CN116761811A - Conjugated polypeptides and vaccines for inducing immune responses - Google Patents

Abstract

Methods and compositions for inducing an immune response in a mammal to one or more antigens are disclosed.

Description

Conjugated polypeptides and vaccines for inducing immune responses

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/027,250 filed on May 19, 2020 and U.S. Provisional Application No. 63/058,362 filed on July 29, 2020, the disclosures of which are hereby incorporated by reference in their entireties for all purposes.

STATEMENT REGARDING INTERESTS IN INVENTS PERFORMED UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Grant No. R01AI118451 awarded by the National Institutes of Health. The government has certain rights in this invention.

background

Traditional vaccine development against previously unknown pathogens takes years, but protecting human health may require a more rapid response of months (1). However, accelerated development is complicated by multiple factors, including the lack of appropriate animal models for emerging pathogens; the risk of antibody-dependent enhancement of infectivity (ADEI), which can occur whenever suboptimal antibody responses are induced (2); and the difficulty of developing new manufacturing processes for subunit, attenuated, or vectored vaccines. In addition, although inducing high titers of neutralizing antibodies (nAbs) seems to be an obvious approach, we do not know what titers of nAbs are protective in practice, nor do we know how this threshold varies at the extremes of age and comorbidity.

With regard to SARS-CoV-2, the definition of the immune correlates of successful vaccination is unclear. Most coronavirus vaccines currently in development target the most variable part of the spike glycoprotein and induce antibody responses only against the virus present in the vaccine. In SARS-CoV-1, escape mutants occurred in the presence of a single nAb against the receptor binding domain (RBD) or a combination of two nAbs in vitro and in mice (2,3). Moreover, as mentioned above, vaccines designed to specifically elicit antibodies must be handled with additional caution due to the potential for ADEI, especially when antibody levels are low (4). Indeed, highly concentrated antisera against SARS-CoV-1 were shown to neutralize viral infectivity, whereas diluted antibodies induced ADEI in human pronuclear cell cultures, resulting in a cytopathic effect 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 were shown to induce non-neutralizing antibodies, and immunized animals were not protected. Instead, they experienced adverse effects such as exacerbated hepatitis, increased morbidity, and a stronger inflammatory response (8,9).

T cell responses elicited by CoV vaccines also play a key role in protection and clearance. It was not possible to clear MERS-CoV infection in T cell-deficient mice, but this was achieved in mice lacking B cells (10). In addition, airway memory CD4 ⁺ T cells were shown to mediate protective immunity against SARS-CoV-1 and MERS-CoV (11). However, most vaccine types do not elicit significant numbers of memory CD4 ⁺ T cells.

CMV vector vaccines can elicit strong antibody responses. Although CMV vaccines elicit weak antibody responses to some transgenes driven by heterologous promoters, CMV infection and vaccination elicit strong antibody responses to proteins expressed under the control of the endogenous pp65b promoter. For example, it has been found that rhesus macaques vaccinated with a CMV vaccine carrying the Ebola virus glycoprotein (GP) under the control of the pp65b promoter can produce GP-specific antibodies (21).

Another important property of CMV vectors for use against emerging pathogens is the ability to be re-administered to previously exposed individuals. Because of this ability, one could conceivably reuse CMV vector vaccines over time to protect against a range of emerging threats.

However, despite the immunological advantages of CMV vaccines when these vaccines are delivered as live viruses, practical barriers have hampered the rapid development of CMV vaccines for human clinical use. One obstacle is the enormous difficulty in large-scale production of uniform test samples from slow-growing and mutable betaherpesviruses (29).

Therefore, there is a need for new, safe, effective and scalable vaccines and vaccination methods that can provide a strong antibody response against pathogens such as SARS-CoV-2 and may also enhance T cell responses. The present disclosure addresses this need and also provides other advantages.

Overview

In one aspect, the present disclosure provides a vaccine for inducing an immune response against 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 immune cells.

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 extracellular domain of a cell adhesion molecule. In some embodiments, the cell adhesion molecule is CD58. In some embodiments, the surface protein is preferentially or only 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, the addition of the lipid anchor is guided 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 a SARS-CoV-2 spike protein. In some embodiments, the multimerization domain is derived from T4fibritin. 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 of the antigen and the ligand or antibody fragment contained in a single polypeptide chain.

In some embodiments, the antibody fragment is an antibody-derived scFv chain, and the VH region and VL region of the scFv are separated by a flexible linker. In some embodiments, the length of the flexible linker is 12 or more amino acids, and the conjugated polypeptide is preferentially bound to the surface protein in a monomeric form. In some embodiments, the length of the flexible linker is shorter than 12 amino acids, and the conjugated polypeptide is preferentially bound to the surface protein in a polymeric form. In some embodiments, the polymer is stabilized by disulfide bonds between monomeric units. In some embodiments, the length of the flexible linker is 5 amino acids. In some embodiments, the conjugated polypeptide also includes a tPA leader sequence. In some embodiments, the length of the tPA leader sequence is 23 amino acids.

In some embodiments, the vaccine further comprises a second antigen from the 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 a SARS-CoV-2 spike glycoprotein or a fragment thereof. In some embodiments, the fragment of the SARS-CoV-2 spike glycoprotein comprises an S1 domain or a receptor binding domain (RBD). In some embodiments, the second antigen comprises a fragment of SARS-CoV-2E protein, M protein, N protein, nsp3 protein, nsp4 protein or nsp6 protein or one of these proteins. In some embodiments, the second antigen comprises a fusion protein comprising a fragment of SARS-CoV-2E protein and M protein or more. 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.

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

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 the extracellular domain 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 antibody-derived scFv chain. 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 guided by a signal sequence. In some embodiments, the signal sequence is derived from CD55. In some embodiments, the transmembrane segment is derived from PDGF receptor, glycophorin A, or SARS-CoV-2 spike protein. In some embodiments, the multimerization domain is derived from T4fibritin. 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 VH region and VL region of the scFv are separated by a flexible linker within the conjugated polypeptide. In some embodiments, the length of the flexible linker is 12 or more amino acids, and wherein the conjugated polypeptide preferentially binds to the surface protein in a monomeric form. In some embodiments, the length of the flexible linker is shorter than 12 amino acids, and the conjugated polypeptide preferentially binds to the surface protein in a polymeric form. In some embodiments, the polymer is stabilized by a disulfide bond. In some embodiments, the length of the flexible linker is 5 amino acids. In some embodiments, the conjugated polypeptide comprises a tPA leader sequence. In some embodiments, the tPA leader sequence is 23 amino acids in length.

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

In some embodiments, the polynucleotide encoding the conjugated polypeptide and/or the second polynucleotide encoding the second antigen are codon optimized. In some embodiments, the polynucleotide encoding the conjugated polypeptide is present in a first expression cassette, wherein the polynucleotide is operably connected to a first promoter, and/or the second polynucleotide encoding the second antigen is present in a second expression cassette, wherein the second polynucleotide is operably connected 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 expression cassette and/or the second expression cassette are present in a vector. In some embodiments, the vector is administered in the form of naked DNA. In some embodiments, the vector is a viral vector. In some such embodiments, the viral vector is a cytomegalovirus (CMV) vector, an adenovirus vector, or an 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.

In some embodiments, the vector is a circular CMV vector, which comprises: (a) a CMV genome or a portion thereof, wherein the CMV genome or a portion thereof contains the first expression cassette or the first expression cassette and the second expression cassette; (b) a bacterial artificial chromosome (BAC) sequence comprising a replication origin; (c) a first terminase complex recognition site (TCRL1) comprising at least two viral direct repeat sequences; and (d) a second terminase complex recognition site (TCRL2) comprising at least two viral direct repeat sequences; wherein the CMV genome or a portion thereof is flanked by TCRL1 and TCRL2, which define a first region of the circular vector extending from TCRL1 to TCRL2 and comprising the CMV genome or a portion thereof; and wherein 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 a portion thereof.

In some embodiments, the vector is a circular CMV vector, which comprises: (a) a CMV genome or a portion thereof, wherein the CMV genome or a portion thereof contains the first expression cassette or the first expression cassette and the second expression cassette; (b) a sequence comprising a replication origin that functions in a unicellular organism; (c) one or more terminase complex recognition loci (TCRLs), which comprise a recombinantly introduced polynucleotide sequence that can be directly cleaved by the HV terminase complex; wherein the CMV genome or a portion thereof is separated from the sequence comprising the replication origin by the TCRL; wherein the CMV genome or a portion thereof is adjacent to the TCRL at at least one end; and the sequence comprising the replication origin is adjacent to the TCRL at at least one end.

In some embodiments, one or more of the 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 conjugated polypeptide comprises a nucleotide sequence selected from the following: 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.

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

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 extracellular domain 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 preferentially or only expressed by T cells. In some embodiments, the antibody fragment is an antibody-derived scFv chain.

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 guided 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 a SARS-CoV-2 spike protein. In some embodiments, the multimerization domain is derived from T4fibritin. 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 antibody fragment is an antibody-derived scFv chain, and the VH region and VL region of the scFv are separated by a flexible linker. In some embodiments, the length of the flexible linker is 12 or more amino acids, and wherein the conjugated polypeptide is preferentially bound to the surface protein in a monomeric form. In some embodiments, the length of the flexible linker is shorter than 12 amino acids, and wherein the conjugated polypeptide is preferentially bound to the surface protein in a polymeric form. In some embodiments, the polymer is stabilized by disulfide bonds between monomeric units. In some embodiments, the length of the flexible linker is 5 amino acids. In some embodiments, the conjugated polypeptide also includes a tPA leader sequence. In some embodiments, the length of the tPA leader sequence is 23 amino acids.

In some embodiments, the pathogen is a virus. In some such embodiments, the virus is SARS-CoV-2. In some embodiments, the antigen comprises a SARS-CoV-2 spike glycoprotein or a fragment thereof. In some embodiments, the fragment of the SARS-CoV-2 spike glycoprotein comprises an S1 domain or a 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.

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).

In some embodiments, the conjugated 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.

In another aspect, the present disclosure provides polynucleotides encoding any of the conjugated polypeptides described herein.

In some embodiments, the polynucleotide is codon optimized. In some embodiments, the polynucleotide comprises the following nucleotide sequence: 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.

In another aspect, the 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.

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

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

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

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.

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

In some embodiments, the vaccine is administered subcutaneously or by electroporation. In some embodiments, the method induces a neutralizing antibody response in the mammal to an antigen present in the conjugated polypeptide, and the neutralizing response is substantially stronger than any antibody-dependent infectivity enhancement (ADEI) induced by the vaccine in the mammal. In some embodiments, the vaccine does not substantially induce ADEI in the mammal. In some embodiments, the method induces CD4 ⁺ and CD8 ⁺ T cell responses to a second antigen.

In some embodiments, the method comprises administering to the mammal by electroporation a DNA priming agent comprising any vector described herein (e.g., a plasmid) encoding any conjugated polypeptide described herein, followed by boosting with any vector described herein (e.g., an adenoviral vector) encoding any antigen described herein (e.g., RBD). In some embodiments, boosting is performed after about 28 days. In some embodiments, the mammal is a human.

Presented herein are numerous embodiments of the present disclosure, including compositions and methods for their preparation and administration.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Stimulation of T cells by 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-γ 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 the anti-CD3 scFv-S1 molecule is able to engage the CD3 complex and activate T cell signaling. The scales on the x- and y-axes range from -10 ³ to 10 ⁵ .

Figure 2. Animation depicting 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 are flanked by BAC sequences, which include the prokaryotic origin of replication oriS.

Figure 3A-3B. CMV vector vaccines lacking UL111A elicit strong CD4 ⁺ and CD8 ⁺ T cell responses at mucosal surfaces. Figure 3A: Balance of CD4 and CD8 responses after adenovirus vector immunization relative to CMV vector immunization. CMV vaccines (individual traces are weak solid lines; median is dark solid line) can induce comparable CD4 and CD8 responses (ratio>1), while adenovirus vectors can induce lower relative frequencies of CD4 responses (dashed lines). Figure 3B: An example of balanced CD4 ⁺ and CD8 ⁺ T cell responses after CMV vector immunization (top), but adenovirus vector immunization is not balanced (bottom). Two weeks after immunization, CD4 ⁺ or CD8 ⁺ cells (left and right, respectively) respond to vaccine antigen stimulation and produce cytokines.

Fig. 4. introduce the terminal enzyme complex recognition seat (TCRL) around BAC replication origin and provide the excellent conversion of CMV BAC genomic DNA external replication virus.Left figure, when using conventional CMV BAC genomic DNA (TR3dIL10), use FuGene 6 transfection 1 microgram CMV BAC genomic DNA to cause only produce 1 plaque, and when using the self-starting construct (TR4dIL10) with CMV TCRL around BAC starting point shown in Figure 2, then produce 20 plaques.Right figure, use calcium phosphate-mediated transfection, a series of comparisons of plaque formation under input DNA amount.The self-starting genome with the TCRL around BAC starting point is all more excellent on each DNA input level.

Figure 5. B cells responding to anti-CD3-linked immunogens receive indiscriminate help.

Figure 6. Subcutaneous vaccination with 100 μg of RhCMVdIL10 vaccine BAC DNA is sufficient to elicit an immune response. Immune responses to pRhCMV-MAGEA4 seen as early as one week after priming are shown.

Figure 7. Protocol overview.

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). Middle, these cassettes were delivered to rhesus macaques as electroporated DNA on day 0 of the vaccination regimen; animals were boosted with adenovirus type 35 (Ad35) vectors around day 28. Bottom, groups of three rhesus macaques received 1 mg of DNA expressing S1, RBD, or s3-RBD at the time of priming and 10 ¹² particles of Ad35/S1 or Ad35/RBD at the time of boosting.

Figure 9. Detection of binding antibodies by endpoint dilution ELISA assay. Responses of individual vaccinated rhesus macaques are represented by thinner lines; the geometric mean responses of each group are represented by thicker lines (black dotted line for S1, gray dashed line for RBD only, and black solid line for s3-RBD). Vaccination with DNA at week 0 and Ad35 at week 4 induced binding antibody responses above the background at week 0 in all subjects for both RBD and s3-RBD constructs. Subjects primed with s3-RBD showed superior responses, with higher means and, in 2/3 cases, higher than the best response seen in the RBD group. At 24 weeks post-vaccination, the geometric mean response for s3-RBD was 10-fold higher than the geometric mean response to RBD.

Figure 10. The reporter virus particle (RVP/pseudovirus) assay tests the inhibitory effect of various dilutions of serum on infection with pseudotyped lentiviral particles carrying the SARS-CoV-2 spike. The curve is then generated and the neutralization titer 50 (NT50) is read as the serum dilution required to obtain 50% inhibition. In this example, vaccination results in high neutralization titers starting from week 5 after the primary immunization.

Figure 11. Results from a longitudinal pseudovirus neutralization assay demonstrate superior induction of neutralizing antibodies by s3-RBD (solid black trace). At week 5 after primary vaccination, the geometric mean titer (GMT) in the s3-RBD group was at least 4-fold higher than that in the RBD group (solid black line relative to dashed gray line). One animal in the s3-RBD group produced a neutralizing antibody titer that exceeded the upper limit of the assay, so the true advantage of s3-RBD is greater. Furthermore, most RBD-only subjects had neutralizing antibody titers below the limit of detection at 24 weeks after vaccination, while all s3-RBD subjects maintained high neutralizing titers at 32 weeks.

Figure 12. Binding antibodies detected by endpoint dilution ELISA assay in rhesus macaques primed with RBD alone, s2-RBD (ie, anti-CD2-RBD conjugated polypeptide), or eDis3-RBD (enhanced diabody anti-CD3-RBD conjugated polypeptide). The response of a single vaccinated rhesus macaque is represented by a thinner line; the geometric mean response of each group is represented by a thicker line (black dotted line for RBD, gray dotted line for s2-RBD, and black solid line for eDis3-RBD). Compared with RBD alone, subjects primed with s2-RBD or eDis3-RBD showed superior responses, reaching higher peaks and remaining at higher levels after 24 weeks.

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 the binding of anti-RBD antibodies to intracellular proteins produced after transfection. (Figure 13A) Negative control cells that were not transfected with any plasmid did not react with anti-RBD antibodies. (Figure 13B) Positive control cells transfected with the expression cassette of RBD (B.1.351) alone produced immunoreactive proteins. (Figure 13C) Cells transfected with the expression cassette of 1dCD58-RBD (B.1.351) produced immunoreactive proteins, indicating that the conjugated polypeptide was successfully produced and has the potential to produce an immune response after vaccination. (FIG. 13D) Cells transfected with the expression cassette of s3-RBD(B.1.351)-PDGFRtm, which is predicted to insert into the cell membrane, produced immunoreactive protein, indicating successful production of the conjugated polypeptide.

Details

1. Introduction

The present disclosure provides methods and compositions for inducing an immune response in a subject. The methods and compositions allow for strong, effective and safe antibodies and/or T cell responses against antigens such as those from viruses such as SARS-CoV-2. Among other things, the methods and compositions also include conjugated polypeptides, which include antigens that are connected to a ligand or antibody fragment that is bound to a surface protein on an immune cell such as a T cell or an antigen presenting cell (APC). In some embodiments, the conjugate is administered in combination with a second antigen (e.g., a second antigen designed to induce a T cell response).

