CN116761811A

CN116761811A - Conjugated polypeptides and vaccines for inducing immune responses

Info

Publication number: CN116761811A
Application number: CN202180061251.9A
Authority: CN
Inventors: 丹尼斯·J·哈蒂根-奥康纳
Original assignee: University of California
Current assignee: University of California
Priority date: 2020-05-19
Filing date: 2021-05-19
Publication date: 2023-09-15

Abstract

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

Description

Conjugated polypeptides and vaccines for inducing immune responses

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application No. 63/027,250 filed on day 19 of 5 in 2020 and U.S. provisional application No. 63/058,362 filed on day 29 of 7 in 2020, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

Statement regarding equity to the application made under federally sponsored research and development

The present application was carried out with government support under foundation number R01AI118451 awarded by the national institutes of health (the National Institutes of Health). The government has certain rights in this application.

Background

Traditional vaccine development against previously unknown pathogens takes years, but protecting human health may require a faster response of months (1). However, accelerated development is complex due to a number of factors, including the lack of appropriate animal models for emerging pathogens; the risk of antibody-dependent enhancement of infectivity (ADEI), which may occur whenever a sub-optimal antibody response is induced (2); and difficulties in developing new manufacturing processes for subunits, attenuated or vector vaccines. In addition, while inducing high titer neutralizing antibodies (nabs) appears to be a clear approach, we do not know what titer of nabs has protective effect in practice, nor how the threshold varies in extreme cases of age and complications.

Regarding SARS-CoV-2, the definition of the immune relevance of successful vaccination is not clear. Most coronavirus vaccines currently under development target the most variable part of the spike glycoprotein and induce only an antibody response against the virus present in the vaccine. In SARS-CoV-1, escape mutants occur in vitro and in mice in the presence of either the nAb of a single anti-Receptor Binding Domain (RBD) or a combination of both nAbs (2, 3). Furthermore, as mentioned above, due to the possible ADEI, especially when the antibody level is low, the vaccine specifically eliciting antibodies has to be handled with additional care (4). In fact, highly concentrated antisera against SARS-CoV-1 proved to neutralize viral infectivity, whereas diluted antibodies caused ADEI in human procaryotic cell cultures, leading to cytopathic effects and increased levels of TNF- α, IL-4 and IL-6 (5-7). Furthermore, vaccine candidates based on full length SARS-CoV-1 spike proved to induce non-neutralizing antibodies and the immunized animals were not protected. Instead, they experience side effects such as exacerbation of hepatitis, increased incidence and a stronger inflammatory response (8, 9).

CoV epidemic diseaseThe shoot-initiated T cell response also plays 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 B cell deficient mice (10). In addition, airway memory CD4 is shown ⁺ T cells mediate protective immunity against SARS-CoV-1 and MERS-CoV (11). However, most vaccine types do not elicit substantial memory of CD4 ⁺ T cells.

CMV vector vaccines can elicit potent 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 endogenous pp65b promoters. For example, it has been found that rhesus monkeys vaccinated with CMV vaccine carrying ebola virus Glycoprotein (GP) can produce GP specific antibodies under the control of pp65b promoter (21).

Another important property of CMV vectors for combating emerging pathogens is the ability to re-administer to previously exposed individuals. Because of this capability, one can imagine that over time CMV vector vaccines are reused against a range of emerging threats.

However, despite the immune advantage of CMV vaccines, practical barriers have hampered the rapid development of CMV vaccines for human clinical use when these vaccines are delivered as live viruses. One obstacle is the great difficulty in large-scale production of uniform test samples from slowly growing and variable beta herpesviruses (29).

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

SUMMARY

In one aspect, the present disclosure provides 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.

In some embodiments, the surface protein is a abundant T cell surface protein involved in signal transduction and/or adhesion. In some embodiments, the enriched T cell surface protein is CD2, CD3, CD4 or CD5. In some embodiments, the enriched T cell surface protein is CD2 or CD3. In some embodiments, the immune cell is a T cell or an Antigen Presenting Cell (APC). In some embodiments, the ligand is an extracellular domain of a cell adhesion molecule. In some embodiments, the cell adhesion molecule is CD58. In some embodiments, the surface protein is expressed preferentially or solely by T cells. In some embodiments, the antibody fragment is an scFv chain of antibody origin.

In some embodiments, the conjugated polypeptide further comprises a lipid anchor (a lipid anchor), a transmembrane segment, a multimerization domain, or any combination of these elements. In some embodiments, the lipid anchor is a glycosyl phosphatidylinositol anchor. In some embodiments, the addition of the lipid anchor is directed by a signal sequence. In some embodiments, the signal sequence is derived from CD55. In some embodiments, the transmembrane segment is derived from 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 IgG1Fc domain. In some embodiments, the conjugated polypeptide is a fusion protein of the antigen and the ligand or antibody fragment contained within a single polypeptide chain.

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

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

In another aspect, the present disclosure provides 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.

In some embodiments of the vaccine, the surface protein is a abundant T cell surface protein involved in signal transduction and/or adhesion. In some embodiments, the enriched T cell surface protein is CD2, CD3, CD4 or CD5. In some embodiments, the enriched T cell surface protein is CD2 or CD3. In some embodiments, the immune cell is a T cell or an Antigen Presenting Cell (APC). In some embodiments, the ligand is an 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 scFv chain of antibody origin. 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 glycosyl phosphatidylinositol anchor. In some embodiments, the addition of the lipid anchor is directed by a signal sequence. In some embodiments, the signal sequence is derived from CD55. In some embodiments, the transmembrane segment is derived from 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 IgG1Fc domain.

In some embodiments, the VH and VL regions of the scFv are separated within the conjugated polypeptide by a flexible linker. In some embodiments, 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. In some embodiments, the flexible linker is shorter than 12 amino acids in length, and the conjugated polypeptide preferentially binds to surface proteins in multimeric form. In some embodiments, the multimer is stabilized by disulfide bonds. In some embodiments, the flexible linker is 5 amino acids in length. In some embodiments, the conjugated polypeptide comprises a tPA leader sequence. In some embodiments, the tPA leader sequence is 23 amino acids in length.

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 within the conjugated polypeptide comprises a SARS-CoV-2 spike glycoprotein or fragment thereof. In some embodiments, the fragment of SARS-CoV-2 spike glycoprotein comprises an S1 domain or a Receptor Binding Domain (RBD). In some embodiments, the second antigen comprises a SARS-CoV-2E protein, an M protein, an N protein, a nsp3 protein, a nsp4 protein, or a nsp6 protein, or a fragment of one of these proteins. In some embodiments, the second antigen comprises a fusion protein comprising a SARS-CoV-2E protein and an M protein or fragment thereof. 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 is codon optimized. In some embodiments, the 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. In some embodiments, the second promoter is a mammalian promoter. In some embodiments, the mammalian promoter is an EF-1 alpha promoter. In some embodiments, the first expression cassette and/or the second expression cassette are present within 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 ^TM . In some embodiments, the vaccine is formulatedFor subcutaneous transfection.

In some embodiments, 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, which define 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 a second region of the circular vector that extends from TCRL1 to TCRL2 and does not comprise the CMV genome or a portion thereof.

In some embodiments, 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 wherein the sequence comprising the origin of replication adjoins the TCRL at least one terminus.

In some embodiments, one or more of the terminal enzyme complex recognition loci comprises a Pac1 site and a Pac2 site. In some embodiments, all of the terminal enzyme 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 the 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 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.

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 conjugate 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 an extracellular domain of a cell adhesion molecule. In some embodiments, the cell adhesion molecule is CD58. In some embodiments, the surface protein is a abundant T cell surface protein involved in signal transduction and/or adhesion. In some embodiments, the ligand or antibody fragment specifically binds to a surface protein that is preferentially or exclusively expressed by T cells. In some embodiments, the antibody fragment is an scFv chain of antibody origin.

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 glycosyl phosphatidylinositol anchor. In some embodiments, the addition of the lipid anchor is directed by a signal sequence. In some embodiments, the signal sequence is derived from CD55. In some embodiments, the transmembrane segment is derived from 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 IgG1Fc domain.

In some embodiments, the antibody fragment is an scFv chain of antibody origin, and the VH and VL regions of the scFv are separated by a flexible linker. In some embodiments, 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. In some embodiments, 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. In some embodiments, the multimer is stabilized by disulfide bonds between monomer units. In some embodiments, the flexible linker is 5 amino acids in length. In some embodiments, the conjugated polypeptide further comprises a tPA leader sequence. In some embodiments, the tPA leader sequence is 23 amino acids in length.

In some embodiments, the pathogen is a virus. In some such embodiments, the virus is SARS-CoV-2. In some embodiments, the antigen comprises SARS-CoV-2 spike glycoprotein or a fragment thereof. In some embodiments, the fragment of 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 CD 4; (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: 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 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 nucleotide sequence of: SEQ ID NO. 2, SEQ ID NO. 7, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26 and SEQ ID NO. 28.

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 adenovirus vector.

In another aspect, the 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 against an antigen present within the conjugated polypeptide, and the neutralizing response is substantially stronger than any antibody dependent infection 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 against the second antigen ⁺ And CD8 ⁺ T cell response.

In some embodiments, the method comprises administering to the mammal a DNA prime comprising any of the vectors (e.g., plasmids) described herein encoding any of the conjugated polypeptides described herein by electroporation, followed by boosting with any of the vectors (e.g., adenovirus vectors) described herein encoding any of the antigens (e.g., RBDs) described herein. In some embodiments, the strengthening is performed after about 28 days. In some embodiments, the mammal is a human.

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

Brief Description of Drawings

FIG. 1T cells were stimulated by anti-CD 3scFv linked to SARS-CoV-2S1 domain. On the left, incubation of T cells in non-stimulated cultures resulted in limited expression of CD69 on the cell surface and negligible interferon-gamma production. On the right, incubation of T cells in the presence of plate-bound anti-CD 3scFv-S1 resulted in increased CD69 and interferon production, indicating that the anti-CD 3scFv-S1 molecule was able to bind to the CD3 complex and activate T cell signaling. The scale on the x-axis and the y-axis is from-10 ³ To 10 ⁵ 。

FIG. 2 depicts the animation of a self-priming RhCMV BAC DNA construct. In the described constructs, each terminal enzyme complex recognition locus (TCRL) consists of two DR repeats. These two TCRL sides a BAC sequence, which includes the origin of replication oriS of the prokaryote.

FIGS. 3A-3B CMV vector vaccine lacking UL111A causes intense CD4 at mucosal surfaces ⁺ And CD8 ⁺ T cell response. Fig. 3A: adenovirus vector immunization was balanced against CD4 and CD8 responses following CMV vector immunization. CMV vaccine (single trace in weak solid line; median in dark solid line) elicits comparable CD4 and CD8 responses (ratio >1) Whereas adenoviral vectors may elicit a lower relative frequency of CD4 responses (dashed line). Fig. 3B: balancing CD4 after CMV vector immunization ⁺ And CD8 ⁺ Examples of T cell responses (upper panel), but adenovirus vector immunity is not balanced (lower panel). Two weeks after immunization, CD4 is shown ⁺ Or CD8 ⁺ Stimulation of vaccine antigens by cells (left and right panels, respectively)In response, cytokines are produced.

FIG. 4. Introduction of a terminal enzyme complex recognition locus (TCRL) around the BAC replication origin provides excellent transformation of CMV BAC genomic DNA replication virus in vitro. The left panel, when using conventional CMV BAC genomic DNA (TR 3dIL 10), transfection of 1 microgram CMV BAC genomic DNA with FuGene 6 resulted in only 1 plaque, while when using the self-priming construct with CMV TCRL around BAC start point (TR 4 dIL) shown in fig. 2, 20 plaques were produced. The right panel, comparison of plaque formation at a series of input DNA amounts using calcium phosphate mediated transfection. The self-starting genome with TCRL around BAC start is superior at every DNA input level.

FIG. 5B cells reactive with anti-CD 3 linked immunogens received no differential help.

FIG. 6A subcutaneous inoculation of 100. Mu.g RhCMVdIL10 vaccine BAC DNA was sufficient to elicit an immune response. Shows the immune response to pRhCMV-MAGEA4 seen one week after early priming.

Fig. 7. Scheme overview.

FIG. 8 expression cassette and experimental design. The upper panel, expression cassette design encoding SARS-CoV-2 spike S1 domain (upper panel, light gray), receptor binding domain (middle panel, gray) or anti-CD 3scFv-RBD fusion protein (S3-RBD, black). In the middle panel, these cassettes were delivered as electroporated DNA to rhesus monkeys on day 0 of the vaccination regimen; about day 28, animals were boosted with adenovirus type 35 (Ad 35) vector. The lower panel shows that three rhesus monkeys in each group received 1mg of S1, RBD, or S3-RBD expressing DNA at the time of priming and 10 at the time of boosting ¹² Ad35/S1 or Ad35/RBD of individual particles.

Figure 9 detection of bound antibodies by end-point dilution ELISA assay. The response of individual vaccinated rhesus monkeys is shown by thinner lines; the geometric mean response of each group is indicated by a thicker line (S1 is a black dot dashed line, RBD is a gray dashed line only, and S3-RBD is a black solid line). Vaccination with DNA at week 0 and Ad35 at week 4 elicited a binding antibody response that exceeded the background at week 0 in all subjects of RBD and s3-RBD constructs. Subjects who were s3-RBD primed exhibited a better response, with a higher average and, in the case of 2/3, a higher than the best response seen in the RBD group. The geometric mean response of s3-RBD was 10-fold higher than that of RBD 24 weeks after vaccination.

Figure 10 report virus particle (RVP/pseudovirus) assay various dilutions of serum were tested for inhibition of infection by pseudotyped lentiviral particles carrying SARS-CoV-2 spike. A curve was then generated and the neutralization titer 50 (NT 50) was read as the serum dilution required to obtain 50% inhibition. In this example, vaccination would result in high neutralization titers starting at week 5 after priming.

FIG. 11. Longitudinal pseudovirus neutralization assay results show that the induction of the s3-RBD neutralizing antibodies is better (black solid trace). At week 5 after the initial immunization, the Geometric Mean Titer (GMT) of the s3-RBD group was at least 4-fold higher than that of the RBD group (solid black versus dashed gray line). The real advantage of s3-RBD is greater because one animal in the s3-RBD group produces neutralizing antibody titers that exceed the upper limit of the assay. Furthermore, most RBD-only subjects had neutralizing antibody titers below the detection limit within 24 weeks after vaccination, while all s3-RBD subjects remained high neutralizing titers within 32 weeks.

FIG. 12 detection of bound antibodies by end-point dilution ELISA assay in rhesus monkeys primed with RBD alone, s2-RBD (i.e., anti-CD 2-RBD conjugated polypeptide) or eDis3-RBD (enhanced diabody anti-CD 3-RBD conjugated polypeptide). The response of individual vaccinated rhesus monkeys is shown by thinner lines; the geometric mean response of each group is indicated by a thicker line (RBD is a dashed black dot line, s2-RBD is a dashed gray dot line, eDis3-RBD is a solid black line). Compared to RBD alone, s2-RBD or edri 3-RBD primed subjects exhibited a better response, which reached a higher peak and remained at a higher level after 24 weeks.

