WO2025111446A1

WO2025111446A1 - Rolling circle amplified dna encoded antigens and methods of use thereof

Info

Publication number: WO2025111446A1
Application number: PCT/US2024/056867
Authority: WO
Inventors: David Weiner; Nicholas TURSI
Original assignee: Wistar Institute of Anatomy and Biology
Current assignee: Wistar Institute of Anatomy and Biology
Priority date: 2023-11-21
Filing date: 2024-11-21
Publication date: 2025-05-30
Anticipated expiration: 2026-05-21

Abstract

Disclosed herein are rolling circle amplification products encoding multiple copies of a disease-associated antigen or a disease-associated antibody or multiple copies of a self¬ assembling nanoparticle comprising a disease-associated antigen domain or a disease-associated antibody. Also disclosed herein are methods of treating or preventing a disease or disorder associated with the encoded antigen or antibody in a subject in need thereof, by administering the rolling circle amplification products, to the subject.

Description

ROLLING CIRCLE AMPLIFIED DNA ENCODED ANTIGENS AND METHODS OF

USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/601,405, filed November 21, 2023, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

A rapid, cost-effective production modality to generate suitable engineered DNA sequences, especially a DNA sequence containing only a promoter and a gene of interest, and devoid of any undesirable DNA sequences that are necessary for maintenance in a bacterium (e g., origin of replication or an antibiotic resistance gene), which can be used for transfection in eukaryotic cells for subsequent RNA and/or protein production is highly desirable. Isothermal DNA amplification techniques such as rolling circle amplification (RCA) may be employed to generate such large quantities of high-quality DNA with less effort, time, and expense, starting from a circular nucleic acid template. For example, RCA enables rapid production of suitable engineered DNA sequences, which may contain only a promoter and a gene of interest.

A need remains in the art for the development of safe and effective vaccines for the treatment of bacterial or viral infection or the treatment or prevention of a disease or disorder associated with bacterial or viral infection such as influenza or COVID-19.

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to an immunogenic composition comprising a Rolling Circle Amplification (RCA) product, wherein the RCA product comprises a nucleic acid molecule encoding multiple copies of a disease-associated antigen, a disease- associated antibody, or a fragment thereof. In some embodiments, the RCA product comprises at least one modified nucleotide or nucleotide analog. In some embodiments, the RCA product comprises a double- stranded concatemeric DNA molecule containing phosphorothioated nucleotides. In one embodiment, the immunogenic composition is generated from RCA of a template nucleic acid molecule. In some embodiments, the template nucleic acid molecule comprises an expression vector.

In one embodiment, the immunogenic composition further comprises a pharmaceutically acceptable excipient.

In one embodiment, the immunogenic composition further comprises an adjuvant.

In some embodiments, the RCA product comprises multiple copies of a nucleic acid molecule encoding a SARS-CoV-2 spike protein receptor binding domain (RBD). In one embodiment, the nucleotide sequence encodes a peptide comprising an amino acid sequence having at least about 90% identity over an entire length of SEQ ID NO:2. In one embodiment, the nucleotide sequence encodes a peptide comprising an amino acid sequence of SEQ ID NO:2. In one embodiment, the nucleotide sequence comprises a nucleotide sequence having at least about 90% identity over an entire length of SEQ ID NO: 1. In one embodiment, the nucleotide sequence comprises SEQ ID NO: 1. In some embodiments, the nucleic acid molecule comprises a rolling circle amplification product generated from rolling circle amplification of SEQ ID NO: 1.

In some embodiments, the RCA product comprises multiple copies of a nucleic acid molecule encoding an influenza HA antigen. In one embodiment, the nucleotide sequence encodes a peptide comprising an amino acid sequence having at least about 90% identity over an entire length of SEQ ID NO:4. In one embodiment, the nucleotide sequence encodes a peptide comprising an amino acid sequence of SEQ ID NO:4. In one embodiment, the nucleotide sequence comprises a nucleotide sequence having at least about 90% identity over an entire length of SEQ ID NO:3. In one embodiment, the nucleotide sequence comprises SEQ ID NO:3. In some embodiments, the nucleic acid molecule comprises a rolling circle amplification product generated from rolling circle amplification of SEQ ID NO:3.

In some embodiment, the RCA product comprises multiple copies of a nucleic acid molecule encoding a SARS-CoV-2 RBD 24mer. In one embodiment, the nucleotide sequence encodes a peptide comprising an amino acid sequence having at least about 90% identity over an entire length of SEQ ID NO:6. In one embodiment, the nucleotide sequence encodes a peptide comprising an amino acid sequence of SEQ ID NO:6. In one embodiment, the nucleotide sequence comprises a nucleotide sequence having at least about 90% identity over an entire length of SEQ ID NO:5. In one embodiment, the nucleotide sequence comprises SEQ ID N0:5. In some embodiments, the nucleic acid molecule comprises a rolling circle amplification product generated from rolling circle amplification of SEQ ID NO: 5.

In some embodiment, the RCA product comprises multiple copies of a nucleic acid molecule encoding a heavy chain of an anti -influenza HA antibody. In one embodiment, the nucleotide sequence encodes a peptide comprising an amino acid sequence having at least about 90% identity over an entire length of SEQ ID NO: 8. In one embodiment, the nucleotide sequence encodes a peptide comprising an amino acid sequence of SEQ ID NO: 8. In one embodiment, the nucleotide sequence comprises a nucleotide sequence having at least about 90% identity over an entire length of SEQ ID NO:7. In one embodiment, the nucleotide sequence comprises SEQ ID NO:7. In some embodiments, the nucleic acid molecule comprises a rolling circle amplification product generated from rolling circle amplification of SEQ ID NO:7.

In some embodiments, the RCA product comprises multiple copies of a nucleic acid molecule encoding a light chain of an anti -influenza HA antibody. In one embodiment, the nucleotide sequence encodes a peptide comprising an amino acid sequence having at least about 90% identity over an entire length of SEQ ID NO: 10. In one embodiment, the nucleotide sequence encodes a peptide comprising an amino acid sequence of SEQ ID NO: 10. In one embodiment, the nucleotide sequence comprises a nucleotide sequence having at least about 90% identity over an entire length of SEQ ID NO:9. In one embodiment, the nucleotide sequence comprises SEQ ID NO:9. In some embodiments, the nucleic acid molecule comprises a rolling circle amplification product generated from rolling circle amplification of SEQ ID NO:9.

In one embodiment, the invention relates to a method of inducing an immune response against SARS Coronavirus 2 (SARS-CoV-2) in a subject in need thereof, the method comprising administering an immunogenic composition comprising an RCA product comprising multiple copies of a nucleic acid molecule encoding a SARS-CoV-2 spike protein receptor binding domain (RBD), a nucleic acid molecule encoding a SARS-CoV-2 spike protein receptor binding domain (RBD), or a peptide comprising a SARS-CoV-2 spike protein receptor binding domain (RBD) to the subject. In some embodiments, the nucleic acid molecule comprises a rolling circle amplification product generated from rolling circle amplification of SEQ ID NO: 1. In one embodiment, the method of administering includes at least one of electroporation and injection. In one embodiment, the invention relates to a method of protecting a subject in need thereof from infection with SARS-CoV-2, the method comprising administering an RCA product comprising multiple copies of a nucleic acid molecule encoding a SARS-CoV-2 spike protein receptor binding domain (RBD), a nucleic acid molecule encoding a SARS-CoV-2 spike protein receptor binding domain (RBD), or a peptide comprising a SARS-CoV-2 spike protein receptor binding domain (RBD) to the subject. In some embodiments, the nucleic acid molecule comprises a rolling circle amplification product generated from rolling circle amplification of SEQ ID NO: 1. In one embodiment, the method of administering includes at least one of electroporation and injection.

In one embodiment, the invention relates to a method of treating a subject in need thereof against SARS-CoV-2, the method comprising administering an RCA product comprising multiple copies of a nucleic acid molecule encoding a SARS-CoV-2 spike protein receptor binding domain (RBD), a nucleic acid molecule encoding a SARS-CoV-2 spike protein receptor binding domain (RBD), or a peptide comprising a SARS-CoV-2 spike protein receptor binding domain (RBD) to the subject. In some embodiments, the nucleic acid molecule comprises a rolling circle amplification product generated from rolling circle amplification of SEQ ID NO: 1. In one embodiment, the method of administering includes at least one of electroporation and injection.

In one embodiment, the invention relates to a method of inducing an immune response against infection with influenza, the method comprising administering an RCA product comprising multiple copies of a nucleic acid molecule encoding an influenza HA antigen, a nucleic acid molecule encoding an influenza HA antigen, or a peptide comprising an influenza HA antigen to the subject. In some embodiments, the nucleic acid molecule comprises a rolling circle amplification product generated from rolling circle amplification of SEQ ID NO:3. In one embodiment, the method of administering includes at least one of electroporation and injection.

In one embodiment, the invention relates to a method of protecting a subject in need thereof from infection with influenza, the method comprising administering an RCA product comprising multiple copies of a nucleic acid molecule encoding an influenza HA antigen, a nucleic acid molecule encoding an influenza HA antigen, or a peptide comprising an influenza HA antigen to the subject. In some embodiments, the nucleic acid molecule comprises a rolling circle amplification product generated from rolling circle amplification of SEQ ID N0:3. In one embodiment, the method of administering includes at least one of electroporation and injection.

In one embodiment, the invention relates to a method of treating a subject in need thereof against influenza, the method comprising administering an RCA product comprising multiple copies of a nucleic acid molecule encoding an influenza HA antigen, a nucleic acid molecule encoding an influenza HA antigen, or a peptide comprising an influenza HA antigen to the subject. In some embodiments, the nucleic acid molecule comprises a rolling circle amplification product generated from rolling circle amplification of SEQ ID NO:3. In one embodiment, the method of administering includes at least one of electroporation and injection.

In one embodiment, the invention relates to a method of inducing an immune response against infection with influenza, the method comprising administering an RCA product comprising multiple copies of a nucleic acid molecule encoding an anti -influenza HA antibody, a nucleic acid molecule encoding an anti-influenza HA antibody, or a peptide comprising an antiinfluenza HA antibody to the subject. In some embodiments, the nucleic acid molecule comprises a rolling circle amplification product generated from rolling circle amplification of SEQ ID NO:7, SEQ ID NO:9, or a combination thereof. In one embodiment, the method of administering includes at least one of electroporation and injection.

In one embodiment, the invention relates to a method of protecting a subject in need thereof from infection with influenza, the method comprising administering an RCA product comprising multiple copies of a nucleic acid molecule encoding an anti-influenza HA antibody, a nucleic acid molecule encoding an anti -influenza HA antibody, or a peptide comprising an anti -influenza HA antibody to the subject. In some embodiments, the nucleic acid molecule comprises a rolling circle amplification product generated from rolling circle amplification of SEQ ID NO:7, SEQ ID NO:9, or a combination thereof. In one embodiment, the method of administering includes at least one of electroporation and injection.

In one embodiment, the invention relates to a method of treating a subject in need thereof against influenza, the method comprising administering an RCA product comprising multiple copies of a nucleic acid molecule encoding an influenza HA antigen, a nucleic acid molecule encoding an influenza HA antigen, or a peptide comprising an influenza HA antigen to the subject. In some embodiments, the nucleic acid molecule comprises a rolling circle amplification product generated from rolling circle amplification of SEQ ID NO:7, SEQ ID NOV, or a combination thereof. In one embodiment, the method of administering includes at least one of electroporation and injection.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

Figure 1A through Figure 1C depict data demonstrating that rolling circle amplification (RCA) DNA delivered via adaptive electroporation (EP) generates germinal center (GC) T follicular helper (Tfh) cell responses. Figure 1A depicts a schematic of the experimental design. Mice were immunized with 2pg of each construct or left naive. Constructs were Flu A/California/04/2009 Hemagglutinin (HA) in pVAX vector (HA pVAX), HA DNA made using RCA low MW (LMW) species (HA RCA LMW), or HA DNA made using RCA high MW (HMW) species (HA RCA HMW). GC Tfh cell responses were measured in the draining lymph nodes 14 days post immunization. Figure IB depicts a representative FACS plot of GC Tfh responses. Figure 1C depicts a bar graph quantifying GC Tfh responses.

Figure 2A and Figure 2B depict data demonstrating that RCA DNA delivered via adaptive EP generates germinal center B (GC B) cell responses. Mice were immunized with 2pg of either HA pVAX, HA RCA LMW, HA RCA HMW, or left naive. Figure 2A depicts a representative FACS plot and bar graph measuring GC B cell responses in draining lymph nodes 14 days post immunization. Figure 2B depicts a representative FACS plot and bar graph measuring antigen-specific GC B cell responses in draining lymph nodes 14 days post immunization.

Figure 3A and Figure 3B depict data quantifying serum titers up to 1 month. Mice were immunized with 2 pg of either HA pVAX, HA RCA LMW, or HA RCA HMW. Figure 3A depicts a line graph measuring serum antibody titers to flu HA over time at 2 weeks and 4 weeks post immunization. Figure 3B depicts a bar graph measuring serum antibody titers to flu HA at 4 weeks post immunization. Figure 4A and Figure 4B depict data demonstrating that RCA DNA delivered via adaptive EP generates adaptive immune responses. Figure 4A depicts a schematic of the experimental design. Mice were immunized with 2pg of either HA pVAX, HA RCA LMW, HA RCA HMW, or left naive. Figure 4B depicts a graph quantifying IFNy-secreting cells in the spleen using ELISpot at 14 days post immunization.

Figure 5A through Figure 5C depict data demonstrating that RCA DNA delivered via adaptive EP generates CD8 T cell responses. Mice were immunized with 2 pg of either HA pVAX, HA RCA LMW, HA RCA HMW, or left naive and CD8+ T cell responses were assessed at 14 days post immunization using intracellular flow cytometry. Figure 5 A depicts a representative FACS plot and a bar graph quantifying CD107a+ effector CD8+ T cells. Figure 5B depicts a representative FACS plot and a bar graph quantifying IFNy + effector CD8+ T cells. Figure 5C depicts a representative FACS plot and a bar graph quantifying TNFa+ effector CD8+ T cells.

Figure 6A and Figure 6B depict data demonstrating that RCA DNA delivered via adaptive EP generates CD4 T cell responses. Figure 6A depicts a schematic of the experimental design. Mice were immunized with 2 pg of either HA pVAX, HA RCA LMW, HA RCA HMW, or left naive and CD8+ T cell responses were assessed at 14 days post immunization using intracellular flow cytometry. Figure 6B depicts a bar graph quantifying IFNy+ effector CD4+ T cells. Figure 6C depicts a bar graph quantifying IL-2+ effector CD4+ T cells. Figure 6D depicts a bar graph quantifying TNFa+ effector CD4+ T cells.

Figure 7A and Figure 7B depict data demonstrating that increased doses of RCA LMW product improve GC responses. Figure 7A depicts a schematic of the experimental design. Mice were immunized with 2pg HA in pVAX, 2pg HA RCA LMW, lOpg HA RCA LMW, or 25pg HA RCA LMW. Figure 7B depicts bar graphs measuring germinal center Tfh (left), total GC B cell (middle), and antigen-specific GC B cell (right) responses in draining lymph nodes 14 days post immunization.

Figure 8A and Figure 8B depict data demonstrating that a moderate dose of HA RCA LMW enhances T cell responses. Figure 8A depicts a schematic of the experimental design. Mice were immunized with 2pg HA in pVAX, 2pg HA RCA LMW, lOpg HA RCA LMW, or 25 pg HA RCA LMW. Figure 8B depicts a graph quantifying IFNy-secreting cells in spleen using ELISpot at 14 days post immunization. Figure 9A through Figure 9D depict data demonstrating that an increased dose of HA RCA LMW product enhances CD8 T cell responses. Mice were immunized with 2pg HA in pVAX, 2pg HA RCA LMW, lOpg HA RCA LMW, or 25 pg HA RCA LMW. T cell responses were assessed in the spleen at day 14 post immunization using flow cytometry. Figure 9A depicts a representative FACS plot quantifying IFNy+ effector CD8+ T cells. Figure 9B depicts a bar graph quantifying IFNy+ CD8+ T cells. Figure 9C depicts a bar graph quantifying CD107a+ effector CD8+ T cells. Figure 9D depicts a bar graph quantifying TNFa+ effector CD8+ T cells.

Figure 10A though Figure IOC depict data demonstrating that an increased dose of HA RCA LMW product dramatically enhances absolute numbers of CD8 T cells. Mice were immunized with 2pg HA in pVAX, 2pg HA RCA LMW, lOpg HA RCA LMW, or 25 g HA RCA LMW. T cell responses were assessed in the spleen at day 14 post immunization using flow cytometry. Figure 10A depicts a bar graph quantifying the number of CD107a+ CD8+ T cells. Figure 10B depicts a bar graph quantifying the number of ZFNy+ CD8+ T cells. Figure IOC depicts a bar graph quantifying the number of TNFa + CD8+ T cells.

