conjugated polypeptide. In some embodiments, the Fc domain is a human IgG1 Fc domain.

[0026] In some embodiments, the antibody fragment is an scFv chain derived from an antibody, and the VH and VL regions of the scFv are separated by a flexible linker. In some embodiments, the flexible linker is 12 amino acids or more in length, and the conjugated polypeptide preferentially binds to the surface protein as a monomer. In some embodiments, the flexible linker is less than 12 amino acids in length, and the conjugated polypeptide preferentially binds to the surface protein as a multimer. In some embodiments, the multimer is stabilized by disulfide bonds between the monomer units. In some embodiments, the flexible linker is 5 amino acids in length. In some embodiments, the conjugated polypeptide further comprises a tPA leader sequence. In some embodiments, the tPA leader sequence is 23 amino acids in length.

[0027] In some embodiments, the pathogen is a virus. In some such embodiments, the virus is SARS-CoV-2. In some embodiments, the antigen comprises the SARS-CoV-2 spike glycoprotein or a fragment thereof. In some embodiments, the fragment of the SARS-CoV-2 spike glycoprotein comprises the S1 domain or the receptor binding domain (RBD). In some embodiments, the conjugated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, and SEQ ID NO:27.

[0028] In another aspect, the present disclosure provides a conjugated polypeptide comprising: (i) a tissue plasminogen activator (tPA) signal sequence; (ii) a single-chain variable fragment (scFv) that specifically binds to CD2, CD3, or CD4; (iii) a flexible linker; and (iv) a SARS-CoV-2 receptor binding domain (RBD).

[0029] In some embodiments, the conjugate polypeptide comprises the amino acid sequence of SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID 25 or SEQ ID NO:27.

[0030] In another aspect, the present disclosure provides a polynucleotide encoding any of the conjugate polypeptides described herein.

[0031] In some embodiments, the polynucleotide is codon optimized. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:28.

[0032] In another aspect, the present disclosure provides an expression cassette comprising any of the polynucleotides described herein. In some embodiments, the expression cassette comprises the nucleotide sequence of SEQ ID NO:8.

[0033] In another aspect, the present disclosure provides a vector comprising any of the polynucleotides or expression cassettes described herein.

[0034] In some embodiments, the vector is a plasmid. In some embodiments, the vector is an adenoviral vector.

[0035] In another aspect, the present disclosure provides any of the diabodies, triabodies, tetrabodies, or dimers described herein.

[0036] In another aspect, the present disclosure provides a vaccine comprising any of the conjugated polypeptides, polynucleotides, antigens, expression cassettes, vectors, diabodies, triabodies, tetrabodies, or dimers described herein.

[0037] In another aspect, the present disclosure provides a method of inducing an immune response against a pathogen in a mammal, comprising administering to the mammal any of the vaccines described herein.

[0038] In some embodiments, the vaccine is administered subcutaneously or by electroporation. In some embodiments, the method induces a neutralizing antibody response in the mammal against the antigen present in the conjugated polypeptide, wherein the neutralizing response is substantially greater than any antibody-dependent enhancement of infection (ADEI) induced in the mammal by the vaccine. In some embodiments, the vaccine does not substantially induce ADEI in the mammal. In some embodiments, the method induces both CD4+ and CD8+ T cell responses against the second antigen.

[0039] In some embodiments, the method comprises administering to the mammal by electroporation a DNA prime containing any of the vectors described herein, e.g., a plasmid, encoding any of the conjugated polypeptides described herein, followed by boosting with any of the vectors described herein, e.g., an adenoviral vector, encoding any of the antigens described herein, e.g., RBD. In some embodiments, the boost occurs about 28 days later. In some embodiments, the mammal is a human.

[0040] Numerous aspects of the disclosure are presented herein, including compositions and methods for preparing and administering the compositions. [Brief explanation of the drawings]

[0041] [Figure 1] Figure 1. Stimulation of T cells with anti-CD3 scFv linked to the SARS-CoV-2 S1 domain. Left: Incubation of T cells in culture without stimulation results in limited CD69 expression on the cell surface and negligible interferon-gamma production. Right: Incubation of T cells in the presence of plate-bound anti-CD3 scFv-S1 results in increased CD69 and interferon production, indicating that anti-CD3 scFv-S1 molecules can bind to the CD3 complex and activate T cell signaling. The scale extends from -103 to 105 on both the x- and y-axes. [Figure 2] Figure 2 is a schematic diagram showing a self-priming RhCMV BAC DNA construct. In the depicted construct, each terminase complex recognition locus (TCRL) consists of two DR repeats. The two TCRLs flank a BAC sequence containing the prokaryotic origin of replication, oriS. [Figure 3] Figures 3A-3B. CMV-vectored vaccines lacking UL111A induce strong CD4+ and CD8+ T cell responses at mucosal surfaces. Figure 3A: Balanced CD4 and CD8 responses after adenoviral-vectored versus CMV-vectored immunization. CMV vaccines (thin solid lines, individual traces; thick solid lines, median) elicited comparable CD4 and CD8 responses (ratios >1), whereas adenoviral vectors elicited lower relative frequencies of CD4 responses (dotted lines). Figure 3B: Example of balanced CD4+ and CD8+ T cell responses after CMV-vectored immunization (top), but not adenoviral-vectored immunization (bottom). Two weeks after immunization, CD4+ or CD8+ cells (left and right, respectively) responding to vaccine antigen stimulation with cytokine production are shown. [Figure 4] Figure 4. Introduction of a terminase complex recognition locus (TCRL) surrounding a BAC replication origin provides superior conversion of CMV BAC genomic DNA to replicating virus in vitro. Left, Transfection of 1 μg of CMV BAC genomic DNA using FuGene6 results in the production of only one plaque when using conventional CMV BAC genomic DNA (TR3dIL10), but 20 plaques when using a self-priming construct with a CMV TCRL (TR4dIL10) surrounding the BAC origin as in Figure 2. Right, Comparison of plaque formation over a range of input DNA amounts using calcium phosphate-mediated transfection. Self-priming genomes with a TCRL surrounding the BAC origin are superior at all DNA input levels. [Figure 5] Figure 5. B cells reactive with anti-CD3-linked immunogens receive promiscuous help. [Figure 6]Figure 6. Subcutaneous vaccination with 100 μg of RhCMVdIL10 vaccine BAC DNA is sufficient to elicit an immune response. Immune responses to pRhCMV-MAGEA4 are shown as early as 1 week after priming. [Figure 7] Figure 7. Protocol overview. [Figure 8] Figure 8. Expression cassettes and experimental design. Top, Design of expression cassettes encoding the SARS-CoV-2 spike S1 domain (top, light gray), receptor-binding domain (middle, gray), or anti-CD3 scFv-RBD fusion protein (s3-RBD, black). Center, These cassettes were delivered to rhesus macaques as electroporated DNA on day 0 of the vaccination protocol, and animals were boosted with adenovirus type 35 (Ad35) vectors on approximately day 28. Bottom, Three macaques per group received 1 mg of DNA expressing S1, RBD, or s3-RBD at the time of prime and 10 Ad35 / S1 or Ad35 / RBD particles at the time of boost. [Figure 9] Figure 9. Binding antibodies detected by endpoint dilution ELISA assay. Responses of individual vaccinated macaques are shown as thinner lines, and the geometric mean responses by group are shown as thicker lines (S1 dotted black line, RBD alone dashed gray line, s3-RBD solid black line). Vaccination with DNA at week 0 and Ad35 at week 4 results in binding antibody responses above background at week 0 in all recipients of RBD and s3-RBD constructs. Recipients of s3-RBD primes show superior responses that are on average higher and in 2 / 3 cases higher than the best response generated in the RBD group. The geometric mean response to s3-RBD is 10-fold higher than the geometric mean response to RBD by 24 weeks post-vaccination. [Figure 10]Figure 10. Reporter viral particle (RVP / pseudovirus) assay tests the inhibition of infection by pseudotyped lentiviral particles bearing a SARS-CoV-2 spike by various dilutions of serum. A curve is then generated and the neutralization titer 50 (NT50) is read as the dilution of serum required to obtain 50% inhibition. In this example, vaccination results in high neutralization titers starting 5 weeks after priming. [Figure 11] Figure 11. Results of the longitudinal pseudovirus neutralization assay demonstrate superior induction of neutralizing antibodies by s3-RBD (solid black line). Five weeks after primary vaccination, geometric mean titers (GMTs) are at least four-fold higher in the s3-RBD group than in the RBD group (solid black line vs. dashed gray line). The true advantage of s3-RBD is greater, as one animal in the s3-RBD group developed neutralizing antibody titers above the upper limit of the assay. Furthermore, most recipients of RBD alone have neutralizing antibody titers below the limit of detection by 24 weeks post-vaccination, whereas all s3-RBD recipients maintain high neutralizing titers through 32 weeks. [Figure 12] Figure 12. Binding antibodies detected by endpoint dilution ELISA assay in macaques primed with RBD alone, s2-RBD (i.e., anti-CD2-RBD conjugated polypeptide), or eDis3-RBD (enhanced diabody anti-CD3-RBD conjugated polypeptide). Responses of individual vaccinated macaques are shown as thinner lines, and geometric mean responses per group are shown as thicker lines (RBD dotted black line, s2-RBD dashed gray line, eDis3-RBD solid black line). Compared to RBD alone, recipients of either s2-RBD or eDis3-RBD prime show superior responses that reach a higher peak and are maintained at higher levels after 24 weeks. [Figure 13]Figures 13A-13D. Host cells transduced with 1dCD58-RBD(B.1.351) or s3-RBD(B.1.351)-PDGFRtm produce immunoreactive RBD. This assay detects binding of anti-RBD antibodies to intracellular proteins produced after transfection. (Figure 13A) Negative control cells not transfected with the plasmid do not react with anti-RBD antibodies. (Figure 13B) Positive control cells transfected with an expression cassette for RBD(B.1.351) alone produce immunoreactive proteins. (Figure 13C) Cells transfected with an expression cassette for 1dCD58-RBD(B.1.351) produce immunoreactive proteins, demonstrating successful production of the conjugated polypeptide and its potential to generate an immune response after vaccination. (Figure 13D) Cells transfected with an expression cassette for s3-RBD(B.1.351)-PDGFRtm, which is predicted to be inserted into the cell membrane, produced immunoreactive protein, indicating successful production of the conjugated polypeptide. DETAILED DESCRIPTION OF THE INVENTION

[0042] Detailed Description 1. Introduction The present disclosure provides methods and compositions for inducing an immune response in a subject. The methods and compositions enable a strong, effective, and safe antibody and / or T cell response to an antigen, such as an antigen derived from a virus such as SARS-CoV-2. The methods and compositions include, among other things, a conjugated polypeptide comprising an antigen linked to a ligand or antibody fragment that binds to a surface protein on an immune cell, such as a T cell or antigen-presenting cell (APC). In some embodiments, the conjugated agent is administered together with a second antigen, for example, a second antigen designed to elicit a T cell response.

[0043] 2. Definition As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0044] A "substantial portion" of a genome indicates that a significant proportion of the genome and the genes contained therein, e.g., 20% of the entire genome and / or 20% of the genes in the genome, is retained, as opposed to a nucleic acid that only contains one or a few genes or elements from the genome, such as, for example, an origin of replication. In the present polynucleotide, the portion includes at least, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the genes present in the genome (i.e., in terms of total nucleotides) or wild-type full-length genome.

[0045] As used herein, the terms "a," "an," or "the" not only include aspects having one member, but also aspects having more than one member. For example, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, reference to "the agent" includes reference to one or more agents known to those of skill in the art, and so forth.

[0046] As used herein, the terms "about" and "approximately" generally refer to an acceptable degree of error for the measured quantity, taking into account the nature or precision of the measurement. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Any reference to "about X" includes at least the value X, 0.8X, 0.81X, 0.82X, 0.83X, 0.84X, 0.85X, 0.86X, 0.87X, 0.88X, 0.89X, 0.9X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, 1.1X, 1.11X, 1.12X, 1.13X, 1.14X, 1.15X, 1.16X, 1.17X, 1.18X, 1.19X, and 1.2X are specifically indicated. Thus, "about X" is intended to teach and provide specification support for a claim limitation of, for example, "0.98X."

[0047] The term "antigen" refers to a molecule or portion thereof that can induce an immune response (e.g., in a subject). While in many cases, an immune response involves the production of antibodies that target or specifically bind to the antigen, as used herein, the term "antigen" also refers to a molecule that induces an immune response other than one that specifically involves the production of antibodies that target the antigen, for example, a cell-mediated immune response that involves the expansion of T cells that target antigen-derived peptides presented on the surface of target cells. In certain embodiments, the antigens of the present disclosure are derived from a pathogen, such that the subject's immune response provides immune defense against the pathogen. In certain embodiments, the pathogen is a virus, such as SARS-CoV-2.

[0048] The term "nucleic acid sequence encoding a peptide" refers, in some embodiments, to a segment of DNA that may be a gene or a portion thereof involved in producing a peptide chain (e.g., an antigen or a fusion protein). A gene generally includes regions preceding and following the coding region (leader and trailer) that are involved in the transcription / translation of the gene product and the regulation of transcription / translation. A gene may also include intervening sequences (introns) between individual coding segments (exons). Leaders, trailers, and introns may include regulatory elements required during the transcription and translation of a gene (e.g., promoters, terminators, translational control sequences, such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary regions, origins of replication, matrix attachment sites, and locus control regions). A "gene product" can refer to either the mRNA or protein expressed from a particular gene.

[0049] The terms "expression" and "expressed" refer to the production of a transcription and / or translation product of a nucleic acid sequence encoding, for example, a protein (e.g., an antigen or a fusion protein). In some embodiments, the terms refer to the production of a transcription and / or translation product encoded by a gene (e.g., a gene encoding an antigen) or a portion thereof. The level of expression of a DNA molecule in a cell can be assessed based either on the amount of corresponding mRNA present in the cell or on the amount of protein encoded by that DNA produced by the cell.

[0050] The term "recombinant," when used with reference to, for example, a polynucleotide, protein, vector, or cell, indicates that the polynucleotide, protein, vector, or cell has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a naturally occurring nucleic acid or protein, or that the cell is derived from a cell so modified. For example, a recombinant polynucleotide contains nucleic acid sequences that are not found within the native (non-recombinant) form of the polynucleotide.

[0051] The term "immune response" refers to any response induced by an antigen (e.g., in a subject), including the induction of immunity against pathogens (e.g., microorganisms such as viruses and bacteria). The immune response induced by the systems, recombinant polynucleotides, compositions, and methods of the present disclosure is typically a desired, intended, and / or protective immune response. The term includes the production of antibodies, e.g., neutralizing antibodies, against the antigen, as well as the development, maturation, differentiation, and activation of immune cells (e.g., B cells and T cells). In some examples, an immune response includes increasing the number or activation of MHC class E and / or class II-restricted CD4+ and / or CD8+ T cells (e.g., in a subject). The term also includes increasing or decreasing the expression or activity of cytokines involved in regulating immune function. As another non-limiting example, an immune response can include increasing the expression or activity of interferon-gamma and / or tumor necrosis factor-alpha (e.g., in a subject).

[0052] Further examples of desired, intended, and / or protective immune responses that can be induced in accordance with the recombinant polynucleotides, compositions, and methods of the present disclosure include class Ia, class Ib, or class II-restricted CD4+ T cells; class Ia, class Ib, or class II-restricted CD8+ T cells; cytokine-producing T cells (e.g., those producing IFN-γ, TNF-α, IL-1-β, IL-2, IL-4, IL-5, IL-10, IL-13, IL-17, IL-18, or IL-23); CD4 regulatory T cells; CD8 regulatory T cells; antigen-specific T follicular helper cells; antibody-producing; NK cells; NKG2C NK cells; CD57 NK cells; FcRγ-negative NK cells; and NK-CTL cells, i.e., those with CD8 T cells that express molecules typical of NK cells, such as NKG2A.

[0053] The term "cytomegalovirus" or "CMV" refers to viruses including members of the Cytomegalovirus genus of viruses (within the order Herpesvirales, family Herpesviridae, subfamily Betaherpesvirinae). The term includes, but is not limited to, human cytomegalovirus (HCMV; also known as human herpesvirus 5 (HHV-5)), simian cytomegalovirus (SCCMV or AGMCMV), baboon cytomegalovirus (BaCMV), owl monkey cytomegalovirus (OMCMV), squirrel monkey cytomegalovirus (SMCMV), and rhesus monkey cytomegalovirus (RhCMV), which infect macaques.

[0054] The term "antigen-presenting cell" or "APC" refers to a cell that displays or presents an antigen or a portion thereof on its surface. Typically, the antigen is displayed or presented in association with a major histocompatibility complex (MHC) molecule. Almost any cell type can function as an APC, and APCs are found in many different tissue types. Professional APCs, such as dendritic cells, macrophages, and B cells, present antigens to T cells in a context that most efficiently results in T cell activation and subsequent proliferation. Many cell types present antigens to cytotoxic T cells.

[0055] An "immune cell" can be any cell of the immune system, including T cells such as helper T cells, CD4+ T cells, CD8+ T cells, TH1, TH2, TH17 and Treg cells, antigen-presenting cells (APCs), B cells, granulocytes including basophils, eosinophils and neutrophils, mast cells, monocytes, macrophages, dendritic cells, and natural killer (NK) cells.

[0056] "Infectious disease antigen" refers to any molecule derived from an infectious disease-causing organism that can induce an immune response (e.g., in a subject). For example, the infectious disease antigen can be derived from a virus, bacterium, fungus, protozoan, helminth, or parasite, and can be, for example, a bacterial wall protein, a viral capsid or structural protein (e.g., a retroviral envelope protein such as an HIV or SIV env protein), or a portion thereof. In some embodiments, the infectious disease antigen is a viral infectious disease antigen from SARS-CoV-2.

[0057] As used herein, the terms "polynucleotide," "nucleic acid," and "nucleotide" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof. The terms include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, and DNA-RNA hybrids, as well as other polymers containing purine and / or pyrimidine bases or other natural, chemically modified, biochemically modified, non-natural, synthetic, or derivatized nucleotide bases. Unless specifically limited, the terms encompass nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses not only the explicitly indicated sequence, but also conservatively modified variants thereof (e.g., degenerate codon substitutions), homologs, and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0058] The terms "vector" and "expression vector" refer to a recombinantly or synthetically produced nucleic acid construct having a series of designated nucleic acid elements that allow for transcription of a particular nucleic acid sequence (e.g., encoding an antigen and / or a fusion protein as described herein) in a host cell or an engineered cell. In some embodiments, a vector comprises a polynucleotide to be transcribed operably linked to a promoter. Other elements that may be present in a vector include those that enhance transcription (e.g., enhancers), those that terminate transcription (e.g., terminators), those that confer a certain binding affinity or antigenicity to a protein (e.g., a recombinant protein) produced from the vector, and those that allow for replication of the vector and its packaging (e.g., into a viral particle). In some embodiments, the vector is a viral vector (i.e., a viral genome or portion thereof). A vector can contain nucleic acid sequences or mutations that, for example, increase tropism and / or modulate immune function. An "expression cassette" comprises a coding sequence operably linked to a promoter and may optionally include a polyadenylation sequence.

[0059] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. All three terms apply to naturally occurring and non-naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids. As used herein, these terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0060] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to vertebrates, preferably mammals, more preferably humans. Mammals include, but are not limited to, murines, mice, rats, monkeys, humans, farm animals, sport animals, and pets. Also included are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro.

[0061] A "ligand" is a molecule that binds to a biomolecule, such as a receptor protein, forms a complex with the biomolecule, such as a receptor protein, thereby changing the conformation of the biomolecule and, consequently, the functional state of the biomolecule. For purposes of this disclosure, a ligand is typically a polypeptide present in a larger conjugated polypeptide together with an antigen. A ligand may be derived from the ectodomain of a cell adhesion protein that interacts with another cell adhesion protein on the surface of a larger molecule, such as an immune cell. An example of a ligand for purposes of this disclosure is the first extracellular domain (or ectodomain) of CD58, designated 1dCD58, which can bind to CD2. For purposes of this disclosure, a ligand is not an antibody, such as a monoclonal antibody.

[0062] As used herein, the term "administering" includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intratumoral, intrathecal, intranasal, intraosseous, or subcutaneous administration to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, intraosseous, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.

[0063] The term "treating" refers to an approach to obtaining beneficial or desired results, including, but not limited to, therapeutic benefit and / or prophylactic benefit. "Therapeutic benefit" refers to any improvement or effect associated with the treatment of one or more diseases, conditions, or symptoms being treated. Therapeutic benefit can also refer to resulting in a cure of one or more diseases, conditions, or symptoms being treated. Furthermore, therapeutic benefit can also refer to increased survival. For prophylactic benefit, the composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even if the disease, condition, or symptom is not yet present.

[0064] The term "therapeutically effective amount" or "sufficient amount" refers to an amount of a system, recombinant polynucleotide, or composition described herein sufficient to produce a beneficial or desired result. A therapeutically effective amount may vary depending on one or more of the subject and disease condition being treated, the subject's weight and age, the severity of the disease condition, the subject's immune status, the mode of administration, etc., which can be readily determined by one of ordinary skill in the art. The specific amount may vary depending on one or more of the particular agent selected, the type of target cell, the location of the target cell in the subject, the dosing regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, and the physical delivery system in which it is carried.

[0065] For the purposes of this specification, effective amount is determined by considerations that may be known in the art.This amount must be effective for achieving desired therapeutic effect in the subject suffering from diseases such as infectious disease or cancer.Desired therapeutic effect can include, for example, improving the undesirable symptoms associated with disease, preventing the onset of such symptoms before they occur, slowing down the progression of symptoms associated with disease, slowing down or limiting any irreversible damage caused by disease, reducing the severity of disease or curing it, or improving survival rate or providing faster recovery from disease.In addition, in the context of preventive treatment, this amount can also be effective for preventing the onset of disease.

[0066] The term "pharmaceutically acceptable carrier" refers to a substance that aids in the administration of an active agent to a cell, organism, or subject. "Pharmaceutically acceptable carrier" also refers to a carrier or excipient that can be included in the composition and does not cause significant adverse toxic effects to the patient. Non-limiting examples of pharmaceutically acceptable carriers include water, sodium chloride (NaCl), saline solution, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings and colorings, liposomes, dispersion media, microcapsules, cationic lipid carriers, isotonicity and absorption delaying agents, and the like. Carriers can also contain or consist of substances to provide stability, sterility, and isotonicity to the formulation (e.g., antimicrobial preservatives, antioxidants, chelating agents, and buffers), to prevent the action of microorganisms (e.g., antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc.), or to provide edible flavors to the formulation. In some instances, a carrier is an agent that facilitates delivery of a polypeptide, fusion protein, or polynucleotide to a target cell or tissue. Those skilled in the art will recognize that other pharmaceutical carriers are also useful in the present methods and compositions.

[0067] The term "vaccine" refers to a biological composition that, when administered to a subject, has the ability to generate adaptive immunity in the subject against a specific pathogen or disease. Typically, one or more antigens, antigen fragments, or polynucleotides encoding antigens or antigen fragments associated with the pathogen or disease of interest are administered to the subject. A vaccine can include, for example, an inactivated or attenuated organism (e.g., a bacterium or virus), a cell, a protein expressed from or on a cell (e.g., a cell surface protein or other protein produced by a cell (e.g., a tumor cell)), a protein produced by an organism (e.g., a toxin), or a part of an organism (e.g., a viral envelope protein or a viral gene encoding various antigens). In some examples, cells are engineered to express proteins such that, when administered as a vaccine, the cells enhance the subject's ability to acquire immunity against a particular cell type (e.g., enhance the subject's ability to acquire immunity against cancer cells) or enhance the subject's ability to acquire immunity against an infectious disease-causing organism, such as a virus, bacterium, fungal organism, protozoan, or helminth. As used herein, the term "vaccine" includes, but is not limited to, the systems and recombinant polynucleotides of the present disclosure, as well as viral particles, host cells and pharmaceutical compositions comprising the systems or recombinant polynucleotides described herein.

[0068] The terms "Pac1 site" and "Pac2 site" refer to cis-acting polynucleotide sequences in the tandem terminal repeats of herpesvirus genomes, including cytomegalovirus genomes, that are recognized by the encapsidation machinery to initiate packaging and direct the cleavage of genome concatemers into single, unit-length genomes (see, e.g., Fields Virology 6th edition, 2013, Knipe and Howley, eds.).

[0069] 3. Vaccines The present disclosure provides vaccines for generating an immune response against an antigen from any type of pathogen. The antigen against which an immune response is generated (e.g., in a subject) depends on the particular disease against which a preventive and / or therapeutic benefit is sought. In some embodiments, the antigen is an infectious disease antigen, such as a viral, bacterial, protozoan, helminth, or fungal pathogen. In certain embodiments, the antigen is a viral antigen, for example, from a coronavirus, such as SARS-CoV-2. In some embodiments, the antigen is a tumor-associated antigen.

[0070] In some embodiments, an immune response (e.g., a desired, intended, or protective immune response in a subject) is induced against a viral antigen (e.g., a viral infectious disease antigen). In some embodiments, an immune response is induced against a bacterial antigen (e.g., a bacterial infectious disease antigen). In some embodiments, an immune response is induced against a fungal antigen (e.g., a fungal infectious disease antigen). In some embodiments, an immune response is induced against a protozoan antigen (e.g., a protozoan infectious disease antigen). In some embodiments, an immune response is induced against a helminth antigen (e.g., a helminth infectious disease antigen). In some embodiments, an immune response is induced against a tumor-associated antigen. In some embodiments, the antigen is a bacterial antigen, a viral antigen, a fungal antigen, a protozoan antigen, a tumor-associated antigen, and / or a helminth antigen. In certain embodiments, the antigen is a viral antigen from a coronavirus, e.g., SARS-CoV-2.

[0071] Vaccines of the present invention can take any of a number of forms, including through the administration of proteins, peptides and nucleic acids, including RNA or DNA, that encode one or more of the antigens described herein.

[0072] Immunogenic conjugates In certain embodiments, the vaccine comprises an "immunogenic conjugate" or "conjugated polypeptide" or "conjugated agent" comprising an antigen linked to a ligand or antibody fragment that binds to a surface protein present on immune cells. For example, the antigen can be linked to an antibody fragment that specifically binds to a protein abundant on the surface of T cells. In some embodiments, the ligand is the ectodomain of a cell adhesion molecule. Any protein abundant on the surface of immune cells, including T cells such as helper T cells, can be targeted by the ligand or antibody fragment. In some embodiments, the protein bound by the ligand or antibody fragment is involved in signal transduction and / or adhesion. Examples of surface proteins that can be bound by the moiety include CD2 (see, e.g., NCBI Gene ID 914 or UNIProt P06729), CD3, including any of the CD3 subunits, i.e., CD3-epsilon (see, e.g., NCBI Gene ID 916 or UNIProt P07766), CD3-gamma (see, e.g., NCBI Gene ID 917 or UNIProt P09693, CD3-delta (see, e.g., NCBI Gene ID 915 or UNIProt P04234), or CD3-zeta (CD247; see, e.g., NCBI Gene ID 919 or UNIProt P20963), CD4 (see, e.g., NCBI Gene ID 920 or UNIProt P01730), and CD5 (see, e.g., NCBI Gene ID 921 or UNIProt P01730). Without being bound by theory, it is believed that in some embodiments, such conjugated polypeptides can cross-link antigen-specific B cells with T cells or other immune cells in the vicinity of the B cells, thereby eliciting stronger antibodies than those obtained by vaccination with the antigen alone.

[0073] The antigen can be any immunogenic antigen derived from a pathogen, i.e., one that can stimulate a B cell (antibody) or T cell immune response and contain one or more epitopes that can be specifically bound by antibodies and / or T cells in a subject. In certain embodiments, the antigen present in the immunogenic conjugate generates a robust antibody response in a subject. For example, in the case of vaccination against a coronavirus such as SARS-CoV-2, the antigen can include a spike glycoprotein or a fragment thereof. In some such embodiments, the fragment comprises the S1 domain, receptor binding domain (RBD), or a fragment thereof (see, e.g., Ou et al., (2020) Nat. Commun. 11(1):1620; Walls et al. (2020) Cell 181(2):281-292; Lan et al. (2020) Nature doi:10.1038 / s41586-020-2180-5; Yuan et al. (2020) Science doi:10.1126 / science.abb7269; NCBI Accession Nos. QIG55857.1, 6VYB_C, 6VYB_B, 6VYB_A, or any of the SARS-CoV-2 spike glycoprotein entries in the NCBI database). In certain embodiments, the antigen comprises the SARS-CoV-2 RBD.

