WO2012040266A2 - Gene-based adjuvants and compositions thereof to increase antibody production in response to gene-based vaccines - Google Patents

Abstract

DNA vectors encoding IL-12, IL-21, and various soluble trimeric TNFSF ligands to increase antibody titers for DNA vaccines are disclosed herein. The DNA vectors include IL-12, IL-21, and TNFSF ligands, either individually or in particular combinations, and increase antibody production to the gpl40 protein of HIV- 1 and increase virus neutralizing antibodies to HIV-1. Compositions, kits, and methods comprising DNA vectors encoding IL-12, IL-21, and various soluble trimeric TNFSF ligands may be utilized in treating disorders or diseases and conditions such as HIV infection and cancer.

Description

GENE-BASED ADJUVANTS AND COMPOSITIONS THEREOF TO INCREASE ANTIBODY PRODUCTION IN RESPONSE TO GENE-BASED VACCINES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of Provisional Application Serial No. 61/386,145 filed September 24, 2010, which is herein incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

[0002] This invention was made with government support under K22-AI-068489 awarded by the National Institutes of Health, the NIH AIDS Reagent and Reference Reagent Program, the NIH Tetramer Core Facility for Gag Tetramer, and SB 15 P30 AI073961 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The invention relates generally to the field of immunology. More particularly, the invention relates to compositions, kits, and methods for increasing antibody production.

BACKGROUND

[0004] Typically vaccines that require antibody production are produced as protein-based vaccines containing adjuvants such as alum. These vaccines produce high titers of antibody to the protein. Vaccines may alternatively use DNA plasmids or virus vectors that express genes of interest encoding the antigen. This has the advantage of producing the protein in the mammalian cell of the vaccine rather than a bacterial system or costly mammalian culture facility. Also, vaccines that are gene-based can produce membrane-spanning proteins that are expressed on the cell surface, as opposed to soluble extracellular proteins.

[0005] As mentioned above, protein vaccines are limited since they are administered as soluble extracellular antigens. In addition, antigens such as HIV-1 Envelope (Env) need to be formed as highly structured complexes to induce neutralizing antibodies that can prevent disease. Envelope is a glycoprotein that is the only viral protein present on the exterior of the HIV-1 virion. The precursor is gpl60. The host cell cleaves gpl60 into gpl20 and gp41. Envelope must be produced on the cell surface as a trimeric complex, which cannot be achieved using protein-based soluble vaccines. One alternative is to link an artificial trimerizing domain onto Envelope to produce soluble trimers as protein vaccines, however recent studies indicate that these trimers do not bind (and therefore are unlikely to generate) neutralizing antibodies. HIV-1 Envelope produced in bacteria is not properly glycosylated. Therefore, a protein based Envelope antigen must be produced using a costly mammalian cell production process.

[0006] DNA vaccines can produce properly glycosylated membrane-bound Envelope antigen that forms trimers which specifically bind neutralizing antibodies. DNA vaccines are also inexpensive to produce and do not require a cold chain for delivery. These are key issues for delivery to resource poor countries. A limitation of DNA vaccines is that they typically are unable to produce high titer antibody responses. Therefore, they cannot generate neutralizing antibodies despite being able to produce membrane-bound, trimeric, glycosylated Envelope. To date there are a very limited number of adjuvants that can increase antibody titers to DNA vaccines.

[0007] DNA vaccines have a number of advantages for the generation of anti-HIV Env antibodies. This includes the ability to generate membrane-bound Env that can form natural trimers. These trimers are expected to facilitate the generation of neutralizing antibodies by hiding the non-neutralizing face of each Env monomer. Despite these advantages, DNA vaccines typically do not induce high antibody titers, especially when compared to vaccination with protein. Even when high antibody titers are generated, the antibody is unable to neutralize virus, perhaps reflecting insufficient B cell maturation and somatic hypermutation that increases antibody-neutralizing activity. The HIV vaccine field is in need of new technologies that can enhance both the titer, and more importantly the neutralizing titer, of vaccines such as DNA vaccines.