Figure 11A through Figure 11C depict data demonstrating that an increased dose of HA RCA LMW product enhances CD4 T cell responses relative to lower doses. Mice were immunized with 2pg HA in pVAX, 2pg HA RCA LMW, lOpg HA RCA LMW, or 25 pg HA RCA LMW. T cell responses were assessed in the spleen at day 14 post immunization using flow cytometry. Figure 11 A depicts a bar graph quantifying IFNy+ effector CD4+ T cells. Figure 1 IB depicts a bar graph quantifying IL2+ effector CD4+ T cells. Figure 11C depicts a bar graph quantifying TNFa+ effector CD4+ T cells.

Figure 12A and Figure 12B depict data demonstrating that there are increases in total activated effector CD8+ T cells with 10 pg HA RCA LMW. Mice were immunized with 2pg HA in pVAX, 2pg HA RCA LMW, lOpg HA RCA LMW, or 25pg HA RCA LMW. Frequencies of effector T cells were assessed in the spleen at day 14 post immunization using flow cytometry. Figure 12A depicts a bar graph quantifying the frequency of effector CD8+ T cells. Figure 12B depicts a bar graph quantifying the frequency of effector CD4+ T cells.

Figure 13 depicts data demonstrating that two separate RCA molecules can be successfully delivered to the same cell in vivo to enable productive biologic synthesis. Mice were immunized with a total of lOOpg of plasmid or RCA DNA encoding human monoclonal antibody (mAb) 2-12C (50pg each heavy chain and light chain) formulated with 12U hyaluronidase per site and bled sequentially over time. At immunization, mice were also administered 200pg of each anti-mouse CD8 (Clone YTS 169.4, BioXCell) and anti-mouse CD4 (Clone GK1.5, BioXCell). An ELISA assay was used to measure human IgG in mouse serum.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to immunogenic compositions comprising an RCA product, wherein the RCA product comprises multiple copies of a nucleic acid molecule encoding a disease-associated antigen or a disease-associated antibody. In one embodiment, the disease-associated antigen comprises a SARS-CoV-2 antigen. In one embodiment, the disease- associated antigen comprises an influenza antigen. In one embodiment, the disease-associated antibody comprises an anti-influenza antibody.

In one embodiment, the SARS-CoV-2 antigen comprises the receptor binding domain (RBD) of the SARS-CoV-2 spike antigen. In one embodiment, the immunogenic composition comprises an RCA product encoding multiple copies of a self-assembling nanoparticle comprising the RBD of the SARS-CoV-2 spike antigen. In some embodiments, the immunogenic composition can be used treat SARS-CoV-2 infection or to prevent or treat a disease or disorder associated with SARS-CoV-2 infection. In one embodiment, the disease or disorder associated with SARS-CoV-2 infection is COVID- 19.

In one embodiment, the influenza antigen comprises a hemagglutinin (HA) antigen. In one embodiment, the immunogenic composition comprises an RCA product encoding multiple copies of a self-assembling nanoparticle comprising the HA antigen. In some embodiments, the immunogenic composition can be used treat influenza infection or to prevent or treat a disease or disorder associated with influenza infection. In one embodiment, the disease or disorder associated with influenza infection is flu.

In one embodiment, the anti-influenza antibody comprises an anti -influenza hemagglutinin (HA) antibody. In one embodiment, the immunogenic composition comprises an RCA product encoding multiple copies of a self-assembling nanoparticle comprising the antiinfluenza HA antibody. In some embodiments, the immunogenic composition can be used treat influenza infection or to prevent or treat a disease or disorder associated with influenza infection. In one embodiment, the disease or disorder associated with influenza infection is flu. The immunogenic composition can elicit both humoral and cellular immune responses that target the encoded antigen. The immunogenic composition can elicit neutralizing antibodies and immunoglobulin G (IgG) antibodies that are reactive with the encoded antigen. The immunogenic composition can also elicit CD8⁺ and CD4⁺ T cell responses that are reactive to the encoded antigen and produce interferon-gamma (IFN-y), tumor necrosis factor alpha (TNF-a), and interleukin-2 (IL-2).

Definitions

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

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

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

“Antibody” as used herein means an antibody of classes IgG, IgM, IgA, IgD or IgE, or fragments, fragments or derivatives thereof, including Fab, F(ab')2, Fd, and single chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies and derivatives thereof. The antibody can be an antibody isolated from the serum sample of mammal, a polyclonal antibody, affinity purified antibody, or mixtures thereof which exhibits sufficient binding specificity to a desired epitope or a sequence derived therefrom. “Antibody fragment” or “fragment of an antibody” as used interchangeably herein refers to a portion of an intact antibody comprising the antigen-binding site or variable region. The portion does not include the constant heavy chain domains (i.e. CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing the three CDRs of the light-chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of the heavy chain variable region.

“Antigen” refers to proteins that have the ability to generate an immune response in a host. An antigen may be recognized and bound by an antibody. An antigen may originate from within the body or from the external environment.

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

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

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

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

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

“Fragment” or “immunogenic fragment” with respect to polypeptide sequences means a polypeptide capable of eliciting an immune response in a mammal that cross reacts with a full length wild type strain SARS-CoV-2 antigen or influenza antigen. Fragments of consensus proteins can comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of a consensus protein. In some embodiments, fragments of consensus proteins can comprise at least 20 amino acids or more, at least 30 amino acids or more, at least 40 amino acids or more, at least 50 amino acids or more, at least 60 amino acids or more, at least 70 amino acids or more, at least 80 amino acids or more, at least 90 amino acids or more, at least 100 amino acids or more, at least 110 amino acids or more, at least 120 amino acids or more, at least 130 amino acids or more, at least 140 amino acids or more, at least 150 amino acids or more, at least 160 amino acids or more, at least 170 amino acids or more, at least 180 amino acids or more, at least 190 amino acids or more, at least 200 amino acids or more, at least 210 amino acids or more, at least 220 amino acids or more, at least 230 amino acids or more, or at least 240 amino acids or more of a consensus protein.

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

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

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

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

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

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

A “peptide,” “protein,” or “polypeptide” as used herein can mean a linked sequence of amino acids and can be natural, synthetic, or a modification or combination of natural and synthetic.

As used herein, the term "phosphorothioated nucleotide" refers to a nucleotide that has an altered phosphate backbone, wherein, the sugar moieties are linked by a phosphorothioate bond. In the phosphate backbone of an oligonucleotide sequence, the phosphorothioate bond contains a sulfur atom as a substitute for a non-bridging oxygen atom. This modification renders the internucleotide linkage resistant to nuclease degradation.

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

As used herein, the term “primer” refers to a short linear oligonucleotide that hybridizes to a target nucleic acid sequence (e.g., a DNA template to be amplified) to prime a nucleic acid synthesis reaction. The primer may be an RNA oligonucleotide, a DNA oligonucleotide, or a chimeric sequence. The primer may contain natural, synthetic, or modified nucleotides. Both the upper and lower limits of the length of the primer are empirically determined. The lower limit on primer length is the minimum length that is required to form a stable duplex upon hybridization with the target nucleic acid under nucleic acid amplification reaction conditions. Very short primers (usually less than 3 nucleotides long) do not form thermodynamically stable duplexes with target nucleic acid under such hybridization conditions. The upper limit is often determined by the possibility of having a duplex formation in a region other than the pre-determined nucleic acid sequence in the target nucleic acid. Generally, suitable primer lengths are in the range of about 3 nucleotides long to about 40 nucleotides long.

As used herein, the term “random primer” refers to a mixture of primer sequences, generated by randomizing a nucleotide at any given location in an oligonucleotide sequence in such a way that the given location may consist of any of the possible nucleotides or their analogues (complete randomization). Thus the random primer is a random mixture of oligonucleotide sequences, consisting of every possible combination of nucleotides within the sequence. For example, a hexamer random primer may be represented by a sequence NNNNNN or (N)6. A hexamer random DNA primer consists of every possible hexamer combinations of 4 DNA nucleotides, A, C, G and T, resulting in a random mixture comprising 46 (4,096) unique hexamer DNA oligonucleotide sequences. Random primers may be effectively used to prime a nucleic acid synthesis reaction when the target nucleic acid's sequence is unknown or for performing a whole-genome amplification reaction. Random primers may also be effective in priming and producing double-stranded rolling circle amplification (RCA) product rather than single-stranded RCA product, depending on the concentration of primer.

As used herein, the term "rolling circle amplification (RCA) product" refers to a nucleic acid amplification product wherein a circular nucleic acid template (e.g., single/double stranded DNA circles) amplifies via a rolling circle amplification reaction mechanism. The rolling circle amplification typically produces concatamers comprising tandem repeat units of the circular nucleic acid template sequence. The rolling circle amplification product DNA may be generated by a linear RCA (LRCA), exhibiting linear amplification kinetics (e.g., RCA using a single, specific primer), or by an exponential RCA (ERCA) exhibiting exponential amplification kinetics. Rolling circle amplification product DNA may also be generated by using multiple primers (multiply primed rolling circle amplification or MPRCA), wherein the rolling circle amplification product DNA is hyper-branched concatamers. In a double-primed RCA, one primer may be complementary, as in the linear RCA, to the circular nucleic acid template, whereas the other may be complementary to the tandem repeat unit nucleic acid sequences of the RCA product DNA. The RCA product DNA may be generated by the RCA in vitro under isothermal conditions using a suitable nucleic acid polymerase such as Phi29 DNA polymerase.

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

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

“Substantially identical” as used herein can mean that a first and second amino acid sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or more amino acids. Substantially identical can also mean that a first nucleic acid sequence and a second nucleic acid sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or more nucleotides.

“Synthetic antibody” as used herein refers to an antibody that is encoded by the recombinant nucleic acid sequence described herein and is generated in a subject.

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

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

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

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

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

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

Rolling Circle Amplification DNA Products

One or more embodiments are directed to rolling circle amplification (RCA) products and methods of use thereof as vaccines for inducing an immune response in a subject. Provided herein are immunogenic compositions, such as vaccines, comprising an RCA product encoding multiple copies of an antigen. Also provided herein are immunogenic compositions comprising an RCA product encoding multiple copies of an antibody. The immunogenic composition can be used to treat a disease or disorder associated with expression of the encoded antigen or antibody. Exemplary diseases and disorders include, but are not limited to viral diseases, bacterial diseases, autoimmune diseases and cancers. In some embodiments, administration of RCA amplified DNA products encoding multiple copies of a disease- associated antigen can significantly induce an immune response of a subject administered the vaccine, thereby protecting against or treating the disease associated with the encoded antigen.

Provided herein are immunogenic compositions, such as vaccines, comprising an RCA product encoding multiple copies of a SARS coronavirus 2 (SARS-CoV-2) antigen, a fragment thereof, a variant thereof, or a combination thereof. The vaccine can be used to treat SARS-CoV-2 infection, thereby treating, preventing, and/or protecting against SARS-CoV-2 based pathologies. In one embodiment, the SARS-CoV-2 based pathology is COVID-19. The vaccine can significantly induce an immune response of a subject administered the vaccine, thereby treating SARS-CoV-2 infection and protecting against COVID-19.

Provided herein are immunogenic compositions, such as vaccines, comprising an RCA product encoding multiple copies of an influenza antigen, a fragment thereof, a variant thereof, or a combination thereof. The vaccine can be used to treat influenza virus infection, thereby treating, preventing, and/or protecting against influenza-based pathologies. In one embodiment, the influenza-based pathology is flu. The vaccine can significantly induce an immune response of a subject administered the vaccine, thereby treating influenza infection and protecting against flu.

In one embodiment, the SARS-CoV-2 antigen comprises the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. In one embodiment, the RCA product encodes multiple copies of a SARS-CoV-2 RBD antigen.

In one embodiment, the influenza antigen comprises a hemagglutinin (HA) antigen. In one embodiment, the RCA product encodes multiple copies of an HA antigen.

Provided herein are immunogenic compositions comprising an RCA product encoding multiple copies of an anti -influenza antibody, a fragment thereof, a variant thereof, or a combination thereof. The immunogenic composition, when administered to a subject in need thereof, can result in the generation of a synthetic antibody in the subject. The synthetic antibody can bind a target molecule (i.e., an antigen) present in the subject. Such binding can neutralize the antigen, block recognition of the antigen by another molecule, for example, a protein or nucleic acid, and elicit or induce an immune response to the antigen. The antibody can be used to treat influenza virus infection, thereby treating, preventing, and/or protecting against influenzabased pathologies. In one embodiment, the influenza-based pathology is flu. In one embodiment, the anti-influenza antibody comprises an anti -influenza HA antibody. In one embodiment, the RCA product encodes multiple copies of an anti-influenza HA antibody.

The RCA product can be generated from the amplification of a circular template nucleic acid molecule. The template nucleic acid molecule can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The template nucleic acid molecule can also include additional sequences that encode linker, leader, or tag sequences that are linked to the encoded antigen by a peptide bond.

In one embodiment, the template nucleic acid molecule encodes a SARS-CoV-2 antigen, an influenza antigen, or an anti-influenza antibody, which is incorporated into a selfassembling peptide nanoparticle (SAPN) viral particle for use in a vaccine of the invention. Selfassembling protein nanoparticles (SAPN) may be formed by the assembly of one or more polypeptide chains comprising at least one antigen and at least one protein oligomerization domain. Without limitation, the SAPN of the invention may self-assemble into a tetrahedron, a cube, an octahedron, a dodecahedron, or an icosahedron. The SAPN of the invention may be used as an efficient means for presenting one or more SARS-CoV-2 antigen or influenza antigen.

In one embodiment, the SAPN of the invention comprises the receptor binding domain of the SARS-CoV-2 spike protein. In one embodiment, the SAPN of the invention comprises a dimer of the receptor binding domain of the SARS-CoV-2 spike protein. In one embodiment, the SAPN of the invention comprises the hemagglutinin antigen. In one embodiment, the SAPN of the invention comprises the anti -influenza hemagglutinin antibody heavy chain, light chain, or combination thereof.

The vaccine can induce a humoral immune response in the subject administered the vaccine. The induced humoral immune response can be specific for the encoded antigen. The induced humoral immune response can be reactive with the encoded antigen. The humoral immune response can be induced in the subject administered the vaccine by about 1.5-fold to about 16-fold, about 2-fold to about 12-fold, or about 3-fold to about 10-fold. The humoral immune response can be induced in the subject administered the vaccine by at least about 1.5- fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 15.5-fold, or at least about 16.0-fold.

The humoral immune response induced by the vaccine can include an increased level of neutralizing antibodies associated with the subject administered the vaccine as compared to a subject not-administered the vaccine. The neutralizing antibodies can be specific for the encoded antigen. The neutralizing antibodies can be reactive with the encoded antigen. The neutralizing antibodies can provide protection against and/or treatment of a viral or bacterial infection and its associated pathologies in the subject administered the vaccine.

The humoral immune response induced by the vaccine can include an increased level of IgG antibodies associated with the subject administered the vaccine as compared to a subject not administered the vaccine. These IgG antibodies can be specific for the encoded antigen. These IgG antibodies can be reactive with the encoded antigen. The level of IgG antibody associated with the subject administered the vaccine can be increased by about 1.5-fold to about 16-fold, about 2-fold to about 12-fold, or about 3-fold to about 10-fold as compared to the subject not administered the vaccine. The level of IgG antibody associated with the subject administered the vaccine can be increased by at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 1 l .O-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 15.5-fold, or at least about 16.0-fold as compared to the subject not administered the vaccine.

The vaccine can induce a cellular immune response in the subject administered the vaccine. The induced cellular immune response can be specific for the encoded antigen. The induced cellular immune response can be reactive to the SARS-CoV-2 antigen. The induced cellular immune response can be reactive to the HA antigen. The induced cellular immune response can include eliciting a CD8⁺ T cell response. The elicited CD8⁺ T cell response can be reactive with the SARS-CoV-2 antigen. The elicited CD8⁺ T cell response can be reactive with the HA antigen. The elicited CD8⁺ T cell response can be polyfunctional. The induced cellular immune response can include eliciting a CD8 T cell response, in which the CD8⁺ T cells produce interferon-gamma (IFN-y), tumor necrosis factor alpha (TNF-a), interleukin-2 (IL-2), or a combination of IFN-y and TNF-a.

The induced cellular immune response can include an increased CD8⁺ T cell response associated with the subject administered the vaccine as compared to the subject not administered the vaccine. The CD8⁺ T cell response associated with the subject administered the vaccine can be increased by about 2-fold to about 30-fold, about 3-fold to about 25-fold, or about

4-fold to about 20-fold as compared to the subject not administered the vaccine. The CD8⁺ T cell response associated with the subject administered the vaccine can be increased by at least about 1.5-fold, at least about 2.0-fold, at least about 3.0-fold, at least about 4.0-fold, at least about 5.0- fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 16.0-fold, at least about 17.0-fold, at least about 18.0-fold, at least about 19.0-fold, at least about 20.0-fold, at least about 21.0-fold, at least about 22.0-fold, at least about 23.0-fold, at least about 24.0-fold, at least about 25.0-fold, at least about 26.0-fold, at least about 27.0-fold, at least about 28.0-fold, at least about 29.0-fold, or at least about 30.0-fold as compared to the subject not administered the vaccine.