[0074] The antigen is linked to a ligand or antibody fragment capable of binding to a surface protein present on immune cells. In some embodiments, the surface protein is an abundant surface protein on T cells involved in signal transduction and / or adhesion. In some embodiments, the surface protein is present on T cells or antigen-presenting cells (APCs). In some embodiments, the surface protein is preferentially expressed by T cells (e.g., compared to other immune cells). In some embodiments, the surface protein is expressed substantially exclusively by T cells (i.e., expressed by T cells and not significantly expressed by other immune cells). In some embodiments, the ligand is a protein that naturally binds to a surface protein on immune cells, such as a natural ligand for an immune cell receptor, or a derivative or fragment of a natural ligand. In some embodiments, the ligand is an extracellular domain (ectodomain) or a cell adhesion molecule, such as CD58. For example, in some embodiments, the ligand is the 95-residue membrane-distal N-terminal domain of CD58 (1dCD58), which is exclusively involved in adhesion to CD2. For purposes of this disclosure, a ligand does not include an antibody, e.g., a monoclonal antibody.

[0075] In some embodiments, the antibody fragment is a fragment of a monoclonal antibody. In some embodiments, the antibody fragment is a chimeric antibody fragment. In some embodiments, the antibody fragment is a humanized antibody fragment. In some embodiments, the antibody fragment is a human antibody fragment. In some embodiments, the antibody fragment is an antigen-binding fragment such as F(ab')2, Fab', Fab, or scFv. The term "antibody fragment" can also encompass multispecific antibodies and hybrid antibodies with dual or multiple antigen or epitope specificities. In some embodiments, the antibody fragment is a nanobody or single-domain antibody (sdAb) comprising a single monomeric variable antibody domain, e.g., a single VHH domain. In certain embodiments, the antibody fragment is an scFv, e.g., an anti-CD2, anti-CD3, or anti-CD4 scFv. For example, in some embodiments, the fragment is an scFv derived from an anti-CD2 antibody, such as LO-CD2a. In some embodiments, the fragment is an scFv derived from an anti-CD3 antibody, such as SP34. In some embodiments, the fragment is an scFv derived from an anti-CD4 antibody, such as hu5A8. In some embodiments, the scFv is derived from a humanized antibody.

[0076] In some embodiments, for example, when the antibody fragment is an scFv, the VH and VL domains of the antibody are separated by a flexible linker. In some embodiments, the flexible linker is 12 or more amino acids, e.g., 15 amino acids, in length. In such embodiments, the VH and VL domains typically fold properly, allowing the conjugated polypeptide to act as a monomer (e.g., bind to a surface protein). In other embodiments, the flexible linker is less than 12 amino acids in length, e.g., 5, 6, 7, 8, 9, 10, or 11 amino acids in length. In such embodiments, the VH and VL regions may have insufficient length to fold properly as a monomer, promoting the formation of multimers, such as diabodies, triabodies, tetrabodies, etc. In some embodiments, the present disclosure includes diabodies, triabodies, or tetrabodies formed between the scFv antibody fragments described herein. In some embodiments, such multimers are stabilized by disulfide bonds between the monomer units.

[0077] Many techniques known in the art can be used to prepare antibody fragments that bind to surface proteins.See, for example, Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., pp.77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2nd ed.1986)).In some embodiments, antibodies are prepared by immunizing one or more animals (such as mice, rabbits, or rats) with an antigen to induce antibody responses.To generate monoclonal antibodies, B cells are fused with myeloma cells, and then screened for antigen specificity.

[0078] Genes encoding the heavy and light chains of the antibody of interest can be cloned from cells; for example, genes encoding monoclonal antibodies can be cloned from hybridomas and used to produce recombinant monoclonal antibodies, from which antibody fragments can be generated. Gene libraries encoding the heavy and light chains of monoclonal antibodies can also be produced from hybridomas or plasma cells. Furthermore, phage or yeast display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, for example, McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992); Lou et al., PEDS 23:311 (2010); and Chao et al., Nature Protocols, 1:755-768 (2006)). Alternatively, antibodies and antibody sequences can be isolated and / or identified using yeast-based antibody display systems, such as those disclosed in, for example, Xu et al., Protein Eng Des Sel, 2013, 26:663-670; WO 2009 / 036379; WO 2010 / 105256; and WO 2012 / 009568. Random combination of heavy and light chain gene products generates a large pool of antibodies with different antigen specificities (see, for example, Kuby, Immunology (3rd ed. 1997)). Techniques for producing single-chain antibodies or recombinant antibodies (U.S. Patent No. 4,946,778, U.S. Patent No. 4,816,567) can also be adapted to produce antibodies.

[0079] In some embodiments, antibody fragments (such as Fab, Fab', F(ab')2, scFv, nanobody, or diabody) are generated. In certain embodiments, the antibody fragment is an scFv (single-chain variable fragment). An scFv is a recombinant polypeptide comprising the variable regions of a light (VL) and a heavy (VH) immunoglobulin chain. In some embodiments, the VH and VL sequences are linked by a flexible linker sequence. See, e.g., Nelson (2010) MAbs. 2(1):77-83. Various techniques have been developed for producing antibody fragments, such as proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., J. Biochem. Biophys. Meth., 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)) and the use of recombinant host cells to produce fragments. For example, antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from E. coli cells and chemically coupled to form F(ab')2 fragments (see, e.g., Carter et al., BioTechnology, 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to those skilled in the art.

[0080] Methods for measuring binding affinity and binding kinetics are known in the art. These methods include, but are not limited to, solid-phase binding assays (e.g., ELISA assays), immunoprecipitation, surface plasmon resonance (e.g., Biacore™ (GE Healthcare, Piscataway, NJ)), kinetic exclusion assays (e.g., KinExA™), flow cytometry, fluorescence-activated cell sorting (FACS), BioLayer interferometry (e.g., Octet™ (ForteBio, Inc., Menlo Park, CA)), and Western blot analysis.

[0081] In some embodiments, the affinity agent is a peptide, for example, a peptide that binds to a T cell surface protein. In some embodiments, the agent is a peptide aptamer. A peptide aptamer is an artificial protein selected or engineered to bind to a specific target molecule. Typically, a peptide contains one or more peptide loops of variable sequence displayed by a protein scaffold. Peptide aptamer selection can be performed using different systems, including the yeast two-hybrid system. Peptide aptamers can also be selected from combinatorial peptide libraries constructed by phage display and other surface display technologies, such as mRNA display, ribosome display, bacterial display, and yeast display. See, for example, Reverdatto et al., 2015, Curr. Top. Med. Chem. 15:1082-1101.

[0082] In some embodiments, the agent is an affimer. Affimers are small, highly stable proteins, typically with a molecular weight of approximately 12-14 kDa, that bind to their target molecules with similar specificity and affinity as antibodies. Generally, affimers display two peptide loops and an N-terminal sequence that can be randomized to bind different target proteins with high affinity and specificity, in a manner similar to monoclonal antibodies. Stabilization of the two peptide loops by a protein scaffold constrains the possible conformations that the peptides can adopt, increasing binding affinity and specificity compared to libraries of free peptides. Affimers and methods for producing them have been described in the art. See, e.g., Tiede et al., eLife, 2017, 6:e24903. Affimers are also commercially available, e.g., from Avacta Life Sciences.

[0083] Antigen and ligand / antibody fragment can be directly or indirectly linked to each other in many ways.For example, in some embodiments, antigen and ligand / antibody fragment are directly (covalently) linked, for example, via a chemical linker or by being in a single fusion protein.Methods for linking polypeptides to each other, for example, for linking antigen to ligand / antibody fragment, are known in the art and are available from commercial suppliers, for example, TriLink BioTechnologies, Vector Laboratories, Kerafast, SydLabs, INTERCHIM protein-protein conjugation kit, etc.

[0084] In certain embodiments, the antigen and the ligand / antibody fragment are present in a single fusion protein. For example, in certain embodiments, the immunogenic conjugate is a fusion protein comprising an antigen and an anti-CD3 scFv antibody fragment. In some embodiments, the fusion protein also comprises a flexible linker separating the antigen and the scFv sequence. The fusion protein can be expressed in vitro, purified, formulated, and administered in protein form using standard molecular biology and pharmaceutical methods, as described in more detail elsewhere herein, or can be administered as a polynucleotide encoding the fusion protein.

[0085] In some embodiments, particularly when the fusion protein is administered via administration of a polynucleotide encoding the fusion protein, the fusion protein comprises a tPA leader sequence, e.g., a 23 amino acid long tPA leader sequence (e.g., UniProt P00750; Kou et al. (2017) Immunol. Lett. 190:51-57; Wang et al. (2011) Appl. Microbiol. Biotech. 91(3):731-740; Delogu et al. (2002) Microbial Immun. Vacc. Doi:10.1128 / IAI.70.1.292-302.2002). In some such embodiments, the tPA leader sequence comprises a 22P / A enhancing mutation (see, e.g., Wang et al. (2011)).

[0086] In some embodiments, the antigen and ligand / antibody fragment present in the conjugated polypeptide or fusion protein are separated by a flexible linker. Linkers suitable for separating protein domains are known in the art and can include, for example, glycine and serine residues, such as 2 to 20 glycine and / or serine residues. In one embodiment, the flexible linker is (Gly4Ser) n a flexible peptide linker, e.g., of the sequence TIFF2026021474000002.tif4128 containing (Gly4Ser)3 linker.

[0087] In some embodiments, the conjugated polypeptide comprises a domain such as a lipid anchor, a transmembrane segment, a multimerization domain, or a combination of two or more of these domains. Examples of lipid anchors include, for example, glycosylphosphatidylinositol anchors. In some embodiments, the addition of a lipid anchor, such as a glycosylphosphatidylinositol anchor, is directed by a signal sequence, such as a signal sequence derived from CD55. Examples of suitable transmembrane segments include, but are not limited to, transmembrane segments derived from the PDGF receptor, glycophorin A, or the SARS-CoV-2 spike protein. Examples of multimerization domains include, for example, a domain derived from T4 fibritin and an Fc domain, such as a human IgG Fc domain. In some embodiments, the present disclosure encompasses dimers or other multimers formed between the conjugated polypeptides that multimerize, for example, T4 fibritin or the Fc domain. In some embodiments, the multimers are stabilized by disulfide bonds between the monomer units. In some embodiments, the Fc or other domain is located at the C-terminus of the conjugated polypeptide.

[0088] In some embodiments, where the vaccine comprises a polynucleotide encoding a fusion protein, and the polynucleotide is present in a viral vector, such as a CMV vector, the coding sequence for the fusion protein is operably linked to a late promoter, e.g., the CMV pp65b promoter. Without being bound by theory, it is believed that expressing an antigen, e.g., an immunogenic conjugate, through a strong late promoter such as pp65b elicits a robust antibody response but only a weak T cell response.

[0089] In some embodiments, the antigen and ligand / antibody fragment (also called "affinity agent") are indirectly linked, i.e., linked through a non-covalent interaction that bridges the two entities. For example, the antigen and ligand / antibody fragment can be linked via a second "bridging" antibody or fragment thereof. In some embodiments, the antigen is a membrane protein embedded in the Nanodisc, and the bridging antibody is a bispecific antibody fragment that binds to both (i) either the antigen itself or a membrane scaffolding protein within the Nanodisc, and (ii) a T-cell surface protein. Nanodiscs are synthetic membrane systems comprising a lipid bilayer surrounded by amphipathic proteins called membrane scaffolding proteins (MSPs). Any Nanodisc system, including any MSP or amphipathic peptide, such as MSP derived from apoA1, can be used in the present methods. In some embodiments, synthetic Nanodiscs are used. The preparation and use of nanodiscs is known in the art, see, e.g., Bayburt et al. (2002) FEBS Letters 584(9):1721-1727; Denisov et al. (2004) J. Am. Chem. Soc. 126(11):3477-3487; Grinkova et al. (2010) PEDS 23(11):843-848; Midtgaard et al. (2016) Soft Matter 10(5):738-752; Larsen et al. (2016) Soft Matter 12(27):5937-5949; Kondo et al. (2016) Colloids and Surfaces B: Biointerfaces 146:423-430; Knowles et al. al. (2009) J. Am. Chem. Soc. 131(22):7484-7485; Oluwole et al. (2017) 33(50):14378-14388; Rouck et al. (2017) FEBS Lett. 591(14):2057-2088; Denisov & Sligar (2016) Nat. Struct. Mol. Biol. 23(6):481-486, the entire disclosures of each of which are incorporated herein by reference.

[0090] In some embodiments, the conjugated polypeptide has the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:27, or the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID It includes an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to NO:27.

[0091] Additional antigens In some embodiments, the vaccine comprises a second antigen, which can be present together with or instead of the immunogenic conjugate.When both antigens are present (i.e., immunogenic conjugate and second antigen), the antigens can be administered together, i.e., in the same vaccine, or independently, for example, separately formulated, at the same time, for example, during a single clinical visit, or at different times, for example, on different days.One or both antigens can be administered in protein form or polynucleotide form (i.e., as a polynucleotide encoding two antigens).In some embodiments, when administered in polynucleotide form, the coding sequence of the immunogenic conjugate and the coding sequence of the second antibody are present in a single vector, and each of them is operably linked to a promoter. In some embodiments, the second antigen is administered as a polynucleotide encoding the antigen operably linked to a constitutive promoter, for example, a mammalian promoter such as EF-1α (e.g., Wang et al. (2017) J. Cell Mol. Med 21(11):3044-3054; Edmonds et al. (1996) J. Cell Sci. 109(11):2705-2714; NCBI Gen ID 1915; the entire disclosures of which are incorporated herein by reference). Without being bound by theory, it is believed that expressing the second antigen in the cytoplasmic compartment of cells elicits robust T cell responses, but with minimal associated antibody responses.

[0092] In some embodiments, when the pathogen is a coronavirus such as SARS-CoV-2, the second antigen comprises an E (envelope protein, see e.g., NCBI Gene ID 43740570), an M (membrane glycoprotein, see e.g., NCBI Gene ID 43740571), or an N (nucleocapsid phosphoprotein, see e.g., NCBI Gene ID 43740575), or a fragment thereof. In some embodiments, the second antigen comprises the E and M proteins of a coronavirus such as SARS-CoV-2, or a fusion protein comprising a fragment of the E and / or M proteins. Other suitable SARS-CoV-2 antigens include nsp3, nsp4, or nsp6, or a fragment thereof. In certain embodiments, the vaccine comprises a codon-optimized coding sequence for a fusion protein comprising the N protein and / or the E and M proteins of SARS-CoV-2 (e.g., as set forth in SEQ ID NOs:3 and 4).

[0093] nucleic acid vaccines In some embodiments, nucleic acid vaccines, such as DNA vaccines, are used to introduce immunogenic conjugates and / or second antigens. Thus, in some embodiments, the present disclosure provides a polynucleotide encoding any of the conjugate polypeptides described herein. In some embodiments, the DNA vaccine is prepared as a DNA vector or plasmid. In some embodiments, the DNA vaccine is prepared as a recombinant virus, for example, by modifying a parent virus to incorporate exogenous genetic material, such as one or more polynucleotides encoding one or more antigens described herein. A non-limiting list of suitable viruses that can be used for the purposes of the present disclosure includes lentiviruses (e.g., HIV, HIV-1, HIV-2, FIV, BIV, EIAV, MW, CAEV, SIV), adenoviruses and adeno-associated viruses, alphaviruses, herpesviruses (e.g., cytomegaloviruses), flaviviruses, and poxviruses. For methods and examples concerning the use of suitable viral vectors, see, e.g., U.S. Patent Nos. 5,219,740, 7,250,299, 7,608,273, 6,465,634, 7,811,812, 5,744,140, ​​8,124,398, 5,173,414, 7,022,519, 7,125,705, 6,905,862, 7,989,425, 6,468,711, 7,015,024, 7,338,662, 5,871,742, and 6,340,462. In such embodiments, the virus is typically recombination competent (ie, capable of replicating in the infected host cell).Modification of such viruses and vectors or plasmids to prepare the DNA vaccines of the present invention can be accomplished using standard molecular biology techniques, for example, as taught in Sambrook et al. (1989) "Molecular Cloning: A Laboratory Manual" (2nd ed. Cold Spring Harbor Press) and Ausubel et al. (Eds.) (2000-2010) "Current Protocols in Molecular Biology" (John Wiley and Sons).

[0094] In some embodiments, the polynucleotide comprises an amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:27, or an amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO: The amino acid sequence encodes an amino acid sequence that contains at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to NO:27. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26 or SEQ ID NO:28, or the nucleotide sequence set forth in SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26 or SEQ ID NO: It includes a nucleotide sequence that contains at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to NO:28.

[0095] In certain embodiments, the DNA vaccine comprises a cytomegalovirus (CMV) vector, e.g., a self-initiating CMV DNA vector (SLCMV), such as those described in U.S. Provisional Application No. 62 / 842,419, filed May 2, 2019, the entire disclosure of which is incorporated herein by reference. Self-initiating CMV vaccines are administered as CMV genomes that "prime" vectored vaccine replication in vivo, resulting in a robust immune response with the unique characteristics associated with CMV infection. SLCMV vectors may comprise one or more of a number of features, including: (i) being based on the Towne HCMV strain, which has been proven safe for use in humans of all ages; (ii) providing an immune response in CMV-seropositive and -seronegative individuals due to CMV's ability to co-infect and unique deletions in the CMV genome; (iii) providing an exceptionally broad and strong T cell response that "paints" the vaccine antigen; (iv) having a proven ability to elicit a balanced response including antibodies, CD4+ T cells, and CD8+ T cells, without reliance on or dominance of any single effector response, and providing localization of responding T cells with an effector memory phenotype to mucosal surfaces; (v) allowing for engineering of new vaccine candidates in weeks to months; and (vi) providing a single manufacturing process based on plasmid production in E. coli.

[0096] As shown in the examples below, in contrast to most vaccine types that do not elicit large numbers of memory CD4+ T cells, this CMV-vectored vaccine does. Furthermore, T cells that respond to CMV-vectored vaccines localize to the airways, among other effector sites, and can be recovered, for example, by bronchoalveolar lavage (12). CMV-responsive T cells recapitulate other essential characteristics of cells shown to be protective against SARS-CoV-1, including CXCR3 expression, IFN-γ production, and IL-10 production (13).

[0097] In SLCMV vectors, utilizing the DNA form of CMV vectors for vaccination requires modification of the current CMV BAC construct so that the BAC backbone can be excised in vivo without recombinase or nuclease expression. CMV has relatively strict packaging constraints due to the need to package a unit-length genome into an icosahedral capsid. In some embodiments, the CMV BAC utilizes an endogenous recombinase gene located within the BAC portion of the DNA construct. Upon transfection into mammalian cells, the recombinase is expressed and excises the BAC replication machinery from the replicating genome. To create a BAC DNA vector suitable for in vivo delivery in humans, the CMV genome ends are rearranged to allow BAC excision without the need for recombinase expression. In particular, the reconstructed BAC construct utilizes the viral terminase complex to remove the bacterial replication origin during the packaging step of CMV replication. In some embodiments, the BAC origin and replication machinery are located between viral direct repeat sequences.

[0098] Thus, in one aspect, the present disclosure provides a self-initiating herpesvirus (HV) (e.g., CMV) recombinant polynucleotide comprising one or more polynucleotides encoding one or more antigens (or conjugated polypeptides) described herein, the polynucleotide comprising: (a) a herpesvirus (HV) genome or a substantial portion thereof; (b) a sequence comprising an origin of replication functional in a unicellular organism; (c) one or more terminase complex recognition loci (TCRLs) comprising recombinantly introduced polynucleotide sequences capable of directing cleavage by an HV terminase complex, wherein the HV genome or portion thereof is separated from the sequence comprising the origin of replication by the TCRL; the HV genome or portion thereof is flanked by the TCRL on at least one end; and the sequence comprising the origin of replication is flanked by the TCRL on at least one end; and (d) one or more polynucleotides encoding one or more antigens operably linked to a promoter. In one embodiment, the recombinant polynucleotide comprises a polynucleotide encoding an immunogenic conjugate (e.g., an antigen fused to a polypeptide that specifically binds to an abundant T cell surface protein) operably linked to a late promoter such as pp65b, and / or a polynucleotide encoding an antigen operably linked to a constitutive promoter such as EF-1α.

[0099] As used herein, the "end" of a particular genomic region or element in a vector, such as a genome or a portion thereof, refers to either end of the region or element beyond which a different region or element (or end of a nucleic acid molecule) exists. For example, in some embodiments, in a circular vector, one end (or end) of the HV genome can be directly adjacent to the first end of a first TCRL element, and the other end (or end) of the HV genome can be directly adjacent to the first end of a second TCRL element. In the same circular vector, one end (or end) of the region containing the origin can be directly adjacent to the second end of the first TCRL element, and the other end (or end) of the region containing the origin can be directly adjacent to the second end of the second TCRL element. In some embodiments, the CMV vector used herein does not contain a viral IL-10 gene.

[0100] It will be understood that the polynucleotide may be circular or linear, and that additional elements may be present, for example, between the HV genome or a substantial portion thereof and the sequence comprising the BAC or YAC origin of replication (e.g., genetic elements present between the TCRL adjacent to the HV genome or a substantial portion thereof and an origin of replication (BAC or YAC) that functions in a unicellular organism).

[0101] Antigen coding sequence The recombinant polynucleotides, e.g., viral vectors, of the present disclosure comprise nucleic acid sequences encoding antigens, e.g., conjugated polypeptides described herein, and / or coronavirus antigens, such as spike proteins E, M, or N, or combinations and / or fragments thereof, as described herein. Rapid advances in the study of various genomes have enabled cloning approaches that can search DNA sequence databases of humans or other model organisms for any gene segments that share a certain percentage of sequence homology with known nucleotide sequences, e.g., known nucleotide sequences encoding antigens. Any DNA sequences so identified can then be obtained by chemical synthesis and / or polymerase chain reaction (PCR) techniques, such as overlap extension. While complete de novo synthesis may be sufficient for short sequences, to obtain larger genes, it may be necessary to further isolate full-length coding sequences from cDNA or genomic libraries of humans or other model organisms using synthetic probes.

[0102] Alternatively, nucleic acid sequences can be isolated from cDNA or genomic DNA libraries (e.g., human or rodent cDNA libraries or human, rodent, bacterial, or viral genomic DNA libraries) using standard cloning techniques such as the polymerase chain reaction (PCR), and homology-based primers can often be derived from known nucleic acid sequences. Commonly used techniques for this purpose are described in standard texts, e.g., Sambrook and Russell, supra.

[0103] cDNA libraries can be commercially available or can be constructed. General methods for isolating mRNA, generating cDNA by reverse transcription, ligating the cDNA into a recombinant vector, transfecting the recombinant host for propagation, screening, and cloning are well known (e.g., Gubler and Hoffman, Gene, 25:263-269 (1983); Ausubel et al., supra). Once an amplified segment of nucleotide sequence is obtained by PCR, the segment can be further used as a probe to isolate the full-length polynucleotide sequence encoding the protein of interest from a cDNA library. A general description of suitable procedures can be found in Sambrook and Russell, supra.

[0104] Similar procedures can be followed to obtain full-length sequences encoding proteins of interest from human or other model organism genomic libraries. Genomic libraries are commercially available or can be constructed according to various art-recognized methods. As a non-limiting example, to construct a genomic library, DNA is first extracted from the organism's tissue. The DNA is then mechanically sheared or enzymatically digested to obtain fragments of approximately 12-20 kb in length. The fragments are then separated from undesired polynucleotide fragments by gradient centrifugation and inserted into bacteriophage λ vectors. These vectors and phages are packaged in vitro. Recombinant phages are analyzed by plaque hybridization as described by Benton and Davis, Science, 196:180-182 (1977). Colony hybridization is performed as described by Grunstein et al., Proc. Natl. Acad. Sci. USA, 72:3961-3965 (1975).

[0105] In certain embodiments, the polynucleotide encoding the antigen (e.g., the immunogenic conjugate and / or other antigen) is present in an expression cassette, i.e., operably linked to one or more promoters. Any promoter capable of driving expression of a polynucleotide in one or more cells of a subject can be used, including inducible and constitutive promoters. In some embodiments, a CMV promoter is used. In certain embodiments, a late viral promoter such as pp65b is used to drive expression of the immunogenic conjugate. In certain embodiments, a constitutive mammalian promoter such as EF1-alpha is used to drive expression of the second antigen described herein. In some embodiments, the EF1-alpha promoter comprises the first intron of the EF1-alpha gene. The vector can include other regulatory elements, such as terminators, translational regulatory elements, such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, border regions, origins of replication, matrix attachment sites, and locus control regions. The use of such elements is well known in the art.

[0106] In some embodiments, the recombinant polynucleotide of the present disclosure contains a nucleic acid sequence encoding a selectable marker. Selectable markers are useful, for example, when a polynucleotide as described herein is recombinantly modified, particularly when it is desirable to screen a population of modified polynucleotides (e.g., using bacterial, yeast, plant, or animal cells) for polynucleotides incorporating a desired modification. Whether a polynucleotide is recombinantly modified within a cell (e.g., bacterial cells, e.g., using Red / ET recombination) or recombinantly modified and then introduced into a cell (e.g., bacterial, yeast, plant, or animal cell) for screening, a selectable marker can be used to identify which cells contain a polynucleotide incorporating a modification of interest. Taking an antibiotic resistance gene as an example of a selectable marker, treating cells containing a recombinant polynucleotide with an antibiotic identifies which cells contain a recombinant polynucleotide incorporating the antibiotic resistance gene (i.e., cells that survive antibiotic treatment should have incorporated the antibiotic resistance gene). If desired, the recombinant polynucleotide can be further screened (e.g., purified from the cells, amplified, and sequenced) to confirm that the desired modification was recombinantly introduced into the polynucleotide at the correct location.

[0107] When the selectable marker is an antibiotic resistance gene, the gene can confer resistance to chloramphenicol, Zeocin, ampicillin, kanamycin, tetracycline, or another suitable antibiotic known to those of skill in the art. In some embodiments, a selectable marker is used that results in a visible phenotype, such as the color of an organism or population of organisms. As a non-limiting example, the phenotype can be examined by growing the organism (e.g., a cell or other organism containing a recombinant polynucleotide) and / or their progeny under conditions that result in the phenotype, which may not be visible under normal growth conditions.

[0108] In some embodiments, the selectable marker used to identify the cell that contains the polynucleotide containing the modification of interest is a fluorescently tagged protein, a chemical stain, a chemical indicator, or a combination thereof.In other embodiments, the selectable marker responds to the change of stimulus, biochemical substance, or environmental condition.In some examples, the selectable marker responds to the concentration of metabolic product, protein product, drug, cell phenotype of interest, cell product of interest, or a combination thereof.

[0109] The size of a recombinant polynucleotide depends on the particular antigen and other proteins encoded, the presence and choice of regulatory sequences and / or expression vectors (e.g., viral vectors), the choice and location of different elements such as TCRL, etc. Furthermore, the size of a recombinant polynucleotide depends on whether the nucleic acid sequences encoding the antigen and other proteins are present within the same recombinant polynucleotide or within separate recombinant polynucleotides.

[0110] In some embodiments, the recombinant polynucleotide is between about 1 kilobase and about 300 kilobases (e.g., about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110 , 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or 300 kilobases in length). In some embodiments, the recombinant polynucleotide is greater than about 300 kilobases in length.