2. Definition

As used herein, the following terms have the meanings ascribed to them unless otherwise specified.

"Majority" of a genome means that a significant proportion of the genome and the genes contained therein (e.g., 20% of the total genome and/or 20% of the genes within the genome) is retained, rather than, for example, nucleic acids containing only one or a few genes or elements (such as replication origins) from the genome. In the polynucleotides of the present application, the portion comprises at least, for example, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the genome (i.e., in terms of total nucleotides) or the genes present in the wild-type full-length genome.

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

As used herein, the terms "about" and "approximately" shall generally mean an acceptable degree of error for a measured quantity given 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" specifically means at least 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, 100X, 101X, 102X, 103X, 104X, 105X, 106X, 107X, 108X, 109X, 110X, 111X, 112X, 113X, 114X, 115X, 116X, 117X, 118X, 119X, 120X, 121X, 122X, 123X, 124X, 125X, 126X, 127X, 128X, 129X, .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. Thus, "about X" is intended to teach and provide written descriptive support for claiming a limitation of, for example, "0.98X."

The term "antigen" refers to a molecule or a portion thereof that can induce an immune response (e.g., in a subject). Although in many cases, the immune response involves the production of antibodies that target or specifically bind to an antigen, as used herein, the term "antigen" also refers to a molecule that induces an immune response, rather than a molecule that specifically involves the production of antibodies that target an antigen, for example, a cell-mediated immune response involving the amplification of T cells that target antigen-derived peptides presented on the surface of a target cell. In certain embodiments, the antigen of the present disclosure is derived from a pathogen so that the immune response of the subject provides immune protection against the pathogen. In certain embodiments, the pathogen is a virus (e.g., SARS-CoV-2).

The term "nucleic acid sequence encoding a peptide" refers to a DNA segment, which in some embodiments may be a gene or a portion thereof that is involved in producing a peptide chain (e.g., an antigen or a fusion protein). A gene will generally include regions before and after the coding region (leader and trailer sequence) that participate in the transcription/translation of a gene product and the regulation of transcription/translation. A gene may also include sequences (introns) between individual coding segments (exons). Leader sequences, trailer sequences, and introns may include regulatory elements (e.g., promoters, terminators, translation regulatory sequences (such as ribosome binding sites and internal ribosome entry sites), enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions, etc.) that are necessary in the transcription and translation of a gene. A "gene product" may refer to mRNA or protein expressed from a specific gene.

The terms "expression" and "expressed" refer to the production of transcription and/or translation products, such as the production of transcription and/or translation products of nucleic acid sequences encoding proteins (e.g., antigens or fusion proteins). In some embodiments, the term refers to the production of transcription and/or translation products encoded by a gene (e.g., a gene encoding an antigen) or a portion thereof. The expression level of a DNA molecule in a cell can be assessed based on the amount of the corresponding mRNA present in the cell or the amount of protein encoded by the DNA produced by the cell.

The term "recombinant" when used with reference to, for example, a polynucleotide, protein, vector or cell, refers to a polynucleotide, protein, vector or cell that has been modified by the introduction of a heterologous nucleic acid or protein or by the alteration of a native nucleic acid or protein, or a cell derived from a cell so modified. For example, a recombinant polynucleotide comprises a nucleic acid sequence that is not found in the native (non-recombinant) form of the polynucleotide.

The term "immune response" refers to any response induced by an antigen (e.g., in a subject), including immune induction against pathogens (e.g., viruses and microorganisms such as bacteria). The immune response induced by the systems, recombinant polynucleotides, compositions and methods of the present disclosure is generally a desired, expected and/or protective immune response. The term includes the production of antibodies (e.g., neutralizing antibodies) against antigens, and the development, maturation, differentiation and activation of immune cells (e.g., B cells and T cells). In some cases, the immune response includes increasing the number or activation of MHC E class 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, the immune response may include increasing the expression or activity of interferon-γ and/or tumor necrosis factor-α (e.g., in a subject).

Other examples of desirable, anticipated and/or protective immune responses that can be induced by the recombinant polynucleotides, compositions and methods of the present disclosure include, but are not limited to, those involving: class Ia, Ib or II restricted CD4 ⁺ T cells; class Ia, Ib or II restricted CD8 ⁺ T cells; cytokine-producing T cells (e.g., T cells that produce IFN-γ, TNF-α, IL-1-β, IL-2, IL-4, IL-5, IL-10, IL-13, IL-17, IL-18 or IL-23); CCR7-CD8 ⁺ T cells (e.g., effector memory cells); CXCR5 ⁺ T cells (i.e., those that home to B cell follicles); CD4 ⁺ regulatory T cells; CD8 ⁺ regulatory T cells; antigen-specific T follicular helper cells; antibody production; NK cells; NKG2C ⁺ NK cells; CD57 ⁺ NK cells; FcR-γ negative NK cells; and NK-CTL cells, i.e., CD8 ⁺ T cells expressing NK cell-typical molecules (such as NKG2A).

The term "cytomegalovirus" or "CMV" refers to viruses that include members of the genus Cytomegalovirus (belonging to 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)) that infects rhesus monkeys, simian cytomegalovirus (SCCMV or AGMCMV), baboon cytomegalovirus (BaCMV), night monkey cytomegalovirus (OMCMV), squirrel monkey cytomegalovirus (SMCMV), and rhesus monkey cytomegalovirus (RhCMV).

The term "antigen presenting cell" or "APC" refers to a cell that displays or presents an antigen or a portion thereof on the cell surface. Typically, the antigen is displayed or presented in the form of a major histocompatibility complex (MHC) molecule. Almost all cell types can serve as APCs, and APCs are found in a large number of different tissue types. Professional APCs (such as dendritic cells, macrophages, and B cells) present antigens to T cells in an environment that most effectively leads to T cell activation and subsequent proliferation. Many cell types present antigens to cytotoxic T cells.

“Immune cells” 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.

"Infectious disease antigen" refers to any molecule derived from an organism that causes an infectious disease that can induce an immune response (e.g., in a subject). For example, an infectious disease antigen can be derived from a virus, bacteria, fungus, protozoa, worm, 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 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.

As used herein, the terms "polynucleotide", "nucleic acid" and "nucleotide" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers. The term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA and DNA-RNA hybrids, and other polymers containing purine and/or pyrimidine bases or other natural, chemically modified, biochemically modified, non-natural, synthetic or derived nucleotide bases. Unless otherwise specified, the term includes nucleic acids containing known analogs of natural nucleotides, which have similar binding properties to the reference nucleic acid. Unless otherwise specified, a specific nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), homologs and complementary sequences as well as explicitly indicated 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)).

The terms "vector" and "expression vector" refer to nucleic acid constructs recombined or synthesized with a series of specific nucleic acid elements that allow transcription of specific nucleic acid sequences (e.g., nucleic acid sequences encoding antigens and/or fusion proteins described herein) in host cells or engineered cells. In some embodiments, the vector includes a polynucleotide to be transcribed, which is operably linked to a promoter. Other elements that may be present in the vector include those that enhance transcription (e.g., enhancers), those that terminate transcription (e.g., terminators), those that confer certain binding affinity or antigenicity to proteins (e.g., recombinant proteins) produced from the vector, and those that can replicate the vector and its packaging (e.g., become viral particles). In some embodiments, the vector is a viral vector (i.e., a viral genome or a portion thereof). The vector may include, for example, a nucleic acid sequence or mutation that increases tropism and/or regulates immune function. An "expression cassette" includes a coding sequence operably linked to a promoter, and an optional polyadenylation sequence.

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

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, mice, rats, apes, humans, farm animals, sports animals and pets. It also includes tissues, cells and their progeny of biological entities obtained in vivo or cultured in vitro.

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

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

The term "treatment" refers to an approach to obtaining a beneficial or desired result, including but not limited to a therapeutic benefit and/or a preventive benefit. "Therapeutic benefit" means any treatment-related improvement or effect on one or more diseases, conditions, or symptoms being treated. Therapeutic benefit may also mean the effect of curing one or more diseases, conditions, or symptoms being treated. In addition, therapeutic benefit may also mean improved survival. For preventive benefit, the composition may be administered to a subject at risk for developing a particular disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even though the disease, condition, or symptom may not yet have appeared.

The term "therapeutically effective amount" or "sufficient amount" refers to an amount of a system, recombinant polynucleotide or composition described herein that is sufficient to produce a beneficial or desired result. The therapeutically effective amount may vary depending on one or more of the following: the subject and the 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 easily determined by one of ordinary skill in the art. The specific amount may vary depending on one or more of the following: the specific agent selected, the target cell type, the location of the target cell in the subject, the dosing regimen followed, whether it is administered in combination with other compounds, the time of administration, and the physical delivery system that carries it.

For the purposes herein, an effective amount is determined by considerations such as those that may be known in the art. The amount must be effective to achieve a desired therapeutic effect in an object suffering from a disease (such as an infectious disease or cancer). The desired therapeutic effect may include, for example, improving undesirable symptoms associated with the disease, preventing the manifestation of these symptoms before these symptoms occur, slowing down the progression of disease-related symptoms, slowing down or limiting any irreversible damage caused by the disease, alleviating the severity of the disease or curing the disease, or increasing the survival rate or providing a faster recovery from the disease. In addition, in the context of preventive treatment, the amount may also effectively prevent the development of the disease.

The term "pharmaceutically acceptable carrier" refers to a substance that helps to apply an active agent to a cell, an organism or an object. "Pharmaceutically acceptable carrier" also refers to a carrier or excipient that can be included in the present composition and does not cause obvious adverse toxicological effects to the patient. Non-limiting examples of pharmaceutically acceptable carriers include water, sodium chloride (NaCl), physiological saline solution, lactated Ringer's solution, normal sucrose, normal glucose, adhesives, fillers, disintegrants, lubricants, coatings, sweeteners, spices and pigments, liposomes, dispersion media, microcapsules, cationic lipid carriers, isotonic agents and absorption delay agents, etc. The carrier may also include or be composed of the following substances: for providing stability, sterility and isotonicity (e.g., antimicrobial preservatives, antioxidants, chelating agents and buffers) for the preparation, for preventing the effects of microorganisms (e.g., antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc.) or for providing the preparation with a material composition of edible flavors, etc. In some cases, a carrier is an agent that facilitates the delivery of a polypeptide, fusion protein or polynucleotide to a target cell or tissue. One skilled in the art will recognize that other pharmaceutical carriers may also be used in the methods and compositions of the present invention.

The term "vaccine" refers to a biological composition that, when applied to an object, has the ability to produce acquired immunity against a specific pathogen or disease in an object. Typically, one or more antigens, antigenic fragments, or polynucleotides encoding antigens or antigenic fragments related to a target pathogen or disease are administered to an object. Vaccines may include, for example, inactivated or attenuated organisms (e.g., bacteria or viruses), cells, proteins expressed by cells or expressed on cells (e.g., cell surface proteins or other proteins produced by cells (e.g., tumor cells), proteins produced by organisms (e.g., toxins), or a part of an organism (e.g., viral envelope proteins or viral genes encoding various antigens). In some cases, cells are engineered to express proteins so that when administered as a vaccine, they enhance the ability of an object to obtain immunity against a specific cell type (e.g., enhancing the ability of an object to obtain immunity against cancer cells), or against an organism (e.g., virus, bacteria, fungal organisms, protozoa, or worms) that causes an infectious disease. As used herein, the term "vaccine" includes, but is not limited to, systems and recombinant polynucleotides disclosed herein, as well as viral particles, host cells, and pharmaceutical compositions comprising systems or recombinant polynucleotides as described herein.

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

3. Vaccines

The present disclosure provides vaccines for generating an immune response to an antigen from any type of pathogen. The antigen (e.g., in a subject) to which an immune response is generated will depend on the specific disease for which prevention and/or treatment benefits are sought. In some embodiments, the antigen is an infectious disease antigen, such as a virus, bacteria, protozoa, worm, or fungal pathogen. In specific embodiments, the antigen is a viral antigen, such as an antigen from a coronavirus (e.g., SARS-CoV-2). In some embodiments, the antigen is a tumor-associated antigen.

In some embodiments, an immune response (e.g., a desired, anticipated, 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, viral, fungal, protozoan, tumor-associated antigen, and/or helminth antigen. In specific embodiments, the antigen is a viral antigen from a coronavirus (e.g., SARS-CoV-2).

The vaccines of the present application may take any of a variety of forms, including by administration of proteins, peptides, and nucleic acids (including RNA or DNA encoding one or more of the antigens described herein).

Immunogenic conjugates

In specific embodiments, the vaccine comprises an "immunogenic conjugate" or "conjugated polypeptide" or "conjugate" comprising an antigen linked to a ligand or antibody fragment that binds to a surface protein present on an immune cell. For example, an antigen can be linked to an antibody fragment that specifically binds to an abundant protein on the surface of a T cell. In some embodiments, the ligand is an extracellular domain of a cell adhesion molecule. Any abundant protein on the surface of an immune cell (including a T cell, such as a helper T cell) can be targeted by a ligand or antibody fragment. In some embodiments, the protein bound by the ligand or antibody fragment participates in signal transduction and/or adhesion. Examples of surface proteins that can be partially bound include CD2 (see, e.g., NCBI gene ID 914 or UNIProt P06729); CD3, including any CD3 subunit, i.e., CD3-ε (see, e.g., NCBI gene ID 916 or UNIProt P07766); CD3-γ (see, e.g., NCBI gene ID 917 or UNIProt P09693; CD3-δ (see, e.g., NCBI gene ID 915 or UNIProt P04234) or CD3-ζ (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 P01731). P06217). Without being bound by the following theory, it is believed that in some embodiments, such conjugated polypeptides can bridge antigen-specific B cells with T cells or other immune cells in the vicinity of the B cells and thereby elicit stronger antibodies than those obtained by vaccination with the antigen alone.

The antigen can be any immunogenic antigen from a pathogen, i.e., containing one or more epitopes that can stimulate a B cell (antibody) or T cell immune response and be specifically bound by antibodies and/or T cells in a subject. In certain embodiments, the antigen present in the immunogenic conjugate produces a strong antibody response in a subject. For example, for vaccination against a coronavirus such as SARS-CoV-2, the antigen may comprise a spike glycoprotein or a fragment thereof. In some such embodiments, the fragment comprises an S1 domain, a 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 numbers QIG55857.1, 6VYB_C, 6VYB_B, 6VYB_A, or any SARS-CoV-2 spike glycoprotein entry in the NCBI database). In specific embodiments, the antigen comprises a SARS-CoV-2 RBD.

Antigen is connected to a ligand or antibody fragment that can bind to a surface protein present on an immune cell. In some embodiments, the surface protein is an abundant surface protein that participates in signal transduction and/or adhesion on a T cell. In some embodiments, the surface protein is present on a T cell or an antigen presenting cell (APC). In some embodiments, the surface protein is preferentially expressed by a T cell (for example, relative to other immune cells). In some embodiments, the surface protein is substantially exclusively expressed by a T cell (i.e., expressed by a T cell, and not significantly expressed by other immune cells). In some embodiments, the ligand is a protein that naturally binds to a surface protein on an immune cell, for example, a natural ligand of an immune cell receptor, or a derivative or fragment of a natural ligand. In some embodiments, the ligand is an extracellular domain (extracellular domain) or a cell adhesion molecule (for example, CD58). For example, in some embodiments, the ligand is a 95-residue membrane distal N-terminal domain (ldCD58) of CD58, which is fully responsible for adhesion to CD2. For purposes of this disclosure, the ligand does not include an antibody (for example, a monoclonal antibody).

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, scFv, etc. The term "antibody fragment" can also include multispecific antibodies and hybrid antibodies, which have two or more antigens or epitope specificities. In some embodiments, the antibody fragment is a nanobody or a single domain antibody (sdAb), which contains a single monomer variable antibody domain (e.g., a single VHH domain). In specific embodiments, the antibody fragment is a scFv (e.g., anti-CD2, anti-CD3 or anti-CD4 scFv). For example, in some embodiments, the fragment is a scFv derived from an anti-CD2 antibody (e.g., LO-CD2a). In some embodiments, the fragment is a scFv derived from an anti-CD3 antibody (e.g., SP34). In some embodiments, the fragment is a scFv derived from an anti-CD4 antibody (e.g., hu5A8). In some embodiments, the scFv is derived from a humanized antibody.

In some embodiments, for example, where the antibody fragment is scFv, the VH domain and VL domain of the antibody are separated by a flexible linker. In some embodiments, the length of the flexible linker is 12 amino acids or longer (e.g., 15 amino acids). In such embodiments, the VH domain and the VL domain are generally able to fold appropriately to allow the conjugated polypeptide to be used as (e.g., bound to a surface protein) monomer. In other embodiments, the length of the flexible linker is less than 12 amino acids (e.g., 5, 6, 7, 8, 9, 10 or 11 amino acids in length). In such embodiments, the VH region and the VL region may have a length that is not enough to fold appropriately as a monomer to promote the formation of a multimer (e.g., a double-chain antibody, a three-chain antibody, a four-chain antibody, etc.). In some embodiments, the present disclosure is included in double-chain antibodies, three-chain antibodies or four-chain antibodies formed between scFv antibody fragments described herein. In some embodiments, such multimers are stabilized by disulfide bonds between monomer units.