The induced cellular immune response can include an increased frequency of CD3⁺CD8⁺ T cells that produce IFN-y. The frequency of CD3 CD8 IFN-y T cells associated with the subject administered the vaccine can be increased by at least about 2-fold, 3 -fold, 4-fold,

5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold as compared to the subject not administered the vaccine.

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

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

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

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

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

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

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

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

The vaccine of the present invention can have features required of effective vaccines such as being safe so the vaccine itself does not cause illness or death; is protective against illness resulting from exposure to live pathogens such as viruses or bacteria; induces neutralizing antibody to prevent invention of cells; induces protective T cells against intracellular pathogens; and provides ease of administration, few side effects, biological stability, and low cost per dose.

The vaccine can further induce an immune response when administered to different tissues such as the muscle or skin. The vaccine can further induce an immune response when administered via electroporation, or injection, or subcutaneously, or intramuscularly.

SARS Coronavirus 2 (SARS-CoV-2) Antigen

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

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

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

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

The SARS-CoV-2 RBD can have an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:6. In some embodiments, the SARS-CoV-2 RBD can be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:6.

The nucleic acid molecule encoding the SARS-CoV-2 RBD antigen can comprise the nucleic acid sequence of SEQ ID NO: 1, which encodes SEQ ID NO:2. The nucleic acid molecule encoding the SARS-CoV-2 RBD antigen can comprise the nucleic acid sequence of SEQ ID N0:5 or SEQ ID NO: 12, which encode SEQ ID NO:6. In some embodiments, the nucleic acid molecule encoding the SARS-CoV-2 RED antigen can comprise a nucleotide sequence that encodes the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:6. In some embodiments, the nucleic acid molecule encoding the SARS-CoV-2 RBD antigen can comprise a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over an entire length of the nucleic acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:5, or SEQ ID NO: 12. In some embodiments, the SARS-CoV-2 RBD antigen can be operably linked to an IgE leader sequence.

Immunogenic fragments of SEQ ID NO:2 or SEQ ID NO:6 can be provided. Immunogenic fragments can comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of SEQ ID NO:2 or SEQ ID NO:6. In some embodiments, immunogenic fragments include a leader sequence, such as for example an immunoglobulin leader, such as the IgE leader. In some embodiments, immunogenic fragments are free of a leader sequence.

Immunogenic fragments of proteins with amino acid sequences homologous to immunogenic fragments of SEQ ID NO:2 or SEQ ID NO:6 can be provided. Such immunogenic fragments can comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:2 or SEQ ID NO:6. In some embodiments, immunogenic fragments include a leader sequence, such as for example an immunoglobulin leader, such as the IgE leader. In some embodiments, immunogenic fragments are free of a leader sequence.

Some embodiments relate to immunogenic fragments of SEQ ID NO: 1, SEQ ID NO:5, or SEQ ID NO: 12. Immunogenic fragments can be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the full length of SEQ ID NO: 1, SEQ ID NO:5, or SEQ ID NO: 12. Immunogenic fragments can comprise at least 95%, at least 96%, at least 97% at least 98% or at least 99% identity to fragments of SEQ ID NO: 1, SEQ ID NO:5, or SEQ ID NO: 12. In some embodiments, immunogenic fragments include sequences that encode a leader sequence, such as for example an immunoglobulin leader, such as the IgE leader. In some embodiments, fragments are free of coding sequences that encode a leader sequence.

Hemagglutinin Antigen

As described above, in one embodiment, the invention relates to a vaccine comprising an RCA amplification product encoding multiple copies of a HA antigen, a fragment thereof, a variant thereof, or a combination thereof.

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

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

The HA antigen can have an amino acid sequence of SEQ ID NO:4. In some embodiments, the HA antigen can be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO:4.

The nucleic acid molecule encoding HA antigen can comprise the nucleic acid sequence of SEQ ID NO:3, which encodes SEQ ID NO:4. In some embodiments, the nucleic acid molecule encoding the HA antigen can comprise a nucleotide sequence that encodes the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the nucleic acid molecule encoding the HA antigen can comprise a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over an entire length of the nucleic acid sequence set forth in SEQ ID NO:3. In some embodiments, the HA antigen can be operably linked to an IgE leader sequence.

Immunogenic fragments of SEQ ID NO:4 can be provided. Immunogenic fragments can comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of SEQ ID NO:4. In some embodiments, immunogenic fragments include a leader sequence, such as for example an immunoglobulin leader, such as the IgE leader. In some embodiments, immunogenic fragments are free of a leader sequence.

Immunogenic fragments of proteins with amino acid sequences homologous to immunogenic fragments of SEQ ID NO:4 can be provided. Such immunogenic fragments can comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:4. In some embodiments, immunogenic fragments include a leader sequence, such as for example an immunoglobulin leader, such as the IgE leader. In some embodiments, immunogenic fragments are free of a leader sequence.

Some embodiments relate to immunogenic fragments of SEQ ID NO:3. Immunogenic fragments can be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the full length of SEQ ID NO:3. Immunogenic fragments can comprise at least 95%, at least 96%, at least 97% at least 98% or at least 99% identity to fragments of SEQ ID NO:3. In some embodiments, immunogenic fragments include sequences that encode a leader sequence, such as for example an immunoglobulin leader, such as the IgE leader. In some embodiments, fragments are free of coding sequences that encode a leader sequence.

Anti -HA Antibody

As described above, in one embodiment, the invention relates to a vaccine comprising an RCA amplification product encoding multiple copies of an anti-influenza antibody, a fragment thereof, a variant thereof, or a combination thereof. In some embodiments, the anti-influenza antibody is an anti-HA antibody. In one embodiment, the immunogenic composition comprises a nucleotide sequence encoding a synthetic antibody. In one embodiment, the immunogenic composition comprises a nucleic acid molecule comprising a first nucleotide sequence encoding a first synthetic antibody and a second nucleotide sequence encoding a second synthetic antibody. In some embodiments, the first synthetic antibody is an antibody heavy chain and the second synthetic antibody is an antibody light chain. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a cleavage domain.

In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an anti-influenza antibody. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a heavy chain of an anti-influenza antibody. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a light chain of an antiinfluenza antibody. In one embodiment, the anti-influenza antibody is an anti -influenza broadly neutralizing antibody.

In one embodiment, the first nucleotide sequence encodes an amino acid sequence at least 90% homologous to SEQ ID NO: 8 and the second nucleotide sequence encodes an amino acid sequence at least 90% homologous to SEQ ID NO: 10. In one embodiment, the first nucleotide sequence encodes SEQ ID NO:8 and the second nucleotide sequence encodes SEQ ID NOTO.

In one embodiment, the first nucleotide sequence comprises a nucleotide sequence at least 90% homologous to SEQ ID NO:7 and the second nucleotide sequence comprises a nucleotide sequence at least 90% homologous to SEQ ID NO:9. In one embodiment, the first nucleotide sequence comprises a nucleotide sequence as set forth in SEQ ID NO:7 and the second nucleotide sequence comprises a nucleotide sequence as set forth in SEQ ID NO:9.

In one embodiment, the first nucleotide sequence comprises a nucleotide sequence at least 90% homologous to SEQ ID NO: 13 and the second nucleotide sequence comprises a nucleotide sequence at least 90% homologous to SEQ ID NO: 14. In one embodiment, the first nucleotide sequence comprises a nucleotide sequence as set forth in SEQ ID NO: 13 and the second nucleotide sequence comprises a nucleotide sequence as set forth in SEQ ID NO: 14.

The immunogenic composition can result in the generation of the synthetic antibody in the subject within at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, or 60 hours of administration of the composition to the subject. The immunogenic composition can result in generation of the synthetic antibody in the subject within at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days of administration of the immunogenic composition to the subject. The immunogenic composition can result in generation of the synthetic antibody in the subject within about 1 hour to about 6 days, about 1 hour to about 5 days, about 1 hour to about 4 days, about 1 hour to about 3 days, about 1 hour to about 2 days, about 1 hour to about 1 day, about 1 hour to about 72 hours, about 1 hour to about 60 hours, about 1 hour to about 48 hours, about 1 hour to about 36 hours, about 1 hour to about 24 hours, about 1 hour to about 12 hours, or about 1 hour to about 6 hours of administration of the immunogenic composition to the subject.

The immunogenic composition, when administered to the subject in need thereof, can result in the generation of the synthetic antibody in the subject more quickly than the generation of an endogenous antibody in a subject who is administered an antigen to induce a humoral immune response. The immunogenic composition can result in the generation of the synthetic antibody at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days before the generation of the endogenous antibody in the subject who was administered an antigen to induce a humoral immune response.

The immunogenic composition of the present invention can have features required of effective compositions such as being safe so that the composition does not cause illness or death; being protective against illness; and providing ease of administration, few side effects, biological stability and low cost per dose.

Recombinant Nucleic Acid Sequence

As described above, the composition can comprise a recombinant nucleic acid sequence. The recombinant nucleic acid sequence can encode the antibody, a fragment thereof, a variant thereof, or a combination thereof. The antibody is described in more detail below.

The recombinant nucleic acid sequence can be a heterologous nucleic acid sequence. The recombinant nucleic acid sequence can include one or more heterologous nucleic acid sequences. The recombinant nucleic acid sequence can be an optimized nucleic acid sequence. Such optimization can increase or alter the immunogenicity of the antibody. Optimization can also improve transcription and/or translation. Optimization can include one or more of the following: low GC content leader sequence to increase transcription; mRNA stability and codon optimization; addition of a kozak sequence (e.g., GCC ACC) for increased translation; addition of an immunoglobulin (Ig) leader sequence encoding a signal peptide; addition of an internal IRES sequence and eliminating to the extent possible cis-acting sequence motifs (i.e., internal TATA boxes).

Recombinant Nucleic Acid Sequence Construct

The recombinant nucleic acid sequence can include one or more recombinant nucleic acid sequence constructs. The recombinant nucleic acid sequence construct can include one or more components, which are described in more detail below.

The recombinant nucleic acid sequence construct can include a heterologous nucleic acid sequence that encodes a heavy chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The recombinant nucleic acid sequence construct can include a heterologous nucleic acid sequence that encodes a light chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The recombinant nucleic acid sequence construct can also include a heterologous nucleic acid sequence that encodes a protease or peptidase cleavage site. The recombinant nucleic acid sequence construct can also include a heterologous nucleic acid sequence that encodes an internal ribosome entry site (IRES). An IRES may be either a viral IRES or an eukaryotic IRES. The recombinant nucleic acid sequence construct can include one or more leader sequences, in which each leader sequence encodes a signal peptide. The recombinant nucleic acid sequence construct can include one or more promoters, one or more introns, one or more transcription termination regions, one or more initiation codons, one or more termination or stop codons, and/or one or more polyadenylation signals. The recombinant nucleic acid sequence construct can also include one or more linker or tag sequences. The tag sequence can encode a hemagglutinin (HA) tag.

Heavy Chain Polypeptide The recombinant nucleic acid sequence construct can include the heterologous nucleic acid encoding the heavy chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The heavy chain polypeptide can include a variable heavy chain (VH) region and/or at least one constant heavy chain (CH) region. The at least one constant heavy chain region can include a constant heavy chain region 1 (CHI), a constant heavy chain region 2 (CH2), and a constant heavy chain region 3 (CH3), and/or a hinge region.

In some embodiments, the heavy chain polypeptide can include a VH region and a CHI region. In other embodiments, the heavy chain polypeptide can include a VH region, a CHI region, a hinge region, a CH2 region, and a CH3 region.

The heavy chain polypeptide can include a complementarity determining region (“CDR”) set. The CDR set can contain three hypervariable regions of the VH region. Proceeding from N-terminus of the heavy chain polypeptide, these CDRs are denoted “CDR1,” “CDR2,” and “CDR3,” respectively. CDR1, CDR2, and CDR3 of the heavy chain polypeptide can contribute to binding or recognition of the antigen.

In one embodiment, the heavy chain polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO: 8.

In one embodiment, the heavy chain polypeptide comprises an amino acid sequence as set forth in SEQ ID NO:8.

In one embodiment, the heavy chain polypeptide is encoded by a nucleotide sequence at least 90% identical to SEQ ID NO:7. In one embodiment, the heavy chain polypeptide is encoded by a nucleotide sequence at least 90% identical to SEQ ID NO: 13.

In one embodiment, the heavy chain polypeptide is encoded by a nucleotide sequence as set forth in SEQ ID NO:7. In one embodiment, the heavy chain polypeptide is encoded by a nucleotide sequence as set forth in SEQ ID NO: 13.

Light Chain Polypeptide

The recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding the light chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The light chain polypeptide can include a variable light chain (VL) region and/or a constant light chain (CL) region. The light chain polypeptide can include a complementarity determining region (“CDR”) set. The CDR set can contain three hypervariable regions of the VL region. Proceeding from N-terminus of the light chain polypeptide, these CDRs are denoted “CDR1,” “CDR2,” and “CDR3,” respectively. CDR1, CDR2, and CDR3 of the light chain polypeptide can contribute to binding or recognition of the antigen.

In one embodiment, the light chain polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO: 10.

In one embodiment, the light chain polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 10.

In one embodiment, the light chain polypeptide is encoded by a nucleotide sequence at least 90% identical to SEQ ID NO:9. In one embodiment, the light chain polypeptide is encoded by a nucleotide sequence at least 90% identical to SEQ ID NO: 14.

In one embodiment, the light chain polypeptide is encoded by a nucleotide sequence as set forth in SEQ ID NO:9. In one embodiment, the light chain polypeptide is encoded by a nucleotide sequence as set forth in SEQ ID NO: 14.

Protease Cleavage Site

The recombinant nucleic acid sequence construct can include heterologous nucleic acid sequence encoding a protease cleavage site. The protease cleavage site can be recognized by a protease or peptidase. The protease can be an endopeptidase or endoprotease, for example, but not limited to, furin, elastase, HtrA, calpain, trypsin, chymotrypsin, trypsin, and pepsin. The protease can be furin. In other embodiments, the protease can be a serine protease, a threonine protease, cysteine protease, aspartate protease, metalloprotease, glutamic acid protease, or any protease that cleaves an internal peptide bond (i.e., does not cleave the N-terminal or C- terminal peptide bond).

The protease cleavage site can include one or more amino acid sequences that promote or increase the efficiency of cleavage. The one or more amino acid sequences can promote or increase the efficiency of forming or generating discrete polypeptides. The one or more amino acids sequences can include a 2A peptide sequence.

Linker Sequence The recombinant nucleic acid sequence construct can include one or more linker sequences. The linker sequence can spatially separate or link the one or more components described herein. In other embodiments, the linker sequence can encode an amino acid sequence that spatially separates or links two or more polypeptides.

Promoter

The recombinant nucleic acid sequence construct can include one or more promoters. The one or more promoters may be any promoter that is capable of driving gene expression and regulating gene expression. Such a promoter is a cis-acting sequence element required for transcription via a DNA dependent RNA polymerase. Selection of the promoter used to direct gene expression depends on the particular application. The promoter may be positioned about the same distance from the transcription start in the recombinant nucleic acid sequence construct as it is from the transcription start site in its natural setting. However, variation in this distance may be accommodated without loss of promoter function.

The promoter may be operably linked to the heterologous nucleic acid sequence encoding the heavy chain polypeptide and/or light chain polypeptide. The promoter may be a promoter shown effective for expression in eukaryotic cells. The promoter operably linked to the coding sequence may be a CMV promoter, a promoter from simian virus 40 (SV40), such as SV40 early promoter and SV40 later promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter may also be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine, human polyhedrin, or human metalothionein.

The promoter can be a constitutive promoter or an inducible promoter, which initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development. The promoter may also be a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic. Examples of such promoters are described in US patent application publication no. US20040175727, the contents of which are incorporated herein in its entirety.

The promoter can be associated with an enhancer. The enhancer can be located upstream of the coding sequence. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine or a viral enhancer such as one from CMV, FMDV, RSV or EBV. Polynucleotide function enhances are described in U.S. Patent Nos. 5,593,972, 5,962,428, and W094/016737, the contents of each are fully incorporated by reference.

Intron

The recombinant nucleic acid sequence construct can include one or more introns. Each intron can include functional splice donor and acceptor sites. The intron can include an enhancer of splicing. The intron can include one or more signals required for efficient splicing.

Transcription Termination Region

The recombinant nucleic acid sequence construct can include one or more transcription termination regions. The transcription termination region can be downstream of the coding sequence to provide for efficient termination. The transcription termination region can be obtained from the same gene as the promoter described above or can be obtained from one or more different genes.

Initiation Codon

The recombinant nucleic acid sequence construct can include one or more initiation codons. The initiation codon can be located upstream of the coding sequence. The initiation codon can be in frame with the coding sequence. The initiation codon can be associated with one or more signals required for efficient translation initiation, for example, but not limited to, a ribosome binding site.

Termination Codon

The recombinant nucleic acid sequence construct can include one or more termination or stop codons. The termination codon can be downstream of the coding sequence. The termination codon can be in frame with the coding sequence. The termination codon can be associated with one or more signals required for efficient translation termination.