[0111] In some embodiments, the recombinant polynucleotide present in the system of the present disclosure is from about 1 kilobase to about 300 kilobases, from about 1 kilobase to about 250 kilobases, from about 1 kilobase to about 200 kilobases, from about 1 kilobase to about 150 kilobases, from about 1 kilobase to about 100 kilobases, from about 1 kilobase to about 50 kilobases, from about 1 kilobase to about 40 kilobases, from about 1 kilobase to about 30 kilobases, from about 1 kilobase to about 20 kilobases, from about 1 kilobase to about 10 kilobases, from about 50 kilobase to about 300 kilobases, from about 50 kilobase to about 250 kilobases. The length is about 50 kilobases to about 200 kilobases, about 50 kilobases to about 150 kilobases, about 50 kilobases to about 100 kilobases, about 100 kilobases to about 300 kilobases, about 100 kilobases to about 250 kilobases, about 100 kilobases to about 200 kilobases, about 100 kilobases to about 150 kilobases, about 150 kilobases to about 300 kilobases, about 150 kilobases to about 250 kilobases, about 150 kilobases to about 200 kilobases, about 200 kilobases to about 300 kilobases, or about 200 kilobases to about 250 kilobases.

[0112] General recombinant technology For example, basic texts disclosing general methods and techniques in the field of recombinant genetics for the preparation, maintenance and cultivation of recombinant vectors or plasmids include Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Ausubel et al., eds., Current Protocols in Molecular Biology (1994).

[0113] For nucleic acids, sizes are given in either kilobases (kb) or base pairs (bp). In some instances, these are estimates obtained from agarose or acrylamide gel electrophoresis, sequenced nucleic acids, or published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. In some instances, protein sizes are estimated from gel electrophoresis, sequenced proteins, derived amino acid sequences, or published protein sequences.

[0114] Non-commercially available oligonucleotides can be chemically synthesized according to the solid-phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Lett. 22:1859-1862 (1981), for example, using an automated synthesizer as described in Van Devanter et al., Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides is carried out using any art-recognized strategy, for example, native acrylamide gel electrophoresis or anion-exchange HPLC as described in Pearson & Reanier, J. Chrom. 255:137-149 (1983).

[0115] The sequence of a protein domain or gene of interest can be confirmed after cloning or subcloning, for example, using the chain termination method of sequencing double-stranded templates of Wallace et al., Gene 16:21-26 (1981).

[0116] Based on sequence homology, degenerate oligonucleotides can be designed as primer sets, and PCR can be performed under appropriate conditions to amplify segments of nucleotide sequences from cDNA or genomic libraries (see, for example, White et al., PCR Protocols: Current Methods and Applications, 1993; Griffin and Griffin, PCR Technology, CRC Press Inc. 1994). The amplified segments can be used as probes to obtain the full-length nucleic acid encoding the protein of interest.

[0117] Once the nucleic acid sequence encoding the protein of interest is obtained, the coding sequence can be further modified by many well-known techniques, such as restriction endonuclease digestion, PCR and PCR-related methods, to generate coding sequences, including mutants and variants derived from wild-type protein.Then, the polynucleotide sequence encoding desired polypeptide can be subcloned into a vector, for example, an expression vector, so that recombinant polypeptide can be produced from the resulting construct.Further modification of coding sequence, such as nucleotide substitution, can then be carried out to change the characteristics of polypeptide.

[0118] A variety of mutagenesis protocols have been established and described in the art and can be easily used to modify the polynucleotide sequence encoding a protein of interest.See, for example, Zhang et al., Proc. Natl. Acad. Sci. USA, 94: 4504-4509 (1997); and Stemmer, Nature, 370: 389-391 (1994).The procedures can be used separately or in combination to generate a set of variants of nucleic acids, and thus variants of the encoded polypeptides.Kits for mutagenesis, library construction and other diversity generation methods are commercially available.

[0119] Examples of mutational methods that generate diversity include site-directed mutagenesis (Botstein and Shortle, Science, 229:1193-1201 (1985)), mutagenesis using uracil-containing templates (Kunkel, Proc. Natl. Acad. Sci. USA, 82:488-492 (1985)), oligonucleotide-directed mutagenesis (Zoller and Smith, Nucl. Acids Res., 10:6487-6500 (1982)), phosphorothioate-modified DNA mutagenesis (Taylor et al., Nucl. Acids Res., 13:8749-8787 (1985)), and mutagenesis using gapped double-stranded DNA (Kramer et al., Nucl. Acids Res., 12:9441-9456 (1984)).

[0120] Other possible methods for generating mutations include point mismatch repair (Kramer et al., Cell, 38:879-887 (1984)), mutagenesis using repair-deficient host strains (Carter et al., Nucl. Acids Res., 13:4431-4443 (1985)), deletion mutagenesis (Eghtedarzadeh and Henikoff, Nucl. Acids Res., 14:5115 (1986)), restriction selection and restriction purification (Wells et al., Phil. Trans. R. Soc. Lond. A, 317:415-423 (1986)), and mutagenesis by total gene synthesis (Nambiar et al., Cell, 38:879-887 (1984)). al., Science, 223:1299-1301 (1984)), double-strand break repair (Mandecki, Proc. Natl. Acad. Sci. USA, 83:7177-7181 (1986)), polynucleotide chain termination mutagenesis (U.S. Patent No. 5,965,408), and error-prone PCR (Leung et al., Biotechniques, 1:11-15 (1989)).

[0121] Codon optimization In some embodiments, a nucleic acid sequence encoding a protein of interest (e.g., an antigen or other protein) is codon-optimized. The term "codon optimization" refers to modifying a nucleic acid sequence to reduce or rebalance codon bias (i.e., preferential use of certain codons, which may vary between species), without changing the encoded amino acid sequence. In some embodiments, codon optimization increases translation efficiency (e.g., of an antigen or other protein). As a non-limiting example, leucine is encoded by six different codons, some of which are rarely used. By rebalancing codon usage (e.g., within a reading frame), a preferred leucine codon can be selected over rarely used codons. A nucleic acid sequence encoding a protein of interest (e.g., an antigen or other protein) is modified to convert rarely used codons into preferred codons.

[0122] Rare codons can be defined, for example, by using a codon usage table derived from the sequenced genome of the host species, i.e., the species in which the protein (e.g., antigen) is expressed. For example, see the codon usage table obtained from Kazusa DNA Research Institute, Japan (www.kazusa.or.jp / codon / ), used in conjunction with software from DNA2.0 (www.dna20.com / ), such as "Gene Designer 2.0" software, at a cutoff threshold of 15%.

[0123] Codon optimization can also be used to adjust GC content, for example, to increase mRNA stability or reduce secondary structure, or to minimize codons that may otherwise result in stretches of sequence that impair expression of a protein of interest (e.g., an antigen or other protein).

[0124] 4. Formulation and vaccination methods subject The methods and compositions can be used for vaccination of any subject, e.g., a human or other mammal, that can benefit from an enhanced immune response to infection, e.g., infection with a coronavirus such as SARS-CoV-2. In some embodiments, the subject is male. In some embodiments, the subject is female. In some embodiments, the subject is an adult (e.g., adult male). In some embodiments, the subject is an adolescent. In some embodiments, the subject is a child. In some embodiments, the subject is over 60, 70, or 80 years of age.

[0125] In some embodiments, the subject has never been infected with a pathogen, such as SARS-CoV-2, and the methods and compositions are used to enhance the subject's immune defenses against the pathogen to prevent future infection. In other embodiments, the subject has already been infected with a pathogen, and the methods are used to enhance the subject's immune response against the pathogen to delay or potentially reverse the original infection.

[0126] Pharmaceutical Compositions The present disclosure provides compositions comprising an immunogenic component (e.g., a DNA vaccine or one or more immunogenic polypeptides) capable of inducing immunity to a targeting agent (e.g., an antigen or an immunogenic conjugate comprising the antigen) and a pharmaceutically acceptable carrier. In some embodiments, the vaccine further comprises one or more adjuvants or compounds. Thus, the present disclosure provides pharmaceutical compositions for inducing an immune response in a subject. In some embodiments, the composition comprises one or more polynucleotides encoding one or more proteins, e.g., coronavirus antigens and / or immunogenic conjugates, and a pharmaceutically acceptable carrier. In some embodiments, the composition comprises one or more polypeptide antigens, e.g., the immunogenic conjugates described herein, and / or coronavirus antigens, e.g., including spike protein, E, M, or N protein, fragments thereof, or combinations thereof, and a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises an adjuvant.

[0127] Composition can be formulated for example, injection, inhalation or local administration, for example, to promote direct exposure of host cell and tissue to immunogenic component.In some embodiments, composition, for example, DNA vaccine, is formulated for subcutaneous injection.In some embodiments, composition, for example, DNA vaccine, is formulated as naked DNA (see, for example, U.S. Patent No. 6,265,387, U.S. Patent No. 6,972,013 and U.S. Patent No. 7,922,709).

[0128] In certain embodiments, DNA vaccines are prepared as DNA vectors or plasmids. In some embodiments, compositions, e.g., DNA vaccines, are prepared as recombinant viruses, for example, by modifying a parent virus to incorporate exogenous genetic material, such as one or more polynucleotides encoding one or more antigens described herein. In some embodiments, the virus is a herpesvirus, such as CMV, adenovirus, or adeno-associated virus (AAV). Self-initiating CMV (SLCMV) vectors can be prepared, for example, by culturing E. coli containing the vector, lysing the cultured bacterial cells, purifying the vector while ensuring that endotoxin is absent or below the pyrogenic threshold (e.g., 5 endotoxin units / kg body weight), and formulating the vector for administration. In some embodiments, proteins encoding the antigens are produced in vitro using standard molecular biology techniques and purified prior to the vaccine formulation described herein.

[0129] In some embodiments, nucleic acid vaccines are formulated with an in vivo transfection agent, which comprises one or more reagents that can protect nucleic acid from degradation in vivo and facilitate the delivery of nucleic acid to cells.Suitable examples of such agents include but are not limited to in vivo-jet PEI™ (Polyplus), TurboFect™ (Thermo Scientific), LIPID™ (Altogen Biosystems), GenJet™ Plus or PepJet™ Plus (SignaGen), DogtorMag™ (OZ Biosciences), Avalanche™ (EZ Biosystems) etc., and can be used according to the manufacturer's instructions.

[0130] The pharmaceutical composition of the present disclosure can include a pharmaceutically acceptable carrier. In certain aspects, the pharmaceutically acceptable carrier is determined in part by the specific composition to be administered as well as the specific method used to administer the composition. Thus, there are a wide variety of formulations of pharmaceutical compositions suitable for use in the present methods and compositions (see, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18TH ED., Mack Publishing Co., Easton, PA (1990)).

[0131] Pharmaceutical compositions often further comprise one or more buffering agents (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose, or dextran), mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxytoluene, butylated hydroxyanisole, etc.), bacteriostats, chelating agents such as EDTA or glutathione, solutes that render the formulation isotonic, hypotonic, or weakly hypertonic with the blood of the recipient, suspending agents, thickening agents, preservatives, flavoring agents, sweetening agents, and coloring compounds, as appropriate.

[0132] Pharmaceutical compositions are administered in a manner compatible with the dosage formulation and in an amount that is therapeutically or prophylactically effective.The amount to be administered depends on various factors, including, for example, individual's age, weight, physical activity, genetic characteristics, general health, sex and diet, the symptoms or diseases to be treated or prevented, and the stage or severity of the symptoms or diseases.In certain embodiments, the size of the dose can also be determined by the existence, nature and extent of any adverse side effects associated with the administration of therapeutic or prophylactic agents in a specific individual.Other factors that can affect the specific dose level and frequency of administration for any specific patient include the activity of the specific compound used, the metabolic stability and duration of action of the compound, the mode and time of administration, and excretion rate.

[0133] In some embodiments, the vaccine comprises an adjuvant, i.e., a compound administered to a subject together with an antigen to enhance the immune response to the antigen.The adjuvant can increase the immunogenicity of the vaccine in any of many ways, and can include inorganic compounds such as salts, for example, aluminum salts, and organic compounds and compound mixtures, including extracts and preparations, for example, Freund's incomplete adjuvant, squalene, MF59, monophosphoryl lipid A, QS-21.

[0134] Generally, for administering a compound (e.g., a vaccine or adjuvant) for therapeutic or prophylactic (e.g., vaccination) purposes, the compound is given in a therapeutically or prophylactically effective dose. In particular, an effective amount of a pharmaceutical composition is an amount sufficient to obtain an enhanced immune response against the antigen or pathogen from which the antigen is derived, and / or an amount sufficient to enhance the subject's immunity against infection from the pathogen or against the spread of an already existing infection in the subject, for example, taking into account any of the parameters or indicators described herein.

[0135] In certain embodiments, the dose may take the form of a solid, semi-solid, lyophilized powder or liquid dosage form, such as, for example, a tablet, pill, pellet, capsule, powder, solution, suspension, emulsion, suppository, retention enema, cream, ointment, lotion, gel, aerosol, foam, etc., preferably in a unit dosage form suitable for simple administration of precise dosage amounts.

[0136] As used herein, the term "unit dosage form" refers to a physically discrete unit (e.g., an ampoule) suitable as a unitary administration for humans and other mammals, each unit containing a predetermined quantity of a therapeutic or prophylactic agent calculated to produce a desired onset, tolerability, and / or therapeutic or prophylactic effect in combination with a suitable pharmaceutical excipient. Additionally, more concentrated dosage forms can be prepared from which more dilute unit dosage forms can then be produced. Thus, more concentrated dosage forms contain substantially greater amounts of the therapeutic or prophylactic compound, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times the amount.

[0137] Methods for preparing such dosage forms are known to those skilled in the art (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra). Dosage forms typically contain a conventional pharmaceutical carrier or excipient and may further contain other medicinal agents, carriers, adjuvants, diluents, tissue penetration enhancers, solubilizers, etc. Appropriate excipients can be tailored to the particular dosage form and route of administration by methods well known in the art (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra).

[0138] Administration In some embodiments, the prevention and / or treatment comprises administering a composition described herein directly to the subject. As a non-limiting example, a pharmaceutical composition (e.g., comprising a vaccine described herein and a pharmaceutically acceptable carrier) can be delivered directly to the subject (e.g., by local injection or systemic administration).

[0139] The compositions of the present disclosure can be administered as a single dose or as multiple doses, for example, two doses administered at intervals of about 1 month, about 2 months, about 3 months, about 6 months, or about 12 months. Other suitable administration schedules can be determined by a medical professional.

[0140] In some embodiments, additional compounds or drugs can be co-administered to the subject to alleviate signs or symptoms of the disease being treated, reduce side effects caused by the induction of an immune response, etc.

[0141] The pharmaceutical composition can be administered to a subject locally or systemically, for example, intraperitoneally, intramuscularly, intraarterially, orally, intravenously, intracranially, intrathecally, intrathecally, intralesionally, intranasally, subcutaneously, intracerebroventricularly, topically, and / or by inhalation. In certain embodiments, the composition is administered by electroporation (e.g., in the case of a priming DNA vaccine) or subcutaneously (e.g., in the case of a booster).

[0142] The vaccine can be administered any number of times, for example, 1, 2, 3, 4, 5 or more times, and after any number of vaccination regimens, for example, every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks. In certain embodiments, the vaccine is administered as a DNA prime using, for example, a plasmid encoding a conjugated polypeptide described herein, followed by a booster, for example, about 4 weeks later, using, for example, an adenoviral vector encoding an antigen. The DNA vaccine can be administered at any of a number of levels, for example, 4 mg of plasmid DNA vector per subject per vaccination, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mg of plasmid DNA vector per subject per vaccination, or 1-10, 1-20, 1-8, 1-7, 2-6, 3-5 mg of plasmid DNA vector per subject per vaccination.

[0143] Assessment of immune response The immune response of a subject receiving a vaccine of the present disclosure can be detected, characterized, or quantified in any of a number of ways.For example, any of the assays described in any of the Examples can be used to detect the presence of the polynucleotide of the present disclosure in the cells of a subject, to detect and characterize antibodies or T cells specific to the present vaccine, or to evaluate the protection provided by the vaccine against infection by a pathogen.In some embodiments, particularly in embodiments in which a nucleic acid vector is used to deliver the present antigen, the presence and level of vector sequence can be evaluated by, for example, measuring the sequence using qPCR from blood or saliva samples from the subject.Nucleic acid levels can also be detected from different tissues of a subject, for example, obtained from biopsy or by washing mucosal tissue.

[0144] In some embodiments, the subject's immune response is assessed by immunophenotyping, e.g., by evaluating T cell memory effector subsets, NK cells with adaptive properties (e.g., FcεRIy low "memory" NK cells), T cells with innate properties (e.g., NKG2A+ cells), or antigen-presenting cells (e.g., monocytes expressing CD80 / 83 / 86). Such cells can be assessed, e.g., using flow cytometry as described in the Examples.

[0145] The subject's immune response can also be evaluated by characterizing the antigen-specific T cell response from the subject.For example, as described in the Examples, PBMC or LNMC cells can be stimulated with one or more antigens from the vaccine, optionally in combination with an inhibitor such as a VL9 peptide or anti-HLA antibody.After an appropriate time, for example, 16 hours, the cells can be evaluated using antibodies against, for example, CD3, CD4, CD8, CCR7, CD95, IL-2, IL-17, IFN-γ, and / or TNF-α.Cytokine-secreting CD4+ and / or CD8+ cells can be evaluated, for example, using flow cytometry.

[0146] In some embodiments, antibodies obtained from subjects can be evaluated by detecting antigen binding, for example, using ELISA. In some embodiments, neutralizing or enhancing antibodies are tested using RVP (reporter virus particle) assay. In certain embodiments, the vaccine induces a strong neutralizing antibody response and a low or non-existent enhancing response. In some embodiments, for example, when vaccination strategies are tested in model animals, antigen stimulation assays using pathogens can be used, as described in the Examples.

[0147] To evaluate the efficacy of the vaccines described herein, any of the parameters or effects described in the Examples or elsewhere herein (e.g., neutralizing antibody production, specific T cell response, protection against pathogens, etc.) can be used. In some embodiments, the vaccines of the present disclosure result in at least about a 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 150%, 200%, 250%, 300% or more increase in any parameter or effect described herein compared to a control value (e.g., a value observed in a subject who has not received a vaccine of the present disclosure). In some embodiments, the vaccines described herein do not substantially induce antibody-dependent enhancement of infection (ADEI) in a subject. In some embodiments, the vaccines induce a neutralizing antibody response in a subject that is substantially greater than any ADEI induced in the subject.

[0148] The present compositions and / or methods can be used to prevent and / or treat any number of diseases. In some embodiments, an infectious disease is prevented and / or treated. In some embodiments, a bacterial infectious disease is prevented and / or treated. In some embodiments, a viral infectious disease is prevented and / or treated. In some embodiments, a fungal infectious disease is prevented and / or treated. In some embodiments, a protozoan infectious disease is prevented and / or treated. In some embodiments, a helminth infectious disease is prevented and / or treated. In some embodiments, cancer is prevented and / or treated. In some embodiments, a bacterial, viral, fungal, protozoan, and / or helminth infectious disease is prevented and / or treated. In some embodiments, a coronavirus infectious disease is prevented and / or treated. In some embodiments, COVID-19 (i.e., a disease caused by SARS-CoV-2 infection) is prevented or treated.

[0149] 5. Kit In another aspect, a kit is provided herein. In some embodiments, the kit comprises a vaccine of the present disclosure (e.g., a vaccine comprising one or more of the present immunogenic conjugates or antigens, or comprising a vector comprising a polynucleotide encoding one or more antigens or immunogenic conjugates of the present disclosure, and optionally a pharmaceutically acceptable carrier). In some embodiments, the kit comprises an adjuvant. In some embodiments, the kit is for inducing an immune response to an antigen, such as a coronavirus spike, E, M, or N protein, a fragment thereof, or a combination thereof. In other embodiments, the kit is for preventing or treating a disease, such as COVID-19. In some embodiments, the kit is for inducing a B cell (i.e., antibody) response to one or more antigens. In some embodiments, the kit is for inducing a T cell response to one or more antigens. In some embodiments, the kit is for inducing a B cell response to one antigen (e.g., an antigen present in an immunogenic conjugate described herein) and a T cell response to a second antigen (e.g., a second antigen described herein).

[0150] The kits of the present disclosure can be packaged in a manner that allows for safe or convenient storage or use (e.g., in a box or other container with a lid). Typically, the kit includes one or more containers, each container storing a specific kit component, such as a reagent, control sample, etc. The choice of container depends on the specific form of its contents, e.g., the kit components in liquid form, powder form, etc. Furthermore, the containers can be made of materials designed to maximize the shelf life of the kit components. As a non-limiting example, light-sensitive kit components can be stored in opaque containers.

[0151] In some embodiments, the kit contains one or more elements, e.g., a syringe, useful for administering the composition (i.e., a pharmaceutical composition described herein) to a subject. In still other embodiments, the kit further includes instructions (e.g., instructions for using the kit to enhance an immune response in a subject to an antigen from a pathogen such as SARS-CoV-2), e.g., including directions (i.e., protocols) for practicing the method. The instructional materials typically include, but are not limited to, written or printed materials. Any medium capable of storing such instructions and communicating such instructions to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD-ROMs), and the like. Such media may include an address to an internet site providing such instructional materials. [Example]

[0152] 6. Working Example The present disclosure will be described in more detail with specific examples.The following examples are provided for illustrative purposes only and are not intended to limit the present disclosure in any way.Those skilled in the art will easily recognize various non-critical parameters that can be changed or modified to produce essentially the same results.

[0153] Example 1. Conjugate polypeptide vaccine comprising a SARS-CoV-2 S1 domain linked to an anti-CD3 single-chain variable fragment This example provides a conjugated polypeptide that can bind to both B cells bearing surface receptors reactive to the SARS-CoV-2 spike protein "S1" domain and T cells bearing the abundant T cell surface protein CD3. When included in an anti-SARS-CoV-2 vaccine regimen, such as those described below, this conjugated polypeptide elicits an antibody response against the S1 domain that can contribute to protection against COVID-19.

[0154] SARS-CoV-2 enters cells through the activity of the spike protein (S), which has receptor-binding (S1) and membrane-fusion (S2) domains. The SARS-CoV-2 spike exhibits many characteristics of conventional class I fusion proteins, including the presence of a characteristic heptad repeat within the fusion domain. Antibodies against the S1 domain can block infection by SARS-CoV-2 by blocking the interaction of the spike protein with its receptor, the ACE2 protein. CD3 is a multimeric protein complex composed of four polypeptide chains (epsilon, gamma, delta, and zeta) that associate with the T cell receptor (TCR) and play a key role in transmitting activation signals from the TCR to the interior of the T cell.

[0155] By fusing the coding sequence for the following protein elements: a tissue plasminogen activator signal sequence (to enable secretion of the conjugated polypeptide from cells), an anti-CD3 scFv derived from a murine anti-CD3 antibody, SP34 (see, e.g., U.S. Patent Application Publication No. 2016 / 0068605 ), a flexible linker, and SARS-CoV-2 S1 (codon-optimized), we generated a conjugated polypeptide (see, e.g., FIG. 5 ) comprising the S1 region of the SARS-CoV-2 spike and a CD3-binding polypeptide. The resulting amino acid sequence is shown as SEQ ID NO. 1. The codon-optimized nucleic acid sequence for this polypeptide (SEQ ID NO. 2) was synthesized and cloned into a pUC19 plasmid downstream of the EF1-α promoter sequence (including its first intron) and upstream of the SV40 polyadenylation sequence.

[0156] To test the CD3-binding function of this conjugated polypeptide, the resulting plasmid was transfected into 293 cells by calcium phosphate precipitation. Supernatant from the transfected cells was applied to tissue culture plates to allow secreted proteins to bind to the plate surface, resulting in an immobilized array of such secreted proteins, which would transmit an activation signal to T cells when bound to the TCR. We applied T cells from rhesus monkeys to the coated or control wells. Staining with fluorescent antibodies and data collection using a flow cytometer revealed that the immobilized conjugated polypeptide activated T cells, resulting in both upregulation of CD69 on the cell surface and intracellular interferon-gamma production (Figure 1). This result confirmed the binding of the conjugated polypeptide to CD3 molecules on the surface of T cells.

[0157] Example 2. Provision of SARS-CoV-2 vaccine candidates This example provides a SARS-CoV-2 vaccine candidate that (i) simultaneously elicits T cell and antibody responses to minimize the risk of antibody-dependent enhancement; (ii) is proven effective in macaques and is ready for future Phase I trials in humans. This vaccine platform combines rapid development (DNA administration) with broad and robust T cell responses and neutralizing antibody responses against the spike S1 domain (CMV vector).

[0158] Traditional vaccine development against previously unknown pathogens takes years, but protecting human health may require a more rapid response, on the scale of months. (1) However, rapid development is hindered by the lack of suitable animal models for newly emerging pathogens, the risk of antibody-dependent enhancement (ADEI), which can occur whenever a suboptimal antibody response is induced, and the difficulty of developing new manufacturing processes for subunit, attenuated, or vectored vaccines.

[0159] The self-initiating CMV DNA platform (SLCMV) mitigates or eliminates these challenging issues, enabling the rapid development of novel vaccines that induce exceptionally broad and robust immune responses localized to mucosal surfaces threatened by SARS-CoV-2. The vaccine is administered as a cytomegalovirus genome that "primes" vectored vaccine replication in vivo, resulting in a robust immune response with the unique characteristics associated with CMV infection. SLCMV features include: (i) it is based on the Towne HCMV strain, which has been proven safe for use in humans of all ages; (ii) immune responses in CMV-seropositive and -seronegative individuals due to CMV's ability to superinfect and unique deletions in the CMV genome; (iii) exceptional breadth and intensity of T cell responses that "paint" the vaccine antigen; (iv) a proven ability to elicit a balanced response including antibodies, CD4+ T cells, and CD8+ T cells, without reliance on or dominance of any single effector response; (v) localization of responding T cells to mucosal surfaces with an effector memory phenotype; (vi) engineering of new vaccine candidates in weeks to months; and (vii) a single manufacturing process based on plasmid production in E. coli.

[0160] Most coronavirus vaccines under development are designed to elicit antibodies. Such vaccine strategies must be undertaken with caution due to the potential for antibody-dependent enhancement of infection (ADEI), especially when antibody levels are low (2). High concentrations of antisera against SARS-CoV-1 have been shown to neutralize the virus, whereas diluted antibodies elicited ADEI in human promonocytic cell cultures. On the other hand, T cell responses often target highly conserved internal proteins and are long-lasting. Indeed, airway memory CD4+ T cells have been shown to mediate protective immunity against SARS-CoV-1 and MERS-CoV. We hypothesize that a self-initiated CMV DNA vaccine against SARS-CoV-2 could provide broad and protective adaptive immunity localized to mucosal surfaces.

[0161] Part 1: Characterize immune responses in rhesus macaques to a self-initiated CMV / SARS-CoV-2 vaccine designed to elicit T cell or antibody responses. Transgenes in CMV-vectored vaccines driven by constitutive promoters elicit robust T cell responses but little or no concomitant antibody responses, whereas transgenes expressed from strong late promoters such as pp65b elicit robust antibody and weak T cell responses. Using these strategies, we generated candidate SLCMV DNA vaccines designed to elicit dominant T cell responses against the SARS-CoV-2 E, M, and N proteins and potent neutralizing antibody responses against the spike S1 domain. The latter candidate expresses the spike S1 domain either unmodified or linked to an anti-CD3 scFv, which physically links anti-S1 antibody-producing B cells to T cells capable of providing support. Adaptive immune responses are tracked over time both systemically and at mucosal surfaces. Neutralizing antibody production versus stimulatory antibody production is monitored using a reporter viral particle (RVP) assay.

[0162] Part 2: Evaluate the protective efficacy of the SLCMV / SARS-CoV-2 vaccine against the Davis isolate of SARS-CoV-2 in rhesus macaques. We will propagate and characterize SARS-CoV-2 isolated from patients and evaluate viral pathogenesis in monkeys. We will test the protective efficacy of the T-cell and B-cell vaccines developed in Part 1 by priming vaccinated animals with SARS-CoV-2. We will test the T- and B-cell vaccines separately and together to examine the relative contribution of each arm of the adaptive immune system to protection versus disease enhancement.

[0163] Part 3: Testing safety and potential efficacy of a self-initiated human CMV vectored vaccine in macaques. A human CMV (HCMV)-vectored vaccine has recently been shown to induce strong effector-memory T cell responses in macaques. Therefore, we will generate an HCMV-vectored vaccine as a candidate for future human clinical trials, develop a GMP process for production, and test an SLHCMV regimen for efficacy in macaques. The HCMV-vectored vaccine used for this in vivo experiment will be selected based on the results of Part 2 of the protection study using SLHCMV.