To prepare antibody fragments that bind to surface proteins, many techniques known in the art can be used. See, for example, Kohler & Milstein, Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp. 77-96, 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 edition 1986)). In some embodiments, antibodies are prepared by immunizing one or more animals (such as mice, rabbits or rats) with an antigen to induce an antibody response. To produce monoclonal antibodies, B cells are fused with myeloma cells, which are then screened for antigen specificity.

The genes encoding the heavy and light chains of the target antibody can be cloned from cells, for example, the genes encoding monoclonal antibodies can be cloned from hybridomas and used to produce recombinant monoclonal antibodies, thereby producing antibody fragments. The gene library encoding the heavy and light chains of monoclonal antibodies can also be made from hybridomas or plasma cells. In addition, phage or yeast display technology can be used to identify antibodies and heteropolymeric Fab fragments that specifically bind to the selected antigen (see, for example, McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992); Lou et al., m PEDS 23:311 (2010); and Chao et al., Nature Protocols, 1:755-768 (2006)). Alternatively, a yeast-based antibody presentation system can be used to isolate and/or identify antibodies and antibody sequences, such as, for example, Xu et al., Protein Eng Des Sel, 2013, 26: 663-670; WO 2009/036379; WO 2010/105256; and those disclosed in WO 2012/009568. The random combination of heavy and light chain gene products produces a large number of antibodies with different antigenic specificities (see, e.g., Kuby, Immunology (3rd edition, 1997)). The technology for producing single-chain antibodies or recombinant antibodies (U.S. Pat. No. 4,946,778, U.S. Pat. No. 4,816,567) can also be used to produce antibodies.

In some embodiments, antibody fragments (such as Fab, Fab', F(ab')2, scFv, nanobodies or diabodies) are produced. In specific embodiments, the antibody fragment is a scFv (single chain variable fragment). scFv is a recombinant polypeptide comprising light ( _VL ) and heavy ( _VH ) immunoglobulin chain variable regions. In some embodiments, the VH sequence and the VL sequence are connected 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 cultures. Other techniques for producing antibody fragments will be apparent to those skilled in the art.

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., ), flow cytometry, fluorescence activated cell sorting (FACS), BioLayer interferometry (eg, Octet ^™ (ForteBio, Inc., Menlo Park, CA)), and western blot analysis.

In some embodiments, the affinity agent is a peptide, such as a peptide bound to a T cell surface protein. In some embodiments, the reagent is a peptide aptamer. A peptide aptamer is an artificial protein that is selected or engineered to bind to a specific target molecule. Typically, a peptide includes a peptide loop of one or more variable sequences displayed by a protein scaffold. Different systems (including yeast two-hybrid systems) can be used to select peptide aptamers. Peptide aptamers can also be selected from a combinatorial peptide library 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.

In some embodiments, the agent is an affimer. Affimers are small, highly stable proteins, typically with a molecular weight of about 12-14 kDa, that bind to their target molecules with specificity and affinity similar to antibodies. Typically, affimers display two peptide loops and an N-terminal sequence that can be randomized to bind to different target proteins with high affinity and specificity in a manner similar to monoclonal antibodies. The stabilization of the two peptide loops by the protein scaffold limits the possible conformations that the peptide can take, which increases binding affinity and specificity compared to free peptide libraries. Affimers and methods for preparing affimers are described in the art. See, for example, Tiede et al., eLife, 2017, 6: e24903. Affimers are also commercially available, for example, from Avacta Life Sciences.

Antigen and ligand/antibody fragment can be directly or indirectly connected to each other in a variety of ways. For example, in some embodiments, antigen and ligand/antibody fragment are directly (covalently) connected, for example, by chemical linker or by virtue of being present in a single fusion protein. Methods for connecting polypeptides to each other, for example, methods for connecting antigens to ligand/antibody fragments are known in the art and can be obtained from commercial suppliers, such as protein-protein conjugation kits from TriLink BioTechnologies, from VectorLaboratories, from Kerafast, from SydLabs, from INTERCHIM, etc.

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

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 length tPA leader sequence (see, e.g., UniProtP00750; 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).

In some embodiments, the antigen and ligand/antibody fragment present in the conjugated polypeptide or fusion protein are separated by a flexible linker. Suitable linkers for separating protein domains are known in the art, and can include, for example, glycine and serine amino groups, for example, 2-20 glycine and/or serine residues. In one embodiment, the flexible linker includes a (Gly4Ser) ₃ linker including a sequence GGGGSGGGGSGGGGS (SEQ ID NO: ₅ ).

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 lipid anchors (such as glycosylphosphatidylinositol anchors) is guided 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 PDGF receptors, glycophorin A or SARS-CoV-2 spike proteins. Examples of multimerization domains include, for example, domains derived from T4fibritin and Fc domains (e.g., human IgG Fc domains). In some embodiments, the present disclosure includes dimers or other polymers formed between the conjugated polypeptides of the present application comprising multimerization (e.g., T4fibritin or Fc domains). In some embodiments, the polymer is stabilized by disulfide bonds between monomeric units. In some embodiments, Fc or other domains are located at the C-terminus of the conjugated polypeptide.

In some embodiments, where the vaccine comprises a polynucleotide encoding a fusion protein, and where the polynucleotide is present in a viral vector (e.g., a CMV vector), the coding sequence for the fusion protein is operably linked to a late promoter (e.g., a CMV pp65b promoter). Without being bound by the following theory, it is believed that expression of an antigen (e.g., an immunogenic conjugate) via a strong late promoter (e.g., pp65b) will elicit a strong antibody response, but only a weak T cell response.

In some embodiments, the antigen and the ligand/antibody fragment (also referred to as an "affinity agent") are indirectly linked, i.e., linked by a non-covalent interaction that bridges the two entities. For example, the antigen and the ligand/antibody fragment can be linked by a second "bridging" antibody or a fragment thereof. In some embodiments, the antigen is a membrane protein embedded in a nanodisc, and the bridging antibody is a bispecific antibody fragment that binds (i) the antigen itself or a membrane scaffold protein in the nanodisc, and (ii) a T cell surface protein. A nanodisc is a synthetic membrane system that includes a lipid bilayer surrounded by an amphipathic protein called a membrane scaffold protein (MSP). Any nanodisc system can be used in the methods of the present application, including any MSP (such as apoA1-derived MSP) or amphipathic peptides. In some embodiments, synthetic nanodiscs are used. The preparation and use of nanodiscs are known in the art and are described, for example, in 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., (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 complete disclosures of each of which are incorporated herein by reference.

In some embodiments, the conjugated polypeptide comprises an amino acid sequence as 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 conjugated polypeptide comprises an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to 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.

Additional antigens

In some embodiments, the vaccine comprises a second antigen, which may be present with or in place of the immunogenic conjugate. When both antigens are present (i.e., the immunogenic conjugate and the second antigen), the antigens may be administered together (i.e., in the same vaccine), or administered independently (e.g., formulated separately and administered simultaneously (e.g., during a single clinical visit) or at different times (e.g., different days)). One or both antigens may be administered in protein form or in 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, each of which is operably linked to a promoter. In some embodiments, the second antigen is administered as a polynucleotide encoding the antigen, which is operably linked to a constitutive promoter, e.g., a mammalian promoter (e.g., EF-1α) (see, 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 the following theory, it is believed that expressing the second antigen in the cytoplasmic compartment of the cell will elicit a strong T cell response, but with minimal accompanying antibody response.

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

Nucleic acid vaccines

In some embodiments, nucleic acid vaccines (e.g., DNA vaccines) are used to introduce immunogenic conjugates and/or second antigens. Therefore, in some embodiments, the present disclosure provides polynucleotides encoding any conjugated polypeptide described herein. In some embodiments, DNA vaccines are prepared as DNA vectors or plasmids. In some embodiments, DNA vaccines are prepared as recombinant viruses, for example, by modifying the parental virus to incorporate exogenous genetic material, for example, 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 slow viruses (e.g., HIV, HIV-1, HIV-2, FIV, BIV, EIAV, MW, CAEV, SIV), adenoviruses and adeno-associated viruses, alpha viruses, herpes viruses (e.g., cytomegalovirus), flaviviruses and poxviruses. For methods and examples of suitable viral vector applications, see, e.g., U.S. Pat. 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 recombinant competent (i.e., capable of propagating in infected host cells). Modification of such viruses and vectors or plasmids for use in preparing the DNA vaccines of the present application can be achieved using standard molecular biology techniques, for example, as taught in Sambrook et al., (1989) "Molecular Cloning: A Laboratory Manual" (2nd edition, Cold Spring Harbor Press) and Ausubel et al. (eds.) (2000-2010) "Current Protocols in Molecular Biology" (John Wiley and Sons).

In some embodiments, the polynucleotide encodes an amino acid sequence as 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 polynucleotide encodes an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to 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. In some embodiments, the polynucleotide comprises a nucleotide sequence as shown 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 polynucleotide comprises a nucleotide sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to 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.

In certain embodiments, the DNA vaccine involves a cytomegalovirus (CMV) vector, such as a self-priming CMV DNA vector ( _SL CMV), as described in U.S. Provisional Application No. 62/842,419, filed May 2, 2019, the entire disclosure of which is incorporated herein by reference. The self-priming CMV vaccine is administered in the form of a CMV genome that "primes" the vector vaccine replication in vivo, thereby generating a strong immune response with unique characteristics associated with CMV infection. _SL CMV vectors may contain one or more of a variety of features, including: (i) being based on the Towne HCMV strain, which has been shown to be safe for use in humans of all ages; (ii) providing an immune response in both CMV seropositive and seronegative individuals due to the superinfectivity of CMV and proprietary deletions in the CMV genome; (iii) providing a very broad and strong T cell response that "paints" the vaccine antigens; (iv) having a demonstrated ability to elicit a balanced response that includes antibodies, CD4 ⁺ T cells, and CD8 ⁺ T cells—without being dependent on or dominating any single effector response; providing localization of responding T cells to mucosal surfaces due to an effector memory phenotype; (v) allowing the engineering of new vaccine candidates within weeks to months; and (vi) providing a single manufacturing method based on plasmid production in E. coli.

Unlike most vaccine types, which do not elicit large numbers of memory CD4 ⁺ T cells, the CMV vector vaccines of the present application do so, as shown in the examples below. In addition, T cells that respond to CMV vector vaccines localize to the airways, among other effector sites, and can be recovered by, for example, bronchoalveolar lavage (12). CMV-responsive T cells recapitulate other essential features of cells that have been shown to be protective against SARS-CoV-1, including CXCR3 expression, IFN-γ production, and IL-10 production (13).

In the _SL CMV vector, vaccination with the CMV vector in its DNA form requires changes to the current CMV BAC construct so that the BAC backbone can be excised without recombinase or nuclease expression in vivo. Due to the need to package the unit length genome into an icosahedral capsid, CMV has relatively strict packaging restrictions. In some embodiments, CMV BAC utilizes an endogenous recombinase gene placed in the BAC portion of the DNA construct. After transfection of mammalian cells, the recombinase is expressed and the BAC replication mechanism is excised from the replicating genome. In order to make the BAC DNA vector suitable for delivery in the human body, the CMV genome ends are reorganized so that BAC can be excised without the need for recombinase expression. In particular, the reconfigured BAC construct utilizes the viral terminase complex to eliminate the bacterial replication origin during the packaging step of CMV replication. In some embodiments, the position of the BAC origin and the replication mechanism is located between the viral direct repeat sequences.

Thus, in one embodiment, the present disclosure provides a self-initiating HV (e.g., CMV) recombinant polynucleotide comprising one or more polynucleotides encoding one or more antigens (or conjugated polypeptides) described herein, and including (a) a herpes virus (HV) genome or a substantial portion thereof; (b) a sequence comprising an origin of replication that functions in unicellular organisms; (c) one or more terminase complex recognition loci (TCRLs) comprising a repeatedly introduced polynucleotide sequence that can be directly cleaved by the HV terminase complex; wherein the HV genome or a portion thereof is separated from the sequence comprising the origin of replication by the TCRL; wherein the HV genome or a portion thereof is adjacent to the TCRL at at least one end; and wherein the sequence comprises an origin of replication adjacent to the TCRL at 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 (e.g., pp65b), and/or a polynucleotide encoding an antigen operably linked to a constitutive promoter (e.g., EF-1α).

As used herein, the "end" of a specific genomic region or element (such as a genome or part thereof) in a vector refers to either end of the region or element, and there are different regions or elements (or ends of nucleic acid molecules) outside the end. For example, in a circular vector, in some embodiments, one end (or end) of the HV genome can be directly adjacent to the first end of the first TCRL element, and the other end (or end) of the HV genome can be directly adjacent to the first end of the second TCRL element. In the same circular vector, one end (or end) of the region containing the starting point can be directly adjacent to the second end of the first TCRL element, and the other end (or end) of the region containing the starting point 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.

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 a sequence comprising a BAC or YAC origin of replication, for example, genetic elements present between a TCRL adjacent to the HV genome or a substantial portion thereof and a (BAC or YAC) origin of replication that functions in a unicellular organism.

Antigen coding sequence

The recombinant polynucleotides (e.g., viral vectors) disclosed herein comprise nucleic acid sequences encoding antigens described herein (e.g., conjugated polypeptides) and/or coronavirus antigens as described herein (e.g., spike protein E protein, M protein, or N protein, or combinations and/or fragments thereof). The rapid progress of various genomic studies has made cloning methods possible, in which any gene segment having a certain percentage of sequence homology with a known nucleotide sequence (e.g., a nucleotide sequence encoding an antigen, etc.) can be searched in human or other model organism DNA sequence databases. Any DNA sequence thus identified can then be obtained by chemical synthesis and/or polymerase chain response (PCR) techniques (e.g., overlap extension method). For short sequences, complete de novo synthesis may be sufficient; and further separation of full-length coding sequences from human or other model organism cDNA or genomic libraries using synthetic probes may be necessary to obtain larger genes.

Alternatively, nucleic acid sequences can be isolated from cDNA or genomic DNA libraries (e.g., human or rodent cDNA or human, rodent, bacterial or viral genomic DNA libraries) using standard cloning techniques such as polymerase chain response (PCR), where homology-based primers are generally available from known nucleic acid sequences. Common techniques for this purpose are described in standard texts, such as Sambrook and Russell, supra.

The cDNA library may be commercially available or may be constructed. The general methods of isolating mRNA, preparing cDNA by reverse transcription, connecting the cDNA into a recombinant vector, and transfecting into a recombinant host for breeding, screening, and cloning are well known (see, e.g., Gubler and Hoffman, Gene, 25:263-269 (1983); Ausubel et al., supra). After obtaining an amplified segment of a nucleotide sequence by PCR, the segment may be further used as a probe to isolate the full-length polynucleotide sequence encoding the target protein from a cDNA library. A general description of suitable procedures may be found in Sambrook and Russell, supra.

A similar procedure can be followed to obtain the full-length sequence encoding the target protein from a human or other model organism genomic library. Genomic libraries are commercially available or can be constructed according to various recognized methods. As a non-limiting example, in order to construct a genomic library, DNA is first extracted from the tissue of the organism. The DNA is then mechanically sheared or enzymatically digested to produce fragments of about 12-20 kb in length. The fragments are then separated from the polynucleotide fragments of undesirable size by gradient centrifugation and inserted into a phage lambda vector. These vectors and phages are packaged in vitro. Recombinant phages are analyzed by plaque hybridization, as described in Benton and Davis, Science, 196: 180-182 (1977). Clonal hybridization is performed as described in Grunstein et al., Proc. Natl. Acad. Sci. USA, 72: 3961-3965 (1975).

In a specific embodiment, the polynucleotide encoding the antigen (e.g., 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 the expression of the polynucleotide in one or more cells of the subject can be used, including inducible promoters and constitutive promoters. In some embodiments, a CMV promoter is used. In a specific embodiment, a late viral promoter (e.g., pp65b) is used to drive the expression of the immunogenic conjugate. In a specific embodiment, a constitutive mammalian promoter (e.g., EF1-α) is used to drive the expression of the second antigen as described herein. In some embodiments, the EF1-α promoter includes the first intron of the EF1-α gene. The vector may contain other regulatory sequences, such as terminators, translation regulatory sequences (e.g., ribosome binding sites and internal ribosome entry sites), enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions. The use of such elements is well known in the art.

In some embodiments, the recombinant polynucleotides of the present disclosure include nucleic acid sequences encoding selective markers. For example, when the polynucleotides described herein are being recombinantly modified, particularly when it is necessary to screen the modified polynucleotide population (e.g., using bacteria, yeast, plant or animal cells) to obtain those that have been incorporated with the desired modification, the selective marker is useful. Whether the polynucleotides are recombinantly modified in cells (e.g., bacterial cells, e.g., using Red/ET recombination), or recombinantly modified and subsequently introduced into cells (e.g., bacteria, yeast, plant or animal cells) for screening, the selective marker can be used to identify those cells containing polynucleotides that have been incorporated with the target modification. Taking antibiotic resistance genes as examples of selective markers, cells containing recombinant polynucleotides treated with antibiotics will identify those cells containing recombinant polynucleotides that have been incorporated with antibiotic resistance genes (i.e., cells that survive after antibiotic treatment must have been incorporated with antibiotic resistance genes). If desired, the recombinant polynucleotides can be further screened (e.g., purified, amplified and sequenced from cells) to verify that the desired modification has been recombinantly introduced into the polynucleotides at the correct position.