Polyadenylation Signal

The recombinant nucleic acid sequence construct can include one or more polyadenylation signals. The polyadenylation signal can include one or more signals required for efficient polyadenylation of the transcript. The polyadenylation signal can be positioned downstream of the coding sequence. The polyadenylation signal may be a SV40 polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, or human P-globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from a pCEP4 plasmid (Invitrogen, San Diego, CA).

Leader Sequence

The recombinant nucleic acid sequence construct can include one or more leader sequences. The leader sequence can encode a signal peptide. The signal peptide can be an immunoglobulin (Ig) signal peptide, for example, but not limited to, an IgG signal peptide and a IgE signal peptide.

Arrangement of the Recombinant Nucleic Acid Sequence Construct

As described above, the recombinant nucleic acid sequence can include one or more recombinant nucleic acid sequence constructs, in which each recombinant nucleic acid sequence construct can include one or more components. The one or more components are described in detail above. The one or more components, when included in the recombinant nucleic acid sequence construct, can be arranged in any order relative to one another. In some embodiments, the one or more components can be arranged in the recombinant nucleic acid sequence construct as described below.

Arrangement 1

In one arrangement, a first recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding the heavy chain polypeptide and a second recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding the light chain polypeptide.

For example, in one embodiment, the first recombinant nucleic acid sequence encodes a heavy chain polypeptide having an amino acid sequence at least 90% homologous to SEQ ID NO:8.

In one embodiment, the first recombinant nucleic acid sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:7. In one embodiment, the first recombinant nucleic acid sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO: 13.

In one embodiment, the second recombinant nucleic acid sequence encodes a light chain polypeptide having an amino acid sequence at least 90% homologous to SEQ ID NO: 10.

In one embodiment, the second recombinant nucleic acid sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:9. In one embodiment, the second recombinant nucleic acid sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO: 14.

The first recombinant nucleic acid sequence construct can be placed in a vector. The second recombinant nucleic acid sequence construct can be placed in a second or separate vector. Placement of the recombinant nucleic acid sequence construct into the vector is described in more detail below.

The first recombinant nucleic acid sequence construct can also include the promoter, intron, transcription termination region, initiation codon, termination codon, and/or polyadenylation signal. The first recombinant nucleic acid sequence construct can further include the leader sequence, in which the leader sequence is located upstream (or 5’) of the heterologous nucleic acid sequence encoding the heavy chain polypeptide. Accordingly, the signal peptide encoded by the leader sequence can be linked by a peptide bond to the heavy chain polypeptide.

The second recombinant nucleic acid sequence construct can also include the promoter, initiation codon, termination codon, and polyadenylation signal. The second recombinant nucleic acid sequence construct can further include the leader sequence, in which the leader sequence is located upstream (or 5’) of the heterologous nucleic acid sequence encoding the light chain polypeptide. Accordingly, the signal peptide encoded by the leader sequence can be linked by a peptide bond to the light chain polypeptide. Accordingly, one example of arrangement 1 can include the first vector (and thus first recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide that includes VH and CHI, and the second vector (and thus second recombinant nucleic acid sequence construct) encoding the light chain polypeptide that includes VL and CL. A second example of arrangement 1 can include the first vector (and thus first recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide that includes VH, CHI, hinge region, CH2, and CH3, and the second vector (and thus second recombinant nucleic acid sequence construct) encoding the light chain polypeptide that includes VL and CL.

Arrangement 2

In a second arrangement, the recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide. The heterologous nucleic acid sequence encoding the heavy chain polypeptide can be positioned upstream (or 5’) of the heterologous nucleic acid sequence encoding the light chain polypeptide. Alternatively, the heterologous nucleic acid sequence encoding the light chain polypeptide can be positioned upstream (or 5’) of the heterologous nucleic acid sequence encoding the heavy chain polypeptide.

The recombinant nucleic acid sequence construct can be placed in the vector as described in more detail below.

The recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding the protease cleavage site and/or the linker sequence. If included in the recombinant nucleic acid sequence construct, the heterologous nucleic acid sequence encoding the protease cleavage site can be positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide. Accordingly, the protease cleavage site allows for separation of the heavy chain polypeptide and the light chain polypeptide into distinct polypeptides upon expression. In other embodiments, if the linker sequence is included in the recombinant nucleic acid sequence construct, then the linker sequence can be positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide. The recombinant nucleic acid sequence construct can also include the promoter, intron, transcription termination region, initiation codon, termination codon, and/or polyadenylation signal. The recombinant nucleic acid sequence construct can include one or more promoters. The recombinant nucleic acid sequence construct can include two promoters such that one promoter can be associated with the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the second promoter can be associated with the heterologous nucleic acid sequence encoding the light chain polypeptide. In still other embodiments, the recombinant nucleic acid sequence construct can include one promoter that is associated with the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

The recombinant nucleic acid sequence construct can further include two leader sequences, in which a first leader sequence is located upstream (or 5’) of the heterologous nucleic acid sequence encoding the heavy chain polypeptide and a second leader sequence is located upstream (or 5’) of the heterologous nucleic acid sequence encoding the light chain polypeptide. Accordingly, a first signal peptide encoded by the first leader sequence can be linked by a peptide bond to the heavy chain polypeptide and a second signal peptide encoded by the second leader sequence can be linked by a peptide bond to the light chain polypeptide.

Accordingly, one example of arrangement 2 can include the vector (and thus recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide that includes VH and CHI, and the light chain polypeptide that includes VL and CL, in which the linker sequence is positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

A second example of arrangement of 2 can include the vector (and thus recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide that includes VH and CHI, and the light chain polypeptide that includes VL and CL, in which the heterologous nucleic acid sequence encoding the protease cleavage site is positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

A third example of arrangement 2 can include the vector (and thus recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide that includes VH, CHI, hinge region, CH2, and CH3, and the light chain polypeptide that includes VL and CL, in which the linker sequence is positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

A forth example of arrangement of 2 can include the vector (and thus recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide that includes VH, CHI, hinge region, CH2, and CH3, and the light chain polypeptide that includes VL and CL, in which the heterologous nucleic acid sequence encoding the protease cleavage site is positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

Expression from the Recombinant Nucleic Acid Sequence Construct

As described above, the recombinant nucleic acid sequence construct can include, amongst the one or more components, the heterologous nucleic acid sequence encoding the heavy chain polypeptide and/or the heterologous nucleic acid sequence encoding the light chain polypeptide. Accordingly, the recombinant nucleic acid sequence construct can facilitate expression of the heavy chain polypeptide and/or the light chain polypeptide.

When arrangement 1 as described above is utilized, the first recombinant nucleic acid sequence construct can facilitate the expression of the heavy chain polypeptide and the second recombinant nucleic acid sequence construct can facilitate expression of the light chain polypeptide. When arrangement 2 as described above is utilized, the recombinant nucleic acid sequence construct can facilitate the expression of the heavy chain polypeptide and the light chain polypeptide.

Upon expression, for example, but not limited to, in a cell, organism, or mammal, the heavy chain polypeptide and the light chain polypeptide can assemble into the synthetic antibody. In particular, the heavy chain polypeptide and the light chain polypeptide can interact with one another such that assembly results in the synthetic antibody being capable of binding the antigen. In other embodiments, the heavy chain polypeptide and the light chain polypeptide can interact with one another such that assembly results in the synthetic antibody being more immunogenic as compared to an antibody not assembled as described herein. In still other embodiments, the heavy chain polypeptide and the light chain polypeptide can interact with one another such that assembly results in the synthetic antibody being capable of eliciting or inducing an immune response against the antigen.

Antibody

As described above, the recombinant nucleic acid sequence can encode the antibody, a fragment thereof, a variant thereof, or a combination thereof. The antibody can bind or react with the antigen, which is described in more detail below.

The antibody may comprise a heavy chain and a light chain complementarity determining region (“CDR”) set, respectively interposed between a heavy chain and a light chain framework (“FR”) set which provide support to the CDRs and define the spatial relationship of the CDRs relative to each other. The CDR set may contain three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3,” respectively. An antigen-binding site, therefore, may include six CDRs, comprising the CDR set from each of a heavy and a light chain V region.

The proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the F(ab) fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site. The enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the F(ab’)2 fragment, which comprises both antigen-binding sites. Accordingly, the antibody can be the Fab or F(ab’)2 The Fab can include the heavy chain polypeptide and the light chain polypeptide. The heavy chain polypeptide of the Fab can include the VH region and the CHI region. The light chain of the Fab can include the VL region and CL region.

The antibody can be an immunoglobulin (Ig). The Ig can be, for example, IgA, IgM, IgD, IgE, and IgG. The immunoglobulin can include the heavy chain polypeptide and the light chain polypeptide. The heavy chain polypeptide of the immunoglobulin can include a VH region, a CHI region, a hinge region, a CH2 region, and a CH3 region. The light chain polypeptide of the immunoglobulin can include a VL region and CL region.

The antibody can be a polyclonal or monoclonal antibody. The antibody can be a chimeric antibody, a single chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, or a fully human antibody. The humanized antibody can be an antibody from a non-human species that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule.

The antibody can be an IgGl antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. The antibody can be a chimera of any of an IgGl antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In some embodiments, the antibody hinge domain is modified. For example, in one embodiment, the antibody includes the amino acid substitution Ser288Pro. In one embodiment, includes the amino acid substitution Ser288Pro prevents IgG4 Fab arm switching.

The antibody can be a bispecific antibody as described below in more detail. The antibody can be a bifunctional antibody as also described below in more detail.

As described above, the antibody can be generated in the subject upon administration of the composition to the subject. The antibody may have a half-life within the subject. In some embodiments, the antibody may be modified to extend or shorten its half-life within the subject. Such modifications are described below in more detail.

The antibody can be defucosylated as described in more detail below.

Self-Assembling NanoParticles

In one embodiment, the invention relates to a vaccine comprising an RCA amplification product encoding multiple copies of a self-assembling nanoparticle comprising an oligomerization domain and further comprising a SARS-CoV-2 antigen, an influenza antigen, an influenza antibody, a fragment thereof, a variant thereof, or a combination thereof. In one embodiment, the invention exploits ferritin, a ubiquitous iron storage protein, that self-assembles into spherical nanoparticles and serves as a scaffold to express a heterologous protein. Therefore, in one embodiment, the oligomerization domain comprises ferritin, or a fragment or variant thereof.

In one embodiment, the invention relates to a nucleic acid molecule encoding a self-assembling nanoparticle comprising an oligomerization domain and further comprising a SARS-CoV-2 antigen, an influenza antigen, an influenza antibody, a fragment thereof, a variant thereof, or a combination thereof. In some embodiments, the nucleic acid molecule encoding the oligomerization domain can comprise a nucleotide sequence that encodes the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6; SEQ ID NO:8, or SEQ ID NO: 10. In some embodiments, the nucleic acid molecule encoding the oligomerization domain can comprise a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over an entire length of the nucleic acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NON, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14. In some embodiments, the nucleotide sequence encoding the oligomerization domain can be operably linked to a sequence encoding at least one linker sequence, such as an LS3 or GGS linker sequence.

Leader Sequence

In some embodiments, the antigen of the invention is operably linked to at least one leader sequence or a pharmaceutically acceptable salt thereof. In some embodiments, the nucleic acid molecules of the invention encoding the antigen are operably linked to at least one nucleotide sequence encoding a leader sequence or a pharmaceutically acceptable salt thereof. In some embodiments, the antigen of the invention lacks a leader sequence. In some embodiments, the nucleic acid molecules of the invention encoding the antigen lack a nucleotide sequence encoding a leader sequence. "Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that can be linked at the amino terminus of a protein set forth herein. Signal peptides/leader sequences typically direct localization of a protein. Signal peptides/leader sequences used herein preferably facilitate secretion of the protein from the cell in which it is produced. Signal peptides/leader sequences are often cleaved from the remainder of the protein, often referred to as the mature protein, upon secretion from the cell. Signal peptides/leader sequences are linked at the N terminus of the protein.

In one embodiment, the leader sequence is the IgE leader sequence comprising the amino acid sequence of MDWTWILFLVAAATRVHS (SEQ ID NO:11). In some embodiments therefore, the leader sequence in the disclosed expressible nucleic acid sequence comprises a sequence encoding SEQ ID NO: 11. Linker Sequence

In some embodiments, the antigen sequences of the invention are operably linked to at least one linker sequence. For example, in some embodiments the peptide comprises a linker between the leader sequence and the antigen sequence. A linker can be either flexible or rigid or a combination thereof. In one embodiment, the linker is a (GGS)n repeat wherein, the GGS is repeated at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more than 10 times.

RCA amplification product

The vaccine can comprise one or more RCA amplification product generated from RCA amplification of a template nucleic acid encoding an antigen or an antibody. In some embodiments, the RCA product comprises a concatemeric double-stranded DNA molecule encoding multiple copies of the antigen or the antibody.

The RCA product may comprise one or more nucleotide analogues, a modified nucleotide, or a combination thereof. The nucleotide analogue may have an altered phosphate backbone, sugar moiety, nucleobase, or combinations thereof. For example, additional nucleotide analogue may be a Locked Nucleic Acid (LNA) nucleotide, or a Peptide Nucleic Acid (PNA). In some embodiments, the RCA amplification product comprises one or more phosphorothioated nucleotides. As used herein, the term "modified nucleotides" refers to nucleotides wherein an additional moiety is attached the nucleotides (e.g., a biotinylated nucleotide). In some embodiments, the RCA product comprises a double-stranded concatemeric DNA containing phosphorothioated nucleotides which provides enhanced nuclease resistance property.

In some embodiments, multiple (e.g., two or more) separate double stranded concatemeric DNA may be employed for in vivo expression, wherein each of the separate double stranded concatemeric DNA includes expression sequences encoding different molecules (e.g., protein, antibody, antigen, mRNA, sgRNA, etc.). In some embodiments, multiple (e.g., two or more) separate double stranded concatemeric DNA may be employed for in vivo antigen or antibody expression, wherein each of the separate double stranded concatemeric DNA includes expression sequences encoding different antigens or antibodies. For example, two RCA product DNAs may be employed, wherein a first RCA product DNA includes a first expression sequence encoding a first antigen or antibody, and a second RCA product DNA includes a second expression sequence encoding a second antigen or antibody, wherein the first antigen or antibody is different from the second antigen or antibody.

In some embodiments, at least one sequence of the tandem repeat sequences of the double-stranded concatemeric DNA includes one or more phosphorothioated nucleotides. In some embodiments, each of the tandem repeat sequences of the double stranded concatemeric DNA comprises one or more phosphorothioated nucleotide. The phosphorothioated nucleotides are incorporated in RCA product DNA by using phosphorothioated dNTPs such as a-S-dATP and a-S- dTTP in RCA reaction. The term "phosphorothioated" nucleotide is interchangeably used hereinafter as a "thioated" nucleotide. The term "total nucleotides" refers to the total number of thioated nucleotides and non-thioated nucleotides in particular nucleic acid sequence. The thioated nucleotides may be incorporated by using one or more thioated dNTPs in a DNA amplification reaction that is used to produce the double- stranded concatemeric DNA. For example, in a DNA amplification reaction, at least a portion of dATP may be substituted with thioated dATP. In some other embodiments, a combination of thioated dATP, dGTP, dCTP, and/or dTTP may be used in the DNA amplification reaction that is used for the generation of the double- stranded concatemeric DNA. A thioated dNTP, such as a-S-dATP is added in a pool of non-thioated dNTP mixture, such as a mixture of dATP, dGTP, dTTP and dCTP. A ratio of thioated dNTP to total dNTP (including thioated and non-thioated dNTPs) is calculated by dividing a concentration of thioated nucleotide added to a reaction mixture containing a mixture of thioated and non-thioated dNTPs by a concentration of the total nucleotides (thioated and non- thioated).

In some embodiments, the double-stranded concatemeric DNA is an RCA product DNA that is generated by rolling circle amplification. The RCA product DNA may be a linear or a branched concatamer. In some embodiments, each of the tandem repeat sequences comprises phosphorothioated nucleotides. The RCA product DNA including phosphorothioated nucleotide exhibits increased stability towards restriction digestion in comparison with an RCA product DNA that does not contain any phosphorothioated nucleotide or with a super coiled plasmid DNA. As used herein, "thioated RCA product DNA" refers to the RCA product DNA comprising at least one phosphorothioated nucleotide. Further, the "non-thioated RCA product DNA" refers to an RCA product DNA which does not contain any phosphorothioated nucleotide. In some embodiments, the ratio of phosphorothioated nucleotides to total nucleotides in the double-stranded concatemeric DNA is in a range of 1: 1600 (i.e., 0.001) to 125: 1600 (i.e., 0.078). In some other embodiments, the ratio of phosphorothioated nucleotides to total nucleotides in the double-stranded concatemeric DNA is in a range of 50: 1600 (or 0.031) to 125: 1600 (or 0.078). In certain embodiments, the ratio of phosphorothioated nucleotides to total nucleotides in the double-stranded concatemeric DNA is in a range of 75: 1600 (or 0.047) to 125: 1600 (or 0.078). In one or more embodiments, the ratio of phosphorothioated nucleotides to total nucleotides in the double-stranded concatemeric DNA is 125: 1600 (or 0.078). In some embodiments, the ratio of phosphorothioated nucleotides to total nucleotides in the doublestranded concatemeric DNA is 1 :40 (i.e., 0.025). In certain embodiments, the ratio of phosphorothioated nucleotides to total nucleotides in the double-stranded concatemeric DNA is 1 : 16 (i.e., 0.062). In one alternate embodiment, the ratio of phosphorothioated nucleotides to total nucleotides in the double-stranded concatemeric DNA is 1 : 1. In such embodiments, doublestranded concatemeric DNA include equal numbers of thioated and non-thioated nucleotides. In some embodiments, all the nucleotides of the double-stranded concatemeric DNA, such as RCA product DNA, are phosphorothioated. In other words, the double- stranded concatemeric DNA contains 100% phosphorothioated nucleotides.