FIGS. 13A-13D. Host cells transduced with 1dCD58-RBD (B.1.351) or s3-RBD (B.1.351) -PDGFRtm produced an immunoreactive RBD. The assay detects the binding of anti-RBD antibodies to intracellular proteins produced after transfection. (FIG. 13A) negative control cells not transfected with any plasmid did not react with the anti-RBD antibody. (FIG. 13B) positive control cells transfected with RBD (B.1.351) alone produced immunoreactive proteins. (FIG. 13C) cells transfected with the expression cassette of 1dCD58-RBD (B.1.351) produced immunoreactive proteins, indicating successful production of the conjugated polypeptide and possibly an immune response following vaccination. (FIG. 13D) cells transfected with the expression cassette for s3-RBD (B.1.351) -PDGFRtm, which was predicted to insert into the cell membrane, produced immunoreactive proteins, indicating successful production of the conjugated polypeptide.

Detailed description of the preferred embodiments

1. Introduction to the invention

The present disclosure provides methods and compositions for inducing an immune response in a subject. The methods and compositions allow for strong, potent and safe antibody and/or T cell responses against antigens such as those from viruses such as SARS-CoV-2. The methods and compositions include, among other things, conjugated polypeptides that include an antigen linked to a ligand or antibody fragment that binds to a surface protein on an immune cell, such as a T cell or Antigen Presenting Cell (APC). In some embodiments, the conjugation agent is administered in combination with a second antigen (e.g., a second antigen designed to elicit a T cell response).

2. Definition of the definition

As used herein, the following terms have the meanings they are given, unless otherwise specified.

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

The term "a/an" or "the" as used herein includes aspects having not only one member but also more than one member. For example, the singular forms "a/an" or "the" include 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.

The terms "about" and "approximately" as used herein shall generally mean the acceptable degree of error of the measured quantity given the nature or accuracy of the measurement. Typically, the exemplary degree of error is within 20% (%) of a given value or range of values, preferably within 10%, and more preferably within 5%. Any reference to "about X" is intended to mean, in particular, at least the values X, 0.8X, 0.81X, 0.82X, 0.83X, 0.84X, 0.85X, 0.86X, 0.87X, 0.88X, 0.89X, 0.9X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, 1.1X, 1.11X, 1.12X, 1.13X, 1.14X, 1.15X, 1.16X, 1.17X, 1.18X, 1.19X and 1.2X. Accordingly, "about X" is intended to teach and provide written description support for claiming a limitation such as "0.98X".

The term "antigen" refers to a molecule or portion thereof capable of inducing 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, e.g., a cell-mediated immune response that involves the expansion of T cells that target antigen-derived peptides presented on the surface of target cells. In certain embodiments, the antigens of the present disclosure are derived from a pathogen such that the immune response of the subject provides immune protection against the pathogen. In a particular embodiment, the pathogen is a virus (e.g., SARS-CoV-2).

The term "peptide-encoding nucleic acid sequence" refers to a DNA segment, which in some embodiments may be a gene or portion thereof involved in the production of a peptide chain (e.g., an antigen or fusion protein). Genes will typically include regions before and after the coding regions (leader and trailer sequences) involved in transcription/translation and regulation of transcription/translation of the gene product. A gene may also include intervening sequences (introns) between individual coding segments (exons). Leader, trailer, and intron sequences may include regulatory elements (e.g., promoters, terminators, translational regulatory sequences (e.g., ribosome binding sites and internal ribosome entry sites), enhancers, silencers, insulators, border elements, origins of replication, matrix attachment sites, locus control regions, and the like) necessary for the transcription and translation of a gene. "Gene product" may refer to mRNA or protein expressed from a particular gene.

The terms "expression" and "expressed" refer to the production of transcriptional and/or translational products, e.g., the production of transcriptional and/or translational products of a nucleic acid sequence encoding a protein (e.g., an antigen or fusion protein). In some embodiments, the term refers to the production of a transcriptional and/or translational product 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 the protein encoded by the DNA produced by the cell.

The term "recombinant" when used in reference to, for example, a polynucleotide, protein, vector, or cell, means a polynucleotide, protein, vector, or cell modified by the introduction of a heterologous nucleic acid or protein or alteration of the 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, as well as the development, maturation, differentiation, and activation of immune cells (e.g., B cells and T cells). In some cases, the immune response includes increasing MHC class E and/or class II restricted CD4 ⁺ And/or CD8 ⁺ Number or activation of T cells (e.g., in a subject). The term also includes increasing or decreasing participation in regulationExpression or activity of cytokines for immune function. As another non-limiting example, the immune response may include increasing the expression or activity of interferon-gamma and/or tumor necrosis factor-alpha (e.g., in a subject).

Other examples of desirable, expected, and/or protective immune responses that can be induced by recombinant polynucleotides, compositions, and methods according to the present disclosure include, but are not limited to, those involving: class Ia, class Ib or class II restricted CD4 ⁺ T cells; class Ia, class Ib or class II restricted CD8 ⁺ T cells; cytokine-producing T cells (e.g., T cells that produce IFN-gamma, TNF-alpha, IL-1-beta, 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 cells 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-gamma negative NK cells; and NK-CTL cells, i.e. CD8 expressing NK cell typical molecules (such as NKG 2A) ⁺ T cells.

The term "cytomegalovirus" or "CMV" refers to a virus comprising members of the genus cytomegalovirus (belonging to the order herpesviridae, subfamily betaherpesviridae). The term includes, but is not limited to, rhesus-infected human cytomegalovirus (HCMV; also known as human herpesvirus 5 (HHV-5)), simian cytomegalovirus (SCCMV or AGMCMV), baboon cytomegalovirus (BaCMV), cynomolgus monkey cytomegalovirus (OMCMV), pink monkey cytomegalovirus (SMCMV), and rhesus 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 surface of the cell. Typically, the antigen is displayed or presented as a Major Histocompatibility Complex (MHC) molecule. Almost all cell types can be used as APCs, and APCs exist in a large number of different tissue types. Professional APCs (e.g., dendritic cells, macrophages and B cells) present antigens to T cells in an environment that most effectively results in T cell activation and subsequent proliferation. Many cell types present antigens to cytotoxic T cells.

An "immune cell" may be any cell of the immune system, including T cells (e.g., 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.

An "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, the infectious disease antigen may be derived from a virus, bacterium, fungus, protozoan, worm or parasite, and may be, for example, a bacterial wall protein, a viral capsid or structural protein (e.g., a retroviral envelope protein, such as an HIV or SIV env protein) or a portion thereof. In some embodiments, the infectious disease antigen is a viral infectious disease antigen from SARS-CoV-2.

As used herein, the terms "polynucleotide," "nucleic acid," and "nucleotide" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof. 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 comprising purine and/or pyrimidine bases or other natural, chemically modified, biochemically modified, non-natural, synthetic or derivatized nucleotide bases. Unless specifically limited, the term includes nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), homologs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which a 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 recombinantly or synthetically produced with a series of specified nucleic acid elements that permit transfer of a particular nucleic acid sequence (e.g., a nucleic acid sequence encoding an antigen and/or fusion protein described herein) in a host cell or engineered cell. In some embodiments, the vector comprises a polynucleotide to be transcribed 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 a certain binding affinity or antigenicity to the protein produced from the vector (e.g., recombinant protein), and those that are capable of replicating the vector and its packaging (e.g., into viral particles). In some embodiments, the vector is a viral vector (i.e., a viral genome or a portion thereof). The vector may comprise, for example, nucleic acid sequences or mutations that increase tropism and/or modulate immune function. An "expression cassette" includes a coding sequence operably linked to a promoter, and optionally a polyadenylation sequence.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the term includes 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 a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, mice, rats, apes, humans, farm animals, sports animals, and pets. Also included are tissues, cells, and their progeny of the biological entity obtained in vivo or cultured in vitro.

A "ligand" is a molecule that binds to a biological molecule (e.g., a receptor protein) and forms a complex, thereby altering the conformation of the biological molecule and thus its functional state. For the purposes of this disclosure, a ligand is typically a polypeptide that is present with an antigen within a larger conjugated polypeptide. The ligand may be derived from a larger molecule, such as the 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 ectodomain) of CD58, referred to as 1dCD58, which can bind CD2. For purposes of this disclosure, the ligand is not an antibody (e.g., a monoclonal antibody).

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

The term "treatment" refers to a method of achieving a beneficial or desired result, including but not limited to therapeutic benefit and/or prophylactic benefit. By "therapeutic benefit" is meant any treatment-related improvement or effect of 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 increased survival. For prophylactic benefit, the composition may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of the disease, even though the disease, condition, or symptom may not have occurred.

The term "therapeutically effective amount" or "sufficient amount" refers to an amount of a system, recombinant polynucleotide, or composition described herein sufficient to produce a beneficial or desired result. The therapeutically effective amount may vary according to one or more of the following: the subject and the disease condition being treated, the weight and age of the subject, the severity of the disease condition, the immune status of the subject, the manner of administration, and the like, as readily determinable by one of ordinary skill in the art. The specific amounts may vary depending on one or more of the following: the particular agent selected, the type of target cell, the location of the target cell in the subject, the dosing regimen to be followed, whether to administer in combination with other compounds, the time of administration, and the physical delivery system in which it is carried.

For purposes herein, an effective amount is determined by considerations such as may be known in the art. The amount must be effective to achieve the desired therapeutic effect in a subject suffering from a disease, such as an infectious disease or cancer. Desirable therapeutic effects may include, for example, improving undesirable symptoms associated with a disease, preventing the manifestation of such symptoms before they occur, slowing the progression of symptoms associated with a disease, slowing or limiting any irreversible damage caused by a disease, lessening the severity of a disease or curing a disease, or increasing survival or providing a more rapid recovery from a disease. In addition, in the context of prophylactic treatment, the amount may also be effective to prevent the progression of the disease.

The term "pharmaceutically acceptable carrier" refers to a substance that facilitates administration of an active agent to a cell, organism, or subject. "pharmaceutically acceptable carrier" also refers to a carrier or excipient that may be included in the compositions of the present application and that does not have a significant adverse toxicological impact on the patient. Non-limiting examples of pharmaceutically acceptable carriers include water, sodium chloride (NaCl), physiological saline solution, ringer's lactate, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors and colors, liposomes, dispersion media, microcapsules, cationic lipid carriers, isotonic and absorption delaying agents, and the like. The carrier may further comprise or consist of: for providing stability, sterility and isotonicity to the formulation (e.g., antimicrobial preservatives, antioxidants, chelating agents and buffers), for preventing the action of microorganisms (e.g., antimicrobial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and the like), or for providing flavorants and the like to the formulation. In some cases, the vector is an agent that facilitates delivery of the polypeptide, fusion protein, or polynucleotide to a target cell or tissue. Those skilled in the art will recognize that other pharmaceutical carriers may also be used in the methods and compositions of the present application.

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

The terms "Pac1 site" and "Pac2 site" refer to cis-acting polynucleotide sequences in the direct terminal repeat sequence of the herpesvirus genome (including the cytomegalovirus genome) that are recognized by a encapsidation mechanism to initiate the packaging and direct cleavage of the genome into single unit length genomes (see, e.g., fields Virology 6 th edition, 2013, knope and Howley editions).

3. Vaccine

The present disclosure provides vaccines for generating an immune response against antigens from any type of pathogen. Antigens against which an immune response is raised (e.g., in a subject) will depend on the particular disease for which a prophylactic and/or therapeutic benefit is sought. In some embodiments, the antigen is an infectious disease antigen, such as a virus, bacterium, protozoan, helminth, or fungal pathogen. In particular 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, expected, 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 a particular embodiment, 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 antigens described herein).

Immunogenic conjugates

In particular embodiments, the vaccine comprises an "immunogenic conjugate" or "conjugate polypeptide" or "conjugate agent" comprising an antigen linked to a ligand or antibody fragment that binds to a surface protein present on an immune cell. For example, the antigen may be linked to an antibody fragment that specifically binds to a protein abundant on the surface of T cells. In some embodiments, the ligand is an extracellular domain of a cell adhesion molecule. Any abundant protein on the surface of immune cells (including T cells, such as helper T cells) can be targeted by the ligand or antibody fragment. In some embodiments, the protein bound by the ligand or antibody fragment is involved in signal transduction and/or adhesion. Examples of surface proteins that may be partially bound include CD2 (see, e.g., NCBI gene ID 914 or UNIProt P06729); CD3, including any CD3 subunit, i.e., CD 3-epsilon (see, e.g., NCBI gene ID 916 or UNIProt P07766); CD 3-gamma (see, e.g., NCBI gene ID 917 or UNIProt P09693; CD 3-delta (see, e.g., NCBI gene ID 915 or UNIProt P04234) or CD 3-zeta (CD 247; 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 P06217). It is believed that in some embodiments, such conjugated polypeptides may bridge antigen-specific B cells with T cells or other immune cells in the vicinity of B cells and thereby elicit stronger antibodies than those obtained by antigen vaccination alone.

An antigen may be any immunogenic antigen from a pathogen, i.e., contains 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 within the immunogenic conjugate produces a strong antibody response in the subject. For example, for vaccination against coronaviruses (such as SARS-CoV-2), the antigen may comprise a spike glycoprotein or 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 a particular embodiment, the antigen comprises SARS-CoV-2RBD.

The antigen is linked to a ligand or antibody fragment capable of binding to a surface protein present on the immune cell. In some embodiments, the surface protein is a abundant surface protein on T cells that is involved in signal transduction and/or adhesion. 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 T cells (e.g., relative to other immune cells). In some embodiments, the surface protein is expressed substantially exclusively by T cells (i.e., expressed by T cells and not significantly expressed by other immune cells). In some embodiments, the ligand is a protein that naturally binds to a surface protein on an immune cell, e.g., is a natural ligand for an immune cell receptor, or is a derivative or fragment of a natural ligand. In some embodiments, the ligand is an extracellular domain (ectodomain) or a cell adhesion molecule (e.g., CD 58). For example, in some embodiments, the ligand is the 95 residue membrane distal N-terminal domain of CD58 (1 dCD 58), which is fully responsible for adhesion to CD2. For the purposes of this disclosure, ligands do not include antibodies (e.g., monoclonal antibodies).

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, or the like. The term "antibody fragment" may also include multispecific antibodies and hybrid antibodies that have two or more antigen or epitope specificities. In some embodiments, the antibody fragment is a nanobody or single domain antibody (sdAb) comprising a single monomeric variable antibody domain (e.g., a single VHH domain). In particular embodiments, the antibody fragment is an scFv (e.g., an anti-CD 2, anti-CD 3, or anti-CD 4 scFv). For example, in some embodiments, the fragment is an scFv derived from an anti-CD 2 antibody (e.g., LO-CD2 a). In some embodiments, the fragment is an scFv derived from an anti-CD 3 antibody (e.g., SP 34). In some embodiments, the fragment is an scFv derived from an anti-CD 4 antibody (e.g., hu5 A8). In some embodiments, the scFv is derived from a humanized antibody.