SUMMARY

[0008] DNA vectors encoding IL-12, IL-21, and various soluble trimeric tumor necrosis factor superfamily ligands (TNFSF ligands) capable of increasing antibody titers and increasing neutralizing antibody levels in response to DNA vaccines are disclosed herein. These DNA vectors may also be referred to as DNA vaccine vectors. The DNA vectors may encode an antigen and at least one molecule selected from the group consisting of IL-12, IL-21 , and TNFSF ligands on a single vector. Alternatively, the antigen and at least one molecule selected from the group consisting of IL-12, IL-21 , and TNFSF ligands may be encoded on a different vector. In an embodiment, the antigen is encoded on one vector while the IL-12 is encoded on a different vector. In another embodiment, a TNFSF ligand is encoded on the IL-12 vector or on a different vector. A "different vector" refers to whether IL-12, IL-21 , and TNFSF ligands are encoded on separate plasmids as opposed to being encoded on one plasmid together. Examples of TNFSF ligands are SP-D-CD40L, SP-D-CD27L, SP-D-4-1BBL, SP-D-BAFF, SP-D-APRIL, and SP-D- GITRL. Surfactant Protein D (SP-D) is used to multimerize a variety of TNFSF molecules. The DNA vectors, either individually or in particular combinations, increase antibody production and increase neutralizing antibody levels in response to vaccines expressing membrane bound HIV-1 gpl40 protein.

[0009] DNA or gene-based vaccines that encode an antigen such as HIV-1 Envelope and at least one molecule of the group consisting of IL-12, IL-21 , or a soluble trimeric TNFSF ligand may be utilized in the compositions, methods, and kits disclosed herein. DNA vectors encoding IL-12, IL-21 , and various TNFSF ligands may be part of a vaccine formulation and act as adjuvants to increase the production of neutralizing antibodies to a disease or condition such as HIV infection. Viral vector vaccines may also be used. Compositions that include a DNA vector encoding at least one molecule selected from the group consisting of IL-12, IL-21 , or a soluble trimeric TNFSF ligand may be used to increase antibody titer in response to the vaccine.

[0010] In an embodiment, a DNA vector expressing membrane -bound HIV-1 gpl40 and adjuvant plasmids expressing at least one molecule selected from the group consisting of IL-12, IL-21 , and various soluble multimeric TNFSF ligands (e.g., SP-D-CD40L, SP-D-CD27L, SP-D- 4-1BBL, SP-D-BAFF, SP-D-APRIL, and SP-D-GITRL) may be used to increase antibody production and increase the level of neutralizing antibody.

[0011] In another embodiment, the DNA vector expressing membrane bound HIV-1 gpl40 and adjuvant plasmids expressing a combination of SP-D-BAFF or SP-D-APRIL and IL-12 may be used to increase HIV-1 neutralizing antibody titers.

[0012] Compositions comprising DNA vectors encoding an antigen and at least one molecule selected from the group consisting of IL-12, IL-21 , and a TNFSF ligand and methods of enhancing antibody production and increasing the level of neutralizing antibody using these compositions may be utilized in treating diseases or conditions such as HIV infection or cancer. Administering a DNA vector encoding at least one molecule selected from the group consisting of IL-12, IL-21, and a TNFSF ligand to a subject increases antibody production and increases the level of neutralizing antibody to the vaccine in the subject. The DNA vector may also include the antigen. The antigen may be HIV-1 gpl40.

[0013] IL-12 and a TNFSF ligand act as a vaccine adjuvant when administered with a vaccine. In another embodiment, IL-21 and a TNFSF ligand act as a vaccine adjuvant when administered with a vaccine. In an embodiment, a vaccine formulation for preventing or treating a disease or condition in a subject may comprise IL-12 and a TNFSF ligand or IL-21 and a TNFSF ligand. In an embodiment, the TNFSF ligand is SP-D-BAFF or SP-D-APRIL.

[0014] Kits for the preparation of a vaccine formulation for preventing or treating a disease or condition in a subject may comprise a vaccine vector encoding at least one molecule selected from the group consisting of IL-12, IL-21, and a TNFSF ligand.

[0015] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0016] As used herein, a "nucleic acid" or a "nucleic acid molecule" means a chain of two or more nucleotides such as R A (ribonucleic acid) and DNA (deoxyribonucleic acid), and chemically-modified nucleotides. A "purified" nucleic acid molecule is one that is substantially separated from other nucleic acid sequences in a cell or organism in which the nucleic acid naturally occurs (e.g., 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% free of contaminants). The terms include, e.g., a recombinant nucleic acid molecule incorporated into a vector, a plasmid, a virus, or a genome of a prokaryote or eukaryote. Examples of purified nucleic acids include cDNAs, fragments of genomic nucleic acids, nucleic acids produced polymerase chain reaction (PCR), nucleic acids formed by restriction enzyme treatment of genomic nucleic acids, recombinant nucleic acids, and chemically synthesized nucleic acid molecules. A "recombinant" nucleic acid molecule is one made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.