In some embodiments, the double-stranded RCA product DNA that is used for in vivo protein expression comprises thioated nucleotides. The RCA product DNA having phosphorothiated nucleotides is produced by rolling circle amplification. In these embodiments, the RCA reactions are supplemented with thioated dNTPs, such as a-S-dATP or a-S-dTTP, into the dNTP mixture for random incorporation of thioated bases into the RCA product DNA while amplification. Protein expression is improved when an RCA product comprising thioated nucleotides is used for in vivo transcription and translation when compared to non-thioated RCA products. In some embodiments, the double-stranded concatemeric DNA, such as an RCA product DNA, may be internally thioated (have a-S-dNTP). In such embodiments, to generate a double-stranded concatemeric DNA that are internally thioated, RCA reactions are supplemented with phosphorothioated nucleotides. The phosphorothioated nucleotides are incorporated into the dNTP mixture for random incorporation of thioated bases into the RCA product DNA during amplification. In some other embodiments, a double-stranded concatemeric DNA, such as an RCA product DNA comprising a phosphorothioated nucleotide may be generated (e.g., thioated, having a-S-dNTP) by employing a thioated primer sequence for the RCA reaction.

The RCA product DNA used for in vivo RNA or protein expression may be processed DNA or unprocessed DNA. The "processing" of the RCA product DNA may include an act of restriction digestion, chemical denaturation, heat denaturation, self-cleaving, or enzymatically cleaving of the RCA product DNA of interest. The "processing" of the RCA product DNA may also include purification of the RCA product DNA of interest. The "processing" of the RCA product may also include physically or enzymatically decreasing the length of the RCA product. This "processing" could include, but is not limited to, examples such as shearing, sonication, treatment using restriction digest, transposase, protelomerase, cas9/CRISPR, or any other enzymatic reaction that could be utilized to decrease the length of the RCA product which contains one or more phosphorothioated nucleotides. In some embodiments, the RCA product DNA can be employed as a DNA template for in vivo protein expression without any purification. In some embodiments, the double-stranded concatemeric DNA may be processed to form linear, or circular DNA template for transfection. The linear concatemeric DNA may be inserted into a plasmid vector before transfecting into the eukaryotic cells. In such embodiments, the linear concatemeric DNA may be subjected to restriction digestion to produce fragmented DNA followed by inserting the fragmented DNA into a plasmid vector using recombination technology. In addition, the linear concatemeric DNA may be treated with a recombinase or pro-telomerase or other enzymes to generate circularized, fragmented DNA. In some embodiments, the RCA product DNA is transfected or introduced into the eukaryotic cells without any further processing. In such embodiments, the RCA product is not subjected to any kind of restriction digestion or self-cleaving to form smaller fragments before using it as a DNA template for in vivo protein expression. In some other embodiments, the RCA product is not subjected to any chemical denaturation or heat denaturation to denature the RCA product DNA before employing the DNA template for introducing into the eukaryotic cells for in vivo protein expression. However, in some embodiments, the RCA product DNA may be separated (e.g., by precipitation) to remove salts or any other contaminants, such as primers or smaller fragmented DNA from the reaction medium before proceeding for transfection.

In some embodiments, the RCA product is generated by a rolling-circle amplification reaction which employs reagents such as a primer, a nucleic acid polymerase, and free nucleotides (dNTPs). The nucleic acid polymerase may be a proofreading nucleic acid polymerase, including, but not limited to, a Phi29 DNA polymerase. In some embodiments, the reagents used in the RCA may be pre-treated e.g., by ultraviolet radiation or de-contaminated by incubating the reagents in presence of a nuclease and its co-factor. During the amplification reaction, the DNA template is replicated by a polymerase in the presence of dNTPs (for example, dATP, dGTP, dCTP or dTTP), modified dNTPs (e.g. thioated dNTPs, such as a-S-dGTP, a-S- dCTP, a-S-dATP, and a-S-dTTP), or combinations thereof. RCA may be performed using commercially available RCA amplification kits such as illustra™ TempliPhi™ Amplification Kit (GE Healthcare Life Sciences).

In some embodiments, the RCA reaction may be performed using a random primer mixture or specific primers. Primer sequences comprising one or more nucleotide analogues may also be used. For example, the nucleotide analogues may include phosphorothioated nucleotide, an inosine, a Locked Nucleic Acid (LNA) nucleotide, a Peptide Nucleic Acid (PNA) nucleotide, 2-amino-deoxyadenosine, 2-thio- deoxy thy mi dine, a polycation nucleotide, Zip Nucleic Acid (ZNA) polycation modified nucleotide, or combinations thereof. In one or more embodiments, the random primer mixture has nuclease-resistant primers (e.g., primer sequences comprising phosphorothioate groups at appropriate positions), random hexamers or a hexamer primer.

In some embodiments, the RCA product DNA is generated by using an RCA reaction having a final concentration of dNTPs in a range of about 10 pM to about 10 mM. In one or more embodiments of RCA reactions, the dNTP concentration is less than 10 mM. In these embodiments, the concentration of dNTPs is kept lower than 10 mM to avoid hydrogel formation from the RCA product and to remain at a concentration below or equal to the amount of divalent cation (e.g. magnesium) present in the reaction buffer. Hydrogel formation may occur after amplification in the presence of a high concentration of dNTPs which may further complicate the downstream manipulation such as pipetting and processing of the RCA product. Hydrogel formation may be observed when dNTP concentration of 50 mM or more is used in the RCA reaction.

In some embodiments, the expression sequence in each of the plurality of tandem repeat sequences may comprise a coding sequence, a non-coding sequence, or a combination thereof. In some embodiments, the expression sequence further comprises a polyA sequence, a translational enhancer sequence, a transcriptional termination sequence, a ribosomal binding site, a translational termination sequence, an insulator sequence, or combinations thereof. The expression sequence may further include a pre-promoter sequence, a sequence for protease cleavage or nucleotide cleavage, a sequence for protein purification, or combinations thereof.

In some embodiments, the expression sequence contains a coding sequence, wherein the coding sequence generates a desired protein in the eukaryotic cell. The coding sequence is a nucleic acid sequence containing a particular gene of interest. In general, the coding sequence comprises a promoter, and an open reading frame (ORF). The coding sequence may optionally include a cap-independent translation element (CITE). In some embodiments, the coding sequence further comprises a ribosomal binding site. The coding sequence may comprise a transcription terminator sequence located outside the open reading frame but within the expression sequence. In one or more embodiments, the open reading frame of the coding sequence comprises a codon-optimized sequence, a purification tag sequence, a protease cleavage site or combinations thereof. In some embodiments, the expression sequence comprises both coding and non-coding sequences.

In some embodiments, each of the plurality of tandem repeat sequences comprises at least one expression sequence. In some embodiments, the at least one expression sequence comprises at least one coding sequence. In such embodiments, the at least one coding sequence of the at least one expression sequence comprises at least one promoter, and at least one open reading frame. In some embodiments, each of the plurality of tandem repeat sequences comprises two or more expression sequences. The two or more expression sequences including coding sequences may code for a same protein or different proteins. In some embodiments, the expression sequence includes at least one promoter that is functionally linked to at least one open reading frame. For example, in one aspect, in an expression sequence, one promoter is functionally linked to one open reading frame. In another aspect, in an expression sequence, one promoter is functionally linked to two different open reading frames. In some embodiments, the expression sequence may include two or more promoters functionally linked to two or more open reading frames.

An expression sequence may include a promoter operably linked to two different open reading frames, such as a first open reading frame and a second open reading frame, each of them coding an expression product (e g., protein, antibody, antigen, mRNA, sgRNA, etc.) that is different from the other. In this example, a single promoter is functionally linked to two open reading frames via a cap-independent translation element. Each of the open reading frames includes translation start and translation stop sequences. A translational termination or stop sequence is required for an expression sequence, otherwise an infinite polyprotein may be synthesized, which is undesirable. However, a transcriptional stop codon may be optional for the first open reading frame leading to the generation of a polycistronic mRNA upon transcription. In such instances, the intervening sequences between the first and second open reading frames may be selected such that upon in vivo expression of one or more encoded products (e.g., protein, antibody, antigen, mRNA, sgRNA etc.), even if a single polycistronic mRNA is produced, it can be converted into to independent products. For example, synthesis of a first protein by translation of the first open reading frame may be followed by a ribosomal slippage to the second translation start sequence of the second open reading frame to initiate the synthesis of a second protein from the second open reading frame. This may be achieved by incorporating "self- cleaving sequences" between the first and second open reading frames. Suitable selfcleaving sequences such as viral P2A motif facilitates the creation of two or more proteins from one single mRNA.

In some embodiments, the expression sequence contains a non-coding sequence, wherein the non-coding sequence generates a desired RNA. Such expression sequence does not contain any coding sequence. The non-coding sequence comprises a promoter and a transcription termination sequence. The non-coding sequence is generally devoid of an open reading frame. The expression sequence that contains a non-coding sequence is also referred to as an RNA expression sequence. In some embodiments, the expression sequence consists essentially of a noncoding sequence. In some other embodiments, the expression sequence includes both the coding and non-coding sequences, wherein the RNA can be generated from a non- coding sequence of an expression sequence. In such embodiments, a desired protein may also be subsequently generated from the coding sequence of the same expression sequence. In some embodiments, the generated RNA may be extracted from the eukaryotic cells for different downstream applications. In one embodiment, the extracted RNA may subsequently be packaged into a lentivirus system to deliver in another cell. The non-coding sequence may include, but is not limited to, a sequence for antisense RNA, a short interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), a microRNA mimic, a transfer RNA (tRNA), a ribosomal RNA (rRNA), or combinations thereof. The non-coding sequence may also include CRISPR RNAs (tracrRNA, crRNA, sgRNA, or gRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), Piwi interacting RNA (piRNA), telomerase RNA, spliceosome RNA, enhancer RNA, retrotransposons, X inactive specific transcript (Xist), RNAs encoded by RNA polymerase I and RNA polymerase III, or combinations thereof.

Any of the suitable promoters known in the art, including, for example, T7 RNA polymerase or CMV promoter sequences, may be used in the methods described herein. Likewise, any of suitable ribosomal binding sites known in the art, including but not limited to, IRES, polyA tracts, species-independent translational leaders (SITS), Kozak consensus sequences, and Shine-Dalgarno sequences may be used.

The open reading frame includes translation start and translation stop sequences. In some embodiments, the open reading frame comprises a codon- optimized sequence for enhancing translation. The open reading frame may comprise an amino -terminal peptide fusion sequence derived from an internal ribosome entry site (IRES) for enhanced ribosome recognition, a tag sequence for purification of the desired protein, or a combination thereof. The CITE may comprise an IRES, a translation enhancing element (TEE), or a combination thereof.

As noted, the open reading frame of the coding sequence may comprise a codon- optimized sequence, wherein the codon optimized sequence is generated by considering different factors, such as codon bias, contextual codon preference, and/or individual codon preference. The codon-optimized sequence of the open reading frame may enhance the rate or quality of translation of the RCA product. Codon optimization generally improves the protein expression from the coding sequence by increasing the translational efficiency of a gene of interest. The functionality of a gene may also be increased by optimizing codon usage within the custom designed gene. In codon optimization embodiments, a codon of low frequency in a species may be replaced by a codon with high frequency, for example, a codon UUA of low frequency may be replaced by a codon CUG of high frequency for leucine. Codon optimization may increase mRNA stability and therefore modify the rate of protein translation or protein folding. Further, codon optimization may customize transcriptional and translational control, modify ribosome binding sites, or stabilize mRNA degradation sites.

The transcription termination sequence is generally situated at the 3 ' end of a gene in a DNA template. The transcription termination sequence provides signal in the newly synthesized mRNA to initiate the process of releasing the mRNA from the transcriptional complex, which can also aid in effective translation of the desired protein product. The insulator sequence generally enhances the efficiency of ribosomal binding or translational initiation. Numerous examples of suitable insulator sequences that exist in the art may be used, including for example, sequences encoding poly-histidine tracts. In some embodiments, the insulator sequence may be determined empirically by inserting spacer sequences around the ribosomal binding site or by optimizing or inserting codons within the N-terminus of the expressed protein.

In some embodiments, the expression sequence comprises a coding sequence, a non-coding sequence, or a combination thereof. The coding sequence comprises a promoter, an open reading frame, and optionally a cap-independent translation element (CITE). The capindependent translation element (CITE) of the coding sequence may be an internal ribosome entry site (IRES), a translation enhancing element (TEE), or a combination thereof. The open reading frame of the coding sequence may be codon-optimized for enhancing translation. The open reading frame may further comprise a tag sequence for purification of the desired protein, an amino -terminal peptide fusion sequence derived from an IRES for enhanced ribosome recognition, or a combination thereof. The expression sequence further comprises a polyA sequence, a transcriptional termination sequence, an insulator sequence, or a combination thereof.

In some embodiments, the expression sequence is a minimalistic expression sequence that is devoid of any extraneous sequences that are required for propagation of a plasmid in a host cell. The minimalistic expression sequence for expressing a desired protein includes, at the minimum, a promoter, a ribosomal binding site, and a translational termination sequence. The minimalistic expression sequence for expressing a desired RNA includes, at the minimum, a promoter, a ribosomal binding site, and a translational termination sequence. In some embodiments, the double-stranded RCA product DNA consists essentially of tandem repeats of a minimalistic expression sequence. In such embodiments, the expression sequence may additionally contain sequences that do not materially affect the in vivo expression of one or more encoded products (e.g., protein, antibody, antigen, mRNA, sgRNA etc.) using the RCA product DNA as a template. For example, it may further include sequences such as a translational enhancer sequence, an insulator sequence, or a transcriptional termination sequence. The minimalistic expression sequence of the RCA product DNA excludes any extraneous sequences, such as antibiotic selection gene, or any other accessory sequences that are required for cloning, selection, screening and/or replication in a host cell. The RCA product may be a linear or a branched concatamer containing tandem repeats of the minimalistic expression sequence. The minimalistic expression sequence of the RCA product DNA may be derived from a DNA mini-circle that includes only minimalistic expression sequence.

The double- stranded concatemeric DNA may further comprise an inosine- containing nucleotide, a Locked Nucleic Acid (LNA) nucleotide, a Peptide Nucleic Acid (PNA) nucleotide, 2-amino-deoxyadenosine, 2-thio-deoxythymidine, a polycation nucleotide, or a combination thereof. In some embodiments, the modified nucleotides, such as inosine-containing nucleotide, a Locked Nucleic Acid (LNA) nucleotide, a Peptide Nucleic Acid (PNA) nucleotide, 2-amino-deoxyadenosine, 2- thio-deoxythymidine, a polycation nucleotide are part of a primer sequence that is employed for rolling circle amplification.

The double-stranded concatemeric DNA may be delivered to a eukaryotic cell by any method, including but not limited to, electroporation, sonoporation, impalefection, transduction, optical transfection, magnetofection, nucleofection, hydrodynamic delivery, heat shock-mediated gene delivery, nanoparticle mediated gene-gun delivery, calcium phosphate- mediated delivery, cationic polymer-mediated delivery, or liposome-mediated delivery.

A variety of methods may be used to prepare a DNA mini-circle template for use with methods of the invention. In some embodiments, a linear DNA template may be circularized to generate a DNA mini-circle template. In one example embodiment, the circularization of the linear DNA template may be effected by an enzymatic reaction, for example, by incubation with a ligation enzyme such as DNA ligase. In some embodiments, the terminal ends of the linear DNA template are hybridized to a nucleic acid sequence such that the terminal ends come in close proximity. Incubating with a ligation enzyme may then effect the circularization of the hybridized linear DNA template to generate a DNA mini-circle. Suitable DNA mini-circle template may also be generated by PCR amplification of a portion of a larger DNA (for example, a genomic DNA, or a DNA from a DNA library) using appropriate PCR primers, followed by circularization of the PCR product. DNA mini-circle may also be generated by chemical synthesis of suitable linear oligonucleotides followed by circularization of the synthesized oligonucleotide. In some embodiments, the synthesized linear oligonucleotides may consist essentially of minimalistic expression sequence and achieve circularization via DNA ligase to generate DNA mini -circle.