In some embodiments, for example, wherein the antibody fragment is an scFv, the VH domain and the VL domain of the antibody are separated by a flexible linker. In some embodiments, the flexible linker is 12 amino acids or more in length (e.g., 15 amino acids). In such embodiments, the VH domain and VL domain are generally capable of folding appropriately to allow the conjugated polypeptide to be used as (e.g., bound to) a surface protein monomer. In other embodiments, the flexible linker is less than 12 amino acids in length (e.g., 5, 6, 7, 8, 9, 10, or 11 amino acids in length). In such embodiments, the VH and VL regions may have a length insufficient to fold properly as monomers to promote the formation of multimers (e.g., diabodies, triabodies, tetrabodies, etc.). In some embodiments, the disclosure comprises a diabody, a triabody, or a tetrabody 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, a number of techniques known in the art may be used. See, e.g., kohler & Milstein, nature 256:495-497 (1975); kozbor et al, immunology Today 4:72 (1983); cole et al, pages 77-96, monoclonal Antibodies and Cancer Therapy, alan r.list, inc. (1985); coligan, current Protocols in Immunology (1991); harlow & Lane, antibodies, ALaboratory Manual (1988); and Goding, monoclonal Antibodies: principles and Practice (2 nd edition 1986)). In some embodiments, antibodies are prepared by immunizing one or more animals (e.g., mice, rabbits, or rats) with an antigen to induce an antibody response. To generate monoclonal antibodies, B cells are fused with myeloma cells, which are subsequently subjected to antigen-specific screening.

Genes encoding the heavy and light chains of the antibody of interest may be cloned from cells, e.g., genes encoding monoclonal antibodies may be cloned from hybridomas and used to produce recombinant monoclonal antibodies, whereby antibody fragments may be produced. The gene libraries encoding the heavy and light chains of monoclonal antibodies can also be made from hybridomas or plasma cells. In addition, phage or yeast display techniques can be used to identify antibodies and heteromeric Fab fragments that specifically bind to a selected antigen (see, e.g., 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, yeast-based antibody presentation systems may 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. Random combinations of heavy and light chain gene products produce a large number of antibodies with different antigen specificities (see, e.g., kuby, immunology (3 rd edition, 1997)). Techniques 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 (e.g., fab ', F (ab') 2, scFv, nanobody, or diabody) are produced. In a particular embodiment, the antibody fragment is an scFv (single chain variable fragment). The scFv is a light (V _L ) And weight (V) _H ) Recombinant polypeptides of immunoglobulin chain variable regions. In some embodiments, the VH sequence and the VL sequence are linked by a flexible linker sequence. See, e.g., nelson (2010) MAbs.2 (1): 77-83. Various techniques have been developed for the production of 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 may be isolated from antibody phage libraries. Alternatively, fab '-SH fragments can be recovered directly from E.coli (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 method, the F (ab') 2 fragment may be isolated directly from the recombinant host cell culture. Other techniques for generating antibody fragments will be apparent to those skilled in the art.

Measurement of binding affinityAnd methods of force 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) ^TM (GE Healthcare, piscataway, NJ)), kinetic exclusion assays (e.g.,) Flow cytometry, fluorescence Activated Cell Sorting (FACS), bioLayer interferometry (e.g., octet ^TM (forte Bio, inc., menlo Park, CA)) and western blot analysis.

In some embodiments, the affinity agent is a peptide, e.g., a peptide that binds to a T cell surface protein. In some embodiments, the agent is a peptide aptamer. Peptide aptamers are artificial proteins that are selected or engineered to bind to a specific target molecule. Typically, a peptide comprises one or more peptide loops of variable sequence displayed by a protein scaffold. The selection of peptide aptamers can be performed using different systems, including yeast two-hybrid systems. Peptide aptamers can also be selected from combinatorial peptide libraries constructed by phage display and other surface display technologies (e.g., mRNA display, ribosome display, bacterial display, and yeast display). See, e.g., reverdatto et al 2015, curr. Top. Med. Chem.15:1082-1101.

In some embodiments, the agent is an affimer. Affimer is a small, highly stable protein, typically having a molecular weight of about 12-14kDa, that binds their target molecules with similar specificity and affinity as antibodies. In general, affimers exhibit two peptide loops and an N-terminal sequence that can be randomized to bind different target proteins with high affinity and specificity in a similar manner as 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. The art describes Affimers and methods for making affimers. See, e.g., tiede et al, eLife,2017,6:e24903.Affimer is also commercially available, for example, from Avacta Life Sciences.

The antigen and ligand/antibody fragments may be linked to each other in a variety of ways, either directly or indirectly. For example, in some embodiments, the antigen and ligand/antibody fragments are directly (covalently) linked, e.g., by a chemical linker or by virtue of being present in a single fusion protein. Methods for linking polypeptides to each other, such as for linking antigens to ligand/antibody fragments, are known in the art and are available from commercial suppliers, such as protein-protein conjugation kits from TriLink BioTechnologies, from Vector Laboratories, from Kerafast, from sydlab, from intershim, and the like.

In particular embodiments, the antigen and ligand/antibody fragments are present within a single fusion protein. For example, in a particular embodiment, the immunogenic conjugate is a fusion protein comprising an antigen and an anti-CD 3scFv 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 proteins can be expressed in vitro, purified, formulated, and administered in protein form using standard molecular biology and pharmaceutical methods, or can be administered as polynucleotides encoding the fusion proteins.

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., uniProt P00750; kou et al, (2017) immunol. Lett.190:51-57; wang et al, (2011) appl. Microbiol. Biotech.91 (3): 731-740; delogu et al, (2002) Microal 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 fragments present within the conjugated polypeptide or fusion protein are separated by a flexible linker. Suitable linkers for isolating protein domains are known in the art and may comprise, for example, glycine and serine amino groups, e.g., 2-20 glycine and/or serine residues. In one embodiment, the flexible linker comprises (Gly 4 Ser) _n Flexible and flexiblePeptide linkers, e.g., comprising the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 5) (Gly 4 Ser) ₃ A linker.

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 the lipid anchor include, for example, glycosyl phosphatidylinositol anchors. In some embodiments, the addition of a lipid anchor (e.g., a glycosyl phosphatidylinositol anchor) is directed by a signal sequence (e.g., a signal sequence derived from CD 55). Examples of suitable transmembrane segments include, but are not limited to, transmembrane segments derived from PDGF receptor, glycophorin A or SARS-CoV-2 spike protein. Examples of multimerization domains include, for example, domains derived from T4fibritin and Fc domains (e.g., human IgG Fc domains). In some embodiments, the disclosure includes dimers or other multimers formed between conjugated polypeptides of the application comprising a multimerization (e.g., a T4fibritin or Fc domain). In some embodiments, the multimer is stabilized by disulfide bonds between monomer units. In some embodiments, the Fc or other domain is located at the C-terminus of the conjugated polypeptide.

In some embodiments, wherein the vaccine comprises a polynucleotide encoding a fusion protein, and wherein the polynucleotide is present within a viral vector (e.g., a CMV vector), the coding sequence of the fusion protein is operably linked to a late promoter (e.g., 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., pp65 b) elicits a strong antibody response, but only a weak T cell response.

In some embodiments, the antigen and ligand/antibody fragment (also referred to as an "affinity agent") are linked indirectly, i.e., by a non-covalent interaction bridging the two entities. For example, the antigen and ligand/antibody fragment may be linked by a second "bridging" antibody or fragment thereof. In some embodiments, the antigen is a membrane protein embedded in a nanodisk (nanodisk), and the bridging antibody is a bispecific antibody fragment that binds to (i) the antigen itself or a membrane scaffold protein in a nanodisk, and (ii) a T cell surface protein. Nanodiscs are a synthetic membrane system that includes a lipid bilayer surrounded by an amphiphilic protein called Membrane Scaffold Protein (MSP). Any nanodisk system may be used in the methods of the application, including any MSP (e.g., apoA 1-derived MSP) or amphiphilic peptide. In some embodiments, synthetic nanodiscs are used. The preparation and use of nanodiscs is known in the art and is described, for example, in Bayburt et al, (2002) FEBS Letters 584 (9): 1721-1727; denisonv 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; denisonv & slide (2016) Nat. Structure. Mol. Biol.23 (6): 481-486; the complete disclosure of each of which is incorporated herein by reference.

In some embodiments, the conjugated polypeptide comprises an amino acid sequence as set forth in seq id no: 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 a conjugated polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity 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., within the same vaccine), or separately (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 the form of a protein or in the form of a polynucleotide (i.e., as a polynucleotide encoding both antigens). In some embodiments, the coding sequence of the immunogenic conjugate and the coding sequence of the second antibody are present in a single vector when administered in the form of a polynucleotide, each of which is operably linked to a promoter. In some embodiments, the second antigen is administered as a polynucleotide encoding the antigen operably linked to a constitutive promoter, e.g., a mammalian promoter (e.g., EF-1 a) (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; their complete disclosure is incorporated herein by reference). Without being bound by the following theory, it is believed that expression of the second antigen in the cytosol of the cell elicits a strong T cell response, but minimal concomitant antibody response.

In some embodiments, when the pathogen is a coronavirus (e.g., 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 fragments thereof. In some embodiments, the second antigen comprises a fusion protein comprising an E protein and an M protein, or fragments of an E protein and/or an M protein, of a coronavirus (e.g., SARS-CoV-2). Other suitable SARS-CoV-2 antigens include fragments of nsp3, nsp4 or nsp6 or more. In particular embodiments, the vaccine comprises a codon optimized coding sequence comprising an N protein and/or comprising a fusion protein comprising the E protein and the M protein of SARS-CoV-2 (e.g., as shown in SEQ ID NOs: 3 and 4).

Nucleic acid vaccine

In some embodiments, a nucleic acid vaccine (e.g., a DNA vaccine) is used to introduce the immunogenic conjugate and/or the second antigen. Thus, in some embodiments, the present disclosure provides polynucleotides encoding any of the conjugated polypeptides described herein. In some embodiments, the DNA vaccine is prepared as a DNA vector or plasmid. In some embodiments, the DNA vaccine is prepared as a recombinant virus, e.g., by modifying a parent virus to incorporate exogenous genetic material, e.g., one or more polynucleotides encoding one or more antigens described herein. A non-limiting list of suitable viruses that may be used for purposes of this disclosure include lentiviruses (e.g., HIV-1, HIV-2, FIV, BIV, EIAV, MW, CAEV, SIV), adenoviruses and adeno-associated viruses, alphaviruses, herpesviruses (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 generally capable of recombining (i.e., capable of propagating in an infected host cell). Modification of such viruses and vectors or plasmids for use in preparing the DNA vaccines of the present application can be accomplished using standard molecular biology techniques, for example, as taught in Sambrook et al, (1989) "Molecular Cloning: A Laboratory Manual" (2 nd edition, cold Spring Harbor Press) and Ausubel et al (editions) (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: 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 a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity 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 set forth in: SEQ ID NO. 2, SEQ ID NO. 7, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26 or SEQ ID NO. 28, or a polynucleotide comprising a nucleotide sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity 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 particular embodiments, the DNA vaccine involves a Cytomegalovirus (CMV) vector, e.g., a self-priming CMV DNA vector _SL CMV), as described in U.S. provisional application No. 62/842,419 filed on 5/2 of 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 "initiates" vector vaccine replication in vivo, producing a strong immune response with unique characteristics associated with CMV infection. _SL The CMV vector may comprise one or more of a variety of features, including: (i) Based on the Towne HCMV strain, this strain has proven safe for use in people of all ages; (ii) Providing an immune response in CMV seropositive and seronegative individuals due to the repeated infection capacity of CMV and the proprietary deletion of the CMV genome; (iii) Providing a very broad and strong T cell response that "profile" vaccine antigens; (iv) Has demonstrated ability to elicit an equilibrium response including antibodies, CD4 ⁺ T cells and CD8 ⁺ T cells-without relying on or dominating any single effector response; providing localization of the responsive T cells to the mucosal surface due to the effector memory phenotype; (v) Allowing engineering of new vaccine candidates over weeks to months; and (vi) providing a single manufacturing method based on plasmid production in E.coli.

And does not induce massive memory CD4 ⁺ Most vaccine types for T cells are different, as shown in the examples below, as is true for the CMV vector vaccine of the application. In addition, T cells that respond to CMV vector vaccines are localized to the airway in other effector sites and can pass through, for example, the bronchiAlveoli lavage for recovery (12). CMV-responsive T cells outline other essential features of cells that exhibit protective effects on SARS-CoV-1, including CXCR3 expression, IFN-gamma production, and IL-10 production (13).

At the position of _SL Among CMV vectors, vaccination with CMV vectors in their DNA form requires modification of the current CMV BAC construct so that BAC backbone can be excised without recombinant or nuclease expression in vivo. CMV has relatively stringent packaging limitations due to the need to package a unit length genome into an icosahedral capsid. In some embodiments, the CMV BAC utilizes an endogenous recombinase gene disposed within 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 replication genome. To adapt the BAC DNA vector for delivery in humans, the CMV genome ends are reorganized to enable excision of the BAC without the need for recombinase expression. In particular, the reconfigured BAC construct utilizes a viral terminal enzyme complex to eliminate the bacterial origin of replication during the packaging step of CMV replication. In some embodiments, the location of the BAC origin and replication mechanism is located between the viral direct repeats.

Thus, in one embodiment, the present disclosure provides a self-priming HV (e.g., CMV) recombinant polynucleotide comprising one or more polynucleotides encoding one or more antigens (or conjugated polypeptides) described herein, and comprising (a) a Herpesvirus (HV) genome or a portion thereof; (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 a repeatedly introduced polynucleotide sequence that can be directly cleaved by the HV terminal enzyme complex; wherein the HV genome or a portion thereof is separated from sequences comprising an origin of replication by TCRL; wherein the HV genome or a portion thereof abuts TCRL at least one terminus; and wherein the sequence comprises an origin of replication adjacent to the TCRL at least one terminus; and (d) one or more polynucleotides encoding one or more antigens operably linked to the 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 a abundant T cell surface protein) operably linked to a late promoter (e.g., pp65 b), and/or a polynucleotide encoding an antigen operably linked to a constitutive promoter (e.g., EF-1α).