[0017] As used herein, the term "IL-12 gene" means an IL-12-encoding nucleic acid sequence; a nucleic acid having sequences from which an IL-12 cDNA can be transcribed; and/or allelic variants and homologs of the foregoing. The terms encompass double-stranded DNA, single-stranded DNA, and RNA.

[0018] As used herein, the term "IL-21 gene" means an IL-21 -encoding nucleic acid sequence; a nucleic acid having sequences from which an IL-21 cDNA can be transcribed; and/or allelic variants and homologs of the foregoing. The terms encompass double-stranded DNA, single-stranded DNA, and RNA.

[0019] As used herein, the term "TNFSF ligand gene" means a TNFSF ligand-encoding nucleic acid sequence; a nucleic acid having sequences from which a TNFSF ligand cDNA can be transcribed; and/or allelic variants and homologs of the foregoing. The terms encompass double-stranded DNA, single-stranded DNA, and RNA.

[0020] As used herein, the term "IL-12 protein" means an expression product of an IL-12 gene or a protein that shares at least 65% (but preferably 75, 80, 85, 90, 95, 96, 97, 98, or 99%) amino acid sequence identity with the foregoing and displays a functional activity of a native IL- 12 protein. A "functional activity" of a protein is any activity associated with the physiological function of the protein.

[0021] As used herein, the term "IL-21 protein" means an expression product of an IL-21 gene or a protein that shares at least 65% (but preferably 75, 80, 85, 90, 95, 96, 97, 98, or 99%) amino acid sequence identity with the foregoing and displays a functional activity of a native IL- 21 protein. A "functional activity" of a protein is any activity associated with the physiological function of the protein.

[0022] As used herein, the term "TNFSF ligand protein" means an expression product of a TNFSF ligand gene or a protein that shares at least 65%> (but preferably 75, 80, 85, 90, 95, 96, 97, 98, or 99%) amino acid sequence identity with the foregoing and displays a functional activity of a native TNFSF ligand protein. A "functional activity" of a protein is any activity associated with the physiological function of the protein.

[0023] As used herein, "protein" and "polypeptide" are used synonymously to mean any peptide-linked chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation. A "fusion protein" is a protein made by translation of an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques. [0024] When referring to a peptide, oligopeptide or protein, the terms "amino acid residue", "amino acid" and "residue" are used interchangably and, as used herein, mean an amino acid or amino acid mimetic joined covalently to at least one other amino acid or amino acid mimetic through an amide bond or amide bond mimetic.

[0025] As used herein, "protein" and "polypeptide" are used synonymously to mean any peptide-linked chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation.

[0026] When referring to a nucleic acid molecule, polypeptide, or infectious pathogen, the term "native" refers to a naturally-occurring (e.g., a wild-type (WT)) nucleic acid, polypeptide, or infectious pathogen.

[0027] As used herein, the term "antigen" or "immunogen" means a molecule that is specifically recognized and bound by an antibody.

[0028] The terms "specific binding" and "specifically binds" refer to that binding which occurs between such paired species as enzyme/substrate, receptor/agonist, antibody/antigen, etc., and which may be mediated by covalent or non-covalent interactions or a combination of covalent and non-covalent interactions. When the interaction of the two species produces a non- covalently bound complex, the binding which occurs is typically electrostatic, hydrogen- bonding, or the result of lipophilic interactions. Accordingly, "specific binding" occurs between a paired species where there is interaction between the two which produces a bound complex having the characteristics of an antibody/antigen or enzyme/substrate interaction. In particular, the specific binding is characterized by the binding of one member of a pair to a particular species and to no other species within the family of compounds to which the corresponding member of the binding member belongs.

[0029] The term "antibody" is meant to include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, humanized antibodies, anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled in soluble or bound form, as well as fragments, regions or derivatives thereof, provided by any known technique, such as, but not limited to, enzymatic cleavage, peptide synthesis or recombinant techniques.

[0030] As used herein, "neutralizing antibodies" are antibodies that bind to the virus particle and neutralize infectivity. Neutralizing antibodies may stop infectivity by blocking endocytosis of the virus, preventing uncoating of the genome, causing aggregation of the virus particles, interfering with binding of the virus to receptors, or causing lysis of the enveloped virus when membranes are interrupted.

[0031] As used herein, "neutralize" means stopping infectivity of a virus.