[0164] significance Rapid vaccine development for emerging infectious disease threats remains an unmet need: traditional vaccine development against previously unknown pathogens is delayed by fundamental biological problems that are not easily resolved. Most importantly, while the induction of high-titer neutralizing antibodies (nAbs) seems an obvious approach, it is unclear what titer of nAbs is actually protective, and how this threshold varies across age extremes and comorbidities. For any emerging pathogen, it is unclear whether inadequate antibody responses, particularly likely in older and younger individuals, could result in antibody-dependent enhancement of infection (ADEI).

[0165] The immune correlates of successful vaccination against SARS-CoV-2 are poorly understood: most coronavirus vaccines currently in development target the most variable portion of the spike glycoprotein and induce antibody responses only against the virus present in the vaccine. SARS-CoV-1 escape mutants have emerged both in vitro and in mice in the presence of a single anti-receptor binding domain (RBD) nAb or a combination of two nAbs (2,3). Vaccines that exclusively elicit antibodies must be approached with caution due to the potential for ADEI, especially when antibody levels are low (4). High concentrations of antisera against SARS-CoV-1 have been shown to neutralize virus infectivity, whereas diluted antibodies caused ADEI in human promonocyte cultures, resulting in cytopathic effects and increased levels of TNF-α, IL-4, and IL-6 (5-7). Vaccine candidates based on the full-length SARS-CoV-1 spike have been demonstrated to induce non-neutralizing antibodies, and immunized animals were not protected. Instead, immunized animals experienced adverse effects such as enhanced hepatitis, increased morbidity, and a stronger inflammatory response (8,9).

[0166] T cell responses elicited by CoV vaccines also play an important role in protection and elimination. Elimination of MERS-CoV infection was not possible in T cell-deficient mice, but was achieved in mice lacking B cells (10). Furthermore, airway memory CD4+ T cells have been shown to mediate protective immunity against SARS-CoV-1 and MERS-CoV (11). Most vaccine types do not induce significant numbers of memory CD4+ T cells, but as shown below, CMV-vectored vaccines do. T cells responding to CMV-vectored vaccines localize to the airways, among other effector sites, and are recovered by bronchoalveolar lavage (12). CMV-responsive T cells recapitulate other essential characteristics of cells shown to be protective against SARS-CoV-1, including CXCR3 expression, IFN-γ production, and IL-10 production (13).

[0167] CMV-vectored vaccines can elicit robust antibody responses: While CMV vaccines elicit weak antibody responses to some transgenes driven by heterologous promoters, CMV infection and vaccination elicit robust antibody responses to proteins expressed under the control of the endogenous pp65b promoter. This promoter is one of the most active promoters in the late stages of CMV infection after DNA replication. For example, four of four rhesus macaques vaccinated with a CMV vaccine carrying the Ebola virus glycoprotein (GP) under the control of the pp65b promoter produced GP-specific antibodies, and GP-specific antibody production was found to be enhanced after a second vaccination (21). The high levels of GP antibodies induced by RhCMV / EBOV-GP and the ability of GP antibodies to undergo IgG class switching indicate the presence of sufficient CD4+ T helper function. Three of these four macaques with the highest anti-GP titers were protected against lethal EBOV challenge.

[0168] A key feature of CMV vectors for use against emerging pathogens is their ability to be re-administered to previously exposed individuals, allowing for the repeated use of CMV-vectored vaccines to protect against a range of emerging threats over time.

[0169] Impracticality of conventional CMV vaccines for use against newly emerging pathogens: Despite the immunological advantages of CMV-vectored vaccines, when these vaccines are delivered as live virus, practical obstacles hinder the rapid development of CMV vaccines for human clinical use. The most significant problem is the extreme difficulty of producing a uniform test article on a large scale from a slow-growing, mutable betaherpesvirus (29).

[0170] technological innovation Vaccination with CMV vectors in nucleic acid form: Although transfection with CMV genomic DNA is the basis of many in vitro techniques, and other researchers have attempted to deliver herpesvirus genomes as plasmids in Salmonella organisms (26), to our knowledge, delivery of naked or chemically complexed CMV genomic DNA as a vaccine has not been attempted. We demonstrate below that administration of CMV BAC DNA results in gene expression, genome replication, viremia, and an immune response.

[0171] Placing a BAC replication origin at the CMV genome end to enable BAC excision by the CMV terminase complex: To utilize CMV vectors in their DNA form for vaccination, the current CMV BAC construct must be modified so that the BAC backbone can be excised in vivo without recombinase or nuclease expression. CMV has relatively strict packaging constraints due to the need to package a unit-length genome into an icosahedral capsid. Current CMV BACs utilize an endogenous recombinase gene located within the BAC portion of the DNA construct. Upon transfection into mammalian cells, the recombinase is expressed and excises the BAC replication machinery from the replicating genome. To generate a BAC DNA vector suitable for in vivo delivery in humans, we reorganized the CMV genome ends to enable BAC excision without the need for recombinase expression. Our reconstructed BAC construct utilizes the viral terminase complex to remove the bacterial replication origin during the packaging step of CMV replication (Figure 2). We relocated the BAC origin and replication machinery from their current location (RhCMV US1 / 2) to between the terminase complex recognition locus (TCRL), which contains the viral direct repeat sequence. We show below that this configuration allows for efficient replication and packaging of the vaccine genome after introduction into host cells in vivo.

[0172] New and improved RhCMV-SIV vaccine: First-generation RhCMV-SIV vectors carry an intact endogenous viral IL-10 gene, which suppresses the host immune response (27-31). We have generated a second-generation RhCMV vector platform, viral IL-10-deficient RhCMV or RhCMVdIL10, that has unique immunological characteristics and can protect wtRhCMV-negative infant macaques, which are not protected by the first-generation vaccine (27).

[0173] Example 3. CMV-vectored vaccines induce exceptionally broad and potent T cell responses in both the CD4 and CD8 compartments. T effector-memory (TEM) cells are the predominant type of T cell at mucosal effector sites (14). CMV infection is associated with lifelong, high-frequency CD4+ and CD8+ T cell responses in the effector-memory compartment that protect against CMV pathology but do not eliminate CMV infection or prevent CMV superinfection (15-19). TEM cells elicited by CMV-vectored vaccines further recognize diverse and rare epitopes, including predominant responses to epitopes restricted by class E and class II major histocompatibility complex (MHC) molecules (20). T cells responding to CMV vaccines recognize more than three times as many peptide epitopes as T cells responding to other vaccine types, resulting in a response that "paints" vaccine antigens and should prevent pathogen escape (20). While most published studies have focused on CD8+ T cell responses, CMV vaccines stimulated equally strong CD4+ T cell responses, a feature not seen with other vectored vaccines (Figure 3A). Importantly, the unprecedented abundance of responding CD4+ T cells in the airways of vaccine recipients (Figure 3B) fits the requirements described for protection against SARS-CoV-1 (11).

[0174] Example 4. DNA-based self-initiated CMV-vectored vaccines can be rapidly designed and manufactured for broad protection against emerging threats. To enable manipulation using prokaryotic genetics, the RhCMV-SIV vaccine is maintained as a circular bacterial artificial chromosome (BAC) containing vector genomic DNA embedded with a BAC replication origin (ori) adjacent to recognition signals for site-specific recombinases or restriction enzymes. Excision of the BAC origin by site-specific recombination in vitro or after transfection is required for efficient replication and packaging. We reengineered these vectors to contain a BAC origin outside the viral genome adjacent to a CMV terminase complex recognition site, allowing automatic excision by CMV terminase as the vector begins replication (Figure 2). Thus, this new vector does not require either in vitro digestion or Cre recombinase expression and is delivered to vaccine recipients as a stable, circular DNA molecule. Upon reaching the recipient's cell nucleus, these CMV vaccine genomes enter the viral replication cycle, producing a cascade of virions that elicit a protective immune response identical to that elicited by CMV-vectored vaccines delivered as virions. Indeed, the resulting vaccine response occurs more quickly and is often stronger than that elicited by virions, most likely due to the administration of 10,000-fold more vaccine genomes (Figure 6).

[0175] Example 5: Characterizing immune responses in rhesus macaques to self-initiated CMV / SARS-CoV-2 vaccines designed to elicit T cell or antibody responses. We hypothesize that a self-primed RhCMV vaccine will generate T cell responses as broad as those administered in virion form, and that linking the SARS-CoV-2 S1 immunogen to an anti-CD3 scFv fragment will increase the rate and potency of the NAb response.

[0176] The rationale for this hypothesis is that we have demonstrated that SLRhCMV vaccines initiate replicating virus that can be detected in the blood for several weeks after administration, and we expect these forms to elicit T cell responses (in group B) as broad as those elicited by conventionally encapsidated CMV (approximately 90 peptides per 1000 compared to 12 peptides for adenoviral vectors).

[0177] For SLCMV vaccines designed to elicit B cell responses (groups C–D), we compare the secreted spike S1 domain with the same protein linked to a CD3-binding scFv fragment (Figure 7). The result is a molecule that can "bridge" S1-specific B cells with any nearby T cells. Our preliminary data show that idiotypes linked to anti-CD3 in bispecific antibodies rapidly elicit highly potent anti-idiotypic antibodies. Therefore, we expect that delivery of the spike S1 domain linked to an anti-CD3 scFv will elicit stronger antibodies than S1 alone.

[0178] We will test immune responses to three candidate vaccine components, all administered 2, 8, and 4 weeks before challenge (Figure 7). The T cell component contains a mixture of SLRhCMV / N and SLRhCMV / EM vaccines. We will evaluate the strength and breadth of T cell responses to these vaccines (Group B) to provide data for subsequent correlations of protection. There are two candidate B cell components (Groups C and D), one of which will be selected for efficacy testing in Example 6 due to the induction of stronger neutralizing antibody responses and / or reduced potentiation. Finally, we will test a regimen consisting of a T cell vaccine (SLRhCMV / N + EM and a selected B cell vaccine, administered together).

[0179] Vaccine construction SLRhCMV / N, SLRhCMV / EM, SLRhCMV / S1, and SLRhCMV / S1-anti-CD3 were generated as self-primed BAC constructs bounded by CMV terminase recognition sites, as shown in Figure 2. All coding sequences are codon-optimized versions of those found in SARS-CoV-2. The N and EM fusion protein cassettes are expressed under the control of the EF-1α promoter, including its first intron, and are recombined into the Rh213 / 214 region of the RhCMV genome, a location we have repeatedly used and observed dominant T cell responses in. The S1 and S1-anti-CD3 scFv cassettes are expressed under the control of the endogenous late RhCMV pp65b promoter and preceded by a 23-amino acid tPA leader sequence to promote efficient secretion. The S1-anti-CD3 fusion uses a humanized scFv region derived from the anti-CD3 clone SP34, a clone we previously used to construct bispecific antibodies.

[0180] Endotoxin-free BAC (vaccine) DNA is purified from E. coli cultures using alkaline lysis with Triton X-114 followed by two sequential isopycnic centrifugations. Endotoxin concentrations are measured using the Limulus amebocyte lysate (LAL) assay to ensure they are below the pyrogenic threshold (5 endotoxin units / kg body weight) (32).

[0181] Vaccine administration Our preliminary data (Figure 6) indicate that subcutaneous vaccination with 100 μg of RhCMVdIL10 vaccine BAC DNA is sufficient to induce an immune response. BAC DNA is formulated using in vivo-jetPEI (Polyplus) according to the manufacturer's instructions. Briefly, 100 μg of DNA (50 μg each of RhCMVdIL10-SIVgag and -SIVenv) and 16 μL of in vivo-jetPEI are separately diluted in 5% glucose solution (1 ml), then mixed and incubated for 15 minutes.

[0182] RhCMV vector replication and egress Vaccine-derived RhCMV DNA will be measured weekly in blood and saliva samples by qPCR using our published protocols (16, 33). These measurements provide an assessment of replication and spread of the viral vector encoding the BAC DNA. Spread to various tissues will be assessed by qPCR at autopsy after SARS-CoV-2 challenge in Example 6.

[0183] Immunophenotyping The most interesting immunophenotypes are the T cell memory-effector subset, which contains the largest proportion of cells responding to RhCMV and RhCMV / SIV vaccines; NK cells with adaptive characteristics (i.e., FcεRIγ-low "memory" NK); T cells with innate characteristics (NKG2A+); and antigen-presenting cells, particularly monocytes, expressing CD80 / 83 / 86. All of these cell populations are altered following infection with wild-type or vaccine-strain RhCMV. All are assessed using a set of three flow cytometry panels used in our previously published study to examine antigen-presenting cells, T cells, and NK cells (34).

[0184] Antigen-specific T cell response Assay wells containing up to 1M PBMC or LNMC cells are stimulated with vehicle (negative control for DMSO toxicity), duplicate RBD, S1, E, M, and / or N peptides (Intavis), or PMA / ionomycin (positive control). One hour before stimulation begins, inhibitors such as VL9 peptide or anti-HLA antibodies are applied and added again along with the peptide stimulation. After 16 hours, cells are stained using a fixable viability stain and antibodies reactive against CD3, CD4, CD8, CCR7, CD95, IL-2, IL-17, IFN-γ, and TNF-α. The percentage of cytokine-secreting CD4+ and CD8+ T cells is determined by cytometry, e.g., on a BD Fortessa or FACSymphony.

[0185] antibody response Induction of antibodies binding to the spike S1 domain will be measured by ELISA on weekly plasma samples according to our published protocol (16). Neutralizing or enhancing antibodies will be tested by RVP assay.

[0186] Combined T-cell and B-cell vaccine (Group E) The binding and neutralizing antibody responses in groups C-D will be fully characterized, and groups with better neutralizing titers and lower or no potentiation will be selected for SARS-CoV-2 challenge (see Example 6). Furthermore, after selecting the best candidates for Nab induction, a T / B combined vaccine group (group E) will be formed. This group will receive a combination vaccination with SLRhCMV / N, SLRhCMV / EM, and the selected B cell vaccine.

[0187] Data interpretation We hypothesize that our SLRhCMV vaccine delivered as DNA will elicit robust T- and B-cell responses, and that the latter will be stronger with antigen linked to anti-CD3 scFv. To determine whether these responding cells are found in the lung, the most relevant tissue, we will perform bronchoalveolar lavage and assay T cells that can be recovered from the airways.

[0188] Based on previously observed ADEIs for other RNA viruses causing respiratory infections, including SARS-CoV-1, it is possible that some animals may exhibit enhanced RVP assays after vaccination. Such results will be important for understanding the immunopathogenesis of COVID-19 and for interpreting the results of the antigen challenge experiments described below.

[0189] statistical analysis The nonparametric Kruskal-Wallis test is used to test for between-group differences in summary measures or outcomes at a single time point. To test for associations between longitudinal outcomes, such as T cell and antibody responses, a generalized linear mixed model (GLMM) is used as the analytical framework, with random effects correcting for within-animal correlations caused by continuous measurements.

[0190] Example 6. Evaluating the protective efficacy of an SLCMV / SARS-CoV-2 vaccine against the Davis isolate of SARS-CoV-2 in rhesus macaques. We hypothesize that strong T cell responses localized in the airways protect against SARS-CoV-2 and further protect against antibody-dependent enhancement. The rationale for this hypothesis is that T cell responses are the body's most important defense against intracellular parasites and, if present at sufficiently high frequencies in viral target tissues, are expected to protect against SARS-CoV-2. Indeed, airway memory CD4+ T cells have previously been shown to mediate protective immunity against both SARS-CoV-1 and MERS-CoV (see, e.g., Zhao, Immunity 44:1379). Furthermore, ADEI is thought to occur through enhanced viral uptake into cells, thereby enabling their utilization and productive replication, and viruses internalized by ADEI should be susceptible to clearance by T cells.

[0191] We developed a uniform challenge and monitoring protocol to ensure consistent data sets for virological, immunological, and pathological studies across the entire study. Vaccinated macaques (groups B–E in Figure 7) were challenged at least 8 weeks after the initial vaccination and subsequently underwent repeated clinical evaluations, radiological examinations, collection of respiratory and mucosal secretions (e.g., by bronchoalveolar lavage, tracheal wash, or nasal lavage), saliva, urine, and stool samples, blood sampling, and tissue collection (see, e.g., www.biorxiv.org / content / 10.1101 / 2020.07.07.191007v1).

[0192] Virus for infection We used a virus stock for animal inoculation, designated 2019-nCOV / USA-CA9 / 2020, generated by growing a SARS CoV-2 isolate from a UC Davis patient. If the virus grows poorly, we instead use SARS CoV-2 isolate USA-WA1 / 2020 (BEI Resources). To infect animals, we instilled a total of approximately 6 x 10 TCID into the conjunctiva, nares, and trachea of ​​anesthetized monkeys in 5 ml of 0.9% sterile saline, mimicking the relevant transmission route of COVID-19.

[0193] Sampling and assay Body temperature, weight, and activity are monitored throughout the study. CBC and serum chemistry are obtained for all blood samples to monitor host response and organ function. The sampling schedule is designed to comprehensively characterize viral shedding, cytokine responses, and adaptive immunity to understand how changes in these parameters reflect pulmonary pathology. This sampling schedule and procedure has been successfully used by us to characterize influenza A infection in macaques. Intensive sampling during the first week allows us to study acute virology and host responses. Because ACE2 is expressed in the gastrointestinal and urogenital tracts of rhesus macaques and humans, in addition to respiratory secretions, we evaluate viral shedding in saliva, urine, and feces. At necropsy (d28), we collect all relevant tissues, including salivary glands, lungs, lymph nodes, kidneys, and intestines, to assess viral localization and immune responses by PCR, molecular histology (IHC, ISH), and cytometry. Tissues are evaluated for gross pathology, histopathology, and tissue vRNA levels. Autopsies are performed by board-certified pathologists.

[0194] Viral RNA will be recovered from respiratory tract samples using Thermo's MagMAX Viral / Pathogen Nucleic Acid Isolation Kit (as recommended by the CDC for COVID-19) and quantified by amplification of a segment of the SARS-CoV-2 nucleoprotein (N) gene. The specific RT-PCR assay used in these studies is under evaluation. The CNPRC team is comparing the sensitivity, specificity, and reproducibility of validated RT-PCR assays from the CDC, UCD Health Clinical labs, Wisconsin NPRC, and private vendors. We will select the most consistent assay. Immunological analysis will be performed as described above in Example 5.

[0195] Data interpretation If our hypothesis is correct, group B animals will be protected against SARS-CoV-2 vaccination despite mounting an immune response composed almost exclusively of T cells. Group C or D animals (as selected in Example 5) may also be protected, but we caution against the possibility of ADEI, which would be indicated by increased viral load, shedding, or pathological findings in animals with moderate antibody titers, whether or not shown to enhance in RVP assays.

[0196] statistical analysis Summary findings or findings assessed at individual time points, e.g., at necropsy, will be evaluated using nonparametric tests, adjusting p-values ​​according to Benjamini and Hochberg. Longitudinal results will be evaluated using linear mixed models (with generalizations if necessary), with random effects to accommodate within-animal dependence caused by continuous measurements.

[0197] Example 7. Testing safety and potential efficacy of a self-initiated human CMV vectored vaccine in macaques. We hypothesize that a SLHCMV vaccine can be produced at a 10-20 gram scale in a GMP process, can be safely administered to macaques, and can protect against challenge with SARS-CoV-2. The rationale for this hypothesis is that, surprisingly, it has recently been shown that attenuated HCMV vectors can induce and maintain T effector-memory responses to inserted antigens in rhesus macaques (see, e.g., Caposio Sci Rep 9:19236). Therefore, the rhesus macaque model may provide a setting for testing the efficacy of candidate SLHCMV vaccines manufactured under GMP conditions against SARS-CoV-2. If the HCMV-vectored vaccine can prove safe and effective in macaques, it would be a candidate for subsequent clinical trials.

[0198] We will engineer SLHCMV versions of all four vaccines administered to macaques in groups B through D in Example 5. Furthermore, after challenge of groups B through E, GMP production of the SLHCMV vaccines that will form part of the optimal regimen will be performed. For example, if animals in group B are best protected against pathology, production of SLHCMV / N and SLHCMV / EM will be performed. The resulting GMP product will be sent to UC Davis for vaccination of macaques and efficacy testing against SARS-CoV-2.

[0199] SLHCMV vaccine genome The engineered HCMV vaccine genome is based on the Towne vaccine strain due to its excellent safety record. The vaccine is orthologous to the RhCMV genome engineered in Davis. The viral interleukin-10 gene is absent in both cases; sequences are inserted in the intergenic region near US28 to elicit T cell responses; instead, it is placed under the control of the pp65b promoter to elicit antibody responses. The BAC carries a codon-optimized SARS-CoV-2 sequence driven by the same promoter.

[0200] The plasmid backbone sequence used allows for maintenance of the plasmid at approximately 1 copy (in DH10B cells using oriS for plasmid maintenance) or approximately 15-30 copies (after induction of TrfA expression and interaction of TrfA with an alternative oriV origin). BAC DNA is purified on a laboratory scale by double sequential CsCl equilibrium gradient centrifugation followed by dialysis into PBS. The integrity of the plasmid preparation is confirmed by restriction enzyme footprinting, PCR amplification of the expression cassette, and deep sequencing after tagmentation.

[0201] GMP production will be performed by a CMO partner. Upstream process development will focus on optimizing transformation and culture conditions to ensure maximum homogeneity of the test article. Downstream process development (purification after cell growth) will focus on adapting the CsCl-dependent process used in the laboratory to iodixanol.

[0202] Testing the optimal SLHCMV vaccine regimen in macaques (group F) The SLHCMV vaccine will be given in the same combination and at the same dose as used in one of groups B to E. Immunoassays, SARS-CoV-2 challenge and pathological assessment will be performed in the same manner.

[0203] Data interpretation Our hypothesis predicts that an SLHCMV vaccine can be manufactured in a GMP process at a scale and purity sufficient to allow eventual testing in a Phase I human trial. Furthermore, we believe that an SLHCMV-vectored vaccine could demonstrate efficacy in a rhesus macaque model. While such a result is perhaps counterintuitive, it is possible based on publications in the literature demonstrating that HCMV can complete its life cycle in rhesus fibroblasts (43) and induce strong TEM responses in macaques.

[0204] The immune response to SLHCMV vaccines in macaques is also of interest because it is likely to reflect the response achieved with minimal spread of the vaccine, as seen with the use of fully inactivated HCMV-vectored vaccines in humans. Despite evidence of some genome replication, HCMV (Towne)-based vaccines should allow for minimal or no systemic spread in macaques. Therefore, contrasting immune responses to RhCMV and Towne-vectored antigens should clarify which aspects of the immune response depend solely on immunomodulation and which depend on vector spread.

[0205] statistical analysis Statistical analysis focused on the characteristics of the vaccine preparation. Our previous experience suggests that single-copy BACs have mutation rates similar to those of other plasmids, i.e., below the level of detection by amplicon sequencing (approximately 0.1%) imposed by the polymerase error rate (46-50). SLHCMV genome replication in macaques was determined by either (i) vaccine virus sequences in plasma 1 week after prime or boost, (ii) gB expression in biopsy tissue from the injection site (HCMV gB-specific Abs; gB, as a late gene, is expressed only after genome replication), or (iii) anamnestic antibody responses to HCMV gB, as defined by a doubling of titers 1 month after boost.

[0206] Example 8. Conjugate polypeptide vaccine comprising a SARS-CoV-2 RBD domain linked to an anti-CD3 single-chain variable fragment The inventors designed a conjugate polypeptide vaccine, designated s3-RBD (SEQ ID NO. 6), which is a fusion protein between a humanized anti-CD3 single-chain variable fragment (scFv) derived from the SP34 clone and the SARS-CoV-2 receptor-binding domain (RBD), both preceded by a tissue plasminogen activator (tPA) signal sequence to enable secretion. The RBD is a segment of the SARS-CoV-2 spike protein involved in binding of the spike protein to the human receptor ACE2 and is frequently targeted by neutralizing antibodies. The s3-RBD has the ability to bind both RBD-responsive B cells (via their cell surface, RBD-specific antigen receptor) and helper T cells (via CD3). While not being bound by the following theory regarding a particular mechanism of action, the inventors hypothesize that engagement of pairs or clusters of these cognate receptors on B and T cells should result in activation of both cell types, subsequently promoting continued B cell development. B cells that receive T cell help are more likely to undergo somatic hypermutation and ultimately develop into producers of high-affinity RBD-specific antibodies.

[0207] To immunize rhesus macaques with a genetic vaccine expressing the s3-RBD, we prepared a codon-optimized open reading frame (OPF) having the nucleotide sequence shown in SEQ ID NO. 7. This sequence was synthesized and then placed into an expression cassette downstream of the human EF-1-α promoter sequence and upstream of the SV40-derived polyadenylation signal (resulting in the complete expression cassette sequence shown in SEQ ID NO. 8) using techniques known to those skilled in the art. The plasmid containing the expression cassette was prepared endotoxin-free at a medium scale for administration as a DNA vaccine. The expression cassette was also cloned into the E1 region of an adenovirus type 35 shuttle plasmid, which allows for the transfer of DNA sequences into an E1-, E3-deleted adenovirus type 35 vector.

[0208] To evaluate the immune response to our s3-RBD immunogen in the context of a genetic vaccine in nonhuman primates, we immunized nine monkeys using an electroporated DNA prime (day 0) and an Ad35-vectored boost (day 28; Figure 8). The immunogens tested were the SARS-CoV-2 S1 domain, the RBD domain alone, or s3-RBD, all expressed from codon-optimized ORFs under the control of the EF-1α promoter. A tPA signal sequence was placed upstream of both the isolated RBD domain and the s3-RBD fusion protein to enable their secretion (Figure 8).

[0209] Assessment of binding antibodies by ELISA during the vaccination protocol demonstrated both the superiority of the RBD domain (compared to the entire S1 domain) for immunization and the superior performance of the s3-RBD construct over RBD alone (Figure 9). Surprisingly, the S1 immunogen performed very poorly, eliciting detectable binding and neutralizing antibodies in only one of three vaccine recipients. Note that the low binding antibody response to S1 was discernible by ELISA in two animals (Figure 9), but was insignificant compared to the much higher responses in the other groups. Neutralizing activity was detected in one of these two S1 recipient animals (Figure 9, dotted black line). Immune responses to the RBD domain alone were detectable at all post-boost time points in all three animals that received the genetic vaccine encoding the RBD domain (Figure 9, dashed gray line). However, binding antibody responses were highest in the group that received s3-RBD (Figure 9, solid black line).

[0210] Neutralizing activity was tested against pseudotyped lentiviral particles, which are lentiviral particles lacking their native envelope protein but instead carrying the SARS-CoV-2 spike protein. An example of the curves generated for one animal is shown in Figure 10. For each curve, the neutralization titer 50 (NT50) is taken as the dilution at which infection is reduced to 50%. In Figure 10, the NT50 is not detectable at the first two time points, but sera from all subsequent time points demonstrate neutralization by reducing the infectivity of the pseudotyped particles to less than 50%. All RBD vaccine recipients demonstrated neutralizing titers in the pseudovirus assay, with animal D2 having a peak titer of 1:6539 (Figure 11), which is approximately the 90th percentile for convalescent individuals (Moore and Klasse, 2020). However, as in the ELISA assay for binding antibodies, recipients of the genetic vaccine expressing s3-RBD achieved the highest neutralizing titers, exceeding all RBD recipient animals in two of three cases at most time points after week 4 (Figure 11). Indeed, the geometric mean titers in s3-RBD recipient animals exceeded those in RBD recipients by at least fourfold. The actual fold increase is higher, as one s3-RBD recipient animal produced antibodies at titers exceeding the maximum quantifiable in the assay (1:10240). The neutralizing antibody responses in s3-RBD recipients also showed impressive durability, with all animals maintaining neutralization through 32 weeks after the initial immunization (Figure 11). In contrast, in two of three RBD-alone recipients, neutralizing activity fell below the limit of detection in our assay by week 24.