As used herein, the "end" of a particular genomic region or element (e.g., a genome or portion thereof) in a vector refers to either end of the region or element beyond which a different region or element (or end of a nucleic acid molecule) is present. For example, in a circular vector, in some embodiments, one end (or terminus) of the HV genome may be immediately adjacent to the first end of the first TCRL element, and the other end (or terminus) of the HV genome may be immediately adjacent to the first end of the second TCRL element. In the same circular vector, one end (or terminus) of the region containing the origin may be immediately adjacent to the second end of the first TCRL element and the other end (or terminus) of the region containing the origin may be immediately adjacent to the second end of the second TCRL element. In some embodiments, the CMV vector as used herein does not comprise a viral IL-10 gene.

It will be appreciated that the polynucleotide may be circular or linear and that there may be additional elements, 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 between TCRL adjacent to the HV genome or a substantial portion thereof and a (BAC or YAC) origin of replication that is functional in a single cell organism.

Antigen coding sequences

The recombinant polynucleotides (e.g., viral vectors) of the present disclosure comprise nucleic acid sequences encoding antigens (e.g., conjugated polypeptides) described herein and/or coronavirus antigens (e.g., spike protein E protein, M protein, or N protein, or combinations and/or fragments thereof) as described herein. Rapid progress in various genomic studies has enabled cloning methods in which databases of DNA sequences of human or other model organisms can be searched for any gene segment that has a certain percentage of sequence homology with known nucleotide sequences, such as those encoding antigens and the like. Any DNA sequence so identified can then be obtained by chemical synthesis and/or Polymerase Chain Reaction (PCR) techniques, such as overlap extension. For short sequences, complete de novo synthesis may be sufficient; further isolation of full-length coding sequences from cDNA or genomic libraries of human or other model organisms using synthetic probes may be necessary to obtain larger genes.

Alternatively, the nucleic acid sequence may be isolated from a cDNA or genomic DNA library (e.g., a human or rodent cDNA or a human, rodent, bacterial or viral genomic DNA library) using standard cloning techniques such as Polymerase Chain Reaction (PCR), wherein primers based on homology are typically obtained from known nucleic acid sequences. Common techniques for this purpose are described in standard text, e.g. Sambrook and Russell, supra.

The cDNA library may be commercially available or may be constructed. General methods for isolating mRNA, preparing cDNA by reverse transcription, ligating cDNA into recombinant vectors, transfecting into recombinant hosts for propagation, 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 the nucleotide sequence by PCR, the segment can be further used as a probe to isolate the full-length polynucleotide sequence encoding the protein of interest from the cDNA library. A general description of suitable procedures can be found in Sambrook and Russell, supra.

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

In certain embodiments, the polynucleotide encoding the antigen (e.g., immunogenic conjugate and/or other antigen) is present within the expression cassette, i.e., operably linked to one or more promoters. Any promoter capable of driving expression of a polynucleotide in one or more cells of a subject may be used, including inducible promoters and constitutive promoters. In some embodiments, a CMV promoter is used. In particular embodiments, late viral promoters (e.g., pp65 b) are used to drive expression of the immunogenic conjugate. In certain embodiments, a constitutive mammalian promoter (e.g., EF1- α) is used to drive expression of the second antigen as described herein. In some embodiments, the EF 1-alpha promoter comprises a first intron of the EF 1-alpha gene. The vector may contain other regulatory sequences such as terminators, translational regulatory sequences (e.g., ribosome binding sites and internal ribosome entry sites), enhancers, silencers, insulators, border elements, origins of replication, matrix attachment sites, locus control regions, and the like. The use of such elements is well known in the art.

In some embodiments, the recombinant polynucleotides of the present disclosure comprise a nucleic acid sequence encoding a selectable marker. For example, selectable markers are useful when polynucleotides described herein are being recombinantly modified, particularly when a population of modified polynucleotides needs to be screened (e.g., using bacterial, yeast, plant, or animal cells) to obtain those that have incorporated the desired modification. Whether the polynucleotide is recombinantly modified within cells (e.g., bacterial cells, e.g., using Red/ET recombination), or is recombinantly modified and subsequently introduced into cells (e.g., bacterial, yeast, plant, or animal cells) for selection, a selectable marker may be used to identify those cells that contain the polynucleotide that has incorporated the modification of interest. Using an example of an antibiotic resistance gene as a selectable marker, treatment of cells containing a recombinant polynucleotide with an antibiotic will recognize those cells that contain a recombinant polynucleotide that has incorporated the antibiotic resistance gene (i.e., cells that survive antibiotic treatment must have incorporated the antibiotic resistance gene). If desired, the recombinant polynucleotide may be further screened (e.g., purified, amplified, and sequenced from the cell) to verify that the desired modification has been recombinantly introduced into the polynucleotide at the correct location.

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

In some embodiments, the selectable marker used to identify cells containing a polynucleotide comprising a modification of interest is a fluorescently labeled protein, a chemical stain, a chemical indicator, or a combination thereof. In other embodiments, the selectable marker is responsive to a stimulus, a biochemical or a change in environmental conditions. In some cases, the selectable marker is responsive to the following concentrations: 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 particular antigen 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 different elements (e.g., TCRL), and the like. In addition, the size of the recombinant polynucleotide will depend on whether the nucleic acid sequences encoding the antigen and other proteins are present within the same recombinant polynucleotide or within separate recombinant polynucleotides.

In some embodiments, the recombinant polynucleotide is about 1 kilobase to about 300 kilobases in length (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 some embodiments, the recombinant polynucleotide is greater than about 300 kilobases in length.

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

General recombinant techniques

Basic text disclosing general methods and techniques in the field of recombinant genetics (e.g., for the preparation, maintenance and culture of recombinant vectors or plasmids), including Sambrook and Russell, molecular Cloning, A Laboratory Manual (3 rd edition, 2001); kriegler, gene Transfer and Expression: A Laboratory Manual (1990); and Ausubel et al, edited Current Protocols in Molecular Biology (1994).

For nucleic acids, the size is 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, the size is expressed in kilodaltons (kDa) or the number of amino acid residues. In some cases, protein size can be estimated from gel electrophoresis, sequenced proteins, deduced amino acid sequences, or published protein sequences.

Non-commercially available oligonucleotides can be chemically synthesized, for example, using an automated synthesizer as described in Van Devanter et al, nucleic Acids Res.12:6159-6168 (1984) according to the solid phase phosphoramide triester method described first by Beaucage & Caruthers, tetrahedron Lett.22:1859-1862 (1981). Purification of the oligonucleotides is performed using any art-recognized strategy, such as natural 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 a chain termination method such as 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 segment as a probe, a full length nucleic acid encoding the protein of interest is obtained.

After obtaining the nucleic acid sequence encoding the protein of interest, the coding sequence may also be modified by a number of known techniques (e.g., restriction enzyme digestion, PCR, and PCR-related methods) to produce a coding sequence, including mutants and variants derived from wild-type proteins. The polynucleotide sequence encoding the desired polypeptide may then be subcloned into a vector (e.g., an expression vector) such that the recombinant polypeptide may be produced from the resulting construct. The coding sequence (e.g., nucleotide substitutions) may also be subsequently modified to alter the properties of the polypeptide.

Various mutation generation schemes are established and described in the art and can be readily used to modify polynucleotide sequences encoding a protein of interest. See, e.g., 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 variants of a set of nucleic acids, and thereby variants encoding polypeptides. Kits for mutagenesis, library construction and other methods of diversity generation are commercially available.

Mutation methods to generate diversity include, for example, site-directed mutagenesis (Botstein and Shortle, science,229:1193-1201 (1985)), mutagenesis using uracil-containing templates (Kunkel, proc. Natl. Acad. Sci. USA,82:488-492 (1985)), oligonucleotide-directed mutagenesis (Zoller and Smith, nucleic acids Res.,10:6487-6500 (1982)), phosphorothioate-modified DNA mutagenesis (Taylor et al, nucleic acids Res.,13:8749-8787 (1985)), and mutagenesis using gapped double-stranded DNA (Kramer et al, nucl. Acids Res.,12:9441-9456 (1984)).

Other possible methods of creating mutations include point mismatch repair (Kramer et al, cell,38:879-887 (1984)), mutagenesis using repair-deficient host strains (Carter et al, nucl. Acids Res.,13:4431-4443 (1985)), deletion mutations (Eghtedarzadeh and Henikoff, nucl. Acids Res.,14:5115 (1986)), restriction selection and restriction purification (Wells et al, phil. Trans. R. Soc. Lond. A,317:415-423 (1986)), 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 (Leung. R.S. 1:15) PCR (Biohnque et al, 1981:15).

Codon optimization

In some embodiments, the nucleic acid sequence encoding the protein of interest (e.g., antigen or other protein) is codon optimized. The term "codon optimization" refers to altering a nucleic acid sequence without altering the encoded amino acid sequence in such a way as to reduce or rebalance the codon bias (i.e., preferentially using specific codons that may vary between species). 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 re-balancing codon usage (e.g., in-frame), a preferred leucine codon may be selected over less frequently used codons. The nucleic acid sequence encoding the protein of interest (e.g., antigen or other protein) is altered such that rarely used codons are converted to preferred codons.

For example, rare codons may be defined by using a codon usage table from the sequenced genome derived from the host species, i.e., the species in which the protein (e.g., antigen) is to be expressed. See, e.g., codon usage tables obtained from Kazusa DNA Research Institute, japan (www.kazusa.or.jp/codon /), used in conjunction with software from DNA 2.0 (www.dna20.com /), e.g., the "Gene Designer 2.0" software, cut-off threshold is 15%.

Codon optimization can also be used to modulate GC content, for example, to increase mRNA stability or reduce secondary structure; or otherwise minimize codons that may lead to sequence extension that impairs expression of the protein of interest (e.g., antigen or other protein).

4. Formulations and vaccination methods

Object(s)

The methods and compositions of the invention can be used for vaccination of any subject (e.g., human or other mammal) that can benefit from an enhanced immune response against 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 an adolescent. 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 been infected with a pathogen (e.g., SARS-CoV-2), and the methods and compositions are used to enhance the subject's immune defenses against the pathogen to prevent future infection. In other embodiments, the subject has been infected with a pathogen, and the method is 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 compositions comprising an immunogenic component (e.g., a DNA vaccine or one or more immunogenic polypeptides) capable of inducing immunity to a targeting agent (e.g., an antigen or immunogenic conjugate comprising an antigen) and a pharmaceutically acceptable carrier. In some embodiments, the vaccine further comprises one or more adjuvants or compounds. Accordingly, the present disclosure provides pharmaceutical compositions for inducing an immune response in a subject. In some embodiments, the composition comprises one or more polynucleotides encoding one or more proteins (e.g., coronavirus antigens and/or immunogenic conjugates) and a pharmaceutically acceptable carrier. In some embodiments, the composition comprises one or more polypeptide antigens (e.g., immunogenic conjugates described herein) and/or a coronavirus antigen comprising, for example, a spike protein, an E protein, an M protein, or an N protein, fragments thereof, or combinations thereof, and a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises an adjuvant.

The compositions may be formulated for, for example, injection, inhalation, or topical administration, e.g., to facilitate direct exposure of host cells and tissues to the 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. patent nos. 6,265,387, 6,972,013, and 7,922,709).

In particular embodiments, the DNA vaccine is prepared as a DNA vector or plasmid. In some embodiments, the composition (e.g., DNA vaccine) is prepared as a recombinant virus, e.g., by modifying a parent 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)). Can prepare self-starting CMV _SL CMV) vectors, e.g., by culturing escherichia coli comprising the vector, lysing the cultured bacterial cells, purifying the vector while ensuring that endotoxin is absent or below a heating threshold (e.g., 5 endotoxin units/kg body weight), and formulating the vector for administration. In some embodiments, the proteins encoding the antigens of the application are in vitro using standard molecular biology techniquesProduced and purified prior to vaccine formulation as described herein.

In some embodiments, nucleic acid vaccines are formulated with, for example, in vivo transfection reagents comprising one or more agents that can protect the nucleic acid from degradation in vivo and facilitate delivery of the nucleic acid to the cells. Suitable examples of such agents include, but are not limited to, in vivo-jetpi ^TM (Polyplus)、TurboFectTM(Thermo Scientific)、LIPID ^TM (Altogen Biosystems)、GenJet ^TM Plus or PepJet ^TM Plus(SignaGen)、DogtorMag ^TM (OZ Biosciences)、Avalanche ^TM (EZ Biosystems), etc., and may be used according to manufacturer's instructions.

The pharmaceutical compositions of the present disclosure may comprise a pharmaceutically acceptable carrier. In certain aspects, the pharmaceutically acceptable carrier is determined in part by the particular composition being administered and the particular method used to administer the composition. Thus, there are a variety of pharmaceutical composition formulations suitable for use in the methods and compositions of the present invention (see, e.g., REMINGTON' S PHARMACEUTICAL SCIENCES, 18 th edition, mack Publishing co., easton, PA (1990)).

Optionally, the pharmaceutical composition will typically further comprise 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, butylhydroxytoluene, butylhydroxyanisole, etc.), bacteriostats, chelating agents (e.g., EDTA) or glutathione, solutes that render the formulation isotonic, hypotonic, or poorly hypertonic with the blood of the recipient, suspending agents, thickening agents, preservatives, flavoring agents, sweeteners, and coloring compounds.

The pharmaceutical composition is administered in a manner compatible with the dosage formulation and in a therapeutically or prophylactically effective amount. The amount administered depends on a variety of factors including, for example, age, weight, physical activity, genetic characteristics, general 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 dose may also be determined by the presence, nature, and extent of any adverse side effects associated with the administration of a therapeutic or prophylactic agent in a particular individual. Other factors that may affect the particular dosage level and frequency of dosage for any particular patient include the activity of the particular compound used, the metabolic stability and length of action of that compound, the mode and time of administration, and the rate of excretion.

In some embodiments, the vaccine comprises an adjuvant, i.e., a compound administered to the subject in combination with the antigen, for enhancing the immune response to the antigen. Adjuvants may increase the immunogenicity of a vaccine in any of a variety of ways and may include inorganic compounds (such as salts, e.g. aluminium salts) as well as organic compounds and mixtures of compounds, including extracts and formulations such as 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, in a therapeutically or prophylactically effective dose. In particular, an effective amount of a pharmaceutical composition is an amount sufficient to obtain an enhanced immune response against 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 subject's immunity to infection from the pathogen or to the spread of infection already present in the subject.

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

As used herein, the term "unit dosage form" refers to physically discrete units (e.g., ampoules) suitable as unitary dosages for humans and other mammals, each unit containing a predetermined quantity of therapeutic or prophylactic agent calculated to produce the desired initial, tolerogenic and/or therapeutic or prophylactic effect, in association with a suitable pharmaceutical excipient. Alternatively, more concentrated dosage forms may be prepared from which more diluted unit dosage forms may 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 of preparing such dosage forms are known to those skilled in the art (see, e.g., REMINGTON' S PHARMACEUTICAL SCIENCES, supra). The dosage form typically includes a conventional pharmaceutical carrier or excipient, and may additionally include other agents, carriers, adjuvants, diluents, tissue penetration enhancers, solubilizing agents, and the like. Suitable excipients can be tailored to the particular dosage form and route of administration by methods well known in the art (see, e.g., the pharmaceutical sciences of Leidmington, supra).