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

In some embodiments, the selective marker used to identify cells containing polynucleotides comprising target modifications is a fluorescently labeled protein, a chemical stain, a chemical indicator, or a combination thereof. In other embodiments, the selective marker is responsive to changes in stimulus, biochemical, or environmental conditions. In some cases, the selective marker is responsive to the concentration of a metabolite, a protein product, a drug, a target cell phenotype, a target cell product, or a combination thereof.

The size of the recombinant polynucleotide will depend on the specific antigens and other proteins being encoded, the presence and selection of regulatory sequences and/or expression vectors (e.g., viral vectors), the selection and location of various elements (such as TCRL), etc. In addition, the size of the recombinant polynucleotide will depend on whether the nucleic acid sequences encoding the antigens and other proteins are present in the same recombinant polynucleotide or in separate recombinant polynucleotides.

In some embodiments, the length of the recombinant polynucleotide is about 1 kilobase to 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.

In some embodiments, the length of the recombinant polynucleotides present in the systems of the present disclosure is about 1 kilobase to about 300 kilobases, about 1 kilobase to about 250 kilobases, about 1 kilobase to about 200 kilobases, about 1 kilobase to about 150 kilobases, about 1 kilobase to about 100 kilobases, about 1 kilobase to about 50 kilobases, about 1 kilobase to about 40 kilobases, about 1 kilobase to about 30 kilobases, about 1 kilobase to about 20 kilobases, about 1 kilobase to about 10 kilobases, about 50 kilobase to about 300 kilobases, about 50 kilobase to about 250 kilobases. bases, about 50 kbases to about 200 kbases, about 50 kbases to about 150 kbases, about 50 kbases to about 100 kbases, about 100 kbases to about 300 kbases, about 100 kbases to about 250 kbases, about 100 kbases to about 200 kbases, about 100 kbases to about 150 kbases, about 150 kbases to about 300 kbases, about 150 kbases to about 250 kbases, about 150 kbases to about 200 kbases, about 200 kbases to about 300 kbases, or about 200 kbases to about 250 kbases.

General recombinant technology

Basic texts that disclose general methods and techniques in the field of recombinant genetics (e.g., those used for the preparation, maintenance, and culture 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).

For nucleic acids, sizes are expressed in kilobases (kb) or base pairs (bp). In some cases, these estimates are derived from agarose or acrylamide gel electrophoresis, sequenced nucleic acids, or published DNA sequences. For proteins, sizes are expressed in kilodaltons (kDa) or the number of amino acid residues. In some cases, protein sizes can be estimated from gel electrophoresis, sequenced proteins, deduced amino acid sequences, or published protein sequences.

Oligonucleotides that are not commercially available can be chemically synthesized, for example, according to the solid phase phosphoramidite method first described by Beaucage & Caruthers, Tetrahedron Lett. 22: 1859-1862 (1981), using an automatic synthesizer as described in Van Devanter et al., Nucleic Acids Res. 12: 6159-6168 (1984). Purification of oligonucleotides is performed using any prior art recognized strategy, such as native acrylamide gel electrophoresis or anion exchange HPLC, as described in Pearson & Reanier, J. Chrom. 255: 137-149 (1983).

After cloning or subcloning, the sequence of the protein domain or gene of interest can be verified using, for example, the chain termination method of double-stranded template sequencing of Wallace et al., Gene 16:21-26 (1981).

Based on sequence homology, degenerate oligonucleotides can be designed as primer sets, and PCR can be performed under appropriate conditions (see, e.g., White et al., PCR Protocols: Current Methods and Applications, 1993; Griffin and Griffin, PCR Technology, CRC Press Inc. 1994) to amplify nucleotide sequence segments from cDNA or genomic libraries. Using the amplified segments as probes, full-length nucleic acids encoding target proteins are obtained.

After obtaining the nucleic acid sequence encoding the target protein, the coding sequence can also be modified by many known techniques (such as restriction endonuclease digestion, PCR and PCR-related methods) to produce a coding sequence, which includes mutants and variants derived from the wild-type protein. The polynucleotide sequence encoding the desired polypeptide can then be subcloned into a vector (such as an expression vector), so that a recombinant polypeptide can be produced from the resulting construct. The coding sequence can then be modified (such as nucleotide substitution) to change the properties of the polypeptide.

A variety of mutation generation protocols are established and described in the art and can be readily used to modify polynucleotide sequences encoding target proteins. See, for example, Zhang et al., Proc. Natl. Acad. Sci. USA, 94: 4504-4509 (1997); and Stemmer, Nature, 370: 389-391 (1994). These procedures can be used alone or in combination to generate a set of nucleic acid variants, and thereby generate variants of encoded polypeptides. Kits for mutagenesis, library construction, and other diversity generation methods are commercially available.

Mutational methods for generating diversity include, for example, site-directed mutagenesis (Botstein and Shortle, Science, 229: 1193-1201 (1985)), mutagenesis using a uracil-containing template (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 nicked double-stranded DNA (Kramer et al., Nucl. Acids Res., 12: 9441-9456 (1984)).

Other possible methods for generating mutations include site 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., 13:4431-4443 (1985)), Res., 14:5115 (1986)), restriction selection and restriction purification (Wells et al., Phil. Trans. R. Soc. Lond. A, 317:415-423 (1986)), mutagenesis by total gene synthesis (Nambiar et al., Science, 223:1299-1301 (1984)), double-strand break repair (Mandecki, Proc. Natl. Acad. Sci. USA, 83:7177-7181 (1986)), mutagenesis by the polynucleotide chain termination method (U.S. Pat. No. 5,965,408), and error-prone PCR (Leung et al., Biotechniques, 1:11-15 (1989)).

Codon optimization

In some embodiments, the nucleic acid sequence encoding the target protein (e.g., antigen or other protein) is codon optimized. The term "codon optimized" refers to changing the nucleic acid sequence without changing the encoded amino acid sequence, reducing or rebalancing the codon bias (i.e., preferentially using specific codons that may vary between species) in this manner. In some embodiments, codon optimization improves (e.g., antigen or other protein) translation efficiency. As a non-limiting example, leucine is encoded by six different codons, some of which are rarely used. By rebalancing the use of codons (e.g., in a reading frame), a preferential leucine codon can be selected instead of a rarely used codon. The nucleic acid sequence encoding the target protein (e.g., antigen or other protein) is changed so that a rarely used codon is converted into a preferred codon.

For example, rare codons can be defined 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 to be expressed. See, e.g., a codon usage table obtained from Kazusa DNA Research Institute, Japan (www.kazusa.or.jp/codon/), used in conjunction with software from DNA 2.0 (www.dna20.com/), such as "Gene Designer 2.0" software, with a cutoff threshold of 15%.

Codon optimization can also be used to adjust GC content, for example, to increase mRNA stability or reduce secondary structure; or otherwise minimize codons that may lead to sequence stretches that impair expression of a protein of interest (eg, an antigen or other protein).

4. Preparation and vaccination method

Object

The methods and compositions of the present invention can be used for vaccination of any subject (e.g., a human or other mammal) that may benefit from an enhanced immune response to infection (e.g., infection by a coronavirus such as SARS-CoV-2). In some embodiments, the subject is a male. In some embodiments, the subject is a female. In some embodiments, the subject is an adult (e.g., an adult male). In some embodiments, the subject is a teenager. In some embodiments, the subject is a child. In some embodiments, the subject is over 60, 70, or 80 years old.

In some embodiments, the subject has not yet been infected with a pathogen (e.g., SARS-CoV-2), and the methods and compositions are used to enhance the subject's immune defense against the pathogen to prevent future infection. In other embodiments, the subject has already been infected with the pathogen, and the methods are used to enhance the subject's immune response to the pathogen to slow or potentially reverse the original infection.

Pharmaceutical composition

The present disclosure provides a composition 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 an antigen) and a pharmaceutically acceptable carrier. In some embodiments, the vaccine further comprises one or more adjuvants or compounds. Therefore, the present disclosure provides a pharmaceutical composition for inducing an immune response in an object. 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., immunogenic conjugates as described herein) and/or comprises, for example, spike proteins, E proteins, M proteins, or N proteins, fragments thereof, or combinations thereof, and a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises an adjuvant.

The composition can be formulated for, e.g., injection, inhalation, or topical administration, e.g., to facilitate direct exposure of host cells and tissues to immunogenic components. In some embodiments, the composition (e.g., DNA vaccine) is formulated for subcutaneous injection. In some embodiments, the composition (e.g., DNA vaccine) is formulated as naked DNA (see, e.g., U.S. Patents 6,265,387, 6,972,013, and 7,922,709).

In a specific embodiment, 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 the parental virus, to incorporate exogenous genetic material (e.g., one or more polynucleotides encoding one or more antigens described herein). In some embodiments, the virus is a herpes virus (e.g., CMV, adenovirus, or adeno-associated virus (AAV)). Self-starting CMV ( _SL CMV) vectors can be prepared, for example, by culturing the intestinal bacteria comprising the vector, lysing the cultured bacterial cells, purifying the vector, while ensuring that endotoxin is not present or is below the pyrogenic threshold (e.g., 5 endotoxin units/kg body weight), and the vectors prepared for administration. In some embodiments, the protein encoding the antigen of the present application is produced in vitro using standard molecular biology techniques, and purified before the vaccine formulation as described herein.

In some embodiments, the nucleic acid vaccine is formulated with, for example, an in vivo transfection agent comprising one or more agents that can protect the nucleic acid from degradation in vivo and facilitate delivery of the nucleic acid to cells. Suitable examples of such agents include, but are not limited to, in vivo-jetPEI ^™ (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.

Pharmaceutical compositions of the present disclosure may include pharmaceutically acceptable carriers. In some aspects, pharmaceutically acceptable carriers are determined in part by the particular composition used and the particular method for applying the composition. Therefore, there are various pharmaceutical composition preparations suitable for the methods and compositions of the present invention (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, 18th edition, Mack Publishing Co., Easton, PA (1990)).

As appropriate, the pharmaceutical composition will generally also include one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextran), mannitol, proteins, polypeptides or amino acids (e.g., glycine), antioxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxytoluene, butylated hydroxyanisole, etc.), bacteriostats, chelating agents (e.g., 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, sweeteners and coloring compounds.

The pharmaceutical composition is applied in a manner compatible with the dosage formulation, and is applied in an amount effective for treatment or prevention. The amount applied depends on a variety of factors, including, for example, age, body weight, physical activity, genetic characteristics, overall health, sex and personal diet, the condition or disease to be treated or prevented, and the stage or severity of the condition or disease. In certain embodiments, the size of the dosage can also be determined by the presence, nature and degree of any adverse side effects of the concomitant therapeutic agent or preventive agent in a specific individual. Other factors that may affect the specific dosage level and dosage frequency of any specific patient include the activity of the specific compound used, the metabolic stability of the compound and the duration of action, the mode of administration and time, and the excretion rate.

In some embodiments, the vaccine comprises an adjuvant, i.e., a compound administered to a subject in combination with an antigen for the purpose of enhancing the immune response to the antigen. Adjuvants can improve the immunogenicity of a vaccine in any of a variety of ways, and can include inorganic compounds (such as salts, e.g., aluminum salts) as well as organic compounds and mixtures of compounds, including extracts and preparations, e.g., Freund's incomplete adjuvant, squalene, MF59, monophosphoryl lipid A, QS-21.

Typically, a compound (e.g., a vaccine or adjuvant) is administered for therapeutic or prophylactic (e.g., vaccination) purposes, and the compound is administered 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 to an antigen or antigen-derived pathogen, e.g., with reference to any parameter or indicator described herein, and/or an amount sufficient to enhance the immunity of a subject to infection from a pathogen or to the spread of an infection already present in the subject.

In certain embodiments, the dosage can take the form of a solid, semisolid, lyophilized powder, or liquid dosage form, such as, for example, tablets, pills, granules, capsules, powders, solutions, suspensions, emulsions, suppositories, retention enemas, creams, ointments, lotions, gels, aerosols, foams, etc., preferably in a unit dosage form suitable for simple administration of precise dosages.

As used herein, the term "unit dosage form" refers to physically discrete units (e.g., ampoules) suitable as single doses for humans and other mammals, each unit containing a predetermined amount of a therapeutic or prophylactic agent calculated to produce the desired onset, tolerability, and/or therapeutic or prophylactic effect in combination with a suitable pharmaceutical excipient. In addition, more concentrated dosage forms can be prepared, from which more dilute unit dosage forms can then be prepared. Thus, a more concentrated dosage form will contain an amount that is substantially greater than, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times the amount of the therapeutic or prophylactic compound.

Methods for preparing such dosage forms are known to those skilled in the art (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra). The dosage forms typically include conventional pharmaceutical carriers or excipients, and may additionally include other agents, carriers, adjuvants, diluents, tissue penetration enhancers, solubilizers, etc. Suitable excipients can be tailored to specific dosage forms and routes of administration by methods well known in the art (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra).

Application

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

The compositions of the present disclosure can be administered in a single dose or in multiple doses, such as two doses administered at intervals of about one month, about two months, about three months, about six months, or about 12 months. Other suitable dosage schedules can be determined by medical practitioners.

In some embodiments, additional compounds or drugs may be co-administered to the subject. Such compounds or drugs may be co-administered for the purpose of alleviating the signs or symptoms of the disease being treated, reducing side effects caused by inducing an immune response, etc.

The pharmaceutical composition of the present application can be administered to a subject locally or systemically, for example, intraperitoneally, intramuscularly, intraarterially, orally, intravenously, intracranially, intrathecally, intraspinally, intralesionally, intranasally, subcutaneously, intracerebroventricularly, topically and/or by inhalation. In a specific embodiment, the composition is administered by electroporation (e.g., for primary DNA vaccines) or subcutaneously (e.g., for boosting).

The vaccine of the present application can be administered multiple times (e.g., 1, 2, 3, 4, 5 or more times) any one time, and can be after any vaccination in multiple vaccination schemes, for example, every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks. In a specific embodiment, the vaccine is administered in the form of a DNA primary immunization (e.g., with a plasmid encoding a conjugated polypeptide as described herein), and then, for example, after about 4 weeks, an adenovirus vector encoding an antigen is strengthened. The DNA vaccine can be administered at any level in a variety of levels, for example, 4 mg of plasmid DNA vector per object per vaccination, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mg of plasmid DNA vector per object per vaccination, or 1-10, 1-20, 1-8, 1-7, 2-6, 3-5 mg of plasmid DNA vector per object per vaccination.

Assessment of immune response

The immune response of the object receiving the vaccine of the present disclosure can be detected, characterized or quantified in any of a variety of ways. For example, any assay described in any embodiment can be used to detect the presence of the polynucleotides of the present disclosure in the detectable object cells, detect and characterize antibodies or T cells specific to the vaccine of the present application, or evaluate the protection provided by the vaccine against pathogen infection. In some embodiments, particularly in embodiments in which nucleic acid vectors are used to deliver the antigens of the present invention, the presence and level of the vector sequence can be assessed, for example, by using qPCR measurement sequences from, for example, blood or saliva samples from the object. The level of nucleic acids can also be detected from different tissues of the object, for example, as obtained from a biopsy or by lavage of mucosal tissue.

In some embodiments, the immune response of the subject is assessed by immunophenotype, for example, by assessing T cell memory effector subsets, NK cells with adaptive properties (e.g., FcεRIγ ^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, for example, using flow cytometry as described in the Examples.

The immune response of the object can also be assessed by characterizing the antigen-specific T cell response from the object. For example, PBMC or LNMC cells can be stimulated with one or more antigens from the vaccine, optionally, as described in the examples, combined with inhibitors (such as VL9 peptides or anti-HLA antibodies). After a suitable amount of time (e.g., 16 hours), cells can be assessed using antibodies for, for example, CD3, CD4, CD8, CCR7, CD95, IL-2, IL-17, IFN-γ and/or TNF-α. CD4 ⁺ and/or CD8 ⁺ cells secreting cytokines can be assessed using, for example, flow cytometry.

In some embodiments, the antibodies obtained from the object can be evaluated, for example, by using ELISA to detect the combination with the antigen. In some embodiments, neutralization or enhancing antibodies are tested using RVP (reporter virus particles). In specific embodiments, the vaccine of the present application triggers a strong neutralizing antibody response and a low or non-existent enhanced response. In some embodiments, for example, wherein the vaccination strategy is tested in a model animal, as described in the examples, a challenge assay using a pathogen can be used.

Any parameter or effect described in the Examples or elsewhere herein (e.g., neutralizing antibody production, specific T cell response, protection against pathogens, etc.) can be used to evaluate the efficacy of the vaccines described herein. In some embodiments, the vaccines of the present disclosure result in at least about 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 relative to a control value (e.g., a value observed in an object not receiving a vaccine of the present disclosure). In some embodiments, the vaccines described herein do not substantially induce antibody-dependent infectivity enhancement (ADEI) in an object. In some embodiments, the neutralizing antibody response induced by the vaccine in an object is significantly greater than any ADEI induced in an object.