[0211] In some reported cases, immune responses to membrane-bound antigens (e.g., because they have a glycosylphosphatidylinositol anchor or a transmembrane segment) are better than those to the same antigen in secreted form. Therefore, we created a conjugated polypeptide containing an anti-CD3 scFv fragment, the RBD of the B.1.351 ("South African") strain of SARS-CoV-2, and a transmembrane segment from the human PDGF receptor. The resulting amino acid sequence is shown as SEQ ID NO. 25. A codon-optimized nucleic acid sequence for this polypeptide (SEQ ID NO. 26) was synthesized and cloned into the pUC19 plasmid downstream of the EF1-α promoter sequence (including its first intron) and upstream of the SV40 polyadenylation sequence.

[0212] We next confirmed that the s3-RBD(B.1.351)-PDGFRtm conjugate polypeptide (SEQ ID NO. 25) contains the immunoreactive RBD(B.1.351) likely required for the induction of anti-RBD(B.1.351) antibody responses in human subjects when expressed in human cells. A plasmid encoding s3-RBD(B.1.351)-PDGFRtm was transfected into the human embryonic kidney cell line, 293, and two days later, the presence of immunoreactive RBD(B.1.351) protein within the cells was confirmed by antibody staining using the following procedure. Assay—On day 1, 350,000 cells were seeded per well in a 6-well plate. On day 0, a polyethyleneimine (PEI) transfection mix was prepared by adding 150 microliters of 0.1 mg / ml PEI in DMEM to an equal volume of 3 mcg DNA in DMEM. The mix was incubated at room temperature for 20 minutes. 2.7 ml of complete medium was added. The medium containing the complexed DNA was added to washed 293 cells in a 6-well plate. The plate was returned to the incubator for overnight incubation. On day 1, the transfection mix was removed from the cells, the cells were washed, and 2 ml of fresh medium was added. On day 2, the cells were fixed with 5% PFA, permeabilized by washing with a buffer containing 0.05% Triton X-100, incubated with an anti-RBD primary antibody for 2 hours, washed again, incubated with an HRP-conjugated secondary antibody for 2 hours, washed, incubated with TrueBlue HRP substrate for 5–15 minutes, and finally quenched in water. The results (Figure 13D) demonstrate that the s3-RBD(B.1.351)-PDGFRtm construct successfully produces a conjugated polypeptide containing immunoreactive RBD(B.1.351).

[0213] Example 9. Conjugate polypeptide vaccine comprising an anti-CD3 single-chain variable fragment and a SARS-CoV-2 RBD domain linked to an antibody Fc region Multivalent antigen display can promote stronger and longer-lasting antibody responses through efficient cross-linking of B cell receptors (BCRs) and improved antigen transport, endocytic uptake, and ultimately presentation (Brinkkemper Vaccines 7, 2019; Tokatlian Science 363:649, 2019). Repeated epitopes have higher effective avidity for BCRs and can cross-link receptors to effectively activate B cells (Cimica Clin Immunol 183:99, 2017; Zabel J Immunol 192:5499, 2014). A spacing of 5–10 nm of 15–20 hapten molecules is ideal for B cell activation (Vogelstein PNAS 79:395, 1982). Multimeric antigens are most commonly produced ex vivo as proteins but can also be produced in vivo via gene vaccines.

[0214] One approach to improving the immunogenicity of vaccine antigens is to present them on nanoparticles (NPs), for example, with diameters of 25–50 nm. Licensed human papillomavirus and HBsAg vaccines contain NPs, as do efforts to create influenza and respiratory syncytial virus (RSV) vaccines (Darricarre J Virol 92, 2018; Hsia Nature 535:136, 2016; Kanekiyo Nat Immunol 20:362, 2019; Marcandalli Cell 176:1420, 2019). Animal studies have shown that NP presentation significantly improves the quantity and quality of Ab responses compared with delivery of the same antigen as a soluble protein (Brinkkemper Vaccines 7, 2019). Thus, NP display of RSV antigens enhanced NAb titers by more than 10-fold (Marcandalli Cell 176:1420, 2019). NP presentation also allows for the creation of multivalent, antigenically mosaic immunogens that can improve NAb breadth by increasing the avidity of interactions, particularly with the most cross-reactive BCRs, as shown for influenza HA (Kanekiyo Nat Immunol 20:362, 2019).

[0215] Immunoglobulin "crystallizable fragments," or Fc, are dimerizable molecules; therefore, when an immunogen is expressed as a fusion protein with Fc, the result is a dimeric molecule that is likely to have greater immunogenicity for the reasons discussed above. The Fc region can also be modified to polymerize into well-defined complexes containing up to 12 fused partners (Mekhaiel Sci Rep 1:124). Furthermore, due to interactions with salvage neonatal Fc receptors and slower renal clearance relative to larger molecules, the presence of an Fc domain significantly increases the plasma half-life of the fusion protein (Roopenian & Akilesh, 2007 and Kontermann, 2011). The combined Fc domain also allows these molecules to interact with Fc receptors (FcRs) found on immune cells, a feature particularly important for the use of these molecules in tumor therapy and vaccines (Nimmerjahn & Ravetch, 2008).

[0216] To enable expression of multimeric s3-RBD and achieve other benefits conferred by the Fc domain, we therefore designed a protein molecule consisting of a conjugate polypeptide vaccine s3-RBD fused at its C-terminus to a human IgG1 Fc domain, termed "s3-RBD-Fc" (SEQ ID NO. 9). We then prepared a codon-optimized ORF capable of expressing s3-RBD-Fc (SEQ ID NO. 10).

[0217] To test the immunogenicity of s3-RBD-Fc, this ORF is engineered into an expression cassette that is introduced into appropriate DNA and adenoviral vectors that are administered to rhesus monkeys.

[0218] Example 10. Dimerized conjugate polypeptide vaccine comprising a SARS-CoV-2 RBD domain linked to an anti-CD3 single-chain variable fragment forming a diabody The s3-RBD comprises an anti-CD3 scFv fragment, which itself contains the heavy and light chain variable regions (VH and VL) derived from the monoclonal antibody SP34. To form a functional monomeric scFv fragment, these VH and VL regions are separated by a flexible linker, which is required to allow the two domains to achieve the proper three-dimensional configuration required for CD3 binding. To allow proper folding of the monomeric scFv, the linker length is typically >12 amino acids, most frequently 15 amino acids (Wang Antibodies 8:43, 2019).

[0219] A shorter linker connecting the two variable domains can determine the formation of multimeric scFv molecules because the shorter linker is insufficiently long to allow the VH and VL regions to fold into a monomer with correct spatial association. Reducing the length of the linker connecting the two variable domains to less than 8-12 residues promotes the assembly of dimeric VH-VL fragments, generating diabodies with two antigen-binding sites (Holliger et al., 1993; Kortt et al., 1994; Aflthan et al., 1995). Further reduction of the linker sequence to fewer than five amino acids has been shown to result in the formation of trimeric or tetrameric molecules (triabodies, tetrabodies) (Iliades et al., 1997; Kortt et al., 1997; Pei et al., 1997; Le Gall et al., 1999; Dolezal et al., 2000; Hudson and Kortt, 1999).

[0220] Therefore, we designed a dimerized form of s3-RBD by shortening the length of the VH-VL linker to 5 amino acids (SEQ ID NO. 11). We prepared a codon-optimized ORF encoding this dimerized form of the conjugated polypeptide immunogen (SEQ ID NO. 12). This codon-optimized ORF was subsequently engineered into an expression cassette and into DNA and adenovirus vectors that were administered to animals for immunogenicity testing.

[0221] Evaluation of reported crystal structures of diabodies revealed significant structural diversity, suggesting structural instability (Kim Sci Rep 6:34515, 2016). In diabody crystal structures, the light chain does not contribute to interactions between the Fv domains, and the two heavy chains form a relatively small interaction interface. It has been proposed that predictable orientation and distance between antigen-binding sites (e.g., CD3 binding sites) is advantageous for using diabodies as general and reliable intermediaries for artificial protein construction. Nevertheless, many diabodies with the simplest designs, i.e., those with linker sequences shortened to five amino acids, possessed interaction interfaces between the Fv domains that appeared too small to form stable structures (Moraga Cell 160:1196, 2015; Perisic Structure 2:1217, 1994). It has been shown that substitution of arginine residues in the EF loop and the introduction of one or more disulfide bridges between the Fv domains can make diabody structures more rigid and predictable.

[0222] Next, we designed a dimerized form of s3-RBD (SEQ ID NO. 13) predicted to have greater stability by shortening the VH-VL linker length to 5 amino acids, substituting the positively charged lysine residue in the EF loop, and introducing a cysteine ​​residue capable of forming a disulfide bridge. We then prepared a codon-optimized ORF (SEQ ID NO. 14) encoding this dimerized form of the conjugated polypeptide immunogen with enhanced stability. This codon-optimized ORF was subsequently engineered into expression cassettes and into DNA and adenoviral vectors administered to animals for immunogenicity testing.

[0223] Plasmid DNA molecules encoding the enhanced dimerized anti-CD3-RBD conjugate polypeptide, also referred to as eDis3-RBD, were administered as a DNA vaccine (by electroporation of 1 mg of DNA on day 0) to three rhesus macaques. Booster immunizations were performed on day 28 using a type 35 adenovector encoding the RBD alone (Figure 12). The antibody responses in these macaques were compared to those obtained using RBD alone for priming and boosting. The results demonstrate both an improved peak antibody response using eDis3-RBD (a 6.3-fold increase in geometric mean) and an increased sustained antibody response that remained 24 weeks post-vaccination (a 5-fold increase in geometric mean; Figure 12, compare the eDis3-RBD mean solid line with the RBD mean dotted line).

[0224] Example 11. Conjugate polypeptide vaccine comprising a SARS-CoV-2 RBD domain linked to an anti-CD2 single-chain variable fragment CD2 is an adhesion molecule found on the surface of T cells and natural killer (NK) cells. CD2 binds to other adhesion molecules expressed on the surface of other cells, including LFA-3 (CD58). CD2 functions as a costimulatory molecule, meaning that signals delivered through CD2 cooperate with signals delivered by TCR ligation to induce cell proliferation and cytokine production in resting T cells. The close association of CD2 with the CD3-TCR complex appears to be essential for optimal T cell responses. CD2 also binds to LFA-3, a key adhesion molecule responsible for antigen-independent cell adhesion, proliferation of naive T helper cells, and induction of IFN-γ production in memory cells. In addition to LFA-3, CD2 can interact with CD48 and CD59.

[0225] By fusing the coding sequence for the following protein elements: a tissue plasminogen activator signal sequence (to enable secretion of the conjugated polypeptide from cells), an anti-CD2 scFv derived from a rat anti-CD2 antibody, LO-CD2a (see, e.g., U.S. Patent No. 6,849,258 ), a flexible linker, and a SARS-CoV-2 RBD (codon-optimized), the inventors created a conjugated polypeptide comprising the RBD of the SARS-CoV-2 spike and a CD2-binding polypeptide. The resulting amino acid sequence is shown as SEQ ID NO. 15. The codon-optimized nucleic acid sequence for this polypeptide (SEQ ID NO. 16) was synthesized and cloned into a pUC19 plasmid downstream of the EF1-α promoter sequence (including its first intron) and upstream of the SV40 polyadenylation sequence.

[0226] Plasmid DNA molecules encoding the anti-CD2-RBD conjugate polypeptide, also referred to as s2-RBD, were administered as a DNA vaccine (by electroporation of 1 mg of DNA on day 0) to two rhesus macaques. A booster immunization was performed on day 28 using a type 35 adenovector encoding the RBD alone (Figure 12). The antibody responses in these macaques were compared to those obtained using RBD alone for priming and boosting. The results demonstrate both an improved peak antibody response using s2-RBD (a 2.8-fold increase in geometric mean) and an increased sustained antibody response that remained 24 weeks post-vaccination (a 5-fold increase in geometric mean; Figure 12, compare the gray mean dashed line for s2-RBD with the black mean dotted line for RBD alone).

[0227] Example 12. Dimerized conjugate polypeptide vaccine comprising a SARS-CoV-2 RBD domain linked to an anti-CD2 single-chain variable fragment forming a diabody A dimerized form of anti-CD2 scFv-RBD was designed by shortening the length of the VH-VL linker to 5 amino acids (SEQ ID NO. 17). The inventors prepared a codon-optimized ORF encoding this dimerized form of the conjugated polypeptide immunogen (SEQ ID NO. 18). This codon-optimized ORF was subsequently engineered into an expression cassette and into DNA and adenovirus vectors that were administered to animals for immunogenicity testing.

[0228] Next, we designed a dimerized form of anti-CD2 scFv-RBD predicted to have greater stability by shortening the VH-VL linker length to 5 amino acids and introducing a cysteine ​​residue capable of forming a disulfide bridge (SEQ ID NO. 19). The anti-CD2 scFv used in this construct does not have a positively charged residue at a critical position in the EF loop, which is predicted to cause instability. We prepared a codon-optimized ORF encoding this dimerized form of anti-CD2 scFv-RBD with increased stability (SEQ ID NO. 20). This codon-optimized ORF was subsequently engineered into an expression cassette and into DNA and adenovirus vectors administered to animals for immunogenicity testing.

[0229] Example 13. Conjugate polypeptide vaccine comprising a SARS-CoV-2 RBD domain linked to the N-terminal domain of LFA-3 that binds to CD2 Conjugated polypeptide immunogens for delivery in genetic vaccines can be designed using any protein sequence that binds to the appropriate cell surface receptor in the vaccine recipient. The 179-residue ectodomain of human CD58 consists of two extracellular immunoglobulin-like domains anchored to the membrane via either a transmembrane segment or a glycosylphosphatidylinositol (GPI) linker (Dustin et al., 1987b; Wallich et al., 1998). The 95-residue membrane-distal N-terminal domain of CD58 (1dCD58) is entirely responsible for adhesion to CD2 (Sun et al.).

[0230] By fusing the coding sequence for the following protein elements: tissue plasminogen activator signal sequence (to enable secretion of the conjugated polypeptide from cells), the first extracellular domain of human CD58 (1dCD58), a flexible linker, and the SARS-CoV-2 RBD (codon-optimized), we created a conjugated polypeptide comprising the RBD of the SARS-CoV-2 spike and a CD2-binding polypeptide. The resulting amino acid sequence is shown as SEQ ID NO. 21. The codon-optimized nucleic acid sequence for this polypeptide (SEQ ID NO. 22) was synthesized and cloned into a pUC19 plasmid downstream of the EF1-α promoter sequence (including its first intron) and upstream of the SV40 polyadenylation sequence.

[0231] The inventors also generated a conjugate polypeptide comprising the RBD of the B.1.351 ("South African") strain of SARS-CoV-2 and a CD2-binding polypeptide by fusing the coding sequence of the tissue plasminogen activator signal sequence, 1dCD58, a flexible linker, and the SARS-CoV-2 RBD (B.1.351) (i.e., the RBD from SARS-CoV-2 strain B.1.351). The resulting amino acid sequence is shown as SEQ ID NO. 23. The codon-optimized nucleic acid sequence for this polypeptide (SEQ ID NO. 24) was synthesized and cloned into a pUC19 plasmid downstream of the EF1-α promoter sequence (including its first intron) and upstream of the SV40 polyadenylation sequence.

[0232] We next confirmed that the 1dCD58-RBD(B.1.351)-conjugated polypeptide (SEQ ID NO. 23) contains the immunoreactive RBD(B.1.351) likely required for the induction of anti-RBD(B.1.351) antibody responses in human subjects when expressed in human cells. A plasmid encoding 1dCD58-RBD(B.1.351) was transfected into the human embryonic kidney cell line, 293, and two days later, the presence of immunoreactive RBD(B.1.351) protein within the cells was confirmed by antibody staining using the following procedure. Assay—On day 1, 350,000 cells were seeded per well in a 6-well plate. On day 0, a polyethyleneimine (PEI) transfection mix was prepared by adding 150 microliters of 0.1 mg / ml PEI in DMEM to an equal volume of 3 mcg DNA in DMEM. The mix was incubated at room temperature for 20 minutes. 2.7 ml of complete medium was added. The medium containing the complexed DNA was added to washed 293 cells in a 6-well plate. The plate was returned to the incubator for overnight incubation. On day 1, the transfection mix was removed from the cells, the cells were washed, and 2 ml of fresh medium was added. On day 2, the cells were fixed with 5% PFA, permeabilized by washing with a buffer containing 0.05% Triton X-100, incubated with an anti-RBD primary antibody for 2 hours, washed again, incubated with an HRP-conjugated secondary antibody for 2 hours, washed, incubated with TrueBlue HRP substrate for 5–15 minutes, and finally quenched in water. The results (Figure 13C) demonstrate that the 1dCD58-RBD(B.1.351) construct successfully produced a conjugated polypeptide containing immunoreactive RBD(B.1.351).

[0233] Example 14. Conjugate polypeptide vaccine comprising a SARS-CoV-2 RBD domain linked to an anti-CD4 single-chain variable fragment CD4 is a glycoprotein found on the surface of helper T cells as well as some monocytes, macrophages, and dendritic cells. As a member of the immunoglobulin superfamily, CD4 contains four immunoglobulin domains, known as D1-D4, exposed on the cell's surface. The D1 domain is responsible for interacting with the β2 domain of MHC class II molecules, which largely determines the biology of helper T cells responding to peptides presented on MHC class II molecules by antigen-presenting cells. CD4 is also the primary entry receptor for HIV-1 envelope glycoproteins.

[0234] By fusing the coding sequence for the following protein elements: a tissue plasminogen activator signal sequence (to enable secretion of the conjugated polypeptide from cells), an anti-CD4 scFv derived from a humanized murine anti-CD4 antibody, hu5A8 (see, e.g., AIDS Res Hum Retro 13:933), a flexible linker, and the SARS-CoV-2 RBD (codon-optimized), we generated a conjugated polypeptide comprising the RBD of the SARS-CoV-2 spike and a CD4-binding polypeptide. The resulting amino acid sequence is shown as SEQ ID NO. 27. The codon-optimized nucleic acid sequence for this polypeptide (SEQ ID NO. 28) was synthesized and cloned into a pUC19 plasmid downstream of the EF1-α promoter sequence (including its first intron) and upstream of the SV40 polyadenylation sequence.

[0235] References for Examples 1-7 TIFF2026021474000003.tif199166TIFF2026021474000004.tif207166TIFF20260214740 00005.tif207166TIFF2026021474000006.tif227166TIFF2026021474000007.tif202166

[0236] References for Examples 8-13 TIFF2026021474000008.tif205166TIFF2026021474000009.tif226166TIFF2026021474000010.tif113166

[0237] Although the foregoing disclosure has been described in some detail by way of illustration and example for clarity of understanding, those skilled in the art will understand that certain changes and modifications may be practiced within the scope of the appended claims. Furthermore, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.

[0238] Exemplary Embodiments Exemplary aspects provided in accordance with the subject matter of this disclosure include, but are not limited to, the following aspects and the claims. 1. A vaccine for inducing an immune response to a pathogen in a mammal, comprising a conjugated polypeptide comprising an antigen from the pathogen linked to a ligand or antibody fragment that specifically binds to a surface protein present on an immune cell. 2. The vaccine of embodiment 1, wherein said surface protein is an abundant T cell surface protein involved in signal transduction and / or adhesion. 3. The vaccine of embodiment 2, wherein said abundant T cell surface protein is CD2, CD3, CD4, or CD5. 4. The vaccine of embodiment 3, wherein the abundant T cell surface protein is CD2 or CD3. 5. The vaccine of any one of aspects 1 to 4, wherein the immune cell is a T cell or an antigen-presenting cell (APC). 6. The vaccine of any one of aspects 1 to 5, wherein the ligand is an ectodomain of a cell adhesion molecule. 7. The vaccine of embodiment 6, wherein the cell adhesion molecule is CD58. 8. The vaccine of any one of aspects 1-7, wherein the surface protein is preferentially expressed by T cells. 9. The vaccine of any one of aspects 1 to 8, wherein the antibody fragment is an scFv chain derived from an antibody. 10. The vaccine of any one of aspects 1-9, wherein the conjugated polypeptide further comprises a lipid anchor, a transmembrane segment, a multimerization domain, or any combination of these elements. 11. The vaccine of embodiment 10, wherein the lipid anchor is a glycosylphosphatidylinositol anchor. 12. The vaccine of embodiment 10 or 11, wherein addition of the lipid anchor is directed by a signal sequence. 13. The vaccine of aspect 12, wherein the signal sequence is derived from CD55. 14. The vaccine of any one of aspects 10-13, wherein the transmembrane segment is derived from a PDGF receptor, glycophorin A, or SARS-CoV-2 spike protein. 15. The vaccine of any one of aspects 10-14, wherein the multimerization domain is derived from T4 fibritin. 16. The vaccine of any one of aspects 10 to 14, wherein the multimerization domain is an Fc domain. 17. The vaccine of aspect 16, wherein the Fc domain is located at the C-terminus of the conjugated polypeptide. 18. The vaccine of aspect 16 or 17, wherein the Fc domain is a human IgG1 Fc domain. 19. The vaccine of any one of aspects 1-18, wherein the conjugated polypeptide is a fusion protein comprising the antigen and the ligand or the antibody fragment within a single polypeptide chain. 20. The vaccine of embodiment 19, wherein the antibody fragment is an scFv chain derived from an antibody, and the VH and VL domains of the scFv are separated by a flexible linker. 21. The vaccine of aspect 20, wherein the flexible linker is 12 amino acids or more in length, and wherein the conjugated polypeptide preferentially binds to the surface protein as a monomer. 22. The vaccine of aspect 20, wherein the flexible linker is less than 12 amino acids in length and the conjugated polypeptide preferentially binds to the surface protein as a multimer. 23. The vaccine of aspect 22, wherein the multimer is stabilized by disulfide bonds between the monomer units. 24. The vaccine of aspect 22 or 23, wherein the flexible linker is 5 amino acids in length. 25. The vaccine of any one of aspects 1-24, wherein the conjugated polypeptide further comprises a tPA leader sequence. 26. The vaccine of embodiment 25, wherein the tPA leader sequence is 23 amino acids in length. 27. The vaccine of any one of aspects 1-26, further comprising a second antigen from the pathogen. 28. The vaccine of any one of aspects 1-27, wherein the pathogen is a virus. 29. The vaccine of aspect 28, wherein the virus is SARS-CoV-2. 30. The vaccine of aspect 29, wherein the antigen present in the conjugated polypeptide comprises the SARS-CoV-2 spike glycoprotein or a fragment thereof. 31. The vaccine of aspect 30, wherein the fragment of the SARS-CoV-2 spike glycoprotein comprises the S1 domain or the receptor binding domain (RBD). 32. The vaccine of any one of aspects 27-31, wherein the second antigen comprises a SARS-CoV-2E, M, N, nsp3, nsp4, or nsp6 protein, or a fragment thereof. 33. The vaccine of aspect 32, wherein the second antigen comprises a fusion protein comprising SARS-CoV-2E and an M protein, or a fragment thereof. 34. The vaccine of any one of aspects 1 to 33, wherein the mammal is a human. 35. The vaccine of any one of aspects 1-34, wherein the vaccine is formulated for electroporation or subcutaneous injection. 36. The vaccine of any one of aspects 1 to 35, wherein the conjugated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, and SEQ ID NO:21. 37. A vaccine for inducing an immune response to a pathogen in a mammal, the vaccine comprising a polynucleotide encoding a conjugate polypeptide comprising an antigen from the pathogen fused to a ligand or antibody fragment that specifically binds to a surface protein present on an immune cell. 38. The vaccine of embodiment 37, wherein the surface protein is an abundant T cell surface protein involved in signal transduction and / or adhesion. 39. The vaccine of embodiment 38, wherein the abundant T cell surface protein is CD2, CD3, CD4, or CD5. 40. The vaccine of embodiment 39, wherein the abundant T cell surface protein is CD2 or CD3. 41. The vaccine of any one of aspects 37 to 40, wherein the immune cell is a T cell or an antigen-presenting cell (APC). 42. The vaccine of any one of aspects 37-41, wherein the ligand is an ectodomain of a cell adhesion molecule. 43. The vaccine of aspect 42, wherein the cell adhesion molecule is CD58. 44. The vaccine of any one of aspects 37-43, wherein the surface protein is preferentially expressed by T cells. 45. The vaccine of any one of aspects 37-44, wherein the antibody fragment is an scFv chain derived from an antibody. 46. ​​The vaccine of any one of aspects 33 to 45, wherein the conjugated polypeptide further comprises a lipid anchor, a transmembrane segment, a multimerization domain, or any combination of these elements. 47. The vaccine of embodiment 46, wherein the lipid anchor is a glycosylphosphatidylinositol anchor. 48. The vaccine of embodiment 46 or 47, wherein addition of the lipid anchor is directed by a signal sequence. 49. The vaccine of embodiment 48, wherein the signal sequence is derived from CD55. 50. The vaccine of any one of aspects 46-49, wherein the transmembrane segment is derived from a PDGF receptor, glycophorin A, or SARS-CoV-2 spike protein. 51. The vaccine of any one of aspects 46-50, wherein the multimerization domain is derived from T4 fibritin. 52. The vaccine of any one of aspects 46 to 50, wherein the multimerization domain is an Fc domain. 53. The vaccine of aspect 52, wherein the Fc domain is located at the C-terminus of the conjugated polypeptide. 54. The vaccine of embodiment 52 or 53, wherein the Fc domain is a human IgG1 Fc domain. 55. The vaccine of any one of aspects 45-54, wherein the VH and VL regions of the scFv are separated by a flexible linker within the conjugated polypeptide. 56. The vaccine of embodiment 55, wherein the flexible linker is 12 amino acids or more in length, and the conjugated polypeptide preferentially binds to the surface protein as a monomer. 57. The vaccine of embodiment 55, wherein the flexible linker is less than 12 amino acids in length, and wherein the conjugated polypeptide preferentially binds to the surface protein as a multimer. 58. The vaccine of embodiment 57, wherein the multimer is stabilized by disulfide bonds. 59. The vaccine of embodiment 57 or 58, wherein the flexible linker is 5 amino acids in length. 60. The vaccine of any one of aspects 37-59, wherein the conjugated polypeptide comprises a tPA leader sequence. 61. The vaccine of embodiment 60, wherein the tPA leader sequence is 23 amino acids in length. 62. The vaccine of any one of aspects 37-61, further comprising a second polynucleotide encoding a second antigen from the pathogen. 63. The vaccine of any one of aspects 37-62, wherein the pathogen is a virus. 64. The vaccine of aspect 63, wherein the virus is SARS-CoV-2. 65. The vaccine of embodiment 64, wherein the antigen present in the conjugated polypeptide comprises the SARS-CoV-2 spike glycoprotein or a fragment thereof. 66. The vaccine of embodiment 65, wherein the fragment of the SARS-CoV-2 spike glycoprotein comprises the S1 domain or the receptor binding domain (RBD). 67. The vaccine of any one of aspects 62-66, wherein the second antigen comprises a SARS-CoV-2E, M, N, nsp3, nsp4, or nsp6 protein, or a fragment thereof. 68. The vaccine of embodiment 67, wherein the second antigen comprises a fusion protein comprising SARS-CoV-2E and an M protein, or a fragment thereof. 69. The vaccine of any one of aspects 37-68, wherein the mammal is a human. 70. The vaccine of any one of aspects 37-69, which is formulated for electroporation or subcutaneous injection. 71. The vaccine of any one of aspects 37-70, wherein the polynucleotide encoding the conjugate polypeptide and / or the second polynucleotide encoding the second antigen are codon-optimized. 72. The polynucleotide encoding the conjugate polypeptide is present in a first expression cassette, the polynucleotide being operably linked to a first promoter; and / or the second polynucleotide encoding the second antigen is present in a second expression cassette, the second polynucleotide being operably linked to a second promoter; The vaccine of any one of aspects 37 to 71. 73. The vaccine of embodiment 72, wherein the second promoter is a mammalian promoter. 74. The vaccine of embodiment 73, wherein the mammalian promoter is an EF-1α promoter. 75. The vaccine of any one of aspects 37-74, wherein the first and / or second expression cassette is in a vector. 76. The vaccine of embodiment 75, wherein the vector is administered as naked DNA. 77. The vaccine of embodiment 75, wherein the vector is a viral vector. 78. The vaccine of embodiment 77, wherein the viral vector is a cytomegalovirus (CMV), adenovirus, or adeno-associated virus (AAV) vector. 79. The vaccine of any one of aspects 37-78, further comprising an in vivo transfection reagent. 80. The vaccine of aspect 79, wherein the in vivo transfection reagent is in vivo-jetPEI™. 81. The vaccine of any one of aspects 37 to 80, which is formulated for subcutaneous transfection. 82. The vector: (a) a CMV genome or a portion thereof containing the first expression cassette, or the first and second expression cassettes; (b) bacterial artificial chromosome (BAC) sequences containing origins of replication; (c) a first terminase complex recognition locus (TCRL1) containing at least two viral tandem repeat sequences; (d) a second terminase complex recognition locus (TCRL2) containing at least two viral direct repeat sequences; a circular CMV vector comprising the CMV genome or portion thereof is flanked by TCRL1 and TCRL2, thereby defining a first region of the circular vector extending from TCRL1 to TCRL2 and comprising the CMV genome or portion thereof; the BAC sequence is located in a second region of the circular vector extending from TCRL1 to TCRL2 and not including the CMV genome or a portion thereof; The vaccine of any one of aspects 78 to 81. 83. The vector: (a) a CMV genome or a portion thereof containing the first expression cassette, or the first and second expression cassettes; (b) a sequence containing an origin of replication that functions in a unicellular organism; (c) one or more terminase complex recognition loci (TCRLs) comprising recombinantly introduced polynucleotide sequences capable of directing cleavage by the HV terminase complex; a circular CMV vector comprising the CMV genome or portion thereof is separated from a sequence comprising the origin of replication by a TCRL; the CMV genome or portion thereof is flanked on at least one end by a TCRL; The sequence containing the replication origin is flanked on at least one end by a TCRL. The vaccine of any one of aspects 78 to 81. 84. The vaccine of embodiment 82 or 83, wherein the one or more terminase complex recognition loci comprise a Pac1 site and a Pac2 site. 85. The vaccine of embodiment 84, wherein all of the terminase complex recognition loci include a Pac1 site and a Pac2 site. 86. The vaccine of any one of aspects 77-85, wherein the first promoter is a viral promoter. 87. The vaccine of embodiment 86, wherein the viral promoter is a pp65b promoter. 88. The vaccine of any one of aspects 78 to 87, wherein the vector is a CMV vector, and the CMV is Towne HCMV. 89. The vaccine of any one of aspects 37 to 88, wherein the polynucleotide encoding the conjugated polypeptide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22. 90. A conjugated polypeptide comprising an antigen derived from a pathogen linked to a ligand or antibody fragment that specifically binds to a surface protein present on an immune cell. 91. The conjugated polypeptide of embodiment 90, wherein the surface protein is an abundant T-cell surface protein involved in signal transduction and / or adhesion. 92. The conjugated polypeptide of embodiment 90 or 91, wherein the surface protein is CD2, CD3, CD4, or CD5. 93. The conjugated polypeptide of embodiment 92, wherein the surface protein is CD2 or CD3. 94. The conjugated polypeptide of any one of embodiments 90-93, wherein the immune cell is a T cell or an antigen-presenting cell (APC). 95. The conjugated polypeptide of any one of embodiments 90 to 94, wherein the ligand is an ectodomain of a cell adhesion molecule. 96. The conjugated polypeptide of embodiment 95, wherein the cell adhesion molecule is CD58. 97. The conjugated polypeptide of any one of embodiments 90-96, wherein the surface protein is preferentially expressed by T cells. 98. The conjugated polypeptide of any one of aspects 90-94 or 95-97, wherein the antibody fragment is an scFv chain derived from an antibody. 99. The conjugated polypeptide of any one of embodiments 90-98, further comprising a lipid anchor, a transmembrane segment, a multimerization domain, or any combination of these elements. 100. The conjugated polypeptide of embodiment 99, wherein the lipid anchor is a glycosylphosphatidylinositol anchor. 101. The conjugated polypeptide of embodiment 99 or 100, wherein addition of the lipid anchor is directed by a signal sequence. 102. The conjugated polypeptide of embodiment 101, wherein the signal sequence is derived from CD55. 103. The conjugated polypeptide of any one of aspects 99 to 102, wherein the transmembrane segment is derived from a PDGF receptor, glycophorin A, or SARS-CoV-2 spike protein. 104. The conjugated polypeptide of any one of aspects 99 to 103, wherein the multimerization domain is derived from T4 fibritin. 105. The conjugated polypeptide of any one of aspects 99 to 103, wherein the multimerization domain is an Fc domain. 106. The conjugated polypeptide of embodiment 105, wherein the Fc domain is located at the C-terminus of the conjugated polypeptide. 107. The conjugated polypeptide of embodiment 105 or 106, wherein the Fc domain is a human IgG1 Fc domain. 108. The conjugated polypeptide of any one of embodiments 98 to 107, wherein the antibody fragment is an scFv chain derived from an antibody, and the VH and VL regions of the scFv are separated by a flexible linker. 109. The conjugate polypeptide of embodiment 108, wherein the flexible linker is 12 amino acids or more in length, and wherein the conjugate polypeptide preferentially binds to the surface protein as a monomer. 110. The conjugate polypeptide of embodiment 109, wherein the flexible linker is less than 12 amino acids in length, and wherein the conjugate polypeptide preferentially binds to the surface protein as a multimer. 111. The conjugated polypeptide of embodiment 110, wherein the multimer is stabilized by disulfide bonds between the monomer units. 112. The conjugated polypeptide of embodiment 110 or 111, wherein the flexible linker is 5 amino acids in length. 113. The conjugated polypeptide of any one of embodiments 90-112, further comprising a tPA leader sequence. 114. The conjugated polypeptide of embodiment 113, wherein the tPA leader sequence is 23 amino acids in length. 115. The conjugated polypeptide of any one of aspects 90-114, wherein the pathogen is a virus. 116. The conjugated polypeptide of embodiment 115, wherein the virus is SARS-CoV-2. 117. The conjugated polypeptide of embodiment 116, wherein the antigen comprises the SARS-CoV-2 spike glycoprotein or a fragment thereof. 118. The conjugated polypeptide of embodiment 117, wherein the fragment of the SARS-CoV-2 spike glycoprotein comprises the S1 domain or the receptor binding domain (RBD). 119. The conjugated polypeptide of any one of embodiments 90 to 118, comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, and SEQ ID NO:21. 120. A conjugated polypeptide comprising: (i) a tissue plasminogen activator (tPA) signal sequence; (ii) a single-chain variable fragment (scFv) that specifically binds to CD2, CD3, or CD4; (iii) a flexible linker; and (iv) a SARS-CoV-2 receptor binding domain (RBD). 121. The conjugated polypeptide of embodiment 120, comprising the amino acid sequence of SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID 25, or SEQ ID NO:27. 122. A polynucleotide encoding the conjugated polypeptide of any one of embodiments 90 to 121. 123. The polynucleotide of embodiment 122, which is codon-optimized. 124. The polynucleotide of embodiment 123, comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:28. 125. An expression cassette comprising the polynucleotide of any one of embodiments 122 to 124. 126. The expression cassette of embodiment 125, comprising the nucleotide sequence of SEQ ID NO:8. 127. A vector comprising the expression cassette of embodiment 125 or 126. 128. The vector of embodiment 127, which is a plasmid. 129. The vector of embodiment 127, which is an adenovirus vector. 130. A diabody comprising the conjugated polypeptide of any one of embodiments 98 to 121. 131. A dimer comprising the conjugated polypeptide of any one of embodiments 99 to 121. 132. A vaccine comprising the conjugated polypeptide of any one of embodiments 90 to 121, the polynucleotide of any one of embodiments 122 to 124, the expression cassette of embodiment 125 or 126, the vector of any one of embodiments 127 to 129, the diabody of embodiment 130, or the dimer of embodiment 131. 133. A method of inducing an immune response against a pathogen in a mammal, comprising administering to the mammal any of the vaccines of aspects 1 to 89 or 132. 134. The method of embodiment 133, wherein the vaccine is administered subcutaneously or by electroporation. 135. The method of embodiment 133 or 134, wherein the method induces a neutralizing antibody response in the mammal against the antigen present in the conjugated polypeptide, wherein the neutralizing response is substantially greater than any antibody-dependent enhancement of infection (ADEI) induced in the mammal by the vaccine. 136. The method of embodiment 135, wherein the vaccine does not substantially induce ADEI in the mammal. 137. The method of any one of embodiments 133-136, wherein both a CD4+ and a CD8+ T cell response is induced against the second antigen. 138. The method of any one of embodiments 133 to 137, comprising administering to the mammal a DNA prime comprising the vector of embodiment 127 or 128 by electroporation, followed by boosting with an adenoviral vector encoding the RBD. 139. The method of embodiment 138, wherein the booster is administered about 28 days later. 140. The method of any one of embodiments 133 to 139, wherein the mammal is a human.