Application of

In some embodiments, preventing and/or treating comprises administering a composition described herein directly 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 may be administered in a single dose or in multiple doses, for example, two doses administered at intervals of about one month, about two months, about three months, about six months, or about 12 months. Other suitable dosing schedules may be determined by a medical practitioner.

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 the induction of an immune response, and the like.

The pharmaceutical composition of the application may be administered to a subject locally or systemically, e.g. intraperitoneally, intramuscularly, intra-arterially, orally, intravenously, intracranially, intrathecally, intraspinal, intralesionally, intranasally, subcutaneously, intraventricular, topically and/or by inhalation. In particular embodiments, the composition is administered by electroporation (e.g., for priming DNA vaccines) or subcutaneously (e.g., for boosting).

The vaccine of the application may be administered any of a number of times (e.g., 1, 2,3,4, 5 or more) and may be administered after any of a number of vaccination protocols, e.g., every 1, 2,3,4, 5, 6, 7, 8, 9, 10 or more weeks. In certain embodiments, the vaccine is administered in the form of a DNA prime (e.g., with a plasmid encoding a conjugated polypeptide as described herein), followed by boosting with an adenovirus vector encoding an antigen, e.g., after about 4 weeks. The DNA vaccine may be administered at any of a variety of levels, for example, 4mg of plasmid DNA vector per subject per vaccination, or 1, 2,3,4, 5, 6, 7, 8, 9, 10 or more mg of plasmid DNA vector per subject per vaccination, or 1-10, 1-20, 1-8, 1-7, 2-6, 3-5mg of plasmid DNA vector per subject per vaccination.

Assessment of immune response

The immune response of a subject receiving a vaccine of the present disclosure may be detected, characterized, or quantified in any of a variety of ways. For example, any of the assays described in any of the examples can be used to detect the presence of a polynucleotide of the disclosure in a detectable subject cell, to detect and characterize antibodies or T cells specific for a vaccine of the application, or to 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 antigens of the application, the presence and level of vector sequences may be assessed, for example, by measuring sequences using qPCR from, for example, blood or saliva samples from a subject. The level of nucleic acid may also be detected from different tissues of the subject, e.g. as obtained from a biopsy or lavage through mucosal tissue.

In some embodiments, the immune response of a subject is assessed by an immunophenotype, e.g., by assessing a T cell memory effector subset, NK cells with adaptive properties (e.g., fce RIy ^{Low and low} "memory" NK cells), T cells with innate properties (e.g. NKG 2A) ⁺ Cells) or antigen presenting cells (e.g., monocytes that express CD 80/83/86). Such cells may be assessed, for example, using flow cytometry as described in the examples.

The immune response of a subject can also be assessed by characterizing an antigen-specific T cell response from the subject. For example, PBMC or LNMC cells may be stimulated with one or more antigens from the vaccine, optionally in combination with an inhibitor (such as a VL9 peptide or anti-HLA antibody) as described in the examples. After a suitable amount of time (e.g., 16 hours), the cells can be assessed using antibodies to, for example, CD3, CD4, CD8, CCR7, CD95, IL-2, IL-17, IFN- γ, and/or TNF- α. Cytokine-secreting CD4 can be assessed using, for example, flow cytometry ⁺ And/or CD8 ⁺ And (3) cells.

In some embodiments, antibodies obtained from a subject may be evaluated, for example, by detecting binding to an antigen using ELISA. In some embodiments, RVP (reporter virus particle) assays are used to test neutralizing or enhancing antibodies. In particular embodiments, the vaccines of the present application elicit a strong neutralizing antibody response and a low or absent enhancing response. In some embodiments, for example, where vaccination strategies are tested in model animals, challenge assays using pathogens may be used as described in the examples.

Any of the parameters or effects described in the examples or elsewhere herein (e.g., neutralizing antibody production, specific T cell responses, protection against pathogens, etc.) can be used to assess the efficacy of the vaccines described herein. In some embodiments, the vaccine of the present disclosure results in an increase of at least about 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 150%, 200%, 250%, 300% or more in any of the parameters or effects described herein relative to a control value (e.g., a value observed in a subject not receiving the vaccine of the present disclosure). In some embodiments, the vaccine described herein does not substantially induce antibody dependent infection enhancement (ADEI) in the subject. In some embodiments, the vaccine induces a neutralizing antibody response in the subject that is significantly greater than any ADEI induced in the subject.

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

5. Kit for detecting a substance in a sample

In another aspect, provided herein are kits. 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 disclosure, or a vector comprising a polynucleotide encoding one or more antigens or immunogenic conjugates of the present disclosure, and optionally a pharmaceutically acceptable carrier). In some embodiments, the kit comprises an adjuvant. In some embodiments, the kit is used to induce an immune response against an antigen (e.g., coronavirus spike protein, E protein, M protein, or N protein, fragments thereof, or combinations thereof). In other embodiments, the kit is for preventing or treating a disease (e.g., covd-19). In some embodiments, the kit is used to induce a B cell (i.e., antibody) response to one or more antigens. In some embodiments, the kit is used to induce a T cell response against one or more antigens. In some embodiments, the kit is used to induce a B cell response to one 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 may be packaged (e.g., in a box or other container with a lid) in a manner that allows for safe or convenient storage or use. Typically, the kits of the application comprise one or more containers, each container storing a particular kit component (e.g., reagent, control sample, etc.). The choice of container will depend on the particular form of its contents, e.g., the kit components in liquid form, powder form, etc. Furthermore, the container may be made of a material designed to maximize the shelf life of the kit components. As a non-limiting example, the light-sensitive kit components may be stored in an opaque container.

In some embodiments, the kit comprises one or more elements (e.g., a syringe) for administering the composition (i.e., the pharmaceutical components described herein) to a subject. In other embodiments, the kit further comprises instructions for use, e.g., comprising instructions (i.e., a regimen) for practicing the methods of the application (e.g., instructions for using the kit to enhance an immune response of a subject to an antigen from a pathogen, such as SARS-CoV-2). Although the illustrative materials generally 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., magnetic disk, tape, film, chip), optical media (e.g., CD ROM), and the like. Such media may include addresses of internet sites that provide such instructional material.

6. Examples

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

Example 1 conjugated polypeptide epidemic comprising SARS-CoV-2 S1 Domain linked to anti-CD 3 Single-chain variable fragment Seedling

This example provides a conjugated polypeptide capable of binding to B cells having a surface receptor reactive with the "S1" domain of SARS-CoV-2 spike protein and T cells having the abundant T cell surface protein CD 3. When included in anti-SARS-CoV-2 vaccine protocols as described below, the conjugated polypeptide elicits an antibody response to the S1 domain, which may aid in protection against COVID-19.

SARS-CoV-2 enters cells through the activity of spike protein (S) with receptor binding (S1) and membrane fusion (S2) regions. SARS-CoV-2 spike shows many of the characteristics of traditional class I fusion proteins, including the presence of different heptad repeats within the fusion domain. Antibodies to the S1 domain may block SARS-CoV-2 infection by blocking the interaction of spike protein with its receptor ACE2 protein. CD3 is a multimeric protein complex consisting of four polypeptide chains (epsilon, gamma, delta and zeta) associated with the T Cell Receptor (TCR) and plays a key role in the transmission of activation signals from the TCR into the T cell interior.

We created a conjugated polypeptide comprising the S1 region of SARS-CoV-2 spike and a CD3 binding polypeptide by fusing the coding sequences of the following protein elements (see, e.g., fig. 5): tissue-type plasminogen activator signal sequence (to allow secretion of conjugated polypeptides from cells), anti-CD 3scFv derived from murine anti-CD 3 antibodies, SP34 (see, e.g., U.S. patent application publication 2016/0068605 A1); a flexible connector; and SARS-CoV-2S1 (codon optimized). The amino acid sequence is shown as SEQ ID NO. 1. The codon-optimized nucleic acid sequence of this polypeptide (SEQ ID NO. 2) was synthesized and cloned downstream of the EF 1-alpha promoter sequence (including its first intron) and upstream of the SV40 polyadenylation sequence in the pUC19 plasmid.

To test the CD3 binding function of the conjugated polypeptide, the resulting plasmid was transfected into 293 cells by calcium phosphate precipitation. Supernatant from transfected cells is applied to the tissue culture plate to allow the secreted proteins to bind to the surface of the plate, thereby forming a fixed array of such secreted proteins, which if bound to the TCR should send an activation signal to the T cells. We applied T cells from rhesus monkeys to either coated wells or control wells. Fluorescent antibody staining and data collection on flow cytometry revealed that the immobilized conjugated polypeptide activated T cells, resulting in CD69 up-regulation on the cell surface and intracellular interferon-gamma production (fig. 1). This result demonstrates the binding of the conjugated polypeptide to CD3 molecules on the surface of T cells.

Example 2 providing SARS-CoV-2 vaccine candidates

This example provides SARS-CoV-2 vaccine candidates that (i) elicit both T cells and antibody responses to minimize the risk of increased antibody dependence; (ii) proved to be effective in rhesus monkeys; and (ii) ready for future phase I testing in humans. The vaccine platform combines rapid development (DNA administration) with a broad, strong T cell response (due to CMV vector) and neutralizing antibody response to spike S1 domain.

Traditional vaccine development against previously unknown pathogens takes years, but protection of human health may require a rapid response on the scale of months (1). However, due to the lack of appropriate animal models for the emerging pathogens; the risk of antibody-dependent enhancement of infectivity (ADEI), which may occur whenever a sub-optimal antibody response is induced (2); and the difficulty of developing new manufacturing processes for subunits, attenuated or vector vaccines, and the acceleration of development becomes complex.

Self-starting CMV DNA platform _SL CMV) alleviates or eliminates these complications, allowing for the rapid development of new vaccines that can elicit abnormally broad and powerful immune responses, limited to mucosal surfaces threatened by SARS-CoV-2. These vaccines are administered as cytomegalovirus genomes, which "start" vector vaccine replication in vivo, resulting in a strong immune response with unique characteristics associated with CMV infection. _SL The CMV features include: (i) Based on the Towne HCMV strain, the strain can be safely used in people of all ages; (ii) CMV seropositive and seronegative individuals develop immune responses due to CMV repeat infectivity and CMV genome-specific deletions; (iii) A very broad and strong T cell response that "delineates" vaccine antigens; (iv) Proven balanced responses (including antibodies, CD 4) ⁺ T cells and CD8 ⁺ T cells); (v) Localization of responsive T cells at mucosal surfaces due to effector memory phenotypes; (vi) Engineering new vaccine candidates over weeks to months; and (vii) a single preparation based on plasmid production in E.coliAnd (5) manufacturing process.

Most coronavirus vaccines under development are designed to elicit antibodies. Due to possible antibody-dependent enhancement (ADEI), such vaccine strategies must be carefully adopted, especially when antibody levels are low (2). Highly concentrated antisera against SARS-CoV-1 proved to neutralize the virus, whereas diluted antibodies caused ADEI in human procaryotic cell cultures. On the other hand, T cell responses are generally targeted to highly conserved internal proteins and have long lifetimes. In fact, airway memory CD4 ⁺ T cells have been shown to mediate protective immunity against SARS-CoV-1 and MERS-CoV. We hypothesize that self-priming CMV DNA vaccines against SARS-CoV-2 can provide broad and protective adaptive immunity that is localized to mucosal surfaces.

Part 1: the immune response in rhesus monkeys was characterized against a self-priming CMV/SARS-CoV-2 vaccine designed to elicit a T cell or antibody response.

Transgenes in a CMV vector vaccine driven by a constitutive promoter elicit a strong T cell response, but little or no concomitant antibody response; transgenes expressed from strong late promoters (e.g., pp65 b) elicit strong antibody responses and weak T cell responses. Using these strategies, we created candidates _SL CMV DNA vaccine designed to elicit a significant T cell response to SARS-CoV-2E proteins, M proteins and N proteins, as well as a strong neutralizing antibody response to spike S1 domain. The latter candidate expresses a spike S1 domain that is unmodified or linked to an anti-CD 3scFv, which physically links the B cell that produces the anti-S1 antibody to a T cell that can provide assistance. Over time, an adaptive immune response then occurs throughout the body and at mucosal surfaces. The production of neutralizing antibodies relative to enhancing antibodies was monitored using a Reporter Virus Particle (RVP) assay.

Part 2: evaluation of Davis isolates against SARS-CoV-2 in rhesus monkeys _SL Protective efficacy of CMV/SARS-CoV-2 vaccine.

SARS-CoV-2 isolated from the patient was cultured and characterized and the pathogenesis of the virus was assessed in monkeys. We tested the protective efficacy of the T cell and B cell vaccines created in section 1 by challenging vaccinated animals with SARS-CoV-2. T cell and B cell vaccines are tested separately or together to test the relative contribution of each branch of the adaptive immune system to protection against disease enhancement.

Part 3: safety and potential efficacy of self-priming human CMV vector vaccines in rhesus monkeys were tested.

Recent studies have shown that Human CMV (HCMV) vector vaccines elicit strong effector memory T cell responses in rhesus monkeys. Thus, we created HCMV vector vaccines as candidates for future human clinical trials, developed GMP procedures for production, and tested in rhesus monkeys _SL Efficacy of HCMV protocol. Will be used according to section 2 _SL Protective study results of RhCMV HCMV vector vaccines were selected for in vivo experiments.

Significance of the invention

Rapid vaccine development against emerging infectious threats is an unmet need: traditional vaccine development against previously unknown pathogens is slowed by fundamental biological problems that are difficult to solve. Most importantly, although induction of high titer neutralizing antibodies (nabs) appears to be an obvious approach, we do not know what titer of nabs has protective effect in practice, nor how the threshold varies in extreme cases of age and complications. For any emerging pathogen, we do not know whether an adverse antibody response is particularly likely to lead to an enhancement of antibody dependent infection (ADEI), especially in the elderly and young.

The immune relevance of successful vaccination against SARS-CoV-2 vaccine is not well defined: most coronavirus vaccines currently under development are directed against the most variable part of the spike glycoprotein and induce only an antibody response 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 both nAbs (2, 3). Due to the possible ADEI, especially in cases of low antibody levels, care must be taken to access the vaccine specifically eliciting antibodies (4). Highly concentrated antisera against SARS-CoV-1 proved to neutralize viral infectivity, whereas diluted antibodies caused ADEI in human procaryotic cell cultures, resulting in cytopathic effects and increased levels of TNF- α, IL-4 and IL-6 (5-7). Vaccine candidates based on full length SARS-CoV-1 spike proved to be capable of inducing non-neutralizing antibodies and the immunized animals were not protected. Instead, they experience side effects such as exacerbation of hepatitis, increased incidence and a stronger inflammatory response (8, 9).