Any number of diseases can be prevented and/or treated using the compositions and/or methods of the present application. In some embodiments, infectious diseases are prevented and/or treated. In some embodiments, bacterial infectious diseases are prevented and/or treated. In some embodiments, viral infectious diseases are prevented and/or treated. In some embodiments, fungal infectious diseases are prevented and/or treated. In some embodiments, protozoan infectious diseases are prevented and/or treated. In some embodiments, helminth infectious diseases are prevented and/or treated. In some embodiments, cancer is prevented and/or treated. In some embodiments, bacterial, viral, fungal, protozoan and/or helminth infectious diseases are prevented and/or treated. In some embodiments, coronavirus infectious diseases are prevented and/or treated. In some embodiments, COVID-19 (i.e., a disease caused by SARS-CoV-2 infection) is prevented or treated.

5. Kit

On the other hand, kits are provided herein. In some embodiments, the kit comprises a vaccine of the present disclosure (e.g., a vaccine comprising one or more immunogenic conjugates or antigens of the present invention, or a vector comprising a polynucleotide encoding one or more antigens or immunogenic conjugates of the present disclosure, and an optional pharmaceutically acceptable carrier). In some embodiments, the kit comprises an adjuvant. In some embodiments, the kit is used to induce an immune response to an antigen (e.g., a coronavirus spike protein, an E protein, an M protein, or an N protein, a fragment or a combination thereof). In other embodiments, the kit is used to prevent or treat a disease (e.g., COVID-19). In some embodiments, the kit is used to induce a B cell (i.e., an antibody) response to one or more antigens. In some embodiments, the kit is used to induce a T cell response to one or more antigens. In some embodiments, the kit is used to induce a B cell response to an antigen (e.g., an antigen present in an immunogenic conjugate as described herein) and a T cell response to a second antigen (e.g., a second antigen as described herein).

The kits of the present disclosure can be packaged in a manner that allows safe or convenient storage or use (e.g., in a box or other container with a lid). Typically, the kits of the present invention include one or more containers, each of which stores a specific kit component (e.g., a reagent, a control sample, etc.). The choice of container will depend on the specific form of its contents, such as a kit component in liquid form, powder form, etc. In addition, the container can be made of a material designed to maximize the shelf life of the kit components. As a non-limiting example, a light-sensitive kit component can be stored in an opaque container.

In some embodiments, the kit includes one or more elements (e.g., a syringe) for administering a composition (i.e., a drug component described herein) to a subject. In other embodiments, the kit also includes instructions for use, for example, instructions (i.e., protocols) for implementing the method of the present application (e.g., instructions for using the kit to enhance the immune response of a subject to an antigen from a pathogen (e.g., SARS-CoV-2)). Although explanatory materials typically include written or printed materials, they are not limited thereto. The present disclosure contemplates any medium capable of storing such instructions and transmitting them to an end user. Such media include, but are not limited to, electronic storage media (e.g., disks, tapes, films, chips), optical media (e.g., CDROMs), etc. Such media may include the address of an Internet website providing such explanatory materials.

6. Examples

The present disclosure will be described in more detail by way of 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 readily recognize that various non-critical parameters that can be changed or modified to produce substantially the same results.

Example 1. Conjugated polypeptide vaccine comprising a SARS-CoV-2 S1 domain linked to an anti-CD3 single chain variable fragment Seedling

This embodiment provides a conjugated polypeptide that is capable of binding to B cells with surface receptors that react to the "S1" domain of the SARS-CoV-2 spike protein and T cells with abundant T cell surface protein CD3. When included in anti-SARS-CoV-2 vaccine regimens such as those described below, the conjugated polypeptide elicits an antibody response to the S1 domain, which may contribute to protection against COVID-19.

SARS-CoV-2 enters cells through the activity of a spike protein (S) that has receptor binding (S1) and membrane fusion (S2) regions. The SARS-CoV-2 spike displays many features of traditional class I fusion proteins, including the presence of distinct heptad repeats within the fusion domain. Antibodies to the S1 domain can block SARS-CoV-2 infection 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 (ε, γ, δ, and ζ) associated with the T cell receptor (TCR) and plays a key role in transmitting activation signals from the TCR to the interior of the T cell.

We created a conjugate polypeptide comprising the S1 region of the SARS-CoV-2 spike and a CD3 binding polypeptide by fusing the coding sequences of the following protein elements (see, e.g., FIG. 5 ): a tissue-type plasminogen activator signal sequence (to allow the conjugate polypeptide to be secreted from cells), an anti-CD3 scFv derived from a mouse anti-CD3 antibody, SP34 (see, e.g., U.S. Patent Application Publication No. 2016/0068605A1); a flexible linker; and SARS-CoV-2S1 (codon-optimized). The resulting amino acid sequence is shown in SEQ ID NO.1. The codon-optimized nucleic acid sequence of the polypeptide (SEQ ID NO.2) 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.

In order to test the CD3 binding function of the conjugated polypeptide, the resulting plasmid was transfected into 293 cells by calcium phosphate precipitation. The supernatant from the transfected cells was applied to a tissue culture plate to allow secreted proteins to bind to the surface of the plate, thereby forming a fixed array of such secreted proteins, which, if combined with TCR, should send activation signals to T cells. We applied T cells from rhesus monkeys to coated wells or control wells. Fluorescent antibody staining and data collection on a flow cytometer revealed that the immobilized conjugated polypeptide activated T cells, resulting in CD69 upregulation and intracellular interferon-γ production on the cell surface (Fig. 1). The result confirmed the combination of the conjugated polypeptide with the CD3 molecules on the T cell surface.

Example 2. Providing SARS-CoV-2 vaccine candidates

This example provides a SARS-CoV-2 vaccine candidate that (i) stimulates both T cell and antibody responses to minimize the risk of antibody-dependent enhancement; (ii) has been shown to be effective in rhesus macaques; and (ii) is ready for future Phase I testing in humans. This vaccine platform combines rapid development (DNA administration) with broad, strong T cell responses (due to the CMV vector) and neutralizing antibody responses to the Spike S1 domain.

Traditional vaccine development against previously unknown pathogens takes years, but protecting human health may require rapid responses on a scale of months (1). However, accelerated development is complicated by the lack of appropriate animal models for emerging pathogens; the risk of antibody-dependent enhancement of infectiousness (ADEI), which can occur whenever suboptimal antibody responses are induced (2); and the difficulty of developing new manufacturing processes for subunit, attenuated, or vectored vaccines.

The Self-Priming CMV DNA Platform ( _SL CMV) alleviates or eliminates these complexities, allowing for the rapid development of new vaccines that elicit exceptionally broad and robust immune responses localized to mucosal surfaces threatened by SARS-CoV-2. These vaccines are administered as cytomegalovirus genomes that “prime” vector vaccine replication in vivo, resulting in a robust immune response with unique characteristics associated with CMV infection. Features of _SL CMV include: (i) being based on the Towne HCMV strain, proven safe for use in people of all ages; (ii) immune responses generated by both CMV seropositive and seronegative individuals due to CMV superinfection capacity and exclusive deletions in the CMV genome; (iii) very broad and strong T-cell responses that “delineate” the vaccine antigens; (iv) a demonstrated ability to elicit a balanced response (including antibodies, CD4 ⁺ T cells, and CD8 ⁺ T cells) without relying on or dominating any single effector response; (v) localization of responding T cells to mucosal surfaces due to an effector memory phenotype; (vi) engineering of new vaccine candidates within weeks to months; and (vii) a single manufacturing process based on plasmid production in Escherichia coli.

Most coronavirus vaccines in development are designed to elicit antibodies. Such vaccine strategies must be approached with caution due to the potential for antibody-dependent enhancement (ADEI), especially when antibody levels are low (2). Highly concentrated antisera against SARS-CoV-1 were shown to neutralize the virus, whereas diluted antibodies elicited ADEI in human pronuclear cell cultures. On the other hand, T cell responses often target highly conserved internal proteins and are long-lived. Indeed, airway memory CD4 ⁺ T cells were shown to mediate protective immunity against SARS-CoV-1 and MERS-CoV. We hypothesized that a self-priming CMV DNA vaccine against SARS-CoV-2 could provide broad and protective adaptive immunity localized to mucosal surfaces.

Part 1: Characterization of immune responses in rhesus macaques to a self-priming CMV/SARS-CoV-2 vaccine designed to elicit T cell or antibody responses.

Transgenes in CMV vector vaccines driven by constitutive promoters elicited strong T cell responses but little or no accompanying antibody responses; transgenes expressed from strong late promoters such as pp65b elicited strong antibody responses and weak T cell responses. Using these strategies, we created candidate _SL CMV DNA vaccines designed to elicit significant T cell responses to SARS-CoV-2 E, M, and N proteins, as well as strong neutralizing antibody responses to the spike S1 domain. The latter candidate expresses the spike S1 domain either unmodified or linked to an anti-CD3 scFv, which physically connects B cells producing anti-S1 antibodies to T cells capable of providing help. Over time, adaptive immune responses ensued systemically and at mucosal surfaces. The production of neutralizing antibodies relative to enhancing antibodies was monitored using a reporter viral particle (RVP) assay.

Part 2: Evaluation of the protective efficacy of the _SL CMV/SARS-CoV-2 vaccine against the Davis isolate of SARS-CoV-2 in rhesus macaques.

SARS-CoV-2 isolated from patients is grown and characterized, and viral pathogenesis is assessed in monkeys. We test the protective efficacy of the T-cell and B-cell vaccines created in Part 1 by challenging the vaccinated animals with SARS-CoV-2. T-cell and B-cell vaccines are tested separately and together to test the relative contribution of each branch of the adaptive immune system to protection versus disease enhancement.

Part 3: Testing the safety and potential efficacy of a self-priming human CMV vector vaccine in rhesus macaques.

It has recently been shown that human CMV (HCMV) vectored vaccines elicit strong effector memory T cell responses in rhesus macaques. Therefore, we created an HCMV vectored vaccine as a candidate for future human clinical trials, developed a GMP process for production, and tested the efficacy of the _SL HCMV regimen in rhesus macaques. The HCMV vectored vaccine used in this in vivo experiment will be selected based on the results of the protective study using _SL RhCMV in Part 2.

Significance

Rapid vaccine development against emerging infectious threats is an unmet need: traditional vaccine development against previously unknown pathogens has been slowed by difficult to address fundamental biological questions. Most importantly, while inducing high titers of neutralizing antibodies (nAbs) seems an obvious approach, we do not know what titers of nAbs are protective in practice, nor how this threshold varies across extremes of age and comorbidity. For any emerging pathogen, we do not know whether poor antibody responses, particularly in the elderly and young, may lead to antibody-dependent enhancement of infectiousness (ADEI).

The immune correlates of successful vaccination against SARS-CoV-2 are unclear: most coronavirus vaccines currently in development target the most variable part of the spike glycoprotein and induce antibody responses only against the virus present in the vaccine. SARS-CoV-1 escape mutants occur 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 specifically elicit antibodies must additionally be approached with caution due to possible ADEI, especially when antibody levels are low (4). Highly concentrated antisera against SARS-CoV-1 were shown to neutralize viral infectivity, while diluted antibodies induced ADEI in human pronuclear cell cultures, resulting in a cytopathic effect and increased levels of TNF-α, IL-4, and IL-6 (5-7). Vaccine candidates based on the full-length SARS-CoV-1 spike were shown to induce non-neutralizing antibodies, and immunized animals were not protected. Instead, they experienced adverse effects such as exacerbated hepatitis, increased morbidity, and a stronger inflammatory response (8,9).

T cell responses elicited by CoV vaccines also play a key role in protection and clearance. Mice deficient in T cells were unable to clear MERS-CoV infection, but mice lacking B cells were able to do so (10). Furthermore, airway memory CD4 ⁺ T cells were shown to mediate protective immunity against both SARS-CoV-1 and MERS-CoV (11). As shown below, most vaccine types did not elicit significant numbers of memory CD4 ⁺ T cells—but CMV-vectored vaccines did. T cells that responded to CMV-vectored vaccines localized to the airways, among other effector sites, and were recovered by bronchoalveolar lavage (12). CMV-responsive T cells recapitulated other essential features of cells shown to be protective against SARS-CoV-1, including CXCR3 expression, IFN-γ production, and IL-10 production (13).

CMV vector vaccines can elicit strong antibody responses: Although CMV vaccines elicit weak antibody responses to some transgenes driven by heterologous promoters, CMV infection and vaccination elicit strong antibody responses to proteins expressed under the control of the endogenous pp65b promoter. The promoter is one of the most active promoters after late DNA replication during CMV infection. For example, it has been found that 4/4 rhesus macaques vaccinated with a CMV vaccine carrying the Ebola virus glycoprotein (GP) under the control of the pp65b promoter produced GP-specific antibodies, which were boosted after a second vaccine administration (21). The high levels of GP antibodies induced by RhCMV/EBOV-GP and its ability to undergo IgG class switching indicate the presence of adequate CD4 ⁺ T helper function. Three of the four rhesus macaques with the highest anti-GP titers were protected against lethal EBOV challenge.

An important property of CMV vectors for use against emerging pathogens is the ability to be re-administered to previously exposed individuals. This capability enables CMV vector vaccines to be used repeatedly to protect against a range of emerging threats over time.

Unpracticality of conventional CMV vaccines for use against emerging pathogens: Despite their immunological advantages, practical barriers have hampered the rapid development of CMV vaccines for human clinical use when those vaccines are delivered as live viruses. The most important issue is the enormous difficulty of large-scale production of a uniform test article from a slow-growing and mutable betaherpesvirus (29).

Innovation

Vaccination with CMV vectors in nucleic acid form: Although transfection with CMV genomic DNA is the cornerstone of many in vitro techniques, and other researchers have experimented with delivering herpesvirus genomes as plasmids in Salmonella organisms (26), to our knowledge, there have been no attempts to deliver naked or chemically complexed CMV genomic DNA as a vaccine. We demonstrate below that gene expression, genome replication, viremia, and immune responses occur after administration of CMV BAC DNA.

Placing the BAC replication origin at the end of the CMV genome to allow excision of the BAC by the CMV terminase complex: In order to utilize CMV vectors in their DNA form for vaccination, it is necessary to change the current CMV BAC construct so that the BAC backbone can be excised without the expression of recombinases or nucleases in vivo. CMV has relatively strict packaging restrictions due to the need to package the unit length genome into an icosahedral capsid. The current CMV BAC utilizes an endogenous recombinase gene placed within the BAC portion of the DNA construct. After transfection of mammalian cells, the recombinase is expressed and the BAC replication machinery is excised from the replicating genome. In order to make the BAC DNA vector suitable for delivery in humans, we reorganized the CMV genome ends so that the BAC can be excised without the need for recombinase expression. Our reconfigured BAC construct utilizes the viral terminase complex to eliminate the bacterial replication origin during the packaging step of CMV replication (Figure 2). We have moved the location of the BAC origin and replication machinery from its current location (RhCMV US1/2) to between the terminase complex recognition site (TCRL) containing the direct repeat sequence of the virus. We show below that this arrangement allows efficient replication and packaging of the vaccine genome following introduction into host cells in vivo.

New and Improved RhCMV-SIV Vaccines: The first-generation RhCMV-SIV vectors carry an intact endogenous viral IL-10 gene that suppresses host immune responses (27-31). We have created a second-generation RhCMV vector platform—viral IL-10-deficient RhCMV or RhCMVdIL10—that has a unique immunological profile and can protect RhCMV-negative infant rhesus macaques, whereas the first-generation vaccine does not (27).

Example 3. CMV vector vaccines elicit uniquely broad and robust T cell responses in both 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). In addition, TEM cells elicited by CMV vector vaccines recognize distinct and unusual epitopes, including a predominant response 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 the number of peptide epitopes as those responding to other vaccine types, resulting in a response that “paints” the vaccine antigens and should prevent pathogen escape (20). Although much of the published work has focused on CD8 ⁺ T cell responses, CMV vaccines stimulate CD4 ⁺ T cell responses of equivalent strength, a feature not seen with other vector vaccines (Figure 3A). Critically, the unique abundance of responsive CD4 ⁺ T cells in the airways of vaccine recipients ( Figure 3B ) meets the requirements described for protection against SARS-CoV-1 ( 11 ).

Example 4. A DNA-based, self-priming CMV vector vaccine can be rapidly engineered and manufactured to broadly protect It is protected from emerging threats.

In order to allow manipulation using prokaryotic genetics, the RhCMV-SIV vaccine is maintained as a circular bacterial artificial chromosome (BAC), which comprises a vector genome DNA with an embedded BAC replication origin (ori), flanked by a recognition signal of a site-specific recombinase or restriction enzyme. In order to effectively replicate and package, it is necessary to excise the BAC origin by site-specific recombination in vitro or after transfection. We have reengineered these vectors to comprise the BAC origin outside the viral genome, and flanked by a CMV terminal enzyme complex recognition seat, thereby allowing automatic excision by the CMV terminal protease when the vector begins to replicate (Fig. 2). Therefore, the new vector does not require in vitro digestion or Cre recombinase expression, and is delivered to the vaccine recipient as a stable circular DNA molecule. After arriving at the recipient nucleus, these CMV vaccine genomes enter the viral replication cycle, and produce a series of viral particles, which cause the same protective immune response as those caused by the CMV vector vaccine delivered as viral particles. In fact, the vaccine responses generated appeared earlier and were generally stronger than those elicited by viral particles, most likely due to the administration of 10,000 times more vaccine genomes (Figure 6).