Template Molecule

In some embodiments, the RCA amplification product is generated from RCA amplification of a template nucleic acid molecule. In some embodiments, the template nucleic acid molecule comprises a vector comprising a nucleotide sequence encoding an expression product (e.g., protein, antibody, antigen, mRNA, sgRNA, etc.). The vector can have a nucleic acid sequence containing an origin of replication. The vector can be a plasmid, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. The vector can be either a selfreplicating extrachromosomal vector or a vector which integrates into a host genome.

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

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

Expression Vectors

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

Circular and Linear Vectors

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

The vector can be pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing DNA encoding the expression product (e.g., protein, antibody, antigen, mRNA, sgRNA, etc.) and enabling a cell to translate the sequence.

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

Promoter, Intron, Stop Codon, and Polyadenylation Signal

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

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

Excipients and other Components of the Vaccine

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

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

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

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

The vaccine may further comprise a genetic vaccine facilitator agent as described in U.S. Serial No. 021,579 filed April 1, 1994, which is fully incorporated by reference.

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

Method of Vaccination

Also provided herein is a method of treating, protecting against, and/or preventing disease in a subject in need thereof by administering the vaccine to the subject. Administration of the vaccine to the subject can induce or elicit an immune response in the subject. The induced immune response can be used to treat, prevent, and/or protect against disease, for example, pathologies relating to SARS-CoV-2 or influenza virus infection. In one embodiment, the pathology relating to SARS-CoV-2 infection is COVID-19. In one embodiment, the pathology relating to influenza infection is flu.

The induced immune response can include an induced humoral immune response and/or an induced cellular immune response. The humoral immune response can be induced by about 1.5-fold to about 16-fold, about 2-fold to about 12-fold, or about 3-fold to about 10-fold. The induced humoral immune response can include IgG antibodies and/or neutralizing antibodies that are reactive to the encoded expression product (e.g., protein, antibody, antigen, mRNA, sgRNA, etc ). The induced cellular immune response can include a CD8⁺ T cell response, which is induced by about 2-fold to about 30-fold, about 3-fold to about25-fold, or about 4-fold to about 20-fold.

The vaccine dose can be between 1 pg to 10 mg active component/kg body weight/time, and can be 20 pg to 10 mg component/kg body weight/time. The vaccine can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of vaccine doses for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

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

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

The vaccine can be administered by methods well known in the art as described in Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997)); Feigner et al. (U.S. Pat. No. 5,580,859, issued Dec. 3, 1996); Feigner (U.S. Pat. No. 5,703,055, issued Dec. 30, 1997); and Carson et al. (U.S. Pat. No. 5,679,647, issued Oct. 21, 1997), the contents of all of which are incorporated herein by reference in their entirety. The DNA of the vaccine can be complexed to particles or beads that can be administered to an individual, for example, using a vaccine gun. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the expression vector.

The vaccine can be delivered via a variety of routes. Typical delivery routes include parenteral administration, e.g., intradermal, intramuscular or subcutaneous delivery. Other routes include oral administration, intranasal, and intravaginal routes. For the DNA of the vaccine in particular, the vaccine can be delivered to the interstitial spaces of tissues of an individual (Feigner et al., U.S. Pat. Nos. 5,580,859 and 5,703,055, the contents of all of which are incorporated herein by reference in their entirety). The vaccine can also be administered to muscle, or can be administered via intradermal or subcutaneous injections, or transdermally, such as by iontophoresis. Epidermal administration of the vaccine can also be employed. Epidermal administration can involve mechanically or chemically irritating the outermost layer of epidermis to stimulate an immune response to the irritant (Carson et al., U.S. Pat. No. 5,679,647, the contents of which are incorporated herein by reference in its entirety).

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

The vaccine can be a liquid preparation such as a suspension, syrup or elixir. The vaccine can also be a preparation for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e g., injectable administration), such as a sterile suspension or emulsion.

The vaccine can be incorporated into liposomes, microspheres or other polymer matrices (Feigner et al., U.S. Pat. No. 5,703,055; Gregoriadis, Liposome Technology, Vols. Ito III (2nd ed. 1993), the contents of which are incorporated herein by reference in their entirety). Liposomes can consist of phospholipids or other lipids, and can be nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

The vaccine can be administered via electroporation, such as by a method described in U.S. Patent No. 7,664,545, the contents of which are incorporated herein by reference. The electroporation can be by a method and/or apparatus described in U.S. Patent Nos. 6,302,874; 5,676,646; 6,241,701; 6,233,482; 6,216,034; 6,208,893; 6,192,270; 6,181,964; 6,150,148; 6,120,493; 6,096,020; 6,068,650; and 5,702,359, the contents of which are incorporated herein by reference in their entirety. The electroporation may be carried out via a minimally invasive device.

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

The MID may inject the vaccine into tissue without the use of a needle. The MID may inject the vaccine as a small stream or jet with such force that the vaccine pierces the surface of the tissue and enters the underlying tissue and/or muscle. The force behind the small stream or jet may be provided by expansion of a compressed gas, such as carbon dioxide through a micro-orifice within a fraction of a second. Examples of minimally invasive electroporation devices, and methods of using them, are described in published U.S. Patent Application No. 20080234655; U.S. Patent No. 6,520,950; U.S. Patent No. 7,171,264; U.S. Patent No. 6,208,893; U.S. Patent NO. 6,009,347; U.S. Patent No. 6,120,493; U.S. Patent No. 7,245,963; U.S. Patent No. 7,328,064; and U.S. Patent No. 6,763,264, the contents of each of which are herein incorporated by reference.

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

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

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

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

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

The MID may consist of a pulse generator and a two or more-needle vaccine injectors that deliver the vaccine and electroporation pulses in a single step. The pulse generator may allow for flexible programming of pulse and injection parameters via a flash card operated personal computer, as well as comprehensive recording and storage of electroporation and patient data. The pulse generator may deliver a variety of volt pulses during short periods of time. For example, the pulse generator may deliver three 15 volt pulses of 100 ms in duration. An example of such a MID is the Eigen 1000 system by Inovio Biomedical Corporation, which is described in U.S. Patent No. 7,328,064, the contents of which are herein incorporated by reference. The MID may be a CELLECTRA (Inovio Pharmaceuticals, Blue Bell PA) device and system, which is a modular electrode system, that facilitates the introduction of a macromolecule, such as a DNA, into cells of a selected tissue in a body or plant. The modular electrode system may comprise a plurality of needle electrodes; a hypodermic needle; an electrical connector that provides a conductive link from a programmable constant-current pulse controller to the plurality of needle electrodes; and a power source. An operator can grasp the plurality of needle electrodes that are mounted on a support structure and firmly insert them into the selected tissue in a body or plant. The macromolecules are then delivered via the hypodermic needle into the selected tissue. The programmable constant-current pulse controller is activated and constant-current electrical pulse is applied to the plurality of needle electrodes. The applied constant-current electrical pulse facilitates the introduction of the macromolecule into the cell between the plurality of electrodes. Cell death due to overheating of cells is minimized by limiting the power dissipation in the tissue by virtue of constant-current pulses. The Cellectra device and system is described in U.S. Patent No. 7,245,963, the contents of which are herein incorporated by reference.

In one embodiment, the electroporation is performed using 0.1 Amp electric constant current square-wave pulses. In one embodiment, DNA products are injected intramuscularly (IM) into the tibialis anterior muscle. IM immunization is then followed by intramuscular adaptive electroporation. In some embodiments, the intramuscular adaptive electroporation comprises two sets of two pulses at 0.1 Amps, with each set of two pulses lasting 52 milliseconds with a 1 second delay.

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

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

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

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

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

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

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

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

Kit

Provided herein is a kit, which can be used for treating a subject using the method of vaccination described above. In one embodiment, the kit can comprise the vaccine. In one embodiment, the kit can comprise a nucleic acid molecule encoding a RBD antigen of the invention. In one embodiment, the kit can comprise a nucleic acid molecule encoding an HA antigen of the invention. In one embodiment, the kit can comprise a nucleic acid molecule encoding an anti-influenza HA antibody of the invention

The kit can also comprise instructions for carrying out the vaccination method described above and/or how to use the kit. Instructions included in the kit can be affixed to packaging material or can be included as a package insert. While instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” can include the address of an internet site which provides instructions.

EXPERIMENTAL EXAMPLES

The following non-limiting Examples serve to illustrate selected embodiments of the invention. It will be appreciated that variations in proportions and alternatives in elements of the components shown will be apparent to those skilled in the art and are within the scope of embodiments of the present invention.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore, point out specific embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

Example 1 : Development SARS-CoV2 and influenza vaccines with improved potency

The data presented here demonstrate the development of vaccines for influenza and a biologic for SARS-CoV-2 prophylaxis.

To explore whether RCA-amplified plasmid DNA could be immunogenic when delivered using adaptive EP, mice were immunized with influenza H1N1 A/California/04/2009 hemagglutinin (HA) in pVAX vector (HA pVAX), or HA DNA amplified using RCA as a low molecular weight species (HA RCA LMW) or high molecular weight species (HA RCA HMW) (Figure 1 A). Germinal center responses were assessed in the draining lymph nodes 14 days post immunization using flow cytometry. HA RCA DNA species delivered via adaptive EP generated activated T follicular helper cell responses, albeit slightly decreased relative to standard plasmid DNA (Figure IB and Figure 1C). Additionally, HA RCA DNA delivered via adaptive EP also generated both total and HA-specific germinal center B cell responses (Figure 2A and Figure 2B).

To assess whether immunization with RCA DNA using EP could elicit antigenspecific antibody responses. Mice were immunized once as in Figure 1A and HA-specific IgG responses were assessed in the serum over time. Robust HA-specific antibody titers to flu HA were observed over time (Figure 3A and Figure 3B).

To determine whether RCA DNA delivered via adaptive EP could generate T cell responses, mice were immunized once with 2pg HA pVAX, HA RCA LMW, or HA RCA HMW and T cell responses were assessed in the spleen at 14 days post immunization (Figure 4A). RCA DNA species generated robust T cell responses as measured using JF y ELISpot (Figure 4B). Additionally, using flow cytometry to profile effector CD8+ and CD4+ T cell populations, immunization with RCA DNA using EP demonstrated strong CD107a+, ZFNy+, and TNFa+ CD8+ T cell responses (Figure 5A-Figure 5C). Similarly, among CD4 T cell responses, RCA DNA delivered using EP generated strong IFNy+, IL2+, and TNFa+ effector CD4+ T cells (Figure 6A-Figure 6D).

To determine whether there is a dose response using increasing amounts of HA RCA species, mice were immunized with increasing doses of HA RCA LMW (2 pg, 10 pg, and 25 pg) and adaptive immune responses were assessed 14 days post immunization in the draining lymph nodes and spleen (Figure 7A). Among GC populations, increasing doses of RCA LMW improved activated Tfh cell responses, total GC B cell responses, and HA-specific GC B cell responses (Figure 7B). T cell responses, as measured using JFNy ELISpot, demonstrated improved T cell responses at lOpg and 25 pg RCA relative to 2pg. However, using flow cytometry, the lOpg dose of HA RCA LMW dramatically enhanced CD8+ T cell responses as measured through intracellular flow cytometry of various effector molecules/cytokines (Figure 9A-Figure 9D). This pattern remained the same when absolute numbers of cytokine positive CD8+ cells were quantified (Figure lOA-Figure 10C). The lOpg dose of HA RCA LMW delivered using EP also enhanced cytokine-secreting effector CD4+ T cell responses as measured through intracellular flow cytometry (Figure 11 A-Figure 1 1 C). Interestingly, increases among total effector CD8+ and CD4+ T cell populations were also observed, irrespective of an effector cytokine (Figure 12A and Figure 12B). Taken together, these data demonstrate that RCA- amplified DNA can be immunogenic when delivered using adaptive EP.

The methods used for the experiments are now described.

Rolling circle amplification of synthetic DNA fragments and plasmids.

For RCA propagation of synthetic DNA fragments, DNA gBlocks were resuspended in TE according to IDT recommendations and approximately 80-1 OOng were digested with BamHI and Bglll, followed by ligation using T4 DNA ligase, in a thermocycler set to cycle between 7.5 °C and 37 °C at 30 sec intervals to facilitate intramolecular ligation. After 200 cycles, the temperature was raised to 70 °C for 10 minutes to heat-inactivate T4 DNA ligase. Exonuclease I and exonuclease III were then added and incubated at 37 °C for 1 hr to degrade any non-circular DNA, followed by heat-inactivation at 80 °C for 20 min. The resulting DNA minicircles were amplified using a modified illustraTM Single Cell GenomiPhiTM DNA Amplification kit (Cytiva, formerly GE Healthcare Life Sciences) in which the random hexamer primers were optionally replaced with proprietary LNA-containing hexamers and 1-thio-dNTPs were optionally spiked into the Amplification Mix to further thioate the RCA product. While not essential, these modifications tend to improve the DNA concentration of the RCA product and corresponding translational yield in cell-free extract. Minicircle RCA reactions were incubated isothermally overnight, then heat-inactivated at 60 °C for 20 minutes. The resulting non-purified double-stranded RCA products were quantified using Quant-iT™ dsDNA High-Sensitivity Assay Kit (Thermo Fisher Scientific) in which several 1 LIL samplings of minicircle RCA product were measured to normalize for sub-sampling error. Finally, a small portion of each minicircle RCA product was digested with PstI enzyme to confirm accurate restriction fragment analysis by gel electrophoresis. Unpurified minicircle RCA products were subsequently stored at 4 °C until use. Similarly, plasmids were amplified using illustra TempliPhi DNA amplification kit (Cytiva, formerly GE Healthcare Life Sciences) and the unpurified product was stored at 4 °C until use. ELISA

ELISA assay to measure serum antibody titers. Plates are coated with H1N1 A/California/07/2009 HA protein (1 pg/mL) overnight at 4°C. The following day, plates are washed and blocked in a solution of 5% milk in PBS. Plates are subsequently washed and incubated with serial dilutions of primary mouse serum for 1 hour at room temperature. Mouse serum is washed off and plates are incubated with HRP-conjugated goat anti-mouse IgG h+1 for 1 hour at room temperature. Plates are washed and subsequently developed with TMB substrate, and the reaction is stopped with IM sulfuric acid. Plates are read at optical density (OD) 450nm and 570nm. Endpoint titers are calculated against naive serum, and represent a titer that is above the average + 4* (Standard deviation).

Flow cytometry for germinal centers.

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

Tfh: lymphocytes single cells live CD19- CD4+ CD44+ PD-lhi CXCR5+

GC B: lymphocytes single cells live CD4- CD19+ CD38- Fas+

Flu specific GC B: lymphocytes single cells live CD4- CD19+ CD38- Fas+ Flu HA++ (two colors, APC and FITC, see above)

ELISpot.

Splenocytes were stimulated with peptides that span the entire H1N1 A/California/07/2009 protein in five pools at 5 pg mL'¹ for 18 hours at 37 degrees C. Mouse IFNy ELISpot plates (Mabtech) were blocked for at least 30 min in RIO media before application of cell suspension. After 18 hours, the plates were subsequently washed in PBS and incubated with biotinylated anti-mouse IFNY f°^r 2 hours at RT. Plates were washed and subsequently incubated with ALP-conjugated Streptavidin for 1 hour at RT. Plates were washed and incubated with filtered BCIP reagent until development of distinct spots. After spot development, plates were washed with tap water and left to dry before image acquisition on a Mabtech Iris imager.

Flow cytometry - Intracellular cytokine staining.

Single cell suspensions from spleens were prepared as described before and stimulated with peptides that span the SARS-CoV-2 Spike protein at 5 pg/mL for 6 hours at 37°C in the presence of 1 :500 protein transport inhibitor (ThermoFisher) and anti-mouse CD107a-FITC (Biolegend). The cells were then incubated with live/dead Fixable Aqua Dead Cell Stain Kit (Biolegend) for 10 minutes at room temperature, and surface stained with cocktail of surface antibodies (CD4, CD8, CD62L, CD44) at RT for 30 minutes. The cells were then fixed and permeabilized according to manufacturer’s instructions for BD Cytoperm/Cytofix kit and stained with a cocktail of intracellular cytokine markers (IFNy, TNFa, IL-2) at 4°C for 30 minutes. Cells were subsequently washed and resuspended in FACS buffer before acquisition on a FACSymphony A3. Data was analyzed using FlowJo. To analyze, cells were “gated” on lymphocytes, single cells, live, CD4- CD8+ CD44+ CD62L- before looking at effector cytokines/ other molecules, and are reported as a frequency of cytokine+ of effector (CD44+ CD62L-).