The CoV vaccine elicited T cell responses also play a critical role in protection and clearance. T cell deficient mice are unlikely to clear MERS-CoV infection, but can be achieved in B cell deficient mice (10). In addition, airway memory CD4 is shown ⁺ T cells mediate protective immunity against SARS-CoV-1 and MERS-CoV (11). As shown below, most vaccine types do not elicit large amounts of memory CD4 ⁺ T cells-but CMV vector vaccines will prime. T cells that respond to CMV vector vaccines are localized to the airways in other effector sites and recovered by bronchoalveolar lavage (12). CMV-responsive T cells outline other essential features of cells that exhibit protective effects on SARS-CoV-1, including CXCR3 expression, IFN-gamma 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 endogenous pp65b promoters. The promoter is one of the most active promoters after late DNA replication of CMV infection. For example, it has been found that in rhesus monkeys vaccinated with CMV vaccine carrying ebola virus Glycoprotein (GP) under control of pp65b promoter, 4/4 produced GP specific antibodies, which were boosted after the second vaccine administration (21). High levels of GP antibodies induced by RhCMV/EBOV-GP and their ability to undergo IgG class conversion indicate the presence of sufficient CD4 ⁺ T auxiliary function. Three of the four rhesus monkeys with the highest anti-GP titers were protected from fatal EBOV attacks.

An important property of CMV vectors for combating emerging pathogens is the ability to reapply to previously exposed individuals. This capability enables repeated use of CMV vector vaccines to prevent a series of emerging threats over time.

The unavailability of conventional CMV vaccines for combating 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 problem is that there is great difficulty in mass production of a unified test sample from a slowly growing and variable beta herpesvirus (29).

Innovative innovation

Vaccination with CMV vector in nucleic acid form: although transfection with CMV genomic DNA is the cornerstone of many in vitro techniques, and other researchers have tried to deliver the herpesvirus genome as a plasmid in salmonella organisms (26), to our knowledge, no attempt has been made to deliver naked or chemically complexed CMV genomic DNA as a vaccine. We demonstrate hereinafter that gene expression, genomic replication, viremia and immune response occur after administration of CMV BAC DNA.

The BAC replication origin was placed at the end of the CMV genome to allow excision of the BAC by the CMV terminal enzyme complex: to vaccinate with CMV vectors in their DNA form, the current CMV BAC construct needs to be altered so that the BAC backbone can be excised without recombinase or nuclease expression in vivo. CMV has relatively stringent packaging limitations due to the need to package a unit length genome into an icosahedral capsid. Current CMV BACs utilize endogenous recombinase genes disposed within 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 replication genome. To make BAC DNA vectors suitable for delivery in humans, we reorganize the CMV genome ends to be able to excise BACs without the need for recombinase expression. Our reconfigured BAC construct utilized a viral-terminator enzyme complex to eliminate bacterial origins of replication during the packaging step of CMV replication (fig. 2). We have moved the position of the BAC origin and replication mechanism from its current position (RhCMV US 1/2) to between the terminal enzyme complex recognition sites (TCRL) containing viral direct repeats. We show hereinafter that this arrangement allows efficient replication and packaging of the vaccine genome following in vivo introduction into host cells.

Novel improved RhCMV-SIV vaccine: the first RhCMV-SIV vector carries the complete endogenous viral IL-10 gene, which suppresses the host immune response (27-31). We have created a second generation RhCMV vector platform-virus IL-10 deficient RhCMV or RhCMVdIL 10-which has unique immune characteristics and can protect RhCMV negative infant rhesus monkeys, whereas the first generation vaccine cannot (27).

Example 3 cmv vector vaccine elicits unique broad and strong T cell responses in the CD4 and CD8 compartments.

T Effector Memory (TEM) cells are the main type of T cells at mucosal effector sites (14). Final high frequency CD4 in CMV infection and effector-memory compartments ⁺ And CD8 ⁺ T cell responses are associated, which prevent CMV pathology, but do not eliminate CMV infection or prevent CMV repeat infection (15-19). In addition, TEM cells primed by CMV vector vaccines recognize distinct and unusual epitopes, which include a major response to epitopes restricted by class E and class II Major Histocompatibility Complex (MHC) molecules (20). T cells that respond to CMV vaccines recognize more than three times the number of peptide epitopes than those that respond to other vaccine types, thus producing a response that "portrays" the vaccine antigen and should prevent the pathogen from escaping (20). Although much of the published work has focused on CD8 ⁺ T cell response, but CMV vaccine stimulates equivalent strength of CD4 ⁺ T cell responses, a feature not seen with other vector vaccines (fig. 3A). Importantly, the vaccinators airway responded to CD4 ⁺ The unique abundance of T cells (FIG. 3B) meets the requirements described for protection of SARS-CoV-1 (11).

Example 4 DNA-based, self-priming CMV vector vaccines can be rapidly engineered and manufactured for broad protection It is free from emerging threats.

To allow manipulation using prokaryotes, the RhCMV-SIV vaccine is maintained as a circular Bacterial Artificial Chromosome (BAC) comprising vector genomic DNA with an embedded BAC replication origin (ori) flanked by recognition signals for site-specific recombinases or restriction enzymes. For efficient replication and packaging, it is necessary to excise the BAC origin by site-specific recombination in vitro or after transfection. We re-engineered these vectors to contain BAC origins outside the viral genome and flanking CMV terminal enzyme complex recognition loci, allowing automatic excision by CMV terminal protease when the vector begins to replicate (fig. 2). Thus, the novel vector does not require in vitro digestion or Cre recombinase expression and is delivered to the vaccine recipient as a stable circular DNA molecule. Upon reaching the recipient cell nucleus, these CMV vaccine genomes enter the viral replication cycle and produce a series of viral particles that elicit the same protective immune responses as those elicited 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 the viral particles, probably due to the administration of more vaccine genomes at multiples of 10,000 (fig. 6).

EXAMPLE 5 characterization of self-priming CMV/SARS-in rhesus monkeys against peptides designed to elicit T cell or antibody responses Immune response of CoV-2 vaccine.

We hypothesize that the self-priming RhCMV vaccine will produce as broad a T cell response as those administered in the form of viral particles, and that the ligation of the SARS-CoV-2S1 immunogen to the anti-CD 3scFv fragment will increase the speed and efficacy of the NAb response.

The rationale for this hypothesis is that we have demonstrated that SLRhCMV vaccine is injected into replicating virus, which can be detected in the blood within weeks after administration, and we expect that these forms elicit T cell responses (in group B) as broadly as those elicited by conventionally packaged CMV (-90 peptides/1000 versus 12 of adenovirus vectors).

With respect to SLCMV vaccines designed to elicit B cell responses (group C-D), we compared secreted spike S1 domains to the same proteins linked to scFv fragments that bind to CD3 (fig. 7). The result is a molecule that can "bridge" S1-specific B cells and any T cells in the vicinity. Our preliminary data indicate that the idiotype linked to anti-CD 3 in bispecific antibodies rapidly elicits very strong anti-idiotype antibodies. Thus, it is desirable to deliver the spike S1 domain linked to an anti-CD 3scFv to elicit antibodies that are stronger than S1 alone.

We tested the immune response to three candidate vaccine components, all administered twice eight weeks and four weeks prior to challenge (figure 7). The T cell fraction comprises a mixture of SLRhCMV/N and SLRhCMV/EM vaccine. We assessed the intensity and breadth of T cell responses to these vaccines (group B) to provide data for later protection correlation checks. There are two candidate B cell components (group C and group 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 a reduction in enhancement. Finally, we tested a regimen consisting of T cell vaccine (SLRhCMV/n+em and selected B cell vaccine administered together).

Vaccine construction

As shown in FIG. 2, SLRhCMV/N, SLRhCMV/EM, SLRhCMV/S1 and SLRhCMV/S1-anti-CD 3 were generated as self-priming BAC constructs bound by CMV terminator recognition sites. 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-1 a promoter (including its first intron) and have been recombined into the Rh213/214 region of the RhCMV genome, we have re-used this position and observed a major T cell response. The S1 and S1-anti-CD 3scFv cassettes were expressed under the control of the endogenous late RhCMV pp65b promoter and expressed prior to the 23-aa tPA leader sequence to promote efficient secretion. S1-anti-CD 3 fusion uses a humanized scFv region derived from anti-CD 3 clone SP34, a clone that we have previously used to construct bispecific antibodies.

Alkaline lysis was performed using Triton X-114, followed by two consecutive isopycnic centrifuges, and BAC (vaccine) DNA free of endotoxin was purified from E.coli cultures. Endotoxin concentrations were measured using a Limulus Amoebocyte Lysate (LAL) assay to ensure that they were below the pyrogenic threshold (5 endotoxin units/kg body weight) (32).

Vaccine administration

Our preliminary data (figure 6) show that vaccination subcutaneously with 100 μg of RhCMVdIL10 vaccine BAC DNA is sufficient to elicit an immune response. BAC DNA was formulated with in vivo-jetPEI (Polyplus) according to manufacturer's instructions. Briefly, 100. Mu.g of DNA (50. Mu.g each of RhCMVdIL10-SIVgag and-SIVenv) and 16. Mu.L of in vivo-jetPEI were diluted into 5% glucose solution (1 ml) respectively, followed by mixing and incubation for 15 minutes.

RhCMV vector replication and shedding

Vaccine-derived RhCMV DNA in blood and saliva samples was measured weekly by qPCR using our published protocol (16, 33). These measurements provide an assessment of replication and transmission of BAC DNA encoding viral vectors. Transmission to various tissues was assessed by qPCR in necropsy following SARS-CoV-2 challenge in example 6.

Immunophenotyping

The immunophenotype of most interest is the T cell memory effector subset, which contains the largest proportion of cells that respond to RhCMV and RhCMV/SIV vaccines; NK cells with adaptive properties (i.e. fceriγ low "memory" NK); t cells with congenital properties (NKG 2A) ⁺ ) The method comprises the steps of carrying out a first treatment on the surface of the And antigen presenting cells (particularly monocytes) expressing CD 80/83/86. All of these cell populations were altered after infection with wild type or vaccine strain RhCMV. All of these were evaluated using a set of three flow cytometer 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 1MPBMC or LNMC cells were stimulated with vehicle (DMSO-toxic negative control), overlapping RBD, S1 peptide, E peptide, M peptide and/or N peptide (Intavis) or PMA/ionomycin (positive control). Inhibitors (e.g., VL9 peptides or anti-HLA antibodies) were applied one hour prior to the start of stimulation, and peptide stimulation was again added. After 16h, cells were stained with fixable live-dead stain and antibodies reactive to CD3, CD4, CD8, CCR7, CD95, IL-2, IL-17, IFN-gamma and TNF-alpha. Cytokine secreting CD4 ⁺ And CD8 ⁺ The fraction of T cells is 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 antibodies or enhancing antibodies are tested by RVP assay.

Combined T-cell and B-cell vaccine (group E)

The binding and neutralizing antibody responses in the C-D group were well characterized and the group with excellent neutralizing titers and little or no enhancement was selected for the SARS-CoV-2 challenge (see example 6). In addition, after selection of the best candidate for inducing Nab, a T/B combination vaccine group (group E) was formed. The group received combined inoculation of SLRhCMV/N, SLRhCMV/EM and selected B cell vaccine.

Interpretation of data

We hypothesize that our SLRhCMV vaccine (delivered in DNA form) elicits strong T cell and B cell responses, and that the latter response is stronger when the antigen is linked to an anti-CD 3 scFv. To determine if these responsive cells are present in the most relevant tissue (i.e., lung), we performed bronchoalveolar lavage and assayed for T cells that can be recovered from the airway.

Some animals may show an enhancement of the RVP assay after vaccination based on the ADEI previously observed with other respiratory tract infection-causing RNA viruses including SARS-CoV-1. Such results are important for understanding the immune pathogenesis of covd-19 and for interpreting the results of the challenge experiments described below.

Statistical analysis

The nonparametric Kruskal-Wallis test was used to test differences in aggregate measurements or results at a single time between groups. For longitudinal results, e.g. for examining the correlation between T cells and antibody responses, a Generalized Linear Mixed Model (GLMM) was used as an analytical framework, where the random effect correction was performed on the correlation in animals caused by continuous measurements.

EXAMPLE 6 evaluation of the protection of SLCMV/SARS-CoV-2 vaccine against SARS-CoV-2Davis isolates in rhesus monkeys Protective effect.

We hypothesize that a strong T cell response localized to the airway can defend against SARS-CoV-2 and additionally prevent resistance toThe body dependence is enhanced. The rationale for this hypothesis is that the T cell response is the most important defense of the organism against intracellular parasites, and if present in the target tissue of the virus at a sufficiently high frequency, would be expected to defend against SARS-CoV-2. Indeed, airway memory CD4 has previously been shown ⁺ T cells 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, thus the virus is selected and allowed to replicate productively-however, viruses internalized due to ADEI should be readily cleared by T cells.

We developed a unified challenge and monitoring scheme so that consistent virologic, immunological and pathological datasets can be utilized in experiments. Rhesus monkeys vaccinated as shown (groups B-E in fig. 7) will be challenged at least 8 weeks after priming with vaccine and subsequently subjected to repeated clinical evaluations; radiography; collecting respiratory and mucosal secretions (e.g., by bronchoalveolar lavage, tracheal irrigation, or nasal irrigation), saliva, urine, and stool; drawing blood; and tissue collection (see, e.g., www.biorxiv.org/content/10.1101/2020.07.07.191007031).

Virus for infection

The viral stock generated by amplifying the SARS CoV-2 isolate we obtained from UC Davis patients will be used for animal vaccination and is designated 2019-nCOV/USA-CA9/2020. If the virus is not grown in sufficient quantity, we will instead use the SARS CoV-2 isolate USA-WA1/2020 (BEI Resources). To infect animals, a total of about 6×10 in 5ml of 0.9% sterile saline was used ⁶ The TCID50 is instilled into the conjunctiva, nostril and trachea of the anesthetized monkey to reproduce the relevant propagation path of covd-19.

Sampling and measuring

Body temperature, body weight and activity were monitored throughout the course. CBC and serum chemistry were obtained for all blood samples to monitor host response and organ function. The sampling schedule is intended to fully describe viral shedding, cytokine responses, and adaptive immunity to understand how changes in these parameters reflect lung pathology. We have successfully used sampling protocols and procedures to characterize influenza a infection in rhesus monkeys. Dense sampling during the first week enabled us to study acute virology and host response. Since ACE2 is expressed in the gastrointestinal and genitourinary tracts of rhesus monkeys and humans, we also 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 intestinal tract to assess viral localization and immune response by PCR, molecular histology (IHC, ISH) and cytometry. The tissues were evaluated for gross pathology, histopathology, and tissue vRNA levels. Necropsy was performed by a committee-certified pathologist.