Example 5. Characterization of self-priming CMV/SARS-CoV-2 antibodies designed to elicit T cell or antibody responses in rhesus monkeys Immune responses to CoV-2 vaccines.

We hypothesized that self-priming RhCMV vaccines would generate T cell responses as broad as those administered in viral particle form and that linking of the SARS-CoV-2S1 immunogen to an anti-CD3 scFv fragment would enhance the speed and potency of NAb responses.

The rationale for this hypothesis was that we had shown that SLRhCMV vaccines were injected with replicating virus that could be detected in the blood weeks after administration, and we expected these formats to elicit T cell responses (in group B) that were as broad as those elicited by conventionally packaged CMV (~90 peptides/1000 versus 12 for adenoviral vectors).

For the SLCMV vaccines designed to elicit B cell responses (Panels C-D), we compared the secreted spike S1 domain to the same protein linked to a scFv fragment that binds to CD3 (Figure 7). The result is a molecule that can "bridge" S1-specific B cells and any nearby T cells. Our preliminary data suggest that the idiotype linked to anti-CD3 in the bispecific antibody rapidly elicits very strong anti-idiotype antibodies. Therefore, we expect to deliver the spike S1 domain linked to the anti-CD3 scFv to elicit stronger antibodies than S1 alone.

We tested the immune response to three candidate vaccine components, all of which were administered twice eight weeks and four weeks before the challenge (Figure 7). The T cell component contained a mixture of SLRhCMV/N and SLRhCMV/EM vaccines. We evaluated the intensity and breadth of the T cell response to these vaccines (Group B) to provide data for future protection correlation examinations. There are two candidate B cell components (Groups C and D), one of which will be selected for the efficacy test in Example 6 due to induction of a stronger neutralizing antibody response and/or enhanced reduction. Finally, we tested a regimen consisting of a T cell vaccine (SLRhCMV/N+EM and selected B cell vaccines, administered together).

Vaccine construction

As shown in Figure 2, SLRhCMV/N, SLRhCMV/EM, SLRhCMV/S1 and SLRhCMV/S1-anti-CD3 were generated as self-priming BAC constructs bound by the CMV terminase recognition site. All coding sequences are codon-optimized versions of those found in SARS-CoV-2. The N protein and EM fusion protein cassettes are expressed under the control of the EF-1α promoter (including its first intron) and have been recombined into the Rh213/214 region of the RhCMV genome, where we have reused this position and observed major T cell responses. The S1 and S1-anti-CD3 scFv cassettes are expressed under the control of the endogenous late RhCMV pp65b promoter and expressed before the 23-aa tPA leader sequence to promote efficient secretion. The S1-anti-CD3 fusion uses a humanized scFv region derived from the anti-CD3 clone SP34, which is a clone we previously used to construct a bispecific antibody.

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

Vaccine administration

Our preliminary data (Fig. 6) show that subcutaneous vaccination with 100 μg RhCMVdIL10 vaccine BAC DNA is sufficient to induce an immune response. According to the manufacturer's instructions, BAC DNA was prepared with in vivo-jetPEI (Polyplus). In brief, 100 μg DNA (50 μg each of RhCMVdIL10-SIVgag and -SIVenv) and 16 μL in vivo-jetPEI were diluted into 5% glucose solution (1 ml), then mixed and incubated for 15 minutes.

RhCMV vector replication and shedding

Using our published protocol, vaccine-derived RhCMV DNA was measured weekly in blood and saliva samples by qPCR (16, 33). These measurements provide an assessment of the replication and spread of BAC DNA-encoded viral vectors. Spread to various tissues was assessed by qPCR in autopsies after SARS-CoV-2 challenge in Example 6.

Immunophenotyping

The immune phenotypes of greatest interest were the T cell memory effector subset, which comprised the largest proportion of cells responding to RhCMV and RhCMV/SIV vaccines; NK cells with adaptive properties (i.e., FcεRIγlow “memory” NK); T cells with innate properties (NKG2A ⁺ ); and antigen-presenting cells (particularly monocytes) expressing CD80/83/86. All of these cell populations were altered following infection with wild-type or vaccine strain RhCMV. All were assessed using a set of three flow cytometric panels used in our previously published work to examine antigen-presenting cells, T cells, and NK cells (34).

Antigen-specific T cell responses

Assay wells containing up to 1 MPBMC or LNMC cells were stimulated with vehicle (negative control for DMSO toxicity), overlapping RBD, S1 peptide, E peptide, M peptide and/or N peptide (Intavis) or PMA/ionomycin (positive control). Inhibitors (such as VL9 peptide or anti-HLA antibody) were applied one hour before the start of stimulation, and peptide stimulation was added again. After 16 h, cells were stained with a fixable live-dead stain and antibodies that react to CD3, CD4, CD8, CCR7, CD95, IL-2, IL-17, IFN-γ and TNF-α. The fraction of CD4 ⁺ and CD8 ⁺ T cells that secrete cytokines was determined by cytometry on, for example, BD Fortessa or FACSymphony.

Antibody response

Induction of spike S1 domain binding antibodies was measured by ELISA on weekly plasma samples according to our published protocol (16). Neutralizing or enhancing antibodies were tested by RVP assay.

Combined T-cell and B-cell vaccines (Group E)

The binding and neutralizing antibody responses in groups C-D were fully characterized, and groups with excellent neutralizing titers and lower or no enhancement were selected for SARS-CoV-2 challenge (see Example 6). In addition, after selecting the best candidate for inducing Nabs, a T/B combination vaccine group (Group E) was formed. This group received a combined vaccination of SLRhCMV/N, SLRhCMV/EM, and selected B cell vaccines.

Interpretation of the data

We hypothesized that our SLRhCMV vaccine (delivered as DNA) elicited robust T- and B-cell responses, and that the latter response was stronger when the antigen was linked to an anti-CD3 scFv. To determine whether these responding cells were present in the most relevant tissue, the lung, we performed bronchoalveolar lavage and assayed T cells that could be recovered from the airways.

Based on ADEI previously observed with other RNA viruses causing respiratory infections, including SARS-CoV-1, some animals may show enhancement in the RVP assay after vaccination. Such results are important both for understanding the immunopathogenesis of COVID-19 and for interpreting the results of the challenge experiments described below.

Statistical analysis

The nonparametric Kruskal-Wallis test was used to test for differences between groups in summary measures or outcomes at a single time. For longitudinal outcomes, such as testing for associations between T cell and antibody responses, generalized linear mixed models (GLMMs) were used as the analytical framework with random effects correction for within-animal correlations caused by continuous measurements.

Example 6. Evaluation of the protection of SLCMV/SARS-CoV-2 vaccines against the SARS-CoV-2 Davis isolate in rhesus monkeys Protective effectiveness.

We hypothesize that a strong T cell response localized in the airways protects against SARS-CoV-2 and additionally prevents 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 the virus's target tissues, would be expected to protect against SARS-CoV-2. In fact, airway memory CD4 ⁺ T cells have previously been shown to mediate protective immunity against SARS-CoV-1 and MERS-CoV (see, e.g., Zhao, Immunity 44:1379). In addition, ADEI is thought to occur due to enhanced uptake of the virus into cells, so that the virus is co-opted and allowed to replicate productively—however, the virus internalized due to ADEI should be easily cleared by T cells.

We developed a unified challenge and monitoring protocol so that consistent virological, immunological, and pathological data sets can be utilized across experiments. Rhesus macaques vaccinated as indicated (groups B-E in Figure 7) will be challenged at least 8 weeks after the primary vaccination and subsequently undergo repeated clinical assessments; radiography; collection of respiratory and mucosal secretions (e.g., by bronchoalveolar lavage, tracheal wash, or nasal wash) as well as saliva, urine, and feces; blood draws; and tissue collections (see, e.g., www.biorxiv.org/content/10.1101/2020.07.07.191007v1).

Viruses used for infection

The virus stock generated by amplifying the SARS CoV-2 isolate we obtained from a UC Davis patient will be used for animal inoculation and is designated 2019-nCOV/USA-CA9/2020. If the growth of this virus is insufficient, we will use the SARS CoV-2 isolate USA-WA1/2020 (BEI Resources) instead. To infect the animals, a total of approximately 6×10 ⁶ TCID50 in 5 ml of 0.9% sterile saline will be instilled into the conjunctiva, nares, and trachea of anesthetized monkeys to reproduce the relevant transmission routes of COVID-19.

Sampling and determination

Temperature, weight, and activity were monitored throughout. CBC and serum chemistry were obtained for all blood samples to monitor host responses and organ function. The sampling schedule was designed to fully characterize viral shedding, cytokine responses, and adaptive immunity to understand how changes in these parameters reflect lung pathology. We have successfully used the sampling protocol and procedures to characterize influenza A infection in rhesus macaques. Intensive sampling during the first week allowed us to study acute virology and host responses. As ACE2 is expressed in the gastrointestinal and urogenital tracts of rhesus macaques and humans, we assessed viral shedding in saliva, urine, and feces in addition to respiratory secretions. At necropsy (day 28), we collected 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 were evaluated for gross pathology, histopathology, and tissue vRNA levels. Necropsies were performed by board-certified pathologists.

Viral RNA was recovered from respiratory samples using Thermo's MagMAX Viral/Pathogen Nucleic Acid Isolation Kit (recommended by the CDC for COVID-19) and quantified by amplifying the SARS-CoV-2 nucleoprotein (N) gene segment. 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 Laboratories, Wisconsin NPRC, and commercial suppliers. We selected the most consistent assay. Immunological analysis was performed as described above in Example 5.

Interpretation of the data

If our hypothesis is correct, then animals in Group B would be protected against SARS-CoV-2 vaccination despite having generated an immune response composed almost entirely of T cells. Animals in Groups C or D (the ones selected in Example 5) would also be protected, but we remain vigilant about the possibility of ADEI, which could be indicated by increased viral load, shedding, or pathological findings in animals with intermediate antibody titers, regardless of whether they show enhancement in the RVP assay.

Statistical analysis

Summary findings or those assessed at individual time points (e.g., at necropsy) were evaluated using nonparametric tests with p values adjusted according to Benjamini and Hochberg. Longitudinal outcomes were evaluated using linear mixed models (generalized when necessary) with random effects accommodating within-animal dependencies arising from serial measurements.

Example 7. Testing the safety and potential efficacy of a self-priming human CMV vector vaccine in rhesus monkeys.

We assume that the SLHCMV vaccine can be produced on a scale of 10-20 grams in the GMP process, can be safely administered to rhesus monkeys, and can prevent SARS-CoV-2 challenges. The rationale for this hypothesis is that, surprisingly, it has recently been shown that weakened HCMV vectors can elicit and maintain T effector memory responses against inserted antigens in rhesus monkeys (see, e.g., Caposio Sci Rep 9:19236). Therefore, the rhesus monkey model can provide an environment in which the effectiveness of the candidate SLHCMV vaccine produced under GMP conditions against SARS-CoV-2 is tested. If the HCMV vector vaccine is proven to be safe and effective in rhesus monkeys, they will become candidates for subsequent clinical trials.

The SLHCMV forms of all four vaccines administered to group B-D rhesus macaques in Example 5 were engineered. In addition, after challenge with group B-E, GMP production was performed for those SLHCMV vaccines that formed part of the best regimen. For example, if the pathological protection of group B animals was the best, production of SLHCMV/N and SLHCMV/EM was performed. The resulting GMP products were sent to UC Davis for rhesus macaque vaccination and efficacy testing against SARS-CoV-2.

SLHCMV vaccine genome

The HCMV vaccine genome being engineered is based on the Towne vaccine strain due to its excellent safety record. This vaccine is orthologous to the RhCMV genome engineered at Davis. The viral interleukin-10 gene is deleted in both cases; the sequence is inserted into the intergenic region near US28 to stimulate a T cell response; and is placed under the control of the pp65b promoter to stimulate an antibody response. The BAC carries a codon-optimized SARS-CoV-2 sequence driven by the same promoter.

The plasmid backbone sequence used allows the plasmid to be maintained at approximately a single copy (in DH10B cells employing oriS to maintain the plasmid) or ~15-30 copies (after induction of TrfA expression and its interaction with the alternative oriV origin). BAC DNA was purified at laboratory scale by double sequential CsCl equilibrium gradient centrifugation followed by dialysis into PBS. The integrity of the plasmid preparation was confirmed by restriction enzyme footprinting, PCR amplification of the expression cassette and deep sequencing after labeling.

GMP manufacturing was performed by a CMO partner. Upstream process development focused on optimizing conversion and culture conditions to ensure maximum homogeneity of the test article. Downstream process development (purification after cell growth) focused on adapting the CsCl-dependent process used in the laboratory to iodixanol. Testing of the optimal SLHCMV vaccine regimen in rhesus monkeys (Group F)

SLHCMV vaccines were administered in the same combination and at the same dose as used in one of the groups B-E. Immunoassays, SARS-CoV-2 challenges, and pathology assessments were performed similarly.

Interpretation of the data

Our hypothesis predicts that the SLHCMV vaccine can be manufactured in a GMP process with sufficient scale and purity to allow final testing in Phase I human trials. In addition, we believe that the SLHCMV vectored vaccine can demonstrate efficacy in the rhesus macaque model. Such a result may be counterintuitive but is possible based on publications in the literature demonstrating that HCMV can complete its life cycle in rhesus macaque fibroblasts (43) and can elicit a robust TEM response in rhesus macaques.

The immune responses of rhesus macaques to SLHCMV vaccines are also of interest because they may reflect responses achieved with minimal vaccine transmission, as seen in humans with fully inactivated HCMV vector vaccines. Despite some evidence of genome duplication, HCMV (Towne)-based vaccines should be able to achieve minimal or no systemic transmission in rhesus macaques. Therefore, comparing immune responses to RhCMV versus Towne vector antigens should reveal which immune responses are solely dependent on immune regulation and which are dependent on vector transmission.

Statistical analysis

Statistical analysis centered on the characteristics of the vaccine formulation. Our previous experience has shown that single-copy BACs have mutation rates similar to other plasmids, that is, below the level of detection of amplicon sequencing due to polymerase error rates (~0.1%) (46-50). SLHCMV genome replication in rhesus macaques was determined by either (i) vaccine virus sequences in plasma one week after priming or boosting, (ii) gB expression in injection site biopsies (HCMV gB-specific Ab; gB is a late gene expressed only after genome replication), or (iii) anamnestic antibody responses to HCMV gB defined by doubling of titers one month after boosting.

Example 8. Conjugated polypeptide vaccine comprising a SARS-CoV-2 RBD domain linked to an anti-CD3 single chain variable fragment Seedling

We designed a conjugate peptide vaccine, named 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 the tissue-type plasminogen activator (tPA) signal sequence to enable secretion. The RBD is a segment of the SARS-CoV-2 spike protein responsible for its binding to the human receptor ACE2 and is a common target for neutralizing antibodies. s3-RBD has the ability to bind to RBD-responsive B cells (via their cell surface, RBD-specific antigen receptor) and helper T cells (via CD3). Without being bound by the following theory about the specific mechanism of action, we hypothesize that engagement of pairs or clusters of these cognate receptors on B cells and T cells should lead to activation of both cell types, driving continued development of B cells. 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.

In order to immunize rhesus monkeys with a gene vaccine expressing s3-RBD, we prepared a codon-optimized open reading frame using the nucleotide sequence given in SEQ ID NO.7. The sequence was synthesized using techniques known to those skilled in the art and then placed in an expression cassette downstream of the human EF-1-α promoter sequence and upstream of the polyadenylation signal from SV40 (obtaining the complete expression cassette sequence given in SEQ ID NO.8). A plasmid containing an expression cassette was prepared that was endotoxin-free at a medium scale for administration as a DNA vaccine. The expression cassette was also cloned into the E1 region of a 35-type adenovirus shuttle plasmid, which allows the DNA sequence to be transferred to a 35-type adenovirus vector with E1 and E3 deletions.

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 vector boost (day 28; Figure 8). The immunogens tested were the SARS-CoV-2 S1 domain, the RBD domain alone, or the s3-RBD, all of which were expressed from a codon-optimized ORF under the control of the EF-1α promoter. The tPA signal sequence was placed upstream of the isolated RBD domain and the s3-RBD fusion protein to enable their secretion (Figure 8).

Evaluation of binding antibodies by ELISA during the vaccination regimen showed the superiority of the RBD domain for immunity (relative to the entire S1 domain), as well as the superior performance of the s3-RBD construct relative to the RBD alone (Figure 9). Surprisingly, the S1 immunogen performed very poorly, stimulating detectable binding and neutralizing antibodies in only one of the three vaccinees. Note that in two animals, the low binding antibody response to S1 was obvious by ELISA (Figure 9); but it was not significant when compared to the much higher response in other groups; neutralizing activity was detected in one of the two S1 test animals (Figure 9, black dotted line). At all time points after the boost, immune responses only to the RBD domain can be detected in all three animals receiving the gene vaccine encoding the RBD domain (Figure 9, gray dotted line). However, in the group receiving s3-RBD, the binding antibody response was the highest (Figure 9, black solid line).