Example 2: Delivery of a biologic using RCA

It was then examined whether RCA could effectively generate a biologic in vivo. A characterized anti -influenza HA head mAb, 2-12C, was used. Two plasmids or RCA molecules, one encoding the 2-12C heavy chain and the other 2-12C light chain, were coformulated and delivered intramuscularly. Serum human IgG levels were monitored over time. Productive secretion of a human antibody into the serum was observed when 2-12C was delivered using RCA DNA (Figure 13). Although to a lower level than plasmid, these data demonstrate that separate RCA molecules could be delivered to the same cell in vivo to potentiate secretion of a biologic.

The methods used for the experiments are now described.

Mouse immunizations Mice were immunized with a total of lOOpg of plasmid or RCA DNA encoding human mAh 2-12C (50pg each heavy chain and light chain) formulated with 12U hyaluronidase per site and bled sequentially over time. At immunization, mice were also administered 200pg of each anti-mouse CD8 (Clone YTS169.4, BioXCell) and anti-mouse CD4 (Clone GK1.5, BioXCell) intraperitoneally.

Human IgG quantification ELISA

Corning half-area high binding ELISA plates were coated with 5 pg mL ¹ goat anti-human IgG-Fc (Bethyl Laboratories, Montgomery, TX, USA) overnight at 4 °C. The following day, each plate was washed with PBS (Corning Inc., Corning, NY, USA) + 0.01% Tween-20 (ThermoFisher, Waltham, MA, USA) (PBS-T) four times (4x). Plates were then blocked with 5% milk in PBS for 2 h at RT. Upon completion of blocking, plates were washed again 4* with PBS-T and samples diluted in 1% NCS in PBS-T were transferred to plates for a 1 h incubation at RT. A standard curve was generated using purified human IgGic (Bethyl Laboratories, Montgomery, TX, USA). Plates were subsequently washed and goat anti-human IgG-Fc HRP-conjugated secondary antibody (minimal cross-reactivity; Bethyl Laboratories) was diluted to 1 : 10,000 and transferred onto plates for 1 h at RT. Plates are washed and subsequently developed with TMB substrate, and the reaction is stopped with IM sulfuric acid. Plates are read at optical density (OD) 450nm and 570nm. Samples are then interpolated to the standard curve to quantify human IgG in serum.

SEQUENCES:

SARS-CoV-2_RBD nucleotide (SEQ ID NO:1) (Leader sequence is underlined)

ATGGACTGGACTTGGATTCTGTTCCTGGTCGCAGCAGCCACTCGGGTGCATAGCCGC

GTGCAGCCCACTGAAAGCATTGTGAGATTCCCTAACATCACCAATCTGTGCCCATTC

GGCGAGGTGTTTAACGCCACACGGTTCGCCAGCGTGTACGCCTGGAACAGGAAGAG

AATCTCCAATTGCGTGGCCGACTACTCTGTGCTGTATAATAGCGCCTCCTTCTCTACC

TTTAAGTGCTACGGCGTGTCTCCCACCAAGCTGAACGACCTGTGCTTCACAAACGTG

TACGCCGACAGCTTTGTGATCAGGGGCGATGAGGTGAGACAGATCGCACCAGGACA

GACCGGCAAGATCGCAGACTACAACTATAAGCTGCCCGACGATTTCACAGGCTGCG

TGATCGCCTGGAATAGCAACAATCTGGATTCCAAAGTGGGCGGCAACTACAATTATC

TGTACAGGCTGTTCAGAAAGAGCAACCTGAAGCCCTTTGAGCGGGACATCTCTACCG

AGATCTACCAGGCCGGCAGCACACCTTGCAACGGCGTGGAGGGCTTCAATTGTTACT

TTCCACTGCAGTCTTATGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCTTATC

GCGTGGTGGTGCTGAGCTTTGAGCTGCTGCACGCACCAGCAACCGTGTGCGGACCTA

AGAAGAGCACAAACCTGGTGAAGAATAAG

(SEQ ID N0:2) amino acid (Leader sequence is underlined)

MDWTWILFLVAAATRVHSRVOPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRIS

NCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKI

ADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGS

TPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVK

NK

Flu_CA09_HA nucleotide (SEQ ID NO:3) (Leader sequence is underlined)

ATGGACTGGACTTGGATTCTTTTTCTCGTGGCTGCAGCAACTAGGGTGCACAGCATG AAGGCCATCCTGGTGGTGCTGCTGTACACCTTCGCCACAGCCAATGCTGACACCCTG

TGCATCGGCTACCACGCCAACAACAGCACAGACACAGTGGACACAGTGCTGGAGAA

GAACGTGACAGTGACCCACTCTGTCAACCTGCTGGAGGACAAGCACAACGGCAAGC

TGTGTAAACTTCGAGGAGTTGCTCCTCTGCACCTGGGCAAATGTAACATTGCTGGCT

GGATTCTGGGCAACCCTGAATGTGAGAGCCTGAGCACAGCCAGCAGCTGGAGCTAC

ATCGTGGAGACCCCTTCTTCTGATAATGGCACCTGCTACCCTGGAGACTTCATCGAC

TATGAAGAGCTGAGAGAGCAGCTGTCCTCTGTTTCTTCTTTTGAAAGATTTGAAATC

TTCCCCAAGACCAGCAGCTGGCCCAACCACGACTCTAACAAAGGAGTCACAGCTGC

CTGTCCTCATGCTGGAGCCAAGTCCTTCTACAAGAACCTCATCTGGCTGGTGAAGAA

GGGCAACAGCTACCCAAAGCTGTCCAAGTCCTACATCAACGACAAGGGCAAGGAGG

TGCTGGTGCTGTGGGGCATCCACCACCCTTCTACATCTGCTGACCAGCAGAGCCTGT

ACCAGAATGCTGATGCCTACGTCTTTGTGGGCAGCAGCAGATACAGCAAGAAGTTC

AAGCCTGAGATCGCCATCAGACCTAAAGTTAGAGATCAAGAAGGACGCATGAACTA

CTACTGGACCCTGGTGGAGCCTGGAGACAAGATCACCTTTGAGGCCACAGGAAACC

TGGTGGTGCCAAGATATGCCTTCGCCATGGAGAGAAATGCTGGCAGCGGCATCATC

ATCTCTGACACACCTGTGCACGACTGCAACACCACCTGCCAGACACCTAAAGGAGC

CATCAACACCAGCCTGCCTTTCCAGAACATCCACCCCATCACCATCGGCAAATGTCC

TAAATACGTGAAGAGCACCAAGCTGCGGCTGGCCACAGGCCTGAGAAACATCCCTT

CCATCCAGAGCAGAGGCCTGTTTGGAGCCATCGCCGGCTTCATCGAGGGCGGCTGG

ACAGGCATGGTGGATGGCTGGTACGGCTACCACCACCAGAATGAGCAGGGCAGCGG

CTATGCTGCTGACCTGAAGAGCACCCAGAACGCCATCGATGAAATCACCAACAAGG

TGAACAGCGTCATCGAGAAGATGAACACCCAGTTCACAGCTGTGGGCAAGGAGTTC

AACCACCTGGAGAAGAGAATTGAAAACCTGAACAAGAAGGTGGATGATGGCTTCCT

GGACATCTGGACCTACAATGCTGAACTGCTGGTGCTGCTGGAAAACGAAAGAACAC

TGGACTACCACGACAGCAATGTGAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTG

AAGAACAACGCCAAGGAAATTGGAAATGGCTGCTTTGAATTCTACCACAAGTGTGA

CAACACCTGCATGGAATCTGTGAAGAATGGCACCTACGACTACCCAAAGTACTCTG

AAGAAGCCAAGCTGAACAGAGAGGAAATCGATGGTGTGAAGCTGGAGAGCACCAG

AATCTACCAGATCCTGGCCATCTACAGCACAGTGGCCAGCAGCCTGGTGCTGGTGGT

GTCCCTGGGCGCCATCTCCTTCTGGATGTGCAGCAACGGCAGCCTGCAGTGCAGAAT CTGCATC (SEQ ID N0:4) amino acid (Leader sequence is underlined)

MDWTWILFLVAAATRVHSMKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKN

VTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVET P S SDNGTC YPGDFID YEELREQLS S VS SFERFEIFPKT S S WPNHD SNKGVT AACPHAGAK SFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVF VGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMER NAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIP SIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVN SVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHD SNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLN REEIDGVKLESTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICI

SARS-CoV-2 nucleotide Wild-type RBD 24mer nanoparticle insert (SEQ ID NO:5)

ATGGATTGGACATGGATACTGTTCCTGGTGGCCGCCGCCACCAGAGTGCACTCTCTG

AGGTTCGGCATCGTGGCCTCCAGGGCCAATCACGCCCTGGTGGGCGGCTCCGGCGG CAACATCACCAATCTGTGCCCTTTCGGCGAGGTGTTTAACGCCACCAGGTTTGCCTC CGTGTACGCCTGGAACCGCACAAGGATCAGCAACTGCGTGGCCGATTATAGCGTGC TGTACAATTCTGCCAGCTTCTCTACCTTTAAGTGCTACGGCGTGAACCCTACCAAGCT GAACGATCTGTGCTTCACCAACGTGTATGCCGATAGCTTCGTGATCCGGGGCGATGA

GGTGCGCCAGATCGCCCCTGGCCAGACAGGCAAGATCGCCGACTACAACTATAAGC TGCCAGATAATTTCACCGGCTGCGTGATCGCCTGGAATTCCAACAATCTGGACTCTA

AGGTGGGCGGCAACTACAACTATCTGTACCGGCTGTTTCGCAAGTCCAACCTGTCTC CATTCGAGAGAGACATCTCCACAGAGATCTATCAGGCCGGCTCCACACCTTGTAACG

GCACCGAGGGCTTCAACTGCTACTTCCCTCTGCAGAGCTATGGCTTCCAGCCTACCA ATGGCGTGGGCTATCAGCCCTACCGGGTGGTGGTGCTGTCTTTTGAGCTGCTGCACG

CCCCTGCCACAGTGTGCGGCCCTGGCGGCTCTGGCGGCTCCGGCGGCTCTGGCGGCA GCGGCGGCGGCCTGAGCAAGGATATCATCAAGCTGCTGAATGAACAGGTCAACAAG GAAATGCAGAGCAGCAACCTGTACATGTCCATGAGCTCCTGGTGCTATACCCACTCT CTGGACGGAGCAGGCCTGTTCCTGTTTGATCACGCCGCCGAGGAGTACGAGCACGC CAAGAAGCTGATCATCTTCCTGAATGAGAACAATGTGCCCGTGCAGCTGACCTCTAT CAGCGCCCCTGAGCACAAGTTCGAGGGCCTGACACAGATCTTTCAGAAGGCCTACG AGCACGAGCAGCACATCTCCGAGTCTATCAACAATATCGTGGACCACGCCATCAAG TCCAAGGATCACGCCACATTCAACTTTCTGCAGTGGTACGTGGCCGAGCAGCACGA GGAGGAGGTGCTGTTTAAGGACATCCTGGATAAGATCGAGCTGATCGGCAACGAGA ATCACGGGCTGTATCTGGCCGACCAGTATGTGAAGGGCATCGCTAAAAGCAGGAAA

TCA

(SEQ ID N0:6) (Leader sequence is underlined)

MDWTWILFLVAAATRVHSLRFGIVASRANHALVGGSGGNITNLCPFGEVFNATRFASV YAWNRTRISNCVADYSVLYNSASFSTFKCYGVNPTKLNDLCFTNVYADSFVIRGDEVRQ IAPGQTGKIADYNYKLPDNFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLSPFERDI STEIYQAGSTPCNGTEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGP GGSGGSGGSGGSGGGLSKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLF

DHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVD HAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSR KS

2-12C_IgG_HC nucleotide (SEQ ID NO:7)

ATGGACTGGACTTGGAGAATCCTCTTTCTGGTCGCTGCTGCCACCGGTACACACGCA GAGGTGCAGCTGGTCCAGAGCGGTGCTGAGGTGAAGAAACCTGGGGAAAGTCTGAA AATCAGTTGCAAAGGATCTGGAAATGGATTCACCACATACTGGATTGGCTGGGTGA GGCAGGTGCCTGGAAAGGGCCTGGAGTGGATGGGCATTATCTATCCAGATGATAGC GACACCCGTTACTCTCCCTCTTTCCAGGGTCAAGTGTCCATCTCTGCTGACAAAAGC

ATTTCAACCGCCTTTCTGCAGTGGAGCTCTCTGAAGGCCAGTGACACAGCTATGTAC TTCTGTGCCCGGCTGGGAGATGTGGAAACCGCCATGGTGGGCCAGGATGCCTTCCAC ATCTGGGGCCAGGGCACCATGGTGACTGTATCCTCGGCGAGCACCAAGGGACCGAG CGTGTTTCCACTGGCGCCCAGCTCGAAGTCCACTAGCGGGGGCACTGCAGCTCTGGG CTGTCTGGTGAAAGACTATTTTCCTGAGCCAGTGACAGTCAGCTGGAACAGTGGGGC

CCTGACGTCAGGCGTCCACACATTCCCAGCTGTGCTGCAGAGTTCTGGCCTCTACTC ACTGTCCTCCGTGGTAACCGTACCCTCCAGCAGCCTGGGTACCCAGACTTACATCTG TAACGTCAACCACAAGCCTTCCAACACAAAGGTGGACAAGAAGGTCGAGCCAAAGA GCTGTGACAAGACCCACACGTGCCCACCTTGCCCTGCCCCAGAGCTGCTGGGTGGGC CGAGTGTCTTCCTCTTCCCCCCTAAACCCAAGGACACCTTGATGATTAGCAGAACAC CGGAAGTTACATGTGTGGTTGTGGATGTAAGCCATGAAGACCCTGAGGTTAAGTTTA ACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAAACAAAGCCTCGGGAGGAG CAATATAACTCCACGTACAGGGTGGTCTCAGTGCTAACGGTGTTACACCAAGACTGG CTCAACGGCAAGGAGTACAAATGCAAAGTGAGCAATAAAGCCCTACCAGCCCCCAT CGAAAAGACCATATCTAAGGCAAAGGGCCAGCCCAGAGAGCCCCAGGTCTACACAC TGCCACCTTCTAGAGATGAGCTCACCAAGAACCAGGTTAGCCTGACCTGCCTGGTGA AAGGCTTTTATCCTTCAGACATCGCAGTGGAATGGGAATCGAACGGGCAACCCGAA AACAACTACAAGACTACCCCTCCAGTCCTGGACAGTGATGGGAGCTTCTTCCTGTAC AGCAAACTGACCGTGGATAAGTCGCGCTGGCAGCAGGGCAATGTGTTCTCCTGCAG CGTCATGCATGAGGCCCTACACAACCATTACACGCAGAAGTCTCTTAGCCTCTCGCC TGGGAAG

2-12C_IgG_HC (SEQ ID NO: 8)

MDWTWRILFLVAAATGTHAEVQLVQSGAEVKKPGESLKISCKGSGNGFTTYWIGWVR QVPGKGLEWMGIIYPDDSDTRYSPSFQGQVSISADKSISTAFLQWSSLKASDTAMYFCAR LGDVETAMVGQDAFHIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK**

2-12C_IgG_LC (SEQ ID NO:9)

ATGGTGCTGCAGACTCAGGTGTTTATCTCCCTCCTCCTCTGGATCAGCGGTGCCTACG GGGAAATAGTGCTGACTCAAAGCCCTGGGACCCTGAGTCTGTCACCAGGAGACAGA GCTACCTTGAGCTGCCGGGCCAGTCAGTCTGTGTCCTCCTCCTTCCTAGCCTGGTACC AGCAGAAGCCAGGCCAGGCACCCCGGCTGCTGATGTACGGAGCCAGCAGGAGAGC GACAGGCATCCCTGACCGCTTTTCGGGCTCAGGAAGCGGCACAGACTTCACTCTGAC CATCTCCAGATTGGAGCCAGAAGACTTTGCTGTGTACTACTGCCAGCAATATGACAG

CTCCCCCTTCACGTTTGGAGGCGGCACCAAGGTGGAGATCAAGAGGACAGTGGCGG

CTCCCAGCGTTTTCATTTTCCCTCCCTCTGATGAACAGCTCAAGAGTGGCACCGCTTC

TGTGGTCTGCCTGCTTAACAACTTCTACCCAAGAGAAGCCAAGGTCCAGTGGAAAGT

CGATAATGCCTTACAGAGCGGCAACAGTCAGGAGTCTGTGACAGAGCAGGACAGCA

AAGATTCCACCTACAGTCTGTCTTCCACACTGACCCTTAGCAAGGCTGACTATGAGA

AGCATAAGGTTTATGCCTGTGAGGTGACCCACCAGGGCCTGTCCTCTCCTGTGACCA

AATCGTTCAATCGTGGAGAGTGT

(SEQ ID NO: 10)

MVLQTQVFISLLLWISGAYGEIVLTQSPGTLSLSPGDRATLSCRASQSVSSSFLAWYQQK

PGQAPRLLMYGASRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDSSPFTFG

GGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN

SQES VTEQD SKD ST YSLS STLTLSKAD YEKHKVYACEVTHQGLS SPVTKSFNRGEC * *

Leader Sequence (SEQ ID NO:11)