Viral RNA was recovered from respiratory samples using Thermo's MagMAX virus/pathogen nucleic acid isolation kit (CDC recommended for COVID-19) and quantified by amplifying the SARS-CoV-2 nucleoprotein (N) gene segment. Specific RT-PCR assays used in these studies are under evaluation. CNPRC team is comparing the sensitivity, specificity and reproducibility of validated RT-PCR assays from CDC, UCD health clinical laboratory, NPRC, wisconsin, and commercial suppliers. We selected the most consistent assay. Immunological analysis was performed as described above in example 5.

Interpretation of data

If our hypothesis is correct, it is resistant to SARS-CoV-2 vaccination, although animals of group B have developed an immune response that is almost entirely composed of T cells. Animals of group C or group D (animals selected in example 5) could also be protected, but our potential for ADEI remained alert, which could be indicated by increased viral load, shedding or pathological findings in medium antibody titer animals, whether or not they exhibited enhancement in the RVP assay.

Statistical analysis

Summarizing those results found or evaluated at individual time points (e.g., at necropsy), evaluation was performed using a non-parametric test, where the p-value was adjusted according to Benjamini and Hochberg. Longitudinal results were assessed using a linear hybrid model (generalized if necessary) in which random effects were adapted to the intra-animal dependence caused by continuous measurements.

Example 7. Safety and potential efficacy of self-priming human CMV vector vaccine in rhesus monkeys were tested.

We hypothesize that the SLHCMV vaccine can be produced in GMP at a scale of 10-20 grams, can safely administer rhesus monkeys, and can prevent SARS-CoV-2 challenges. The rationale for this hypothesis is that, surprisingly, it has recently been shown that attenuated HCMV vectors can elicit and maintain T-effect memory responses against inserted antigens in rhesus monkeys (see, e.g., caposio Sci Rep 9:19236). Thus, the rhesus model can provide an environment in which candidate SLHCMV vaccines produced under GMP conditions can be tested for efficacy against SARS-CoV-2. If HCMV vector vaccines were proven safe and effective in rhesus monkeys, they would be candidates for subsequent clinical trials.

The SLHCMV form of example 5 was engineered to administer all four vaccines for group B-D rhesus monkeys. In addition, GMP production was performed on those SLHCMV vaccines that formed part of the best regimen after the B-E group challenge. For example, if group B animals are best pathologically protected, production of SLHCMV/N and SLHCMV/EM is performed. The resulting GMP product was sent to UC Davis for rhesus 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. The vaccine is orthologous to the RhCMV genome engineered at Davis. The viral interleukin-10 gene is deleted in both cases; inserting a sequence into the intergenic region near US28 to elicit a T cell response; and placed under the control of the pp65b promoter to elicit an antibody response. BAC carries the codon-optimized SARS-CoV-2 sequence driven by the same promoter.

The plasmid backbone sequences used allowed the plasmid to be maintained at approximately a single copy (in DH10B cells where oriS was used to maintain the plasmid) or 15-30 copies (after induction of TrfA expression and its interaction with alternative oriV origins). BAC DNA was purified on a laboratory scale by dual sequential CsCl equilibrium gradient centrifugation and then dialyzed into PBS. The integrity of the plasmid preparation was confirmed by restriction enzyme footprint, PCR amplification of the expression cassette and deep sequencing after labeling.

GMP production is performed by CMO partners. Upstream process development focused on optimizing transformation and culture conditions to ensure maximum homogeneity of the test sample. Downstream process development (purification after cell growth) focused on adaptation of the CsCl-dependent process used in the laboratory to iodixanol. Testing optimal SLHCMV vaccine protocols in rhesus monkeys (group F)

The SLHCMV vaccine was vaccinated in the same combination and same dose as used in one of the B-E groups. Immunoassays, SARS-CoV-2 challenge and pathology assessment were performed as well.

Interpretation of data

Our hypothesis predicts that the SLHCMV vaccine is produced in GMP process at sufficient scale and purity to allow final testing in phase I human trials. In addition, we believe that the SLHCMV vector vaccine may prove effective in rhesus models. Such results may be counterintuitive, but it is possible that HCMV, according to publications in the literature, could prove to complete its life cycle in rhesus fibroblasts (43) and could elicit a strong TEM response in rhesus.

The immune responses of rhesus monkeys to SLHCMV vaccines are also of interest, as they may reflect the responses achieved with minimal vaccine transmission, as seen with fully inactivated HCMV vector vaccines in humans. Despite some evidence of genome replication, HCMV (Towne) based vaccines should be able to be transmitted with minimal or no systemization in rhesus monkeys. Thus, comparing the immune responses of RhCMV to Towne vector antigens should reveal which immune responses are only dependent on immune modulation in quality and which depend on vector transmission.

Statistical analysis

Statistical analysis is centered on the characteristics of the vaccine formulation. Our previous experience shows that single copy BACs have mutation rates similar to other plasmids, i.e., lower than the level of amplicon sequencing detection (0.1%) due to polymerase error rate (46-50). SLHCMV genome replication in rhesus monkeys is determined by any one of the following: (i) vaccine viral sequences in plasma one week after priming or boosting, (ii) gB expression from injection site biopsies (HCMV gB specific Ab; gB is expressed as late gene only after genome replication), or (iii) memory antibody responses to HCMV gB defined by doubling the titer one month after boosting.

Example 8 conjugated polypeptide epidemic comprising SARS-CoV-2RBD Domain linked to anti-CD 3 Single-chain variable fragment Seedling

We designed a conjugated polypeptide vaccine, designated s3-RBD (SEQ ID NO. 6), which is a fusion protein derived from the SP34 clone between a humanized anti-CD 3 single chain variable fragment (scFv) and the SARS-CoV-2 Receptor Binding Domain (RBD), both preceded by a tissue plasminogen activator (tPA) signal sequence, to achieve secretion. RBD is a segment of 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 RBD-responsive B cells (via their cell surface, RBD-specific antigen receptors) and helper T cells (via CD 3). Without being bound by the following theory regarding specific mechanisms of action, we hypothesize that the engagement of pairs or clusters of these cognate receptors on B cells and T cells should result in activation of both cell types, thereby driving the continued development of B cells. B cells that receive T cell help are more likely to undergo somatic hypermutation and eventually develop into high affinity RBD-specific antibody producers.

To immunize rhesus monkeys with a gene vaccine expressing s3-RBD, we prepared codon optimized open reading frames with the nucleotide sequence set forth in SEQ id No. 7. This 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-alpha promoter sequence and upstream of the polyadenylation signal from SV40 (giving the complete expression cassette sequence given in SEQ ID NO. 8). Plasmids containing the expression cassette were prepared, which were free of endotoxin at medium scale, for administration as DNA vaccines. The expression cassette was also cloned into the E1 region of a type 35 adenovirus shuttle plasmid which allowed transfer of the DNA sequence into an E1, E3 deleted type 35 adenovirus vector.

To evaluate the immune response to our s3-RBD immunogen in the context of a genetic vaccine in non-human primates, we immunized 9 monkeys with electroporation DNA prime (day 0) and Ad35 vector boost (day 28; fig. 8). The immunogens tested were SARS-CoV-2 S1 domain, RBD domain alone or s3-RBD, all expressed from codon optimized ORFs under the control of EF-1 alpha promoter. The tPA signal sequence was placed upstream of the isolated RBD domain and the s3-RBD fusion protein to enable secretion (FIG. 8).

Evaluation of binding antibodies by ELISA during vaccination protocol showed superiority of RBD domain for immunization (relative to the whole S1 domain), and superior performance of S3-RBD construct relative to RBD alone (fig. 9). Surprisingly, the S1 immunogen performed very poorly, eliciting detectable binding and neutralizing antibodies in only one of the three vaccinators. Note that in both animals, the low binding antibody response to S1 was evident by ELISA (fig. 9); but not significant when compared to the much higher responses in the other groups; neutralization activity was detected in one of the two S1 test animals (fig. 9, black dot dashed line). At all post-boost time points, immune responses directed only against RBD domains were detectable in all three animals receiving the gene vaccine encoding RBD domains (fig. 9, grey dashed line). However, the binding antibody response was highest in the group receiving s3-RBD (FIG. 9, black solid line).

Neutralization activity was tested against pseudotyped lentiviral particles, which are lentiviral particles lacking their native envelope protein but carrying the SARS-CoV-2 spike protein. An example of a curve generated for one animal is shown in fig. 10. Each curve takes the median titer 50 (NT 50) as the dilution at which infection was reduced to 50%. In fig. 10, no NT50 was detected at the first two time points, but serum from all subsequent time points showed neutralization by reducing the infectivity of pseudotyped particles to below 50%. All RBD vaccinators exhibited neutralizing titers in the pseudovirus assay, with animal D2 having a peak titer of 1:6539 (fig. 11), which is approximately the 90 th percentile of the convalescence population (Moore and Klasse, 2020). However, as with the ELISA assay for binding antibodies, subjects expressing the s3-RBD gene vaccine obtained the highest neutralization titers at most time points after the fourth week, which exceeded all RBD test animals in 2 out of 3 (fig. 11). In fact, the geometric mean titer of the s3-RBD tested animals exceeded that of the RBD subjects by at least a factor of 4. The actual fold increase was higher because one s3-RBD subject animal produced antibody titers that exceeded the maximum quantifiable in the assay (1:10240). The neutralizing antibody response in s3-RBD subjects also showed an impressive persistence, with all animals remaining neutralized for 32 weeks after the first immunization (fig. 11). In contrast, neutralization activity decreased below the detection limit we assayed after 24 weeks in only 2/3 RBD subjects.

In some reported cases, the immune response to membrane-associated antigens (e.g., because those antigens carry a glycosylphosphatidyl inositol anchor or transmembrane segment) is superior to the immune response to the same antigen in its secreted form. Thus, we created a conjugated polypeptide comprising an anti-CD 3scFv fragment, namely RBD of the b.1.351 ("south africa") strain of SARS-CoV-2, and a transmembrane segment from the human PDGF receptor. The amino acid sequence is shown as SEQ ID NO. 25. The codon-optimized nucleic acid sequence of this polypeptide (SEQ ID NO. 26) was synthesized and cloned downstream of the EF 1-alpha promoter sequence (including its first intron) and upstream of the SV40 polyadenylation sequence in the pUC19 plasmid.

We next verified that the s3-RBD (B.1.351) -PDGFRtm conjugated polypeptide (SEQ ID NO. 25) contains immunoreactive RBD (B.1.351) when expressed in human cells, which may be necessary to induce an anti-RBD (B.1.351) antibody response in human subjects. The plasmid encoding s3-RBD (b.1.351) -PDGFRtm was transfected into human embryonic kidney cell line 293 and after two days the presence of immunoreactive RBD (b.1.351) proteins in the cells was verified by antibody staining using the following procedure. On day 1 of the assay, 350,000 cells were seeded into each well in 6-well plates. On day 0, a Polyethyleneimine (PEI) transfection mixture was prepared by adding 150 microliters of 0.1mg/ml PEI in DMEM to 3mcg of DNA in an equal volume of DMEM; incubating the mixture at room temperature for 20 minutes; 2.7ml of complete medium was added; adding the medium with the composite DNA to washed 293 cells in a 6-well plate; and plates were returned to the incubator overnight for incubation. On day 1, the transfection mixture was removed from the cells, the cells were washed, and 2ml of fresh medium was added. On day 2, cells were fixed with 5% PFA, washed in 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 truebue HRP substrate for 5-15 minutes, and finally quenched in water. The results (FIG. 13D) demonstrate that the s3-RBD (B.1.351) -PDGFRtm construct successfully produced conjugated polypeptides that include immunoreactive RBD (B.1.351).

Example 9 SARS-CoV-2RBD Domain comprising an anti-CD 3 Single-chain variable fragment and an antibody Fc region Conjugated polypeptide vaccine of (2)

Multivalent antigen display can drive a stronger, longer lasting antibody response through efficient cross-linking of B Cell Receptors (BCR) and improved antigen transport, endocytic uptake and final presentation (Brinkkemper Vaccines 7,2019;Tokatlian Science 363:649,2019). The repeat epitope has a higher effective affinity for BCR and can cross-link receptors, effectively activating B cells (Cimica Clin Immunol 183:99,2017;Zabel J Immunol 192:5499,2014). A spacing of 15-20 hapten molecules of 5-10nm is an ideal choice for B cell activation (Vogelstein PNAS 79:395, 1982). Multimeric antigens are most commonly produced ex vivo as proteins, but may also be produced by in vivo genetic vaccines.

For example, one approach to increasing the immunogenicity of vaccine antigens is to present them on Nanoparticles (NPs) 25-50nm in diameter. Licensed human papillomavirus and HBsAg vaccines involve NP 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 number and quality of Ab responses compared to delivering the same antigen as the soluble protein (Brinkkemper Vaccines 7,2019). Thus, NP display of RSV antigen increased NAb titers by more than 10-fold (Marcandali Cell 176:1420, 2019). NP presentation also allows the generation of multivalent antigen chimeric immunogens, which can increase the breadth of NAb by increasing the avidity of specific interactions with the most cross-reactive BCR, as shown by influenza HA (Kanekiyo Nat Immunol 20:362, 2019).

Immunoglobulin "crystallizable fragments" or fcs are dimeric molecules; thus, when the immunogen is expressed as a fusion protein with Fc, the result is that the dimer molecule may be more immunogenic for the reasons described above. The Fc region can also be modified to polymerize into a well-defined complex comprising 12 fusion partners (Mekhaiel Sci Rep 1:124). Furthermore, the presence of the Fc domain significantly increases the plasma half-life of the fusion protein due to its interaction with the salvaged neonatal Fc receptor and the slower renal clearance of the larger molecule (roobenian & Akilesh,2007 and Kontermann, 2011). The attached Fc domains also enable these molecules to interact with Fc receptors (fcrs) found on immune cells, a feature particularly important for their use in tumor therapy and vaccines (Nimmerjahn & Ravetch, 2008).

To allow expression of the multimeric s3-RBD and achieve other benefits conferred by the Fc domain, we therefore designed a protein molecule consisting of the conjugated polypeptide vaccine s3-RBD fused at its C-terminus to the human IgG1Fc domain, known as "s3-RBD-Fc" (SEQ ID NO. 9). Next, we prepared a codon optimized ORF (SEQ ID NO. 10) capable of expressing s 3-RBD-Fc.

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

Example 10 SARS-CoV-2RBD Structure comprising an anti-CD 3 Single-chain variable fragment linked to form a diabody Domain dimerized conjugated polypeptide vaccine

The s3-RBD comprises an anti-CD 3scFv fragment, which itself comprises the heavy and light chain variable regions VH and VL from monoclonal antibody SP 34. To form a functional monomeric scFv fragment, these VH regions 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. To allow correct folding of the monomeric scFv, the linker is typically >12 amino acids in length, and most commonly 15 amino acids (Wang Antibodies8:43,2019).