Neutralization activity was tested against pseudotyped lentiviral particles, which are lentiviral particles that lack their native envelope proteins but carry the SARS-CoV-2 spike protein. An example of a curve generated for one animal is shown in Figure 10. Each curve takes the neutralization titer 50 (NT50) as the dilution at which the infection is reduced to 50%. In Figure 10, no NT50 was detected at the first two time points, but sera from all subsequent time points showed neutralization by reducing the infectivity of the pseudotyped particles to less than 50%. All RBD vaccine recipients showed neutralizing titers in the pseudovirus assay, with animal D2 having a peak titer of 1:6539 (Figure 11), which is approximately the 90th percentile of the recovered population (Moore and Klasse, 2020). However, as with the ELISA assay for binding antibodies, subjects with the genetic vaccine expressing s3-RBD achieved the highest neutralizing titers at most time points after the fourth week, which exceeded all RBD test animals in 2 of 3 cases (Figure 11). In fact, the geometric mean titers of s3-RBD subjects exceeded those of RBD subjects by at least 4-fold. The actual increase was higher because one s3-RBD subject produced an antibody titer that exceeded the maximum quantifiable in the assay (1:10240). The neutralizing antibody responses in s3-RBD subjects also showed impressive durability, with all animals remaining neutralizing 32 weeks after the first immunization (Figure 11). In contrast, in only 2/3 of the RBD subjects did the neutralizing activity drop below the detection limit of our assay after 24 weeks.

In some reported cases, immune responses to membrane-associated antigens (e.g., because those antigens carry a glycosylphosphatidylinositol anchor or a transmembrane segment) are superior to immune responses to secreted forms of the same antigen. Therefore, we created a conjugated polypeptide comprising 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 in SEQ ID NO.25. The codon-optimized nucleic acid sequence of the polypeptide was synthesized (SEQ ID NO.26) and cloned into the pUC19 plasmid downstream of the EF1-α promoter sequence (including its first intron) and upstream of the SV40 polyadenylation sequence.

We next verified that the s3-RBD(B.1.351)-PDGFRtm conjugate polypeptide (SEQ ID NO.25) contained immunoreactive RBD(B.1.351) when expressed in human cells, which may be necessary for inducing anti-RBD(B.1.351) antibody responses in human subjects. The plasmid encoding s3-RBD(B.1.351)-PDGFRtm was transfected into the human embryonic kidney cell line 293, and the presence of immunoreactive RBD(B.1.351) protein in the cells was verified two days later by antibody staining using the following procedure. On day 1 of the assay, 350,000 cells were seeded into each well in a 6-well plate. On day 0, a polyethylenimine (PEI) transfection mixture was prepared by adding 150 microliters of 0.1 mg/ml PEI in DMEM to 3 mcg DNA in an equal volume of DMEM; the mixture was incubated at room temperature for 20 minutes; 2.7 ml of complete medium was added; the medium with complexed DNA was added to the washed 293 cells in a 6-well plate; and the plate was returned to the incubator for overnight incubation. On day 1, the transfection mixture 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, washed in a buffer containing 0.05% Triton X-100 for permeabilization, incubated with anti-RBD primary antibody for 2 hours, washed again, incubated with HRP-conjugated secondary antibody for 2 hours, washed, incubated with TrueBlue HRP substrate for 5-15 minutes, and finally quenched in water. The results ( FIG. 13D ) demonstrated that the s3-RBD(B.1.351)-PDGFRtm construct successfully produced a conjugated polypeptide including an immunoreactive RBD(B.1.351).

Example 9. SARS-CoV-2 RBD domain linked to an anti-CD3 single chain variable fragment and an antibody Fc region Conjugated peptide vaccine

Multivalent antigen display can drive stronger and longer-lasting antibody responses through efficient cross-linking of B cell receptors (BCRs) and improved antigen trafficking, endocytic uptake, and final presentation (Brinkkemper Vaccines 7, 2019; Tokatlian Science 363:649, 2019). Repeated epitopes have higher effective affinity for BCRs and can cross-link receptors to effectively activate B cells (Cimica Clin Immunol 183:99, 2017; Zabel J Immunol 192:5499, 2014). 15–20 hapten molecules with a spacing of 5–10 nm are ideal for B cell activation (Vogelstein PNAS 79:395, 1982). Polymeric antigens are most commonly produced as proteins in vitro, but can also be produced by in vivo genetic vaccines.

For example, one approach to improving the immunogenicity of vaccine antigens is to present them on nanoparticles (NPs) with a diameter of 25–50 nm. Licensed human papillomavirus and HBsAg vaccines involve NPs, as do efforts to create influenza and respiratory syncytial virus (RSV) vaccines (Darricarrere J Virol 92, 2018; Hsia Nature 535:136, 2016; Kanekiyo Nat Immunol 20:362, 2019; Marcandalli Cell 176:1420, 2019). Studies in animals have shown that NP presentation significantly improves the quantity and quality of Ab responses compared to delivering the same antigen as a soluble protein (Brinkkemper Vaccines 7, 2019). Thus, NP display of RSV antigens increased NAb titers by more than 10-fold (Marcandalli Cell 176:1420, 2019). NP presentation also allows the generation of multivalent antigen chimeric immunogens, which can improve the breadth of NAbs by increasing the avidity for specific interactions with the most cross-reactive BCRs, as shown for influenza HA (Kanekiyo Nat Immunol 20:362, 2019).

The immunoglobulin "crystallizable fragment" or Fc is a dimeric molecule; therefore, when the immunogen is expressed as a fusion protein with Fc, the result is a dimeric molecule that may be more immunogenic for the reasons mentioned above. The Fc region can also be modified to polymerize into a well-defined complex containing 12 fusion partners (Mekhaiel Sci Rep 1:124). In addition, the presence of the Fc domain significantly increases the plasma half-life of the fusion protein due to its interaction with salvaged neonatal Fc receptors, as well as slower renal clearance of larger molecules (Roopenian & Akilesh, 2007 and Kontermann, 2011). The attached Fc domain also enables these molecules to interact with Fc receptors (FcR) found on immune cells, a feature that is particularly important for their use in tumor therapy and vaccines (Nimmerjahn & Ravetch, 2008).

To allow the expression of multimeric s3-RBD and realize the other benefits conferred by the Fc domain, we therefore designed a protein molecule consisting of the conjugated peptide vaccine s3-RBD fused at its C-terminus to the human IgG1 Fc domain, referred to as "s3-RBD-Fc" (SEQ ID NO.9). Next, we prepared a codon-optimized ORF capable of expressing s3-RBD-Fc (SEQ ID NO.10).

To test the immunogenicity of s3-RBD-Fc, the ORF was engineered into an expression cassette that was transferred into appropriate DNA and adenoviral vectors, which were administered to rhesus monkeys.

Example 10. SARS-CoV-2 RBD structure comprising an anti-CD3 single-chain variable fragment linked to a double-chain antibody Dimerization conjugated peptide vaccine

s3-RBD contains an anti-CD3 scFv fragment, which itself contains the heavy and light chain variable regions VH and VL from the monoclonal antibody SP34. In order to form a functional monomeric scFv fragment, these VH and VH regions are separated by a flexible linker, which is required to allow the two domains to achieve the appropriate three-dimensional configuration required for CD3 binding. In order to allow the correct folding of the monomeric scFv, the linker length is usually >12 amino acids, and the most common is 15 amino acids (Wang Antibodies8:43,2019).

Shorter linkers connecting the two variable domains can determine the formation of multimeric scFv molecules, as shorter linkers are not long enough to allow monomeric folding with the VH and VL regions properly associated in space. Reduction of the linker length connecting the two variable domains to less than 8–12 residues favors the dimeric assembly of the VH-VL fragments, resulting in 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 less than five amino acids has been shown to result in tri- 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).

Therefore, we designed a dimeric form of s3-RBD by reducing the length of the VH-VL linker to five amino acids (SEQ ID NO.11). We prepared a codon-optimized ORF encoding this dimeric form of the conjugated polypeptide immunogen (SEQ ID NO.12). This codon-optimized ORF was continuously engineered into expression cassettes and into DNA and adenoviral vectors, which were administered to animals for immunogenicity testing.

When evaluating the crystal structure of the reported double-chain antibody, it is obvious that the double-chain antibody structure is unstable (Kim Sci Rep 6:34515,2016). In the crystal structure of the double-chain antibody, the light chain does not contribute to the interaction between the Fv domains, and the two heavy chains form a relatively small interaction interface. In order to use double-chain antibodies as a general and reliable mediator of artificial protein assemblies, it is advantageous to propose that the predictable direction and distance between antigen binding sites (such as CD3 binding sites) will be. Nevertheless, many of the simplest designed double-chain antibodies (i.e., the linker sequence is reduced to 5 amino acids) have an interaction interface between the Fv domains, which seems to be too small to have a stable structure (Moraga Cell 160:1196,2015; Perisic Structure 2:1217,1994). The results show that by replacing the arginine residues in the EF loop and introducing one or more disulfide bridges between the Fv domains, the double-chain antibody structure can be made more rigid and predictable.

Next, we designed a dimeric form of s3-RBD with greater predicted stability (SEQ ID NO. 13) by reducing the length of the VH-VL linker to 5 amino acids, replacing positively charged lysine residues in the EF loop, and introducing cysteine residues that can form disulfide bridges. We prepared a codon-optimized ORF encoding this dimeric form of the conjugated polypeptide immunogen with enhanced stability (SEQ ID NO. 14). This codon-optimized ORF was sequentially engineered into expression cassettes and into engineered DNA and adenoviral vectors, which were administered to animals for immunogenicity testing.

Plasmid DNA molecules encoding enhanced dimerization anti-CD3-RBD conjugated polypeptides (also referred to as eDis3-RBD) were administered as DNA vaccines to three rhesus monkeys (by electroporating 1 mg DNA on day 0); booster immunization was provided on day 28 with a 35-type adenocarcinoma vector encoding only RBD (Figure 12). The antibody responses in these rhesus monkeys were compared with those obtained using only RBD for primary immunization and boosting. The results showed that the peak antibody response using eDis3-RBD was improved (geometric mean increased by 6.3 times), and the persistent antibody response remaining 24 weeks after vaccination was also improved (geometric mean increased by 5 times; Figure 12, compare the solid black average line of eDis3-RBD with the black dotted average line of RBD only).

Example 11. Conjugated polypeptide vaccine comprising a SARS-CoV-2 RBD domain linked to an anti-CD2 single chain variable fragment Seedling

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 acts as a co-stimulatory molecule, meaning that signals sent via CD2 cooperate with signals sent through TCR engagement to induce cell proliferation and cytokine production in resting T cells. The close association of CD2 with the CD3-TCR complex appears to be critical for optimal T cell responses. CD2 is also an important adhesion molecule that binds to LFA-3 and thereby leads to antigen-independent cell adhesion, expansion of naive helper T cells, and induction of IFN-γ in memory cells. In addition to LFA-3, CD2 can also interact with CD48 and CD59.

We created a conjugate polypeptide comprising the RBD of the SARS-CoV-2 spike and a CD2-binding polypeptide by fusing the coding sequences of the following protein elements: a tissue-type plasminogen activator signal sequence (to allow the conjugate polypeptide to be secreted from cells), an anti-CD2 scFv derived from the rat anti-CD2 antibody LO-CD2a (see, e.g., U.S. Pat. No. 6,849,258); a flexible linker; and SARS-CoV-2 RBD (codon-optimized). The resulting amino acid sequence is shown in SEQ ID NO. 15. The codon-optimized nucleic acid sequence of the polypeptide (SEQ ID NO. 16) was synthesized and cloned into the downstream of the EF1-α promoter sequence (including its first intron) and upstream of the SV40 polyadenylation sequence in the pUC19 plasmid.

Plasmid DNA molecules encoding anti-CD2-RBD conjugated polypeptides (also referred to as s2-RBD) were administered as DNA vaccines to two rhesus monkeys (by electroporating 1 mg DNA on day 0); booster immunization was provided on day 28 with a 35-type adenocarcinoma vector encoding only RBD (Figure 12). The antibody responses in these rhesus monkeys were compared with those obtained using only RBD for primary and booster. The results showed that the peak antibody response using s2-RBD was improved (geometric mean increased by 2.8 times), and the persistent antibody response remaining 24 weeks after vaccination was improved (geometric mean increased by 5 times; Figure 12, compare the gray dashed line mean of s2-RBD with the black dotted line mean of RBD only).

Example 12. SARS-CoV-2 RBD structure comprising an anti-CD2 single-chain variable fragment linked to a double-chain antibody Dimerization conjugated peptide vaccine

By reducing the length of the VH-VL linker to five amino acids, a dimeric form of the anti-CD2 scFv-RBD was designed (SEQ ID NO. 17). We prepared a codon-optimized ORF encoding this dimeric form of the conjugated polypeptide immunogen (SEQ ID NO. 18). This codon-optimized ORF was continuously engineered into expression cassettes and into DNA and adenoviral vectors, which were administered to animals for immunogenicity testing.

Next, we designed a dimeric form of anti-CD2scFv-RBD with greater predicted stability by reducing the length of the VH-VL linker to 5 amino acids and introducing cysteine residues that can form disulfide bridges (SEQ ID NO.19). The anti-CD2scFv used in this construct does not have positively charged residues at key positions in the EF loop, which is predicted to cause instability. We prepared a codon-optimized ORF that encodes this enhanced stability, dimeric form of anti-CD2scFv-RBD (SEQ ID NO.20). This codon-optimized ORF was continuously engineered into expression cassettes and engineered into DNA and adenoviral vectors, which were administered to animals for immunogenicity testing.

Example 13. SARS-CoV-2 RBD domain comprising an N-terminal domain linked to LFA-3 that binds to CD2 Conjugated peptide vaccine

Conjugated polypeptide immunogens for delivery in genetic vaccines can be designed using any protein sequence that will bind to the appropriate cell surface receptor in the vaccine receptor. The 179 residue extracellular domain of human CD58 consists of two extracellular immunoglobulin-like domains, which are anchored to the membrane by transmembrane segments or glycosylphosphatidylinositol (GPI) linkers (Dustin et al., 1987b; Wallich et al., 1998). The 95 residue membrane distal N-terminal domain of CD58 (ldCD58) is entirely responsible for adhesion to CD2 (Sun et al.).

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

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

We next verified that the 1dCD58-RBD(B.1.351) conjugated polypeptide (SEQ ID NO.23) contained immunoreactive RBD(B.1.351) when expressed in human cells, which may be necessary for inducing anti-RBD(B.1.351) antibody responses in human subjects. The plasmid encoding 1dCD58-RBD(B.1.351) was transfected into the human embryonic kidney cell line 293, and the presence of immunoreactive RBD(B.1.351) protein in the cells was verified two days later by antibody staining using the following procedure. On day 1 of the assay, 350,000 cells per well were seeded in a 6-well plate. On day 0, a polyethylenimine (PEI) transfection mixture was prepared by adding 150 microliters of 0.1 mg/ml PEI in DMEM to 3 mcg DNA in an equal volume of DMEM; the mixture was incubated at room temperature for 20 minutes; 2.7 ml of complete medium was added; the medium with complexed DNA was added to the washed 293 cells in a 6-well plate; and the plate was returned to the incubator for overnight incubation. On day 1, the transfection mixture 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, washed in a buffer containing 0.05% TritonX-100 for permeabilization, incubated with anti-RBD primary antibody for 2 hours, washed again, incubated with HRP-conjugated secondary antibody for 2 hours, washed, incubated with TrueBlue HRP substrate for 5-15 minutes, and finally quenched in water. The results ( FIG. 13C ) demonstrated that the 1dCD58-RBD(B.1.351) construct successfully produced a conjugated polypeptide including an immunoreactive RBD(B.1.351).

Example 14. Conjugated polypeptide vaccine comprising a SARS-CoV-2 RBD domain linked to an anti-CD4 single chain variable fragment Seedling

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, it contains four immunoglobulin domains, referred to as D1 to D4, which are exposed on the cell surface. The D1 domain facilitates interaction with the β2 domain of the MHC class II molecule, which defines much of the biology of helper T cells, which respond to peptides presented on MHC class II molecules by antigen presenting cells. CD4 is also the major entry receptor for the HIV-1 envelope glycoprotein.

We created a conjugate polypeptide comprising the RBD of the SARS-CoV-2 spike and a CD4 binding polypeptide by fusing the coding sequences of the following protein elements: a tissue-type plasminogen activator signal sequence (to allow secretion of the conjugate polypeptide from cells); an anti-CD4 scFv derived from the humanized mouse anti-CD4 antibody hu5A8 (see, e.g., AIDS Res Hum Retro 13:933); a flexible linker; and SARS-CoV-2 RBD (codon optimized). The resulting amino acid sequence is shown in SEQ ID NO.27. The codon-optimized nucleic acid sequence of the polypeptide (SEQ ID NO.28) was synthesized and cloned into the downstream of the EF1-α promoter sequence (including its first intron) and upstream of the SV40 polyadenylation sequence in the pUC19 plasmid.

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Although the above disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, those skilled in the art will appreciate that certain changes and modifications may be implemented within the scope of the appended claims. In addition, each reference provided herein is incorporated herein by reference in its entirety to the same extent as if each reference was individually incorporated herein by reference.

Exemplary embodiments

Exemplary embodiments provided in accordance with the presently disclosed subject matter include, but are not limited to, the claims and the following embodiments:

1. A vaccine for inducing an immune response against 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 immune cells.

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

3. The vaccine of embodiment 2, wherein the 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 embodiments 1-4, wherein the immune cell is a T cell or an antigen presenting cell (APC).