MDWTWILFLVAAATRVHS

SARS-CoV-2 Wild-type RBD 24mer nanoparticle (SEQ ID NO: 12) (Insert is bolded. Leader sequence is underlined)

GCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTA

TTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGT

TACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT

GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACG

TCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCA

TATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTA

TGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTC

ATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGG

TTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTT

GGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACG

CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCT AACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGA

GACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCGCCACC

ATGGATTGGACATGGATACTGTTCCTGGTGGCCGCCGCCACCAGAGTGCACTC

TCTGAGGTTCGGCATCGTGGCCTCCAGGGCCAATCACGCCCTGGTGGGCGGCT

CCGGCGGCAACATCACCAATCTGTGCCCTTTCGGCGAGGTGTTTAACGCCACC

AGGTTTGCCTCCGTGTACGCCTGGAACCGCACAAGGATCAGCAACTGCGTGGC

CGATTATAGCGTGCTGTACAATTCTGCCAGCTTCTCTACCTTTAAGTGCTACGG

CGTGAACCCTACCAAGCTGAACGATCTGTGCTTCACCAACGTGTATGCCGATA

GCTTCGTGATCCGGGGCGATGAGGTGCGCCAGATCGCCCCTGGCCAGACAGGC

AAGATCGCCGACTACAACTATAAGCTGCCAGATAATTTCACCGGCTGCGTGATC

GCCTGGAATTCCAACAATCTGGACTCTAAGGTGGGCGGCAACTACAACTATCT

GTACCGGCTGTTTCGCAAGTCCAACCTGTCTCCATTCGAGAGAGACATCTCCAC

AGAGATCTATCAGGCCGGCTCCACACCTTGTAACGGCACCGAGGGCTTCAACT

GCTACTTCCCTCTGCAGAGCTATGGCTTCCAGCCTACCAATGGCGTGGGCTATC

AGCCCTACCGGGTGGTGGTGCTGTCTTTTGAGCTGCTGCACGCCCCTGCCACA

GTGTGCGGCCCTGGCGGCTCTGGCGGCTCCGGCGGCTCTGGCGGCAGCGGCG

GCGGCCTGAGCAAGGATATCATCAAGCTGCTGAATGAACAGGTCAACAAGGAA

ATGCAGAGCAGCAACCTGTACATGTCCATGAGCTCCTGGTGCTATACCCACTCT

CTGGACGGAGCAGGCCTGTTCCTGTTTGATCACGCCGCCGAGGAGTACGAGCA

CGCCAAGAAGCTGATCATCTTCCTGAATGAGAACAATGTGCCCGTGCAGCTGA

CCTCTATCAGCGCCCCTGAGCACAAGTTCGAGGGCCTGACACAGATCTTTCAG

AAGGCCTACGAGCACGAGCAGCACATCTCCGAGTCTATCAACAATATCGTGGA

CCACGCCATCAAGTCCAAGGATCACGCCACATTCAACTTTCTGCAGTGGTACGT

GGCCGAGCAGCACGAGGAGGAGGTGCTGTTTAAGGACATCCTGGATAAGATCG

AGCTGATCGGCAACGAGAATCACGGGCTGTATCTGGCCGACCAGTATGTGAAG

GGCATCGCTAAAAGCAGGAAATCATGATAACTCGAGTCTAGAGGGCCCGTTTAAA

CCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTC

CCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAAT

GAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTG

GGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATG

CGGTGGGCTCTATGGCTTCTACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATT GCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGG

CTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGCTCTGATCAAGAGACA

GGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCC

GCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCT

GATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACC

GACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCT

GGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAA

GGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTG

CTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTG

ATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGT

ACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGG

GCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGG

ATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCC

GCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACA

TAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCT

TCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCT

TCTTGACGAGTTCTTCTGAATTATTAACGCTTACAATTTCCTGATGCGGTATTTTCTC

CTTACGCATCTGTGCGGTATTTCACACCGCATCAGGTGGCACTTTTCGGGGAAATGT

GCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATG

AGACAATAACCCTGATAAATGCTTCAATAATAGCACGTGCTAAAACTTCATTTTTAA

TTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAA

CGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCT

TGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTA

CCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACT

GGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGC

CACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTA

CCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGA

TAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCC

CAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAG

AAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAG

GGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTT ATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTC

AGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGG

CCTTTTGCTGGCCTTTTGCTCACATGTTCTT

2-12C_IgG_HC_pVaxl (SEQ ID NO: 13) (Gene insert in bold, vector the rest)

GCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTA

TTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGT

TACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT

GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACG

TCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCA

TATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTA

TGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTC

ATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGG

TTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTT

GGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACG

CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCT

AACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGA

GACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCGCCACC

ATGGACTGGACTTGGAGAATCCTCTTTCTGGTCGCTGCTGCCACCGGTACACA

CGCAGAGGTGCAGCTGGTCCAGAGCGGTGCTGAGGTGAAGAAACCTGGGGAA

AGTCTGAAAATCAGTTGCAAAGGATCTGGAAATGGATTCACCACATACTGGATT

GGCTGGGTGAGGCAGGTGCCTGGAAAGGGCCTGGAGTGGATGGGCATTATCT

ATCCAGATGATAGCGACACCCGTTACTCTCCCTCTTTCCAGGGTCAAGTGTCCA

TCTCTGCTGACAAAAGCATTTCAACCGCCTTTCTGCAGTGGAGCTCTCTGAAGG

CCAGTGACACAGCTATGTACTTCTGTGCCCGGCTGGGAGATGTGGAAACCGCC

ATGGTGGGCCAGGATGCCTTCCACATCTGGGGCCAGGGCACCATGGTGACTGT

ATCCTCGGCGAGCACCAAGGGACCGAGCGTGTTTCCACTGGCGCCCAGCTCGA

AGTCCACTAGCGGGGGCACTGCAGCTCTGGGCTGTCTGGTGAAAGACTATTTT

CCTGAGCCAGTGACAGTCAGCTGGAACAGTGGGGCCCTGACGTCAGGCGTCCA

CACATTCCCAGCTGTGCTGCAGAGTTCTGGCCTCTACTCACTGTCCTCCGTGGT

AACCGTACCCTCCAGCAGCCTGGGTACCCAGACTTACATCTGTAACGTCAACCA CAAGCCTTCCAACACAAAGGTGGACAAGAAGGTCGAGCCAAAGAGCTGTGACA

AGACCCACACGTGCCCACCTTGCCCTGCCCCAGAGCTGCTGGGTGGGCCGAGT

GTCTTCCTCTTCCCCCCTAAACCCAAGGACACCTTGATGATTAGCAGAACACCG

GAAGTTACATGTGTGGTTGTGGATGTAAGCCATGAAGACCCTGAGGTTAAGTT

TAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAAACAAAGCCTCGGG

AGGAGCAATATAACTCCACGTACAGGGTGGTCTCAGTGCTAACGGTGTTACAC

CAAGACTGGCTCAACGGCAAGGAGTACAAATGCAAAGTGAGCAATAAAGCCCT

ACCAGCCCCCATCGAAAAGACCATATCTAAGGCAAAGGGCCAGCCCAGAGAGC

CCCAGGTCTACACACTGCCACCTTCTAGAGATGAGCTCACCAAGAACCAGGTT

AGCCTGACCTGCCTGGTGAAAGGCTTTTATCCTTCAGACATCGCAGTGGAATG

GGAATCGAACGGGCAACCCGAAAACAACTACAAGACTACCCCTCCAGTCCTGG

ACAGTGATGGGAGCTTCTTCCTGTACAGCAAACTGACCGTGGATAAGTCGCGC

TGGCAGCAGGGCAATGTGTTCTCCTGCAGCGTCATGCATGAGGCCCTACACAA

CCATTACACGCAGAAGTCTCTTAGCCTCTCGCCTGGGAAGTGATAACTCGAGTC

TAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCC

ATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACT

GTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCT

ATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATA

GCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTACTGGGCGGTTTTATGGAC

AGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCT

GCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCA

AGCTCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATT

GCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACA

ACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCC

GGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGC

AGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGT

TGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATC

TCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGC

GGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATC

GCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTG

GACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAG CATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATAT

CATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGC

GGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGG

CGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCG

CATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAATTATTAACGCTTACAATTTC

CTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATCAGGTGGC

ACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAA

ATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAGCACGTGC

TAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCAT

GACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAA

GATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACA

AAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTT

TTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTG

TAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCT

CTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGG

TTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGG

TTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTAC

AGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTAT

CCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAA

ACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATT

TTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCT

TTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTT

2-12C_IgG_LC_pVaxl (SEQ ID NO: 14) (Gene insert in bold, vector the rest)

GCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTA

TTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGT

TACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT

GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACG

TCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCA

TATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTA

TGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTC ATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGG

TTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTT

GGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACG

CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCT

AACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGA

GACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCGCCACC

ATGGTGCTGCAGACTCAGGTGTTTATCTCCCTCCTCCTCTGGATCAGCGGTGCC

TACGGGGAAATAGTGCTGACTCAAAGCCCTGGGACCCTGAGTCTGTCACCAGG

AGACAGAGCTACCTTGAGCTGCCGGGCCAGTCAGTCTGTGTCCTCCTCCTTCCT

AGCCTGGTACCAGCAGAAGCCAGGCCAGGCACCCCGGCTGCTGATGTACGGAG

CCAGCAGGAGAGCGACAGGCATCCCTGACCGCTTTTCGGGCTCAGGAAGCGGC

ACAGACTTCACTCTGACCATCTCCAGATTGGAGCCAGAAGACTTTGCTGTGTAC

TACTGCCAGCAATATGACAGCTCCCCCTTCACGTTTGGAGGCGGCACCAAGGT

GGAGATCAAGAGGACAGTGGCGGCTCCCAGCGTTTTCATTTTCCCTCCCTCTG

ATGAACAGCTCAAGAGTGGCACCGCTTCTGTGGTCTGCCTGCTTAACAACTTCT

ACCCAAGAGAAGCCAAGGTCCAGTGGAAAGTCGATAATGCCTTACAGAGCGGC

AACAGTCAGGAGTCTGTGACAGAGCAGGACAGCAAAGATTCCACCTACAGTCT

GTCTTCCACACTGACCCTTAGCAAGGCTGACTATGAGAAGCATAAGGTTTATGC

CTGTGAGGTGACCCACCAGGGCCTGTCCTCTCCTGTGACCAAATCGTTCAATC

GTGGAGAGTGTTGATAACTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCC

TCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCT

TGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCAT

CGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCA

AGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATG

GCTTCTACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCG

CCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCA

AGGATCTGATGGCGCAGGGGATCAAGCTCTGATCAAGAGACAGGATGAGGATCGTT

TCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGA

GGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGT

TCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTG

CCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGC GTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTA TTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAA GTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGC

CCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGC

CGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCG

AACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACC CATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTC

ATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACC

CGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTAC GGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCT

TCTGAATTATTAACGCTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTG

CGGTATTTCACACCGCATCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTA

TTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTG

ATAAATGCTTCAATAATAGCACGTGCTAAAACTTCATTTTTAATTTAAAAGGATCTA GGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTC

CACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTT

CTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGT TTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCG

CAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAAC TCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCA

GTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAG

GCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAAC GACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTC

CCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGA

GCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTT TCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCT

ATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTT GCTCACATGTTCTT

It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the invention, which is defined solely by the appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the invention, may be made without departing from the spirit and scope thereof.

Claims

CLAIMS What is claimed is:

1. An immunogenic composition comprising a rolling circle amplification (RCA) product, wherein the RCA product comprises a nucleic acid molecule encoding multiple copies of a disease-associated antigen, a disease-associated antibody, or a fragment thereof.

2. The immunogenic composition of claim 1, wherein the disease-associated antigen comprises a SARS-CoV-2 spike protein receptor binding domain (RBD), wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: a. a nucleotide sequence encoding a peptide comprising an amino acid sequence having at least about 90% identity over an entire length of SEQ ID NO:2 or SEQ ID NO:6; and b. a nucleotide sequence encoding a peptide comprising an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:6.

3. The immunogenic composition of claim 2, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: a. a nucleotide sequence having at least about 90% identity over an entire length of the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO:5; and b. a nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO: 5.

4. The immunogenic composition of claim 1, wherein the disease-associated antigen comprises a hemagglutinin (HA) antigen, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: a. a nucleotide sequence encoding a peptide comprising an amino acid sequence having at least about 90% identity over an entire length of SEQ ID NO 4; b. a nucleotide sequence encoding a peptide comprising an amino acid sequence of SEQ ID NO:4.

5. The immunogenic composition of claim 4, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: a. a nucleotide sequence having at least about 90% identity over an entire length of the nucleic acid sequence of SEQ ID NO:3; and b. a nucleic acid sequence of SEQ ID NO:3.

6. The immunogenic composition of claim 1, wherein the disease-associated antibody comprises an anti-influenza HA antibody, wherein the nucleic acid molecule comprises a nucleotide sequence encoding at least one of a heavy chain or a light chain of the anti-influenza HA antibody selected from the group consisting of: a. a nucleotide sequence encoding an anti-influenza HA heavy chain comprising an amino acid sequence having at least about 90% identity over an entire length of SEQ ID NO: 8; b. a nucleotide sequence encoding an anti-influenza HA light chain comprising an amino acid sequence having at least about 90% identity over an entire length of SEQ ID NO: 10; c. a nucleotide sequence encoding an anti-influenza HA heavy chain comprising an amino acid sequence of SEQ ID NO:8; and d. a nucleotide sequence encoding an anti-influenza HA light chain comprising an amino acid sequence of SEQ ID NO: 10.

7. The immunogenic composition of claim 6, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: a. a nucleotide sequence encoding an anti-influenza HA heavy chain comprising a nucleotide sequence having at least 90% identity to SEQ ID NO: 7; b. a nucleotide sequence encoding an anti-influenza HA light chain comprising a nucleotide sequence having at least 90% identity to SEQ ID NO: 9; c. a nucleotide sequence encoding an anti-influenza HA heavy chain comprising a nucleotide sequence of SEQ ID NO:7; and d. a nucleotide sequence encoding an anti-influenza HA heavy chain comprising a nucleotide sequence of SEQ ID NO:9.

8. The immunogenic composition of claim 1, wherein the RCA product comprises at least one modified nucleotide or nucleotide analog.

9. The immunogenic composition of claim 1, wherein the RCA product comprises a double-stranded concatemeric DNA molecule containing phosphorothioated nucleotides.

10. The immunogenic composition of claim 1, further comprising a pharmaceutically acceptable excipient.

11. The immunogenic composition of claim 1, further comprising an adjuvant.

12. A nucleic acid molecule comprising one or more nucleotide sequence encoding a disease-associated antigen, a disease-associated antibody, or a fragment thereof, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: a. a nucleotide sequence encoding a peptide comprising an amino acid sequence having at least about 90% identity over an entire length of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NOTO; and b. a nucleotide sequence encoding a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO: 10.

13. The nucleic acid molecule of claim 12, wherein the nucleic acid molecule comprises at least one copy of a nucleotide sequence selected from the group consisting of: a. the nucleotide sequence having at least about 90% identity over an entire length of the nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NON; and b. the nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:9.

14. The nucleic acid molecule of claim 12, wherein the nucleic acid molecule comprises an expression vector.

15. The nucleic acid molecule of claim 14, wherein the nucleic acid molecule comprises an RCA product comprising multiple copies of the nucleotide sequence encoding the antigen.

16. The nucleic acid molecule of claim 15, wherein the RCA product comprises at least one modified nucleotide or nucleotide analog.

17. The nucleic acid molecule of claim 16, wherein the RCA product comprises a double-stranded concatemeric DNA molecule containing phosphorothioated nucleotides.

18. A method of inducing an immune response against a disease associated antigen in a subject in need thereof, the method comprising administering an immunogenic composition of claim 1 or a nucleic acid molecule of claim 12 to the subject.

19. The method of claim 18, wherein the disease-associated antigen is a SARS-CoV-2 spike protein receptor binding domain.

20. The method of claim 18, wherein the disease-associated antigen is a hemagglutinin (HA) antigen.

21. The method of claim 18, wherein the disease-associated antibody is an antiinfluenza HA antibody.

22. The method of claim 18, wherein administering includes at least one of electroporation and injection.

23. A method of protecting a subject in need thereof from a disease or disorder associated with a viral infection, the method comprising administering an immunogenic composition of claim 1 or a nucleic acid molecule of claim 12 to the subject.

24. The method of claim 23, wherein the disease or disorder is COVID-19.

25. The method of claim 23, wherein the disease or disorder is flu.

26. The method of claim 23, wherein administering includes at least one of electroporation and injection.

27. A method of treating a subject in need thereof against a disease or disorder, the method comprising administering an immunogenic composition of claim 1 or a nucleic acid molecule of claim 12 to the subject.

28. The method of claim 27, wherein the disease or disorder is COVID-19.

29. The method of claim 27, wherein the disease or disorder is flu.

30. The method of claim 27, wherein administering includes at least one of electroporation and injection.