The shorter linker linking the two variable domains may determine the formation of the multimeric scFv molecule because the shorter linker has a length insufficient to allow for monomer folding with spatially correct association of the VH and VL regions. The reduction of the linker length linking the two variable domains below 8-12 residues facilitates dimer assembly of VH-VL fragments, resulting in a diabody 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 produce tri-or tetrameric molecules (tri-chain antibodies, tetra-chain antibodies) (Iliades et al, 1997; kortt et al, 1997; pei et al, 1997; le gal et al, 1999; dolezal et al, 2000; hudson and Kortt, 1999).

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

In assessing the crystal structure of the reported diabodies, significant structural diversity was evident, indicating that the diabodies were structurally unstable (Kim Sci Rep 6:34515, 2016). In the crystal structure of diabodies, the light chain does not contribute to the interaction between Fv domains, and the two heavy chains form a relatively small interaction interface. In order to use diabodies as a general and reliable mediator of artificial protein assemblies, it would be advantageous to propose a predictable direction and distance between antigen binding sites (e.g. CD3 binding sites). Nonetheless, many of the simplest designed diabodies (i.e., linker sequences reduced to 5 amino acids) have an interaction interface between Fv domains that appears to be too small to have a stable structure (Moraga Cell 160:1196,2015;Perisic Structure 2:1217,1994). The results indicate that diabody structures can be made more rigid and predictable by substituting arginine residues in the EF loop and introducing one or more disulfide bridges between Fv domains.

Next, we designed an s3-RBD dimer form 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). Such codon optimized ORFs are engineered consecutively into expression cassettes and into engineered DNA and adenovirus vectors, which are administered to animals for immunogenicity testing.

Plasmid DNA molecules encoding enhanced dimeric anti-CD 3-RBD conjugated polypeptides (also known as eDis 3-RBD) were administered as DNA vaccines to three rhesus monkeys (by electroporation of 1mg of DNA on day 0); boost was provided with RBD-only type 35 glandular vector on day 28 (fig. 12). The antibody responses in these rhesus monkeys were compared to those obtained using RBD alone for priming and boosting. The results showed that the peak antibody response with eDis3-RBD was improved (6.3 fold increase in geometric mean) and the persistent antibody response remaining 24 weeks after vaccination was also improved (5 fold increase in geometric mean; FIG. 12, compare the solid black mean line of eDis3-RBD with the black dot-dashed mean line of RBD alone).

EXAMPLE 11 conjugated polypeptide epidemic comprising SARS-CoV-2RBD Domain linked to anti-CD 2 Single-chain variable fragment Seedling

CD2 is an adhesion molecule that is present 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 (CD 58). CD2 functions as a co-stimulatory molecule, meaning that the signal sent via CD2 cooperates with the signal sent by 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 thus leads to antigen-independent cell adhesion, expansion of naive helper T cells, and induction of IFN-gamma in memory cells. In addition to LFA-3, CD2 can also interact with CD48 and CD 59.

We created a conjugated polypeptide comprising RBD and CD2 binding polypeptides of SARS-CoV-2 spike by fusing the coding sequences of the following protein elements: tissue-type plasminogen activator signal sequence (to allow secretion of conjugated polypeptide from cells), anti-CD 2scFv derived from the rat anti-CD 2 antibody LO-CD2a (see, e.g., U.S. patent No. 6,849,258); a flexible connector; and SARS-CoV-2RBD (codon optimized). The amino acid sequence is shown in SEQ ID NO. 15. The codon-optimized nucleic acid sequence of this polypeptide (SEQ ID NO. 16) was synthesized and cloned downstream of the EF 1-alpha promoter sequence (including its first intron) and upstream of the SV40 polyadenylation sequence in the pUC19 plasmid.

Plasmid DNA molecules encoding anti-CD 2-RBD conjugated polypeptides (also known as s 2-RBD) were administered as DNA vaccine to two rhesus monkeys (by electroporation of 1mg of DNA on day 0); boost was provided with RBD-only type 35 glandular vector on day 28 (fig. 12). The antibody responses in these rhesus monkeys were compared to those obtained using RBD alone for priming and boosting. The results showed improved peak antibody responses using s2-RBD (2.8 fold increase in geometric mean) and improved persistent antibody responses remaining 24 weeks after vaccination (5 fold increase in geometric mean; FIG. 12, compare the grey dashed mean of s2-RBD with the black dashed mean of RBD alone).

Example 12 SARS-CoV-2RBD Structure comprising an anti-CD 2 Single-chain variable fragment linked to form a diabody Domain dimerized conjugated polypeptide vaccine

By reducing the length of the VH-VL linker to five amino acids, the dimeric form of the anti-CD 2scFv-RBD was designed (SEQ ID NO. 17). We prepared a codon optimized ORF that encodes this dimeric form of the conjugated polypeptide immunogen (SEQ ID NO. 18). Such codon optimized ORFs were engineered consecutively into expression cassettes and into DNA and adenovirus vectors, which were administered to animals for immunogenicity testing.

Next, we designed a dimeric form of anti-CD 2scFv-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-CD 2scFv used in this construct had no positively charged residues at critical positions in the EF loop, which was predicted to lead to instability. We prepared a codon optimized ORF that encodes this enhanced stable, dimeric form of anti-CD 2scFv-RBD (SEQ ID NO. 20). Such codon optimized ORFs are engineered consecutively into expression cassettes and into DNA and adenovirus vectors, which are administered to animals for immunogenicity testing.

Example 13 SARS-CoV-2RBD Domain comprising an N-terminal Domain attached to LFA-3 that binds CD2 Conjugated polypeptide vaccine of (2)

Conjugated polypeptide immunogens for delivery in a genetic vaccine can be designed using any protein sequence that will bind to the appropriate cell surface receptor of 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 a transmembrane segment or Glycosyl Phosphatidylinositol (GPI) linker (Dustin et al, 1987b; wallich et al, 1998). The 95-residue membrane distal N-terminal domain of CD58 (1 dCD 58) is fully responsible for adhesion to CD2 (Sun et al).

We created a conjugated polypeptide comprising RBD and CD2 binding polypeptides of SARS-CoV-2 spike by fusing the coding sequences of the following protein elements: a tissue-type plasminogen activator signal sequence (to allow secretion of conjugated polypeptide from cells), the first extracellular domain 1dCD58 of human CD58; a flexible connector; and SARS-CoV-2RBD (codon optimized). The amino acid sequence is shown as SEQ ID NO. 21. The codon-optimized nucleic acid sequence of this polypeptide (SEQ ID NO. 22) was synthesized and cloned downstream of the EF 1-alpha 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 of strain b.1.351 ("south africa") SARS-CoV-2 and a CD2 binding polypeptide by fusing the coding sequence of the tissue plasminogen activator signal sequence 1dCD58, the flexible linker, and the SARS-CoV-2RBD (b.1.351) (i.e., the RBD from strain b.1.351). The amino acid sequence is shown as SEQ ID NO. 23. The codon-optimized nucleic acid sequence of this polypeptide (SEQ ID NO. 24) was synthesized and cloned downstream of the EF 1-alpha 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) contains immunoreactive RBDs (B.1.351) when expressed in human cells, which may be necessary to induce an anti-RBD (B.1.351) antibody response in human subjects. The plasmid encoding 1dCD58-RBD (B.1.351) was transfected into human embryonic kidney cell line 293 and after two days the presence of immunoreactive RBD (B.1.351) proteins in the cells was verified by antibody staining using the following procedure. On day 1 of the assay, 350,000 cells per well were seeded in 6-well plates. On day 0, a Polyethyleneimine (PEI) transfection mixture was prepared by adding 150 microliters of 0.1mg/ml PEI in DMEM to 3mcg of DNA in an equal volume of DMEM; incubating the mixture at room temperature for 20 minutes; 2.7ml of complete medium was added; adding the culture medium with the composite DNA to 293 cells washed in a 6-well plate; and the plates were returned to the incubator for overnight incubation. On day 1, the transfection mixture was removed from the cells, the cells were washed, and 2ml of fresh medium was added. On day 2, cells were fixed with 5% PFA, washed in 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 truebue HRP substrate for 5-15 minutes, and finally quenched in water. The results (FIG. 13C) demonstrate that the 1dCD58-RBD (B.1.351) construct successfully produced conjugated polypeptides that include immunoreactive RBDs (B.1.351).

EXAMPLE 14 SARS-CoV-comprising a single-chain variable fragment linked to anti-CD 4Conjugated polypeptide plague of 2RBD domain Seedling

CD4 is a glycoprotein that is present on the surface of helper T cells as well as some monocytes, macrophages and dendritic cells. As a member of the immunoglobulin superfamily, it comprises four immunoglobulin domains, designated D1 to D4, exposed on the cell surface. The D1 domain contributes to interactions with the β2 domain of MHC class II molecules, which defines most 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 primary entry receptor for HIV-1 envelope glycoprotein.

We created a conjugated polypeptide comprising RBD and CD4 binding polypeptides of SARS-CoV-2 spike by fusing the coding sequences of the following protein elements: a tissue-type plasminogen activator signal sequence (to allow secretion of conjugated polypeptides from cells); anti-CD 4scFv derived from humanized mouse anti-CD 4 antibody hu5A8 (see, e.g., AIDS Res Hum Retro 13:933); a flexible connector; and SARS-CoV-2RBD (codon optimization). The amino acid sequence is shown as SEQ ID NO. 27. The codon-optimized nucleic acid sequence of this polypeptide (SEQ ID NO. 28) was synthesized and cloned downstream of the EF 1-alpha promoter sequence (including its first intron) and upstream of the SV40 polyadenylation sequence in the pUC19 plasmid.

Reference to examples 1-7

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

Exemplary embodiments

Exemplary embodiments provided according to 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 an immune cell.

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

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

4. The vaccine of embodiment 3, wherein the enriched 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 glycosyl phosphatidylinositol 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 IgG1Fc 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 and VL regions 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 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 conjugated polypeptide further comprises a tPA leader sequence.

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

27. The vaccine of any one of 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 SARS-CoV-2 spike glycoprotein or a fragment thereof.

31. The vaccine of embodiment 30, wherein the fragment of 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-2E protein, M protein, N protein, nsp3 protein, nsp4 protein, or a fragment of nsp6 protein or above.

33. The vaccine of embodiment 32, wherein the second antigen comprises a fusion protein comprising SARS-CoV-2E 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 an immune cell.

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

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

40. The vaccine of embodiment 39, wherein the enriched 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 glycosyl phosphatidylinositol 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 PDGF receptor, glycophorin a or 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 IgG1Fc domain.

55. The vaccine of any one of embodiments 45-54, wherein the VH and VL regions of the scFv are separated within the conjugated polypeptide by a flexible linker.

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 surface proteins 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 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 conjugated polypeptide comprises a tPA leader sequence.

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

62. The vaccine of any one of 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 SARS-CoV-2 spike glycoprotein or a fragment thereof.

66. The vaccine of embodiment 65, wherein the fragment of 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-2E protein, M protein, N protein, nsp3 protein, nsp4 protein, or a fragment of nsp6 protein or above.

68. The vaccine of embodiment 67, wherein the second antigen comprises a fusion protein comprising a SARS-CoV-2E protein and an M protein or fragment 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 conjugated polypeptide and/or the second polynucleotide encoding the second antigen is codon optimized.

72. The vaccine of any one of embodiments 37-71, 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 embodiment 72, wherein the second promoter is a mammalian promoter.

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

75. The vaccine of any one of embodiments 37-74, wherein the first expression cassette and/or the second expression cassette is present within 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 said 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 ^TM 。

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

(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 embodiments 82 or 83, wherein one or more terminal enzyme complex recognition loci comprise a Pac1 site and a Pac2 site.

85. The vaccine of embodiment 84, wherein all of the terminal enzyme 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 said surface protein is a 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 said surface protein is CD2 or CD3.

94. The conjugated polypeptide of any of embodiments 90-93, wherein the immune cell is a T cell or an Antigen Presenting Cell (APC).

95. The conjugated polypeptide of any of embodiments 90-94, wherein the ligand is an extracellular domain of a cell adhesion molecule.

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

97. The conjugated polypeptide of any of embodiments 90-96, wherein the surface protein is preferentially expressed by T cells.

98. The conjugated polypeptide of any of embodiments 90-94 or 95-97, wherein said antibody fragment is an antibody-derived scFv chain.

99. The conjugate polypeptide of any of embodiments 90-98, wherein the conjugate 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 said lipid anchor is a glycosyl phosphatidylinositol anchor.

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

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

103. The conjugated polypeptide of any of embodiments 99-102, wherein said transmembrane segment is derived from PDGF receptor, glycophorin a or SARS-CoV-2 spike protein.

104. The conjugated polypeptide of any of embodiments 99-103, wherein the multimerization domain is derived from T4fibritin.

105. The conjugated polypeptide of any of embodiments 99-103, wherein the multimerization domain is an Fc domain.

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

107. The conjugated polypeptide of embodiment 105 or 106, wherein said Fc domain is a human IgG1Fc domain.

108. The conjugated polypeptide of any of embodiments 98-107, wherein said antibody fragment is an scFv chain of antibody origin, and wherein the VH region and the VL region of said scFv are separated by a flexible linker.

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

110. The conjugate polypeptide of embodiment 109, 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 embodiment 110, wherein the multimer is stabilized by disulfide bonds between monomer units.

112. The conjugate polypeptide of embodiment 110 or 111, wherein the flexible linker is 5 amino acids in length.

113. The conjugate polypeptide of any one of embodiments 90-112, wherein the conjugate polypeptide further comprises a tPA leader sequence.

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

115. The conjugated polypeptide of any of embodiments 90-114, wherein said pathogen is a virus.

116. The conjugated polypeptide of embodiment 115, wherein said virus is SARS-CoV-2.

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

118. The conjugated polypeptide of embodiment 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 any 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 CD 4; (iii) a flexible linker; and (iv) a SARS-CoV-2 Receptor Binding Domain (RBD).

121. The conjugated polypeptide of embodiment 120, comprising the amino acid sequence of: SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25 or SEQ ID NO. 27.

122. A polynucleotide encoding the conjugated polypeptide of any of embodiments 90-121.

123. The polynucleotide of embodiment 122, wherein said 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. The expression cassette of embodiment 125 comprising the nucleotide sequence of SEQ ID NO. 8.

127. A vector comprising the expression cassette of embodiment 125 or 126.

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

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

130. A diabody comprising the conjugated polypeptide of any of embodiments 98-121.

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

132. A vaccine comprising the conjugate polypeptide of any one of embodiments 90-121, the polynucleotide of any one of embodiments 122-124, the expression cassette of embodiments 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 embodiments 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 higher than any antibody dependent infection enhancement (ADEI) caused by a 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 against the second antigen ⁺ And CD8 ⁺ T cell response.

138. The method of any of embodiments 133-137, wherein the method comprises administering to the mammal a DNA prime comprising the vector of embodiment 127 or 128 by electroporation, followed by boosting with an adenovirus vector encoding an RBD.

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

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

Informal sequence listing

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Claims

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:

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

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.

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.

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.