[0239] Unofficial sequence listing TIFF2026021474000011.tif209170TIFF2026021474000012.tif221170TIFF2026021474000013.t if221170TIFF2026021474000014.tif213170TIFF2026021474000015.tif221170TIFF20260214740 00016.tif221170TIFF2026021474000017.tif221170TIFF2026021474000018.tif221170TIFF202 6021474000019.tif221170TIFF2026021474000020.tif222170TIFF2026021474000021.tif233170

[0240] Sequence information SEQUENCE LISTING <110> THE REGENTS OF THE UNIVERSITY OF CALIFORNIA <120> CONJUGATE POLYPEPTIDES AND VACCINES FOR INDUCING IMMUNE RESPONSES <150> US 63 / 058,362 <151> 2020-07-29 <150> US 63 / 027,250 <151> 2020-05-19 <160> 29 <170> PatentIn version 3.5 <210> 1 <211> 962 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 1 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Ala Ser Glu Val Gln Leu Val Glu Ser Gly Gly 20 25 30 Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser 35 40 45 Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro 50 55 60 Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn 65 70 75 80 Tyr Ala Thr Tyr Tyr Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser 85 90 95 Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Lys 100 105 110 Thr Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg His Gly Asn Phe Gly 115 120 125 Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 130 135 140 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 145 150 155 160 Gly Gly Ser Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser 165 170 175 Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val 180 185 190 Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala 195 200 205 Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro 210 215 220 Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu 225 230 235 240 Ser Gly Val Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp 245 250 255 Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 260 265 270 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val 275 280 285 Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 290 295 300 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 305 310 315 320 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 325 330 335 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 340 345 350 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 355 360 365 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 370 375 380 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 385 390 395 400 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 405 410 415 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 420 425 430 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 435 440 445 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 450 455 460 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 465 470 475 480 Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 485 490 495 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 500 505 510 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 515 520 525 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 530 535 540 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 545 550 555 560 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 565 570 575 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 580 585 590 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 595 600 605 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 610 615 620 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 625 630 635 640 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 645 650 655 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 660 665 670 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 675 680 685 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 690 695 700 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 705 710 715 720 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 725 730 735 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 740 745 750 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 755 760 765 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 770 775 780 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 785 790 795 800 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 805 810 815 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 820 825 830 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 835 840 845 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 850 855 860 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 865 870 875 880 Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile 885 890 895 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 900 905 910 Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 915 920 925 Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 930 935 940 Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala 945 950 955 960 Ser Gln <210> 2 <211> 2889 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 2 atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60 tcggctagcg aggtgcagct cgttgagagt ggtgggggtc tggtccaacc cggtgggagt 120 ctccgcttgt catgtgcagc ttctggcttc acatttaaca cctatgccat gaactgggtc 180 cggcaggccc ccggcaaggg acttgaatgg gttgcacgga tccgctcaaa atacaacac 240 tacgcgactt actacgctgc ctcagtcaag ggtcgcttca cattagtcg cgacgactct 300 aagaatagtc tttatttgca gatgaatagt ctcaagaccg aggacactgc ggtatattat 360 tgcgcacggc acggaaattt tggcaattca tatgttagtt ggttcgctta ctgggggcaa 420 ggcacgctcg ttacggtctc aagtggtggt ggcggttcag ggggagggg ctctggaggg 480 ggcggttctc aaaccgttgt cacccaggaa ccaagcctga ccgtaagccc tggtgggacc 540 gtgactctca cgtgtagaag ttctacagga gctgttacta cctcaaacta cgctaactgg 600 gtacaacaaa aacccggtca agcaccaagg ggactgatag gcggaacaaa caaaagggcg 660 ccgggtactc cggcacgatt ttccggatct cttcttggag gaaaggcagc gttgactttg 720 tctggagttc agcccgagga tgaggcagag tattattgcg cactctggta tagtaacctc 780 tgggtcttcg gcggaggaac taagcttacg gtccttgggg gtggcggcag tggcggtggc 840 gggtctggag gtgggggttc cgttaatctc acgaccagga cccaattgcc tcccgcgtat 900 actaactctt tcacgagggg agtctactat cctgacaaag tatttaggtc ttcagtgctg 960 catagtacac aagacctgtt ccttccgttt ttcagcaacg tgacttggtt ccacgctata 1020 cacgtctcag ggacgaatgg aacaaagcgc ttcgataatc cggttttgcc atttaatgat 1080 ggtgtctatt tcgcatccac agaaaagtcc aacattatca gggggtggat ctttggtacg 1140 acgctggata gcaaaacaca gtccctcctt atcgtcaaca atgccacgaa tgtggtgatt 1200 aaggtttgcg aatttcaatt ttgtaacgac ccttttcttg gcgtatatta tcataaaaac 1260 aacaagtcct ggatggaaag cgaattccgc gtatacagtt ccgcaaacaa ctgtacattt 1320 gaatatgtga gccaaccttt tctgatggac ctggagggca aacagggcaa ctttaaaaat 1380 ttgagagagt tcgtcttcaa aaatattgat ggatatttca agatttatag taagcatacg 1440 cccataaatc ttgtccggga tctgccgcag ggttttagcg ctctcgaacc cttggtagac 1500 ctcccgattg gtataaacat caccaggttt cagacccttc ttgcgttgca ccgcagctat 1560 ctcacgccag gcgatagtag ttcaggttgg actgccggag cagcagccta ctacgtaggc 1620 taccttcaac ctagaacgtt tctgttgaaa tataatgaaa atggtacaat cacagacgcg 1680 gtcgactgcg cactggaccc gctgagcgaa accaaatgta cgctcaagtc cttcaccgta 1740 gagaaaggca tctaccagac ttctaatttc cgagtgcagc cgacggagtc aatcgtgaga 1800 ttccctaaca taactaattt gtgtccattt ggcgaagtgt tcaatgcaac cagattcgcc 1860 tccgtctatg cgtggaatcg aaaaagaatt tcaaactgcg tagcggatta ttctgtcttg 1920 tacaatagtg cctcctttag tacgttcaag tgttatgggg tgtcaccaac gaagttgaat 1980 gatctttgtt tcacgaatgt ttacgctgat tcatttgtaa tacgcggaga cgaagttaga 2040 caaatcgcac cagggcagac aggcaagatc gcggattata attataagct gccagacgac 2100 ttcactgggt gcgttatcgc atggaactcc aacaacttgg atagtaaagt gggcgggaat 2160 tacaactacc tgtatagact tttccgaaag tccaatttga agccattcga aagggacatt 2220 tctactgaaa tatatcaagc gggatcaaca ccttgcaacg gagtggaagg gttcaactgc 2280 tactttccgc tgcaatctta tgggtttcaa ccgactaatg gagtcgggta tcagccttac 2340 agagttgttg ttctttcctt tgagctgttg catgccccgg caaccgtatg tgggcccaag 2400 aaatctacaa acctcgttaa gaataaatgc gtgaatttca acttcaatgg tctcaccggg 2460 acgggggtcc tgaccgaaag taacaagaaa tttctgccct ttcagcaatt cggaagagac 2520 atcgcggaca ctacagacgc cgttcgggac ccgcagactc tcgaaattct tgacatcacg 2580 ccgtgttcat tcggaggcgt ttccgtgatt acaccaggaa cgaataccag caatcaagtg 2640 gcagtgttgt atcaagatgt taattgcact gaagtgcctg tcgctatcca cgcggaccag 2700 ctcacgccta cgtggagggt gtattcaaca ggaagcaacg tgttccaaac acgagcgggt 2760 tgtcttatag gggcggagca cgtgaacaat agttacgaat gtgatatacc gataggggct 2820 gggatatgtg cgtcttatca aacacagacg aatagcccca ggcgcgctcg aagtgtggca 2880 agccaatag 2889 <210> 3 <211> 1260 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 3 atgagcgata acggccccca gatcagagg atgcaccta ggatacatt tggaggtccg 60 tcagacagca ctggctccaa ccagaatggc gagcggtctg gcgcgcggtc taagcaagg 120 agaccacaag gtctcccgaa taacacggcc tcctgtttta cagcgctcac ccagcacggt 180 aaagaggacc tgaagttcccc taggggcaa ggtgtaccga ttaataccaa cagctccccc 240 gatgaccaga ttggttatta tagagagct acagacgca tacggggtgg agatgggaag 300 atgaaggacc tctccctcg gtggtattt tattatctg gcaccggacc ggaagccggg 360 ctcccctacg gcgccaataa ggacggtata atatgggtcg ccactgaggg tgccctcaat 420 accccgaagg accacattgg cactcgaaac ccggcaaca acgcagctat tgtcctgcaa 480 ctcccacagg gtaccacgct cccgaaggt ttttatgccg aagggtcg cggggttca 540 caggctagca gtcgaagctc atctcggagc cgaatagct caggaattc aacacccgga 600 agctccagag gcacaagccc tgcgcggatg gcagggaacg gaggcgatgc tgccctggcc 660 ctccttctct tggatagact taatcagctg gatccaaaa tgtcaggaaa gggccagcaa 720 caacaggtc agacagtgac caaaaaatcc gccgcagagg ccagtagaa acctagacag 780 aagcgaactg ctacaaaggc ctataatgta actcaggcgt tcggacggcg aggccctgaa 840 caaacccagg gcaatttcgg tgaccagaa ctcatagac agggactga ctacaaacat 900 tggccccaga ttgcacaatt tgcccatcc gcctcagcct tctcggaat gtctcgcatc 960 gggatggagg taacaccgag cgggacctgg ctcacataca caggtgcgat aaagctcgat 1020 gaaagatc ccaattta agaccaagtg atactctta ataagcacat agacgcctat 1080 aaaaccttcc cgcccactga gccaaagaaaaaaagcgga cgagacacaa 1140 gcccttccgc aaagacagaa aaaacagcaa acggttacat tgctcctgc ggcagacctg 1200 gatgattttt ccaacagct tcagcaatct atgtctagcg cagatagtac ccaggcgtaa 1260 <210> 4 <211> 891 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 4 atggctgaca gcaatgggac gatcacagtc gaggaactca agaaactgtt ggagcagtgg 60 aatttggtga taggcttctt gttcttgacg tggatatgct tgctgcagtt tgcctatgcg 120 aatagaaacc gcttcttgta tataatcaaa ctgatatttc tctggttgct ctggcccgtc 180 acgcttgcat gttttgtttt ggcggccgtt taccggatca actggattac agggggaatt 240 gcaatagcga tggcatgtct ggtaggattg atgtggctgt cctactttat tgcgtcattt 300 cgattgtttg cacgcactcg gtccatgtgg tcctttaatc cagaaaccaa tatactgctc 360 aacgtaccat tgcatgggac tatactgacg cgacctcttt tggaatcaga gctggtgata 420 ggtgcagtga tactgagagg gcatctccga atagcggggc accacctggg cagatgtgac 480 atcaaagacc ttccaaaaga aatcacggtg gccacgtcaa ggacattgtc atattacaaa 540 cttggcgcat cccaaagagt agcaggagat tctggtttcg cggcatactc taggtacagg 600 atcgggaatt acaaactgaa cacagatcat tcctctagtt ccgacaacat agcccttttg 660 gtacagatgt actctttcgt atcagaggag actggtactc ttatagttaa ttctgtgctg 720 ctctttcttg cgtttgtcgt ctttctgctt gtaacacttg ctattcttac tgcgttgaga 780 ctctgtgcct attgttgtaa tatagtcaat gtatctttgg taaagcctag tttttatgtg 840 tatagccggg tcaaaaacct taactccagc cgagttcccg accttttggt t 891 <210> 5 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 5 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 6 <211> 510 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 6 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Ala Ser Glu Val Gln Leu Val Glu Ser Gly Gly 20 25 30 Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser 35 40 45 Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro 50 55 60 Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn 65 70 75 80 Tyr Ala Thr Tyr Tyr Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser 85 90 95 Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Lys 100 105 110 Thr Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg His Gly Asn Phe Gly 115 120 125 Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 130 135 140 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 145 150 155 160 Gly Gly Ser Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser 165 170 175 Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val 180 185 190 Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala 195 200 205 Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro 210 215 220 Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu 225 230 235 240 Ser Gly Val Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp 245 250 255 Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 260 265 270 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg 275 280 285 Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu 290 295 300 Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr 305 310 315 320 Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val 325 330 335 Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser 340 345 350 Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser 355 360 365 Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr 370 375 380 Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly 385 390 395 400 Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly 405 410 415 Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro 420 425 430 Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro 435 440 445 Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr 450 455 460 Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val 465 470 475 480 Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro 485 490 495 Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 500 505 510 <210> 7 <211> 1533 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 7 atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agctttcgtt 60 tcggctagcg aggtgcagct cgttgagagt ggtgggggtc tggtccaacc cggtgggagt 120 ctccgcttgt catgtgcagc ttctggcttc acatttaaca cctatgccat gaactgggtc 180 cggcaggccc ccggcaaggg acttgaatgg gttgcacgga tccgctcaaa atacaacac 240 tacgcgactt actacgctgc ctcagtcaag ggtcgcttca cattagtcg cgacgactct 300 aagaatagtc tttatttgca gatgaatagt ctcaagaccg aggacactgc ggtatattat 360 tgcgcacggc acggaaattt tggcaattca tatgttagtt ggttcgctta ctgggggcaa 420 ggcacgctcg ttacggtctc aagtggtggt ggcggttcag ggggagggg ctctggaggg 480 ggcggttctc aaaccgttgt cacccaggaa ccaagcctga ccgtaagccc tggtgggacc 540 gtgactctca cgtgtagaag ttctacagga gctgttacta cctcaaacta cgctaactgg 600 gtacaacaaa aacccggtca agcaccaagg ggactgatag gcggaacaaa caaaagggcg 660 ccgggtactc cggcacgatt ttccggatct cttcttggag gaaaggcagc gttgactttg 720 tctggagttc agcccgagga tgaggcagag tattattgcg cactctggta tagtaacctc 780 tgggtcttcg gcggaggaac taagcttacg gtccttgggg gtggcggcag tggcggtggc 840 gggtctggag gtgggggttc ccgagtgcag ccgacggagt caatcgtgag attccctaac 900 ataactaatt tgtgtccatt tggcgaagtg ttcaatgcaa ccagattcgc ctccgtctat 960 gcgtggaatc gaaaaagaat ttcaaactgc gtagcggatt attctgtctt gtacaatagt 1020 gcctccttta gtacgttcaa gtgttatggg gtgtcaccaa cgaagttgaa tgatctttgt 1080 ttcacgaatg tttacgctga ttcatttgta atacgcggag acgaagttag acaaatcgca 1140 ccagggcaga caggcaagat cgcggattat aattataagc tgccagacga cttcactggg 1200 tgcgttatcg catggaactc caacaacttg gatagtaaag tgggcgggaa ttacaactac 1260 ctgtatagac ttttccgaaa gtccaatttg aagccattcg aaagggacat ttctactgaa 1320 atatatcaag cgggatcaac accttgcaac ggagtggaag ggttcaactg ctactttccg 1380 ctgcaatctt atgggtttca accgactaat ggagtcgggt atcagcctta cagagttgtt 1440 gttctttcct ttgagctgtt gcatgccccg gcaaccgtat gtgggcccaa gaaatctaca 1500 aacctcgtta agaataaatg cgtgaatttc tag 1533 <210> 8 <211> 2996 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 8 cgtgaggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt 60 tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg 120 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa 180 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa 240 gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt 300 gaattacttc cacctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg 360 ggtgggagag ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg 420 cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg 480 ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt 540 tctggcaaga tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg 600 gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc 660 tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg 720 tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg 780 caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat 840 ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct 900 ttccgtcctc agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc 960 tcgattagtt ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg 1020 cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga 1080 tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc 1140 agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtgaggaatt agctggcgcg 1200 ccttcccgcc accatggatg caatgaagag agggctctgc tgtgtgctgc tgctgtgtgg 1260 agcagtcttc gtttcggcta gcgaggtgca gctcgttgag agtggtgggg gtctggtcca 1320 acccggtggg agtctccgct tgtcatgtgc agcttctggc ttcacattta acacctatgc 1380 catgaactgg gtccggcagg cccccggcaa gggacttgaa tgggttgcac ggatccgctc 1440 aaaatacaac aactacgcga cttactacgc tgcctcagtc aagggtcgct tcaccattag 1500 tcgcgacgac tctaagaata gtctttattt gcagatgaat agtctcaaga ccgaggacac 1560 tgcggtatat tattgcgcac ggcacggaaa ttttggcaat tcatatgtta gttggttcgc 1620 ttactggggg caaggcacgc tcgttacggt ctcaagtggt ggtggcggtt cagggggagg 1680 gggctctgga gggggcggtt ctcaaaccgt tgtcacccag gaaccaagcc tgaccgtaag 1740 ccctggtggg accgtgactc tcacgtgtag aagttctaca ggagctgtta ctacctcaaa 1800 ctacgctaac tgggtacaac aaaaacccgg tcaagcacca aggggactga taggcggaac 1860 aaacaaaagg gcgccgggta ctccggcacg attttccgga tctcttcttg gaggaaaggc 1920 agcgttgact ttgtctggag ttcagcccga ggatgaggca gagtattatt gcgcactctg 1980 gtatagtaac ctctgggtct tcggcggagg aactaagctt acggtccttg ggggtggcgg 2040 cagtggcggt ggcgggtctg gaggtggggg ttcccgagtg cagccgacgg agtcaatcgt 2100 gagattccct aacataacta atttgtgtcc atttggcgaa gtgttcaatg caaccagatt 2160 cgcctccgtc tatgcgtgga atcgaaaaag aatttcaaac tgcgtagcgg attattctgt 2220 cttgtacaat agtgcctcct ttagtacgtt caagtgttat ggggtgtcac caacgaagtt 2280 gaatgatctt tgtttcacga atgtttacgc tgattcattt gtaatacgcg gagacgaagt 2340 tagacaaatc gcaccagggc agacaggcaa gatcgcggat tataattata agctgccaga 2400 cgacttcact gggtgcgtta tcgcatggaa ctccaacaac ttggatagta aagtgggcgg 2460 gaattacaac tacctgtata gacttttccg aaagtccaat ttgaagccat tcgaaaggga 2520 catttctact gaaatatatc aagcgggatc aacaccttgc aacggagtgg aagggttcaa 2580 ctgctacttt ccgctgcaat cttatgggtt tcaaccgact aatggagtcg ggtatcagcc 2640 ttacagagtt gttgttcttt cctttgagct gttgcatgcc ccggcaaccg tatgtgggcc 2700 caagaaatct acaaacctcg ttaagaataa atgcgtgaat ttctagtagc ctgcaggacg 2760 ccacagctct gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa 2820 acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact 2880 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata 2940 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatct 2996 <210> 9 <211> 752 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 9 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Ala Ser Glu Val Gln Leu Val Glu Ser Gly Gly 20 25 30 Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser 35 40 45 Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro 50 55 60 Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn 65 70 75 80 Tyr Ala Thr Tyr Tyr Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser 85 90 95 Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Lys 100 105 110 Thr Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg His Gly Asn Phe Gly 115 120 125 Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 130 135 140 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 145 150 155 160 Gly Gly Ser Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser 165 170 175 Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val 180 185 190 Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala 195 200 205 Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro 210 215 220 Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu 225 230 235 240 Ser Gly Val Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp 245 250 255 Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 260 265 270 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg 275 280 285 Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu 290 295 300 Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr 305 310 315 320 Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val 325 330 335 Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser 340 345 350 Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser 355 360 365 Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr 370 375 380 Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly 385 390 395 400 Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly 405 410 415 Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro 420 425 430 Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro 435 440 445 Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr 450 455 460 Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val 465 470 475 480 Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro 485 490 495 Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly Gly 500 505 510 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr 515 520 525 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 530 535 540 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 545 550 555 560 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 565 570 575 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 580 585 590 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 595 600 605 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 610 615 620 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 625 630 635 640 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 645 650 655 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 660 665 670 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 675 680 685 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 690 695 700 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 705 710 715 720 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 725 730 735 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 740,745,750 <210> 10 <211> 2259 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 10 atggatgcca tgaagagagg attgtgttgc gtacttctcc tttgcggggc agctttgtg 60 tcagctagtg aagttcaact cgtcgaatca ggtggaggcc ttgtgcaacc gggaggttcc 120 ttgcggcttt catgtgcagc gtcaggcttt accttcaata catatgccat gaactgggtc 180 cgccaggccc cgggcaaggg tctcgaatgg gtagcgcgca tccgcagcaa gtacaataat 240 tatgcgacct attatgcagc ctcagtaaaa gggaggttca ccatatctcg ggacgactct 300 aaaatagcc tctatctcca gatgaatagt cttaaaactg aggatacggc ggtgtactat 360 tgcgcccggc atggcaactt tgggaatagc tatgtctcat ggtttgccta ttggggccag 420 ggaacgctgg tgaccgtttc aagcggagga ggcggatctg gcggcggagg ttcaggtggc 480 ggcggttcac aaacagtcgt gacccaggaa cccagcctta ccgtctctcc cggagggaca 540 gttacactga cgtgcagatc aagtaccggc gccgtcacta cttctaacta cgctaattgg 600 gttcaacaaa agccgggtca agcaccacga ggtttgatag gggggacaaa taaaagggct 660 ccaggcaccc ccgctcgctt ctccgggagt ctcttggggg gcaaagccgc gctgacgttg 720 tctggagtcc agcctgagga tgaagccgag tattactgcg ctctttggta ctctaacctg 780 tgggtctttg ggggaggcac gaaacttaca gtcttgggtg gcggtggttc aggcggtggt 840 ggaagcggag gcggcggttc tagggtgcag ccgacagaaa gtatcgtccg gtttccgaac 900 attaccaatt tgtgtccctt cggcgaagtc tttaatgcta caaggtttgc cagcgtctac 960 gcatggaatc gcaaaagaat aagcaattgc gtagccgatt attcagtatt gtacaattcc 1020 gcttcttttt caacattcaa atgttatggg gtgtccccta cgaagttgaa tgacctgtgt 1080 ttcacgaatg tctacgcaga ttcttttgtc atacgcggcg acgaagtgag acaaattgca 1140 cccggccaaa ccggcaaaat agcggactat aactataagc tccccgacga tttcaccggg 1200 tgcgtcatag cctggaacag caacaacctc gattcaaaag tcggaggcaa ctacaactat 1260 ctttacaggt tgttccggaa gtcaatctg aagccttg aacggtat ctcaactgaa 1320 atttatcaag ccggatctac tccgtgcaat ggagtcgaag gatttaattg ctactttccc 1380 cttcagtcct atggttttca accacgaat ggcgtcggtt accagcccta cagagtcgtt 1440 gttttgtcat tcgaactgct gcacgcgcca gcaacggtat gcggccccaa gaaagcacg 1500 aatttggtga aaaataatg cgttaacttt ggaggtgggg gatccggcgg aggaggtagt 1560 ggagggggag gcagtgacaa aacacacc tgtccgccct gccccgcccc tgagctcctg 1620 gggggaccta gcgtcttttt gttccacca aagcccaaag acacacttat gatatctcgg 1680 acaccggaag taacttgtgt agttgtagac gtatcacatg aggaccccga ggtcaattc 1740 aattggtacg tcgatggtgt cgaagtgcac atgcaaaa cgaaaccgcg agaggagcaa 1800 tataacagta cgtatcgcgt cgtcagcgtg ttgacggtac tccatcaaga ttggctcaat 1860 ggaaaggagt ataagtgcaa ggtatccac aaggccctc ccgcacctat agaaagacg 1920 atttcaaag ccaaggcca accccgagag cccaagtat atacactccc accctcgg 1980 gatgagctga cgaaaaacca ggttagtctc acttgccctcg tcaagggatt ttacccctcc 2040 gatatagcgg tggagtggga gtccaatggc aaccggaga acaactaa aacgactcct 2100 cctgtactcg attcagacgg atctttcttt ttgtacagta agcttactgt tgacaatcc 2160 aggtggcaac aagggaatgt attcagctgt agtgtaatgc acgaggcatt gcacaaccac 2220 tacactcaga aatctctcag tctgtcccca ggaagtag 2259 <210> 11 <211> 500 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 11 Met Asp Ala Met Lys Arg Gly Leu Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Wing Val Phe Val Ser Wing Ser Glu Val Gln Leu Val Glu Ser Gly Gly 20 25 30 Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser 35 40 45 Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro 50 55 60 Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn 65 70 75 80 Tyr Ala Thr Tyr Tyr Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser 85 90 95 Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Lys 100 105 110 Thr Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg His Gly Asn Phe Gly 115 120 125 Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 130 135 140 Thr Val Ser Ser Gly Gly Gly Gly Ser Gln Thr Val Val Thr Gln Glu 145 150 155 160 Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg 165 170 175 Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln 180 185 190 Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys 195 200 205 Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly 210 215 220 Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu 225 230 235 240 Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly 245 250 255 Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 260 265 270 Gly Gly Gly Gly Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe 275 280 285 Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr 290 295 300 Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys 305 310 315 320 Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe 325 330 335 Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr 340 345 350 Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln 355 360 365 Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu 370 375 380 Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu 385 390 395 400 Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg 405 410 415 Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr 420 425 430 Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr 435 440 445 Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr 450 455 460 Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro 465 470 475 480 Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys 485 490 495 Cys Val Asn Phe 500 <210> 12 <211> 1503 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 12 atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 120. tcggctagcg aagtccaact tgtcgaatca ggtgggggac tggtacaacc agggggctcc ctccgcctta gctgcgcagc gtccggggttc acgtttaaca cttacgcaat gaactgggtc cggcaagcac ccggtaaagg tttggagtgg gtggcacgca ttagatccaa attack tatgctacgt actatgcggc tagtgtgag ggaaggttta ctatatcaag agatgacagt aaaaacagtc tctacctcca gatgaactca ctgaaaaccg aggataccgc agtctattat tgcgcgcgcc acggtaattt tggcaattct tacgtgagct ggtttgctta ctggggccag 420 gggactctcg tcaccgtcag ctctggtggt ggtggtagcc agacggtagt tactcaggag 480 ccaagcctga cggtcagtcc gggcggaca gttaccctga catgcaggtc ttccaccggg 540 gcggtcacca cttctaacta tgcgaactgg gtacaacaga aaccaggaca agcaccaga gggcttatcg ggggcacgaa taaacgagca cctggaactc cggcaaggtt ctctggaagc ctgcttgggg gcaaggcggc gctcactctt tcaggtgtgc aaccggaaga cgaagcagaa 720 tattactgtg cattgtggta ctccaacctc tgggtgttcg gtggcgggac caaactcaca 780 gtgctgggcg gtggtgggag cgggggtggg ggttctggtg gtggaggtag ccgagtgcag 840 ccgacggagt caatcgtgag attccctaac ataactaatt tgtgtccatt tggcgaagtg 900 ttcaatgcaa ccagattcgc ctccgtctat gcgtggaatc gaaaaagaat ttcaaactgc 960 gtagcggatt attctgtctt gtacaatagt gcctccttta gtacgttcaa gtgttatggg 1020 gtgtcaccaa cgaagttgaa tgatctttgt ttcacgaatg tttacgctga ttcatttgta 1080 atacgcggag acgaagttag acaaatcgca ccagggcaga caggcaagat cgcggattat 1140 aattataagc tgccagacga cttcactggg tgcgttatcg catggaactc caacaacttg 1200 gatagtaaag tgggcgggaa ttacaactac ctgtatagac ttttccgaaa gtccaatttg 1260 aagccattcg aaagggacat ttctactgaa atatatcaag cgggatcaac accttgcaac 1320 ggagtggaag ggttcaactg ctactttccg ctgcaatctt atgggtttca accgactaat 1380 ggagtcgggt atcagcctta cagagttgtt gttctttcct tgagctgtt gcatgccccg 1440 gcaaccgtat gtgggcccaa gaatctaca aacctcgtta agaataaatg cgtgaatttc 1500 tag 1503 <210> 13 <211> 500 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 13 Met Asp Ala Met Lys Arg Gly Leu Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Wing Val Phe Val Ser Wing Ser Glu Val Gln Leu Val Glu Ser Gly Gly 20 25 30 Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser 35 40 45 Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro 50 55 60 Gly Lys Gly Leu Glu Trp Val Ala Arg With Arg Ser Lys Tyr Asn Asn 65 70 75 80 Tyr Ala Thr Tyr Tyr Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser 85 90 95 Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Thr 100 105 110 Thr Cys Asp Thr Ala Val Tyr Tyr Cys Ala Arg His Gly Asn Phe Gly 115 120 125 Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 130 135 140 Thr Val Ser Ser Gly Gly Gly Gly Ser Gln Thr Val Val Thr Gln Glu 145 150 155 160 Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg 165 170 175 Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln 180 185 190 Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys 195 200 205 Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly 210 215 220 Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu 225 230 235 240 Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly 245 250 255 Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 260 265 270 Gly Gly Gly Gly Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe 275 280 285 Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr 290 295 300 Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys 305 310 315 320 Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe 325 330 335 Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr 340 345 350 Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln 355 360 365 Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu 370 375 380 Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu 385 390 395 400 Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg 405 410 415 Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr 420 425 430 Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr 435 440 445 Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr 450 455 460 Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro 465 470 475 480 Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys 485 490 495 Cys Val Asn Phe 500 <210> 14 <211> 1503 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 14 atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60 tcggctagcg aggttcaatt ggtggaatct ggaggggggc ttgtccaacc cggagggtcc 120 ttgagactga gttgtgcagc gagcggtttc acatttaaca cttacgccat gaactgggtt 180 aggcaagccc ccgggaaggg actggagtgg gttgccagaa tcagatcaaa atacaataat 240 tatgcaacat actatgcagc tagtgtcaag ggaagattca ccataagccg agacgactct 300 aagaatagcc tgtatctgca aatgaatagt ctcacgactt gcgatactgc cgtatactac 360 tgtgcacgcc atggaaattt cggcaacagc tatgtttcct ggtttgccta ttggggacaa 420 ggcacgctcg tcactgtttc ttcaggagga ggtggcagtc aaacggttgt gacccaagaa 480 ccctcgctca ctgtgtcacc aggaggaact gtgacgctca catgccgatc ctccaccggt 540 gccgtcacta cgagcaatta tgctaactgg gtacagcaaa agccagggca agcacctcga 600 ggcctgatcg gtggcacgaa taaacgagcg cctggtacgc cagcccgctt ttctggatct 660 cttctgggcg gaaaagcagc cctgactctc tccggggtac agccggagga cgaagcagag 720 tactactgcg ctctttggta tagcaacctt tgggtgttcg gtggtggtac gaaactcact 780 gttctcggtg gagggggtag cgggggtggc gggagtggtg gcggaggttc acgagtgcag 840 ccgacggagt caatcgtgag attccctaac ataactaatt tgtgtccatt tggcgaagtg 900 ttcaatgcaa ccagattcgc ctccgtctat gcgtggaatc gaaaaagaat ttcaaactgc 960 gtagcggatt attctgtctt gtacaatagt gcctccttta gtacgttcaa gtgttatggg 1020 gtgtcaccaa cgaagttgaa tgatctttgt ttcacgaatg tttacgctga ttcatttgta 1080 atacgcggag acgaagttag acaaatcgca ccagggcaga caggcaagat cgcggattat 1140 aattataagc tgccagacga cttcactggg tgcgttatcg catggaactc caacaacttg 1200 gatagtaaag tgggcgggaa ttacaactac ctgtatagac ttttccgaaa gtccaatttg 1260 aagccattcg aaagggacat ttctactgaa atatatcaag cgggatcaac accttgcaac 1320 ggagtggaag ggttcaactg ctactttccg ctgcaatctt atgggtttca accgactaat 1380 ggagtcgggt atcagcctta cagagttgtt gttctttcct ttgagctgtt gcatgccccg 1440 gcaaccgtat gtgggcccaa gaaatctaca aacctcgtta agaataaatg cgtgaatttc 1500 tag 1503 <210> 15 <211> 506 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 15 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Ala Ser Gln Val Gln Leu Val Gln Ser Gly Ala 20 25 30 Glu Val Gln Arg Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser 35 40 45 Gly Tyr Ile Phe Thr Glu Tyr Tyr Met Tyr Trp Val Arg Gln Ala Pro 50 55 60 Gly Gln Gly Leu Glu Leu Val Gly Arg Ile Asp Pro Glu Asp Gly Ser 65 70 75 80 Ile Asp Tyr Val Glu Lys Phe Lys Lys Lys Val Thr Leu Thr Ala Asp 85 90 95 Thr Ser Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Asp 100 105 110 Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Lys Phe Asn Tyr Arg Phe 115 120 125 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met 145 150 155 160 Thr Gln Ser Pro Pro Ser Leu Leu Val Thr Leu Gly Gln Pro Ala Ser 165 170 175 Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser Ser Gly Asn Thr 180 185 190 Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser Pro Gln Pro Leu 195 200 205 Ile Tyr Leu Val Ser Lys Leu Glu Ser Gly Val Pro Asp Arg Phe Ser 210 215 220 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Gly Val Glu 225 230 235 240 Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Phe Thr His Tyr Pro 245 250 255 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly 260 265 270 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Val Gln Pro Thr 275 280 285 Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly 290 295 300 Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg 305 310 315 320 Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser 325 330 335 Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu 340 345 350 Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg 355 360 365 Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala 370 375 380 Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala 385 390 395 400 Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr 405 410 415 Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp 420 425 430 Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val 435 440 445 Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro 450 455 460 Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe 465 470 475 480 Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr 485 490 495 Asn Leu Val Lys Asn Lys Cys Val Asn Phe 500 505 <210> 16 <211> 1521 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 16 atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60 tcggctagcc aggtccaact tgttcaatca ggagctgagg tacagcgacc gggggcatct 120 gttaaggtca gttgcaaagc ttcaggctat atattcactg aatactatat gtattgggtc 180 cggcaagcac ctgggcaggg cctggaactc gtcggtagaa ttgaccctga ggatggctct 240 attgactatg tcgaaaaatt taaaaaaaaa gtgacgctca cagcggacac tagcagctca 300 actgcttaca tggagctttc cagcttgacc tctgatgata ctgcggtcta ctactgtgcg 360 cgagggaagt ttaactatag gtttgcatac tggggccaag gaacgctggt aaccgtcagt 420 tcaggaggtg gaggaagcgg cggtggaggg agcggaggcg gaggtagcga tgtggtgatg 480 actcaatctc caccttccct gctcgtaacg ctcggccaac cagcctccat aagctgtcgg 540 tcctcacaga gcctgctcca ttcttctggt aacacttatc ttaactggct tctgcaaaga 600 ccgggtcaat caccccaacc tcttatctat ctggtatcta aactggaatc cggggttccc 660 gatcgattca gcgggtcagg cagtggaacg gactttacct tgaaaatttc aggggtagag 720 gcggaagacg taggggcta ctactgtatg caattcactc attacccgta tacgtttgga 780 caagggacta agctggaaat taaaggcggg ggaggttctg ggggaggtgg gagcggggga 840 gggggttccc gagtgcagcc gacggagtca atcgtgagat tccctaacat aactaatttg 900 tgtccatttg gcgaagtgtt caatgcaacc agattcgcct ccgtctatgc gtggaatcga 960 aaaagaattt caaactgcgt agcggattat tctgtcttgt acaatagtgc ctcctttagt 1020 acgttcaagt gttatggggt gtcaccaacg aagttgaatg atctttgttt cacgaatgtt 1080 tacgctgatt catttgtaat acgcggagac gaagttagac aaatcgcacc agggcagaca 1140 ggcaagatcg cggattataa ttataagctg ccagacgact tcactgggtg cgttatcgca 1200 tggaactcca acaacttgga tagtaaagtg ggcgggaatt acaactacct gtatagactt 1260 ttccgaaagt ccaatttgaa gccattcgaa agggacattt ctactgaaat atatcaagcg 1320 ggatcaacac cttgcaacgg agtggaaggg ttcaactgct actttccgct gcaatcttat 1380 gggtttcaac cgactaatgg agtcgggtat cagccttaca gagttgttgt tctttccttt 1440 gagctgttgc atgccccggc aaccgtatgt gggcccaaga aatctacaaa cctcgttaag 1500 aataaatgcg tgaatttcta g 1521 <210> 17 <211> 496 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 17 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Ala Ser Gln Val Gln Leu Val Gln Ser Gly Ala 20 25 30 Glu Val Gln Arg Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser 35 40 45 Gly Tyr Ile Phe Thr Glu Tyr Tyr Met Tyr Trp Val Arg Gln Ala Pro 50 55 60 Gly Gln Gly Leu Glu Leu Val Gly Arg Ile Asp Pro Glu Asp Gly Ser 65 70 75 80 Ile Asp Tyr Val Glu Lys Phe Lys Lys Lys Val Thr Leu Thr Ala Asp 85 90 95 Thr Ser Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Asp 100 105 110 Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Lys Phe Asn Tyr Arg Phe 115 120 125 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 130 135 140 Gly Ser Asp Val Val Met Thr Gln Ser Pro Pro Ser Leu Leu Val Thr 145 150 155 160 Leu Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu 165 170 175 His Ser Ser Gly Asn Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly 180 185 190 Gln Ser Pro Gln Pro Leu Ile Tyr Leu Val Ser Lys Leu Glu Ser Gly 195 200 205 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 210 215 220 Lys Ile Ser Gly Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met 225 230 235 240 Gln Phe Thr His Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu 245 250 255 Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 260 265 270 Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr 275 280 285 Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser 290 295 300 Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr 305 310 315 320 Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly 325 330 335 Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala 340 345 350 Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly 355 360 365 Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe 370 375 380 Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val 385 390 395 400 Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu 405 410 415 Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser 420 425 430 Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln 435 440 445 Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg 450 455 460 Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys 465,470,475,480 Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 485 490 495 <210> 18 <211> 1491 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 18 atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agctttcgtt 60 tcggctagcc aggtgcaact tgttcagagt ggtgcggagg ttcagcgacc cggcgcgagc 120 gtaaaggtga gctgcaaggc tagcggttat atttttactg agtattatat gtattgggtt 180 cggcaagctc ctggccaagg gctcgaactc gtaggcagaa ttgacccaga ggacggcagt 240 atcgattatg tagagaagtt caaaaagaag gtaacactta cagcagatac gtcaagttcc 300 actgcgtaca tggaactctc aagtctgacc agtgatgata cagcagttta ctattgtgcg 360 cggggtaagt tcaattatag gtttgcgtac tggggacaag gtacactggt taccgtcagc 420 tctggaggtg gtgggagcga cgtcgttatg actcagagtc cgccatcact cctggtgacg 480 ctgggtcaac cagcttctat cagttgccgg agctcccagt cactcctgca ttcatcaggg 540 aacacgtacc tgaattggct ccttcagcgg ccaggacaat ctccccaacc tctgatttat 600 cttgtttcca agcttgagag cggcgtccca gatcgatttt ctgggtccgg tagtggtacg 660 gacttcactt tgaaaatatc cggggttgaa gcagaagacg ttggggttta ctattgcatg 720 cagttcacac attatccgta tacatttggg caagggacca aacttgaaat aaaagggggc 780 ggcggatctg ggggtggtgg tagtggtggt ggtggctctc gagtgcagcc gacggagtca 840 atcgtgagat tccctaacat aactaatttg tgtccatttg gcgaagtgtt caatgcaacc 900 agattcgcct ccgtctatgc gtggaatcga aaaagaattt caaactgcgt agcggattat 960 tctgtcttgt acaatagtgc ctcctttagt acgttcaagt gttatggggt gtcaccaacg 1020 aagttgaatg atctttgttt cacgaatgtt tacgctgatt catttgtaat acgcggagac 1080 gaagttagac aaatcgcacc agggcagaca ggcaagatcg cggattataa ttataagctg 1140 ccagacgact tcactgggtg cgttatcgca tggaactcca acaacttgga tagtaaagtg 1200 ggcgggaatt acaactacct gtatagactt ttccgaaagt ccaatttgaa gccattcgaa 1260 agggacattt ctactgaaat atatcaagcg ggatcaacac cttgcaacgg agtggaaggg 1320 ttcaactgct actttccgct gcaatcttat gggtttcaac cgactaatgg agtcgggtat 1380 cagccttaca gagttgttgt tctttccttt gagctgttgc atgccccggc aaccgtatgt 1440 gggcccaaga aatctacaaa cctcgttaag aataaatgcg tgaatttcta g 1491 <210> 19 <211> 496 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 19 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Ala Ser Gln Val Gln Leu Val Gln Ser Gly Ala 20 25 30 Glu Val Gln Arg Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser 35 40 45 Gly Tyr Ile Phe Thr Glu Tyr Tyr Met Tyr Trp Val Arg Gln Ala Pro 50 55 60 Gly Gln Gly Leu Glu Leu Val Gly Arg Ile Asp Pro Glu Asp Gly Ser 65 70 75 80 Ile Asp Tyr Val Glu Lys Phe Lys Lys Lys Val Thr Leu Thr Ala Asp 85 90 95 Thr Ser Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Cys 100 105 110 Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Lys Phe Asn Tyr Arg Phe 115 120 125 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 130 135 140 Gly Ser Asp Val Val Met Thr Gln Ser Pro Pro Ser Leu Leu Val Thr 145 150 155 160 Leu Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu 165 170 175 His Ser Ser Gly Asn Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly 180 185 190 Gln Ser Pro Gln Pro Leu Ile Tyr Leu Val Ser Lys Leu Glu Ser Gly 195 200 205 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 210 215 220 Lys Ile Ser Gly Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met 225 230 235 240 Gln Phe Thr His Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu 245 250 255 Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 260 265 270 Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr 275 280 285 Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser 290 295 300 Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr 305 310 315 320 Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly 325 330 335 Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala 340 345 350 Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly 355 360 365 Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe 370 375 380 Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val 385 390 395 400 Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu 405 410 415 Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser 420 425 430 Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln 435 440 445 Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg 450 455 460 Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys 465 470 475 480 Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 485 490 495 <210> 20 <211> 1491 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 20 atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60 tcggctagcc aggtccagct cgtacaaagc ggagctgaag tccagaggcc gggagcgagc 120 gtaaaggtct cttgtaaagc ctctggatac atttttacgg agtactacat gtattgggtt 180 aggcaggcac ctggtcaagg tttggaattg gtcgggagaa tagaccctga agatgggtca 240 atagattatg tcgaaaaatt caagaagaaa gtaaccctca cagcagatac aagctcaagc 300 actgcttata tggaactttc tagcctcacc tcttgcgaca ccgcagttta ttactgtgcc 360 agagggaaat ttaattacag gtttgcctac tggggacaag gaacgttggt aacggtatca 420 tccggcggtg gtggatcaga cgtggttatg acacagtccc caccgagctt gcttgttact 480 ctcgggcagc ctgccagcat ctcatgcaga tcatctcaga gccttcttca ttcatctggc 540 aacacctacc ttaattggct gctccaacga ccgggtcagt ccccccaacc cttgatatac 600 ttggttagta aactcgaaag tggagttccg gacagattct ctggatctgg aagcggaact 660 gattttaccc tcaagatatc cggcgtagag gcggaagatg tcggggttta ctattgtatg 720 780 ggaggttctg gaggcgggg cagcggtggt ggaggttccc gagtgcagcc gacggagtca 840 atcgtgagat tccctaacat aactaatttg tgtccatttg gcgaagtgtt caatgcaacc 900 agattcgcct ccgtctatgc gtggaatcga aaaagaattt caaactgcgt agcggattat 960 tctgtcttgt aacatagtgc ctcctttagt acgttcaagt gttatggggt gtcaccaacg 1020 aagttgaatg atctttgttt cacgaatgtt tacgctgatt catttgtaat acgcggagac 1080 gaagttagac aaatcgcacc agggcagaca ggcaagatcg cggattataa ttataagctg 1140 ccagacgact tcactgggtg cgttatcgca tggaactcca acaacttgga tagtaaagtg 1200 ggcgggaatt acaactacct gtatagactt ttccgaaagt ccaatttgaa gccattcgaa 1260 agggacattt ctactgaaat atatcaagcg ggatcaacac cttgcaacgg agtggaaggg 1320 ttcaactgct actttccgct gcaatcttat gggttcaac cgactaatgg agtcggtt 1380 cagccttaca gagttgttgt tctttccttt gagctgttgc atgccccggc aaccgtagt 1440 gggcccaaga atctacaaa cctcgttaag aaaatgcg tgaatttcta g 1491 <210> 21 <211> 356 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 21 Met Asp Ala Met Lys Arg Gly Leu Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Ala Ser Phe Ser Gln Gln Ile Tyr Gly Val Val 20 25 30 Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn Val Pro Leu Lys Glu 35 40 45 Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala Glu Leu Glu Asn Ser 50 55 60 Glu Phe Arg Ala Phe Ser Ser Phe Lys Asn Arg Val Tyr Leu Asp Thr 65 70 75 80 Val Ser Gly Ser Leu Thr Ile Tyr Asn Leu Thr Ser Ser Asp Glu Asp 85 90 95 Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr Asp Thr Met Lys Phe Phe 100 105 110 Leu Tyr Val Leu Glu Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe 130 135 140 Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr 145 150 155 160 Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys 165 170 175 Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe 180 185 190 Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr 195 200 205 Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln 210 215 220 Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu 225 230 235 240 Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu 245 250 255 Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg 260 265 270 Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr 275 280 285 Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr 290 295 300 Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr 305 310 315 320 Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro 325 330 335 Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys 340 345 350 Cys Val Asn Phe 355 <210> 22 <211> 1071 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 22 atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60 tcggctagct tcagtcagca aatctacggt gtcgtgtatg gcaatgtgac atttcacgtg 120 ccgagtaacg tccctctcaa ggaggttttg tggaagaagc agaaggataa ggtggccgag 180 ctggagaact ctgagtttcg ggctttctcc tctttcaaaa atcgagtata cctcgataca 240 gtcagtgggt ctctgaccat atataacctt acttcctctg atgaggacga atatgagatg 300 gaatctccga acataacaga tactatgaag ttcttcctct atgtactcga atcagggggt 360 ggtgggtctg gagggggcgg ttcaggggga ggcgggagtc gagtgcagcc gacggagtca 420 atcgtgagat tccctaacat aactaatttg tgtccatttg gcgaagtgtt caatgcaacc 480 agattcgcct ccgtctatgc gtggaatcga aaaagaattt caaactgcgt agcggattat 540 tctgtcttgt acaatagtgc ctcctttagt acgttcaagt gttatggggt gtcaccaacg 600 aagttgaatg atctttgttt cacgaatgtt tacgctgatt catttgtaat acgcggagac 660 gaagttagac aaatcgcacc agggcagaca ggcaagatcg cggattataa ttataagctg 720 ccagacgact tcactgggtg cgttatcgca tggaactcca acacttgga tagtaagtg 780 ggcgggaatt acactacct gtatagactt ttccgaagt ccaatttgaa gccattcgaa 840 agggacattt ctactgaat atatcaagcg ggatcacac cttgcaacgg agtggaaggg 900 ttcaactgct actttccgct gcaatcttat gggttcaac cgactaatgg agtcggt 960 cagccttaca gagttgttgt tctttccttt gagctgttgc atgccccggc aaccgtagt 1020 gggcccaaga atctacaaa cctcgttaag aaaatgcg tgaatttcta g 1071 <210> 23 <211> 356 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 23 Met Asp Ala Met Lys Arg Gly Leu Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Ala Ser Phe Ser Gln Gln Ile Tyr Gly Val Val 20 25 30 Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn Val Pro Leu Lys Glu 35 40 45 Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala Glu Leu Glu Asn Ser 50 55 60 Glu Phe Arg Ala Phe Ser Ser Phe Lys Asn Arg Val Tyr Leu Asp Thr 65 70 75 80 Val Ser Gly Ser Leu Thr Ile Tyr Asn Leu Thr Ser Ser Asp Glu Asp 85 90 95 Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr Asp Thr Met Lys Phe Phe 100 105 110 Leu Tyr Val Leu Glu Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe 130 135 140 Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr 145 150 155 160 Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys 165 170 175 Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe 180 185 190 Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr 195 200 205 Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln 210 215 220 Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu 225 230 235 240 Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu 245 250 255 Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg 260 265 270 Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr 275 280 285 Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr 290 295 300 Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr 305 310 315 320 Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro 325 330 335 Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys 340 345 350 Cys Val Asn Phe 355 <210> 24 <211> 1071 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 24 atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60 tcggctagct tcagtcagca aatctacggt gtcgtgtatg gcaatgtgac atttcacgtg 120 ccgagtaacg tccctctcaa ggaggttttg tggaagaagc agaaggataa ggtggccgag 180 ctggagaact ctgagtttcg ggctttctcc tctttcaaaa atcgagtata cctcgataca 240 gtcagtgggt ctctgaccat atataacctt acttcctctg atgaggacga atatgagatg 300 gaatctccga acataacaga tactatgaag ttcttcctct atgtactcga atcagggggt 360 ggtgggtctg gagggggcgg ttcaggggga ggcgggagtc gagtgcagcc gacggagtca 420 atcgtgagat tccctaacat aactaatttg tgtccatttg gcgaagtgtt caatgcaacc 480 agattcgcct ccgtctatgc gtggaatcga aaaagaattt caaactgcgt agcggattat 540 tctgtcttgt acaatagtgc ctcctttagt acgttcaagt gttatggggt gtcaccaacg 600 aagttgaatg atctttgttt cacgaatgtt tacgctgatt catttgtaat acgcggagac 660 gaagttagac aaatcgcacc agggcagaca ggcaacatcg cggattataa ttataagctg 720 ccagacgact tcactgggtg cgttatcgca tggaactcca acaacttgga tagtaaagtg 780 ggcgggaatt acaactacct gtatagactt ttccgaaagt ccaatttgaa gccattcgaa 840 agggacattt ctactgaaat atatcaagcg ggatcaacac cttgcaacgg agtgaaaggg 900 ttcaactgct actttccgct gcaatcttat gggtttcaac cgacttatgg agtcgggtat 960 cagccttaca gagttgttgt tctttccttt gagctgttgc atgccccggc aaccgtatgt 1020 gggcccaaga aatctacaaa cctcgttaag aataaatgcg tgaatttcta g 1071 <210> 25 <211> 560 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 25 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Ala Ser Glu Val Gln Leu Val Glu Ser Gly Gly 20 25 30 Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser 35 40 45 Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro 50 55 60 Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn 65 70 75 80 Tyr Ala Thr Tyr Tyr Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser 85 90 95 Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Lys 100 105 110 Thr Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg His Gly Asn Phe Gly 115 120 125 Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 130 135 140 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 145 150 155 160 Gly Gly Ser Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser 165 170 175 Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val 180 185 190 Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala 195 200 205 Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro 210 215 220 Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu 225 230 235 240 Ser Gly Val Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp 245 250 255 Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 260 265 270 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg 275 280 285 Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu 290 295 300 Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr 305 310 315 320 Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val 325 330 335 Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser 340 345 350 Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser 355 360 365 Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr 370 375 380 Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly 385 390 395 400 Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly 405 410 415 Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro 420 425 430 Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro 435 440 445 Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr 450 455 460 Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val 465,470,475,480 Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro 485 490 495 Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Ala 500 505 510 Val Gly Gln Asp Thr Gln Glu Val Ile Val Val Pro His Ser Leu Pro 515 520 525 Phe Lys Val Val Val Ile Ser Ala Ile Leu Ala Leu Val Val Leu Thr 530 535 540 Ile Ile Ser Leu Ile Ile Leu Ile Met Leu Trp Gln Lys Lys Pro Arg 545,550,555,560 <210> 26 <211> 1683 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 26 atggacgcta tgaagagggg tctctgctgt gtcctccttc tgtgcggtgc cgtctttgtg 60 tctgcctctg aagtacaatt ggtggagtct gggggaggcc ttgtccaacc aggcgggtct 120 ttgcgactgt catgcgccgc ctctgggtt acattcaaca catatgctat gaattgggtc 180 cgacaagcgc cgggcaaagg actggaatgg gtcgcccgca ttcgctcaaa gtataacaat 240 tatgccacat attacgctgc gtcagtgaaa ggacggttca caatatcacg agacgattct 300 aaaaactcac tgtatctcca aatgaatagc ctcaaaacgg aggacactgc agtcttattat 360 tgcgctcgac atggcaattt tggaatagc tatgtatcct ggtttgcgta ctggggccaa 420 gggacgctgg tgactgtttc aagcggtggc ggcggatccg gtggaggg cagcggcggt 480 ggtggctccc aaaccgttgt cacccaagag ccttctctca cagtgtcccc tggtggcaca 540 gtgactttga cttgtaggtc ttctacaggc gcggtcacga ccagtaatta tgctaattgg 600 gtccagcaga aaccggggca ggcgccaaga ggtctcatag gggggacaaa taaacgcgcg 660 ccgggtacgc ctgcacgatt cagcggatc ttgctgggcg gcaaggcagc cttacgctc 720 tccggggttc agcccgaaga cgaggcagaa tactattgtg cgctgtggta tagtaatctt 780 tgggtatttg gaggaggtac aaaattgacc gtgttgggtg gaggaggatc cggggggaggc 840 ggaagcgggg gtggcggttc tcgagtgcag ccgacggagt caatcgtgag attccctaac 900 ataactaatt tgtgtccatt tggcgaagtg ttcaatgcaa ccagattcgc ctccgtctat 960 gcgtggaatc gaaaaagaat ttcaaactgc gtagcggatt attctgtctt gtacaatagt 1020 gcctccttta gtacgttcaa gtgttatggg gtgtcaccaa cgaagttgaa tgatctttgt 1080 ttcacgaatg tttacgctga ttcatttgta atacgcggag acgaagttag acaaatcgca 1140 ccagggcaga caggcaacat cgcggattat aattataagc tgccagacga cttcactggg 1200 tgcgttatcg catggaactc caacaacttg gatagtaaag tgggcgggaa ttacaactac 1260 ctgtatagac ttttccgaaa gtccaatttg aagccattcg aaagggacat ttctactgaa 1320 atatatcaag cgggatcaac accttgcaac ggagtgaaag ggttcaactg ctactttccg 1380 ctgcaatctt atgggtttca accgacttat ggagtcgggt atcagcctta cagagttgtt 1440 gttctttcct ttgagctgtt gcatgccccg gcaaccgtat gtgggcccaa gaaatctaca 1500 aacctcgtta agaataaatg cgtgaatttc aacgctgtgg ggcaagatac tcaggaggtc 1560 atcgttgtcc cacattcact gccttttaag gtcgttgtga tatccgcaat tcttgcgctg 1620 gtggtactta caataataag cctcataatc cttataatgc tctggcaaaa gaagccacgc 1680 tag 1683 <210> 27 <211> 509 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 27 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Ala Ser Gln Val Gln Leu Gln Gln Ser Gly Pro 20 25 30 Glu Val Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser 35 40 45 Gly Tyr Thr Phe Thr Ser Tyr Val Ile His Trp Val Arg Gln Lys Pro 50 55 60 Gly Gln Gly Leu Asp Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly 65 70 75 80 Thr Asp Tyr Asp Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp 85 90 95 Thr Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu 100 105 110 Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Lys Asp Asn Tyr Ala Thr 115 120 125 Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 145 150 155 160 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 165 170 175 Glu Arg Val Thr Met Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 180 185 190 Thr Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 195 200 205 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 210 215 220 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 225 230 235 240 Ile Ser Ser Val Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 245 250 255 Tyr Tyr Ser Tyr Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 260 265 270 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg 275 280 285 Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu 290 295 300 Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr 305 310 315 320 Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val 325 330 335 Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser 340 345 350 Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser 355 360 365 Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr 370 375 380 Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly 385 390 395 400 Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly 405 410 415 Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro 420 425 430 Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro 435 440 445 Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr 450 455 460 Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val 465 470 475 480 Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro 485 490 495 Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn 500 505 <210> 28 <211> 1533 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 28 atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60 tcggctagcc aggtacagct ccaacagtcc ggtccagagg tcgtgaaacc tggtgcatcc 120 gttaaaatga gttgcaaagc ttccggttat acgttcacat cttacgttat ccattgggtt 180 cgccagaaac cgggtcaggg acttgactgg atcggttata taaatccgta caatgatggc 240 accgactacg acgagaaatt caaaggaag gctacactta cgtctgatac gagtacctca 300 actgcttaca tggaactttc ctctttgagg tctgaagaca cggcagttta ctattgtgcc 360 agaaaaag ataattatgc cacgggagct tggttcgcat actgggggca aggaacattg 420 gttacggtaa gtagtgggg aggcgggaagt ggggcggag gctcaggtgg tggggggttca 480 gatatgtga tgacacagag cccggacagtga cttgcagtga gtctcgggcga acgcgttacc 540 atgaattgta agagtgtca gtctctcttg tatagcacta atcaaaagaa ttatttggca 600 tggtaccaac agaagccggg tcagtctcct aaactgctga tttactgggc gtcaacacgg 660 gagtctggag ttccggatcg attctccgga tctggaagcg gtactgactt caccctcacg 720 atctcaagtg tgcaagcgga ggacgttgcg gtgtattact gccaacagta ttattcttac 780 cgaacattcg gcggtgggac caagctggaa ataaaagggg gtggcggcag tggcggtggc 840 gggtctggag gtgggggttc ccgagtgcag ccgacggagt caatcgtgag attccctaac 900 ataactaatt tgtgtccatt tggcgaagtg ttcaatgcaa ccagattcgc ctccgtctat 960 gcgtggaatc gaaaaagaat ttcaaactgc gtagcggatt attctgtctt gtacaatagt 1020 gcctccttta gtacgttcaa gtgttatggg gtgtcaccaa cgaagttgaa tgatctttgt 1080 ttcacgaatg tttacgctga ttcatttgta atacgcggag acgaagttag acaaatcgca 1140 ccagggcaga caggcaagat cgcggattat aattataagc tgccagacga cttcactggg 1200 tgcgttatcg catggaactc caacaacttg gatagtaaag tgggcgggaa ttacaactac 1260 ctgtatagac ttttccgaaa gtccaatttg aagccattcg aaagggacat ttctactgaa 1320 atatatcaag cgggatcaac accttgcaac ggagtggaag ggttcaactg ctactttccg 1380 ctgcaatctt atgggtttca accgactaat ggagtcgggt atcagcctta cagagttgtt 1440 gttctttcct ttgagctgtt gcatgccccg gcaaccgtat gtgggcccaa gaaatctaca 1500 aacctcgtta agaataaatg cgtgaatttc tag 1533 <210> 29 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 29 Gly Gly Gly Gly Ser 1 5