6. The vaccine of any one of embodiments 1-5, wherein the ligand is an extracellular domain 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 embodiments 1-7, wherein the surface protein is preferentially expressed by T cells.

9. The vaccine of any one of embodiments 1-8, wherein the antibody fragment is an antibody-derived scFv chain.

10. The vaccine of any one of embodiments 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 the addition of the lipid anchor is directed by a signal sequence.

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

14. The vaccine of any one of embodiments 10-13, wherein the transmembrane segment is derived from PDGF receptor, glycophorin A, or SARS-CoV-2 spike protein.

15. The vaccine of any one of embodiments 10-14, wherein the multimerization domain is derived from T4fibritin.

16. The vaccine of any one of embodiments 10-14, wherein the multimerization domain is an Fc domain.

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

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

19. The vaccine of any one of embodiments 1-18, wherein the conjugated polypeptide is a fusion protein comprising an antigen and a ligand or antibody fragment within a single polypeptide chain.

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

21. The vaccine of embodiment 20, wherein the flexible linker is 12 or more amino acids in length, and wherein the conjugated polypeptide preferentially binds to the surface protein in monomeric form.

22. The vaccine of embodiment 20, wherein the flexible linker is shorter than 12 amino acids in length, and wherein the conjugated polypeptide preferentially binds to the surface protein in a multimeric form.

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

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

25. The vaccine of any one of embodiments 1-24, wherein the conjugate 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 embodiments 1-26, further comprising a second antigen from a pathogen.

28. The vaccine of any one of embodiments 1-27, wherein the pathogen is a virus.

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

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

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

32. The vaccine of any one of embodiments 27-31, wherein the second antigen comprises SARS-CoV-2 E protein, M protein, N protein, nsp3 protein, nsp4 protein or nsp6 protein or a fragment thereof.

33. A vaccine as described in embodiment 32, wherein the second antigen comprises a fusion protein comprising SARS-CoV-2 E protein and M protein or fragments thereof.

34. The vaccine of any one of embodiments 1-33, wherein the mammal is a human.

35. The vaccine of any one of embodiments 1-34, wherein the vaccine is formulated for electroporation or subcutaneous injection.

36. The vaccine of any one of embodiments 1-35, 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 against a pathogen in a mammal, the vaccine comprising a polynucleotide encoding a conjugated polypeptide comprising an antigen from the pathogen fused to a ligand or antibody fragment that specifically binds to a surface protein present on immune cells.

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 embodiments 37-40, wherein the immune cell is a T cell or an antigen presenting cell (APC).

42. The vaccine of any one of embodiments 37-41, wherein the ligand is an extracellular domain of a cell adhesion molecule.

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

44. The vaccine of any one of embodiments 37-43, wherein the surface protein is preferentially expressed by T cells.

45. The vaccine of any one of embodiments 37-44, wherein the antibody fragment is an antibody-derived scFv chain.

46. The vaccine of any one of embodiments 33-45, 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 the 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 embodiments 46-49, wherein the transmembrane segment is derived from a PDGF receptor, glycophorin A, or a SARS-CoV-2 spike protein.

51. The vaccine of any one of embodiments 46-50, wherein the multimerization domain is derived from T4fibritin.

52. The vaccine of any one of embodiments 46-50, wherein the multimerization domain is an Fc domain.

53. The vaccine of embodiment 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 embodiments 45-54, wherein the VH region and the VL region 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 or more amino acids in length, and wherein the conjugated polypeptide preferentially binds to the surface protein in monomeric form.

57. The vaccine of embodiment 55, wherein the flexible linker is shorter than 12 amino acids in length, and wherein the conjugated polypeptide preferentially binds to the surface protein in a multimeric form.

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 embodiments 37-59, wherein the conjugate 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 embodiments 37-61, further comprising a second polynucleotide encoding a second antigen from a pathogen.

63. The vaccine of any one of embodiments 37-62, wherein the pathogen is a virus.

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

65. The vaccine of embodiment 64, wherein the antigen present within 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 an S1 domain or a receptor binding domain (RBD).

67. The vaccine of any one of embodiments 62-66, wherein the second antigen comprises SARS-CoV-2 E protein, M protein, N protein, nsp3 protein, nsp4 protein or nsp6 protein or a fragment thereof.

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

69. The vaccine of any one of embodiments 37-68, wherein the mammal is a human.

70. The vaccine of any one of embodiments 37-69, wherein the vaccine is formulated for electroporation or subcutaneous injection.

71. The vaccine of any one of embodiments 37-70, wherein the polynucleotide encoding the conjugate polypeptide and/or the second polynucleotide encoding the second antigen is codon optimized.

72. The vaccine of any one of embodiments 37-71, wherein the polynucleotide encoding the conjugated polypeptide is present in a first expression cassette, wherein the polynucleotide is operably linked to a first promoter, and/or a second polynucleotide encoding the second antigen is present in a second expression cassette, wherein the second polynucleotide is operably linked to a second promoter.

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 the EF-1α promoter.

75. The vaccine of any one of embodiments 37-74, wherein the first expression cassette and/or the second expression cassette are present in a vector.

76. The vaccine of embodiment 75, wherein the vector is administered in the form of 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) vector, an adenovirus vector, or an adeno-associated virus (AAV) vector.

79. The vaccine of any one of embodiments 37-78, further comprising an in vivo transfection reagent.

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

81. The vaccine of any one of embodiments 37-80, wherein the vaccine is formulated for subcutaneous transfection.

82. The vaccine of any one of embodiments 78-81, wherein the vector is a circular CMV vector comprising:

(a) a CMV genome or a portion thereof, wherein the CMV genome or a portion thereof contains the first expression cassette or the first expression cassette and the second expression cassette;

(b) a bacterial artificial chromosome (BAC) sequence containing an origin of replication;

(c) a first terminal enzyme complex recognition locus (TCRL1) comprising at least two viral direct repeat sequences; and

(d) a second terminal enzyme complex recognition locus (TCRL2) comprising at least two viral direct repeat sequences;

wherein the CMV genome or a portion thereof is flanked by TCRL1 and TCRL2, thereby defining a first region of a circular vector extending from TCRL1 to TCRL2 and comprising the CMV genome or a portion thereof; and

Wherein the BAC sequence is located in the second region of the circular vector extending from TCRL1 to TCRL2 and not comprising the CMV genome or a part thereof.

83. The vaccine of any one of embodiments 78-81, wherein the vector is a circular CMV vector comprising:

(a) a CMV genome or a portion thereof, wherein the CMV genome or a portion thereof contains the first expression cassette or the first expression cassette and the second expression cassette;

(b) a sequence comprising an origin of replication that functions in unicellular organisms;

(c) one or more terminase complex recognition loci (TCRLs) comprising a recombinantly introduced polynucleotide sequence capable of being directly cleaved by the HV terminase complex;

wherein the CMV genome or a portion thereof is separated from a sequence comprising an origin of replication by TCRL;

wherein the CMV genome or a portion thereof is adjacent to a TCRL at at least one end; and

The sequence comprising the replication origin is flanked by a TCRL at at least one end.

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 comprise a Pac1 site and a Pac2 site.

86. The vaccine of any one of embodiments 77-85, wherein the first promoter is a viral promoter.

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

88. The vaccine of any one of embodiments 78-87, wherein the vector is a CMV vector, and wherein the CMV is Towne HCMV.

89. The vaccine of any one of embodiments 37-88, 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 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 90-94, wherein the ligand is an extracellular domain 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 embodiments 90-94 or 95-97, wherein the antibody fragment is an antibody-derived scFv chain.

99. The conjugated polypeptide of any one of embodiments 90-98, wherein the conjugated polypeptide further comprises 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 the 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 embodiments 99-102, wherein the transmembrane segment is derived from a PDGF receptor, glycophorin A, or a SARS-CoV-2 spike protein.

104. The conjugated polypeptide of any one of embodiments 99-103, wherein the multimerization domain is derived from T4 fibritin.

105. The conjugated polypeptide of any one of embodiments 99-103, 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. A conjugated polypeptide as described in any of embodiments 98-107, wherein the antibody fragment is an antibody-derived scFv chain, and wherein the VH region and VL region of the scFv are separated by a flexible linker.

109. The conjugated polypeptide of embodiment 108, wherein the flexible linker is 12 or more amino acids in length, and wherein the conjugated polypeptide preferentially binds to the surface protein in monomeric form.

110. The conjugated polypeptide of embodiment 109, wherein the length of the flexible linker is shorter than 12 amino acids, and wherein the conjugated polypeptide preferentially binds to the surface protein in a multimeric form.

111. The conjugated polypeptide of embodiment 110, wherein the polymer is stabilized by disulfide bonds between 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, wherein the conjugated polypeptide further comprises 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 embodiments 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 a 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 an S1 domain or a receptor binding domain (RBD).

119. The conjugated polypeptide of any one of embodiments 90-118, 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 following amino acid sequence: 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 90-121.

123. The polynucleotide of embodiment 122, wherein the polynucleotide 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 122-124.

126. An expression cassette as described in embodiment 125, which comprises 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, wherein the vector is a plasmid.

129. The vector of embodiment 127, wherein the vector is an adenoviral vector.

130. A double-chain antibody comprising the conjugated polypeptide of any one of embodiments 98-121.

131. A dimer comprising the conjugated polypeptide of any one of embodiments 99-121.

132. A vaccine comprising the conjugated polypeptide of any one of embodiments 90-121, the polynucleotide of any one of embodiments 122-124, the expression cassette of embodiment 125 or 126, the vector of any one of embodiments 127-129, 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, the method comprising administering to the mammal the vaccine of any one of embodiments 1-89 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 a mammal against an antigen present in the conjugated polypeptide, and wherein the neutralizing response is significantly greater than any antibody-dependent infectious enhancement (ADEI) caused by the vaccine in the mammal.

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

137. The method of any one of embodiments 133-136, wherein the method induces CD4 ⁺ and CD8 ⁺ T cell responses to the second antigen.

138. The method of any one of embodiments 133-137, wherein the method comprises administering to a 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 boosting is performed after about 28 days.

140. The method of any one of embodiments 133-139, wherein the mammal is a human.

Informal Sequence Listing

Claims (140)

1. A vaccine for inducing an immune response in a mammal against a pathogen, 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.

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

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

4. The vaccine of claim 3, wherein the enriched 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 extracellular domain 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 antibody-derived scFv chain.

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. The vaccine of claim 10, wherein the lipid anchor is a glycosyl phosphatidyl inositol anchor.

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

15. The vaccine of claim 10, wherein the multimerization domain is derived from T4fibritin.

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

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

18. 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 antibody fragment within a single polypeptide chain.

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

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

22. The vaccine of claim 20, wherein the flexible linker is shorter than 12 amino acids in length, and wherein the conjugated polypeptide preferentially binds to the surface protein in multimeric form.

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

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

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

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

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

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

31. The vaccine of claim 30, wherein the fragment of SARS-CoV-2 spike glycoprotein comprises an S1 domain or a Receptor Binding Domain (RBD).

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

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

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

35. The vaccine of claim 1, wherein the vaccine is formulated for electroporation or subcutaneous injection.

36. 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 in a mammal against a pathogen, the vaccine comprising a polynucleotide encoding a conjugated 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 claim 37, wherein the surface protein is a abundant T cell surface protein involved in signal transduction and/or adhesion.

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

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

41. The vaccine of claim 37, wherein the immune cell is a T cell or an Antigen Presenting Cell (APC).

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

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

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

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

46. 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. The vaccine of claim 46, wherein the lipid anchor is a glycosyl phosphatidyl inositol anchor.

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

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

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

51. The vaccine of claim 46, wherein the multimerization domain is derived from T4fibritin.

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

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

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

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

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

57. The vaccine of claim 55, wherein the flexible linker is shorter than 12 amino acids in length, and wherein the conjugated polypeptide preferentially binds to the surface protein in multimeric form.

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

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

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

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

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

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

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

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

66. The vaccine of claim 65, wherein the fragment of SARS-CoV-2 spike glycoprotein comprises an S1 domain or a Receptor Binding Domain (RBD).

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

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

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

70. The vaccine of claim 37, wherein the vaccine is formulated for electroporation or subcutaneous injection.

71. The vaccine of claim 37 or 62, wherein the polynucleotide encoding the conjugated polypeptide and/or the second polynucleotide encoding the second antigen is codon optimized.

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

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

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

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

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

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

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

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

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

81. The vaccine of claim 37, wherein the vaccine is formulated for subcutaneous transfection.

82. The vaccine of claim 78, wherein the vector is a circular CMV vector comprising:

(a) A CMV genome or a portion thereof, wherein the CMV genome or a portion thereof comprises the first expression cassette or the first expression cassette and the second expression cassette;

(b) A Bacterial Artificial Chromosome (BAC) sequence comprising an origin of replication;

(c) A first terminal enzyme complex recognition site (TCRL 1) comprising at least two viral direct repeats; and

(d) A second terminal enzyme complex recognition site (TCRL 2) comprising at least two viral direct repeats;

Wherein the CMV genome or a portion thereof is flanked by TCRL1 and TCRL2, thereby defining a first region of a circular vector extending from TCRL1 to TCRL2 and comprising the CMV genome or a portion thereof; and is also provided with

Wherein the BAC sequence is located in a second region of the circular vector that extends from TCRL1 to TCRL2 and does not comprise the CMV genome or a portion thereof.

83. The vaccine of claim 78, wherein the vector is a circular CMV vector comprising:

(a) A CMV genome or a portion thereof, wherein the CMV genome or a portion thereof comprises the first expression cassette or the first expression cassette and the second expression cassette;

(b) A sequence comprising an origin of replication that is functional in a single cell organism;

(c) One or more terminal enzyme complex recognition loci (TCRL) comprising recombinantly introduced polynucleotide sequences capable of being directly cleaved by the HV terminal enzyme complex;

wherein the CMV genome or a portion thereof is separated from the sequence comprising the origin of replication by TCRL;

wherein the CMV genome or a portion thereof abuts TCRL at least one terminus; and is also provided with

Wherein the sequence comprising the origin of replication adjoins a TCRL at least one terminus.

84. The vaccine of claim 82, wherein one or more of the terminal enzyme complex recognition loci comprises a Pac1 site and a Pac2 site.

85. The vaccine of claim 84, wherein all of the terminal enzyme complex recognition loci comprise a Pac1 site and a Pac2 site.

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

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

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

89. The vaccine of claim 37, 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, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26 and SEQ ID NO. 28.

90. A conjugated polypeptide comprising an antigen 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 claim 90, wherein the surface protein is a abundant T cell surface protein involved in signal transduction and/or adhesion.

92. The conjugate polypeptide of claim 90, wherein the surface protein is CD2, CD3, CD4, or CD5.

93. The conjugate polypeptide of claim 92, wherein the surface protein is CD2 or CD3.

94. The conjugated polypeptide of claim 90, wherein the immune cell is a T cell or an Antigen Presenting Cell (APC).

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

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

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

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

99. The conjugate polypeptide of claim 90, wherein the conjugate polypeptide further comprises a lipid anchor, a transmembrane segment, a multimerization domain, or any combination of these elements.

100. The conjugate polypeptide of claim 99, wherein the lipid anchor is a glycosyl phosphatidyl inositol anchor.

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

102. The conjugate polypeptide of claim 101, wherein the signal sequence is derived from CD55.

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

104. The conjugate polypeptide of claim 99, wherein the multimerization domain is derived from T4 fibritin.

105. The conjugate polypeptide of claim 99, wherein the multimerization domain is an Fc domain.

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

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

108. The conjugated polypeptide of claim 98, wherein the antibody fragment is an antibody-derived scFv chain, and wherein the VH region and the VL region of the scFv are separated by a flexible linker.

109. The conjugate polypeptide of claim 108, wherein the flexible linker is 12 or more amino acids in length, and wherein the conjugate polypeptide preferentially binds to the surface protein in monomeric form.

110. The conjugate polypeptide of claim 108, wherein the flexible linker is shorter than 12 amino acids in length, and wherein the conjugate polypeptide preferentially binds to the surface protein in multimeric form.

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

112. The conjugate polypeptide of claim 110, wherein the flexible linker is 5 amino acids in length.

113. The conjugate polypeptide of claim 90, wherein the conjugate polypeptide further comprises a tPA leader sequence.

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

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

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

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

118. The conjugate polypeptide of claim 117, wherein the fragment of SARS-CoV-2 spike glycoprotein comprises an S1 domain or a Receptor Binding Domain (RBD).

119. 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 CD 4; (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 NO. 25 or SEQ ID NO. 27.

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

123. The polynucleotide of claim 122, wherein said polynucleotide is codon optimized.

124. The polynucleotide of claim 123, comprising a nucleotide sequence selected from the group consisting of seq id nos: 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 claim 122.

126. The expression cassette of claim 125 comprising the nucleotide sequence of SEQ ID No. 8.

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

128. The vector of claim 127, wherein the vector is a plasmid.

129. The vector of claim 127, wherein the vector is an adenovirus vector.

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

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

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

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

135. The method of claim 133, wherein the method induces a neutralizing antibody response in the mammal against an antigen present within the conjugated polypeptide, and wherein the neutralizing response is significantly higher than any antibody dependent infection enhancement (ADEI) induced by the vaccine in the mammal.

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

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

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

139. The method of claim 138, wherein the strengthening is performed after about 28 days.

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