Claims

1. A vaccine for inducing an immune response against a pathogen in a mammal, comprising a conjugated polypeptide comprising an antigen derived from the pathogen linked to a ligand or antibody fragment that specifically binds to a surface protein present on an immune cell.

2. 2. The vaccine of claim 1, wherein the surface protein is an abundant T cell surface protein involved in signal transduction and / or adhesion.

3. 3. The vaccine of claim 2, wherein the abundant T cell surface protein is CD2, CD3, CD4 or CD5.

4. 4. The vaccine of claim 3, wherein the abundant T cell surface protein is CD2 or CD3.

5. The vaccine of claim 1, wherein the immune cell is a T cell or an antigen-presenting cell (APC).

6. The vaccine of claim 1, wherein the ligand is an ectodomain of a cell adhesion molecule.

7. The vaccine of claim 6, wherein the cell adhesion molecule is CD58.

8. The vaccine of claim 1, wherein the surface protein is preferentially expressed by T cells.

9. The vaccine of claim 1, wherein the antibody fragment is an scFv chain derived from the antibody.

10. The vaccine of claim 1, wherein the conjugated polypeptide further comprises a lipid anchor, a transmembrane segment, a multimerization domain, or any combination of these elements.

11. 11. The vaccine of claim 10, wherein the lipid anchor is a glycosylphosphatidylinositol anchor.

12. 11. The vaccine of claim 10, wherein the addition of the lipid anchor is directed by a signal sequence.

13. The vaccine of claim 12, wherein the signal sequence is derived from CD55.

14. 11. The vaccine of claim 10, wherein the transmembrane segment is derived from a PDGF receptor, glycophorin A, or SARS-CoV-2 spike protein.

15. 11. The vaccine of claim 10, wherein the multimerization domain is derived from T4 fibritin.

16. The vaccine of claim 10, wherein the multimerization domain is an Fc domain.

17. 17. The vaccine of claim 16, wherein the Fc domain is located at the C-terminus of the conjugated polypeptide.

18. 17. The vaccine of claim 16, wherein the Fc domain is a human IgG1 Fc domain.

19. The vaccine of claim 1, wherein the conjugated polypeptide is a fusion protein comprising the antigen and the ligand or the antibody fragment within a single polypeptide chain.

20. 20. The vaccine of claim 19, wherein the antibody fragment is an scFv chain derived from an antibody, and the VH and VL regions of the scFv are separated by a flexible linker.

21. 21. The vaccine of claim 20, wherein the flexible linker is 12 amino acids or more in length and the conjugated polypeptide preferentially binds to the surface protein as a monomer.

22. 21. The vaccine of claim 20, wherein the flexible linker is less than 12 amino acids in length and the conjugated polypeptide preferentially binds to the surface protein as a multimer.

23. 23. The vaccine of claim 22, wherein the multimer is stabilized by disulfide bonds between the monomer units.

24. 23. The vaccine of claim 22, wherein the flexible linker is 5 amino acids in length.

25. The vaccine of claim 1, wherein the conjugated polypeptide further comprises a tPA leader sequence.

26. 26. The vaccine of claim 25, wherein the tPA leader sequence is 23 amino acids in length.

27. 10. The vaccine of claim 1, further comprising a second antigen from the pathogen.

28. 2. The vaccine of claim 1, wherein the pathogen is a virus.

29. 29. The vaccine of claim 28, wherein the virus is SARS-CoV-2.

30. 30. The vaccine of claim 29, wherein the antigen present in the conjugated polypeptide comprises the SARS-CoV-2 spike glycoprotein or a fragment thereof.

31. 31. The vaccine of claim 30, wherein the fragment of the SARS-CoV-2 spike glycoprotein comprises the S1 domain or the receptor binding domain (RBD).

32. 30. The vaccine of claim 29, wherein the second antigen comprises a SARS-CoV-2E, M, N, nsp3, nsp4 or nsp6 protein, or a fragment thereof.

33. 33. The vaccine of claim 32, wherein the second antigen comprises a fusion protein comprising SARS-CoV-2E and an M protein, or a fragment thereof.

34. The vaccine of claim 1, wherein the mammal is a human.

35. 10. The vaccine of claim 1, formulated for electroporation or subcutaneous injection.

36. 2. The vaccine of claim 1, wherein the conjugated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 and SEQ ID NO:

27.

37. A vaccine for inducing an immune response against a pathogen in a mammal, comprising a polynucleotide encoding a conjugate polypeptide comprising an antigen derived from the pathogen fused to a ligand or antibody fragment that specifically binds to a surface protein present on an immune cell.

38. 38. The vaccine of claim 37, wherein the surface protein is an abundant T cell surface protein involved in signal transduction and / or adhesion.

39. 39. The vaccine of claim 38, wherein the abundant T cell surface protein is CD2, CD3, CD4 or CD5.

40. 40. The vaccine of claim 39, wherein the abundant T cell surface protein is CD2 or CD3.

41. 38. The vaccine of claim 37, wherein the immune cell is a T cell or an antigen-presenting cell (APC).

42. 38. The vaccine of claim 37, wherein the ligand is an ectodomain of a cell adhesion molecule.

43. 43. The vaccine of claim 42, wherein the cell adhesion molecule is CD58.

44. 38. The vaccine of claim 37, wherein the surface protein is preferentially expressed by T cells.

45. 38. The vaccine of claim 37, wherein the antibody fragment is an scFv chain derived from the antibody.

46. 38. The vaccine of claim 37, wherein the conjugated polypeptide further comprises a lipid anchor, a transmembrane segment, a multimerization domain, or any combination of these elements.

47. 47. The vaccine of claim 46, wherein the lipid anchor is a glycosylphosphatidylinositol anchor.

48. 47. The vaccine of claim 46, wherein the addition of the lipid anchor is directed by a signal sequence.

49. 49. The vaccine of claim 48, wherein the signal sequence is derived from CD55.

50. 47. The vaccine of claim 46, wherein the transmembrane segment is derived from a PDGF receptor, glycophorin A, or SARS-CoV-2 spike protein.

51. 47. The vaccine of claim 46, wherein the multimerization domain is derived from T4 fibritin.

52. 47. The vaccine of claim 46, wherein the multimerization domain is an Fc domain.

53. 53. The vaccine of claim 52, wherein the Fc domain is located at the C-terminus of the conjugated polypeptide.

54. 53. The vaccine of claim 52, wherein the Fc domain is a human IgG1 Fc domain.

55. 46. ​​The vaccine of claim 45, wherein the VH and VL regions of the scFv are separated by a flexible linker within the conjugated polypeptide.

56. 56. The vaccine of claim 55, wherein the flexible linker is 12 amino acids or more in length and the conjugated polypeptide preferentially binds to the surface protein as a monomer.

57. 56. The vaccine of claim 55, wherein the flexible linker is less than 12 amino acids in length and the conjugated polypeptide preferentially binds to the surface protein as a multimer.

58. 58. The vaccine of claim 57, wherein the multimer is stabilized by disulfide bonds.

59. 58. The vaccine of claim 57, wherein the flexible linker is 5 amino acids in length.

60. 38. The vaccine of claim 37, wherein the conjugated polypeptide comprises a tPA leader sequence.

61. 61. The vaccine of claim 60, wherein the tPA leader sequence is 23 amino acids in length.

62. 38. The vaccine of claim 37, further comprising a second polynucleotide encoding a second antigen from the pathogen.

63. 38. The vaccine of claim 37, wherein the pathogen is a virus.

64. 64. The vaccine of claim 63, wherein the virus is SARS-CoV-2.

65. 65. The vaccine of claim 64, wherein the antigen present in the conjugated polypeptide comprises the SARS-CoV-2 spike glycoprotein or a fragment thereof.

66. 66. The vaccine of claim 65, wherein the fragment of the SARS-CoV-2 spike glycoprotein comprises the S1 domain or the receptor binding domain (RBD).

67. 63. The vaccine of claim 62, wherein the second antigen comprises a SARS-CoV-2E, M, N, nsp3, nsp4 or nsp6 protein, or a fragment thereof.

68. 68. The vaccine of claim 67, wherein the second antigen comprises a fusion protein comprising SARS-CoV-2E and an M protein, or a fragment thereof.

69. 38. The vaccine of claim 37, wherein the mammal is a human.

70. 38. The vaccine of claim 37, formulated for electroporation or subcutaneous injection.

71. 63. The vaccine of claim 37 or 62, wherein the polynucleotide encoding the conjugate polypeptide and / or the second polynucleotide encoding the second antigen are codon-optimized.

72. the polynucleotide encoding the conjugated polypeptide is present in a first expression cassette, the polynucleotide being operably linked to a first promoter; and / or the second polynucleotide encoding the second antigen is present in a second expression cassette, the second polynucleotide being operably linked to a second promoter; 63. The vaccine of claim 37 or 62.

73. 73. The vaccine of claim 72, wherein the second promoter is a mammalian promoter.

74. 74. The vaccine of claim 73, wherein the mammalian promoter is an EF-1α promoter.

75. 73. The vaccine of claim 72, wherein the first and / or second expression cassette is present in a vector.

76. 76. The vaccine of claim 75, wherein the vector is administered as naked DNA.

77. 76. The vaccine of claim 75, wherein the vector is a viral vector.

78. 78. The vaccine of claim 77, wherein the viral vector is a cytomegalovirus (CMV), adenovirus, or adeno-associated virus (AAV) vector.

79. 38. The vaccine of claim 37, further comprising an in vivo transfection reagent.

80. 80. The vaccine of claim 79, wherein the in vivo transfection reagent is in vivo-jetPEI™.

81. 38. The vaccine of claim 37, formulated for subcutaneous transfection.

82. The vector is (a) a CMV genome or a portion thereof containing the first expression cassette, or the first and second expression cassettes; (b) bacterial artificial chromosome (BAC) sequences containing origins of replication; (c) the first terminase complex recognition locus (TCRL1) containing at least two viral tandem repeat sequences; (d) a second terminase complex recognition locus (TCRL2) containing at least two viral direct repeat sequences; a circular CMV vector comprising the CMV genome or portion thereof is flanked by TCRL1 and TCRL2, thereby defining a first region of the circular vector extending from TCRL1 to TCRL2 and comprising the CMV genome or portion thereof; the BAC sequence is located in a second region of the circular vector extending from TCRL1 to TCRL2 and not including the CMV genome or a portion thereof; 79. The vaccine of claim 78.

83. The vector is (a) a CMV genome or a portion thereof containing the first expression cassette, or the first and second expression cassettes; (b) a sequence containing an origin of replication that functions in unicellular organisms; (c) one or more terminase complex recognition loci (TCRLs) comprising recombinantly introduced polynucleotide sequences capable of directing cleavage by the HV terminase complex; a circular CMV vector comprising the CMV genome or portion thereof is separated from a sequence comprising the origin of replication by a TCRL; the CMV genome or portion thereof is flanked on at least one end by a TCRL; The sequence containing the replication origin is flanked on at least one end by a TCRL.

79. The vaccine of claim 78.

84. 83. The vaccine of claim 82, wherein the one or more terminase complex recognition loci include a Pac1 site and a Pac2 site.

85. 85. The vaccine of claim 84, wherein all of the terminase complex recognition loci include a Pac1 site and a Pac2 site.

86. 78. The vaccine of claim 77, wherein the first promoter is a viral promoter.

87. 87. The vaccine of claim 86, wherein the viral promoter is a pp65b promoter.

88. 79. The vaccine of claim 78, wherein the vector is a CMV vector, and the CMV is Towne HCMV.

89. 38. The vaccine of claim 37, wherein the polynucleotide encoding the conjugate polypeptide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26 and SEQ ID NO:

28.

90. A conjugated polypeptide comprising an antigen derived from a pathogen linked to a ligand or antibody fragment that specifically binds to a surface protein present on an immune cell.

91. 91. The conjugated polypeptide of claim 90, wherein said surface protein is an abundant T cell surface protein involved in signal transduction and / or adhesion.

92. 91. The conjugated polypeptide of claim 90, wherein said surface protein is CD2, CD3, CD4 or CD5.

93. 93. The conjugated polypeptide of claim 92, wherein said surface protein is CD2 or CD3.

94. 91. The conjugated polypeptide of claim 90, wherein the immune cell is a T cell or an antigen-presenting cell (APC).

95. 91. The conjugated polypeptide of claim 90, wherein the ligand is an ectodomain of a cell adhesion molecule.

96. 96. The conjugated polypeptide of claim 95, wherein said cell adhesion molecule is CD58.

97. 91. The conjugated polypeptide of claim 90, wherein said surface protein is preferentially expressed by T cells.

98. 91. The conjugated polypeptide of claim 90, wherein the antibody fragment is an scFv chain derived from an antibody.

99. 91. The conjugated polypeptide of claim 90, further comprising a lipid anchor, a transmembrane segment, a multimerization domain, or any combination of these elements.

100. 100. The conjugated polypeptide of claim 99, wherein said lipid anchor is a glycosylphosphatidylinositol anchor.

101. 100. The conjugated polypeptide of claim 99, wherein the addition of the lipid anchor is directed by a signal sequence.

102. 102. The conjugated polypeptide of claim 101, wherein said signal sequence is derived from CD55.

103. 100. The conjugated polypeptide of claim 99, wherein said transmembrane segment is derived from a PDGF receptor, glycophorin A, or SARS-CoV-2 spike protein.

104. 100. The conjugated polypeptide of claim 99, wherein said multimerization domain is derived from T4 fibritin.

105. 100. The conjugated polypeptide of claim 99, wherein said multimerization domain is an Fc domain.

106. 106. The conjugated polypeptide of claim 105, wherein the Fc domain is located at the C-terminus of the conjugated polypeptide.

107. 106. The conjugated polypeptide of claim 105, wherein said Fc domain is a human IgG1 Fc domain.

108. 99. The conjugated polypeptide of claim 98, wherein the antibody fragment is an scFv chain derived from an antibody, the VH and VL regions of the scFv being separated by a flexible linker.

109. 109. The conjugated polypeptide of claim 108, wherein said flexible linker is 12 amino acids or more in length and said conjugated polypeptide preferentially binds to said surface protein as a monomer.

110. 109. The conjugated polypeptide of claim 108, wherein said flexible linker is less than 12 amino acids in length and said conjugated polypeptide preferentially binds to said surface protein as a multimer.

111. 111. The conjugated polypeptide of claim 110, wherein the multimer is stabilized by disulfide bonds between the monomer units.

112. 111. The conjugated polypeptide of claim 110, wherein said flexible linker is 5 amino acids in length.

113. 91. The conjugated polypeptide of claim 90, further comprising a tPA leader sequence.

114. 114. The conjugated polypeptide of claim 113, wherein said tPA leader sequence is 23 amino acids in length.

115. 91. The conjugated polypeptide of claim 90, wherein the pathogen is a virus.

116. 116. The conjugated polypeptide of claim 115, wherein the virus is SARS-CoV-2.

117. 117. The conjugated polypeptide of claim 116, wherein the antigen comprises the SARS-CoV-2 spike glycoprotein or a fragment thereof.

118. 118. The conjugated polypeptide of claim 117, wherein said fragment of the SARS-CoV-2 spike glycoprotein comprises the S1 domain or the receptor binding domain (RBD).

119. 91. The conjugated polypeptide of claim 90, comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 and SEQ ID NO:

27.

120. A conjugated polypeptide comprising: (i) a tissue plasminogen activator (tPA) signal sequence; (ii) a single-chain variable fragment (scFv) that specifically binds to CD2, CD3, or CD4; (iii) a flexible linker; and (iv) a SARS-CoV-2 receptor binding domain (RBD).

121. The conjugated polypeptide of claim 120, comprising the amino acid sequence of SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID 25 or SEQ ID NO:

27.

122. 122. A polynucleotide encoding the conjugated polypeptide of claim 90 or claim 121.

123. 123. The polynucleotide of claim 122, which is codon optimized.

124. The polynucleotide of claim 123, comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26 and SEQ ID NO:

28.

125. 123. An expression cassette comprising the polynucleotide of claim 122.

126. 126. The expression cassette of claim 125, comprising the nucleotide sequence of SEQ ID NO:

8.

127. 126. A vector comprising the expression cassette of claim 125.

128. 128. The vector of claim 127, which is a plasmid.

129. 128. The vector of claim 127, which is an adenoviral vector.

130. 99. A diabody comprising the conjugated polypeptide of claim 98.

131. 100. A dimer comprising the conjugated polypeptide of claim 99.

132. 132. A vaccine comprising the conjugated polypeptide of claim 90 or claim 120, the polynucleotide of claim 122, the expression cassette of claim 125, the vector of claim 127, the diabody of claim 130, or the dimer of claim 131.

133. 136. A method of inducing an immune response against a pathogen in a mammal, comprising administering to the mammal a vaccine according to any one of claims 1, 37 or 132.

134. 134. The method of claim 133, wherein the vaccine is administered subcutaneously or by electroporation.

135. 134. The method of claim 133, wherein said method induces a neutralizing antibody response in said mammal against said antigen present in said conjugated polypeptide, said neutralizing response being substantially greater than any antibody-dependent enhancement of infection (ADEI) induced in said mammal by said vaccine.

136. 136. The method of claim 135, wherein the vaccine does not substantially induce ADEI in the mammal.

137. The method of claim 133, wherein the method induces both a CD4+ and a CD8+ T cell response against the second antigen.

138. 134. The method of claim 133, comprising administering to said mammal a DNA prime comprising the vector of claim 125 by electroporation, followed by boosting with an adenoviral vector encoding the RBD.

139. 139. The method of claim 138, wherein said booster is administered about 28 days later.

140. 134. The method of claim 133, wherein the mammal is a human.

Images