[0032] As used herein the term "adjuvant" means any material which modulates to enhance the humoral and/or cellular immune response.

[0033] As used herein, the terms "displayed" or "surface exposed" are considered to be synonyms, and refer to antigens or other molecules that are present (e.g., accessible to immune site recognition) at the external surface of a structure such as a cell.

[0034] As used herein, "vaccine" includes all prophylactic and therapeutic vaccines.

[0035] As used herein, "viral vector" refers to a vector that uses live viruses to carry DNA encoding an antigen to a cell.

[0036] As used herein, the term "biologic" refers to a wide range of medicinal products such as vaccines, blood and blood components, allergenics, somatic cells, genes expressing a product in gene therapy, tissues, and recombinant therapeutic proteins created by recombinant DNA technology, antibodies, synthetic drugs, and long peptides (polypeptides), synthetic compounds, and (glycol)proteins.

[0037] By the phrase "immune response" is meant induction of antibody and/or immune cell-mediated responses specific against an antigen or antigens or allergen(s) or drug or biologic. The induction of an immune response depends on many factors, including the immunogenic constitution of the challenged organism, the chemical composition and configuration of the antigen or allergen or drug or biologic, and the manner and period of administration of the antigen or allergen or drug or biologic. An immune response has many facets, some of which are exhibited by the cells of the immune system (e.g., B-lymphocytes, T-lymphocytes, macrophages, and plasma cells). Immune system cells may participate in the immune response through interaction with an antigen or allergen or other cells of the immune system, the release of cytokines and reactivity to those cytokines. Immune responses are generally divided into two main categories - humoral and cell-mediated. The humoral component of the immune response includes production of antibodies specific for an antigen or allergen or drug or biologic. The cell- mediated component includes the generation of delayed-type hypersensitivity and cytotoxic effector cells against the antigen or allergen. [0038] As used herein, the term "treatment" is defined as the application or administration of a therapeutic agent to a patient, or application or administration of the therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease, or the predisposition toward disease.

[0039] The terms "patient", "subject", and "individual" are used interchangeably herein, and mean a mammalian subject who is to be treated, who has been treated, or who is being considered for treatment, with human patients being preferred. In some cases, the methods, kits, and compositions described herein find use in experimental animals, in veterinary applications, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters, as well as non-human primates.

[0040] Although compositions, kits, and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable compositions, kits, and methods are described below. All publications, patent applications, and patents mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. The particular embodiments discussed below are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG. 1 shows a four trimer form of CD40L, SP-D-CD40L, formed by disulfide bonds at the N-terminus of the trimeric protein. CD40L is a TNFSF ligand.

[0042] FIG. 2 shows a graph of CD4+ T cell proliferation showing that supernatants from

AD293 cells transfected with SP-D-CD40L (**), SP-D-CD27L (**), SP-D-GITRL (***), SP-D-

LIGHT (***), SP-D-41BBL (***), SP-D-RANKL (**) induce proliferation of T cells.

[0043] FIG. 3 shows a vaccination protocol in which BALB/c mice (n=4) were vaccinated intramuscularly at Days 1, 14 and 28 with gpl40-CD5-opt + IL-12 or IL-21 without various SP-

D-TNFSF ligands (SP-D-TNFSFL) or with SP-D-TNFSFL and sacrificed after 2 weeks. FIG. 3 shows the vaccination protocol for the data in FIGS. 4A-4D. [0044] FIGS. 4A-4D show antibody secretion measured by ELISA. IgGl and IgGl + IgG2a antibody response to Env protein induced by gpl40-CD5-opt + IL-12 or IL-21 with or without SP-D-TNFSFL was measured.

[0045] FIG. 5 shows a vaccination protocol in which BALB/c mice (n=10) were vaccinated intramuscularly at Days 1, 14 and 28 with gpl40-CD5-opt + IL-12 without SP-D-TNFSFL or with various SP-D-TNFSFL and sacrificed after 2 weeks. FIG. 5 shows the vaccination protocol for the data in FIGS. 6A-6D.

[0046] FIGS. 6A-6D show antibody secretion measured by ELISA. IgG2 and IgGl + IgG2a antibody response to Env protein induced by gpl40-CD5-opt + IL-12 or IL-21 with or without SP-D-TNFSFL was measured.

[0047] FIGS. 7A-7C show the results of an ELISpot assay indicating that IFN-γ, IL-2 and IL-4 secretion of Env specific splenocytes was induced by the combination of gpl40 + IL-12 + SP-D-TNFSFL.

[0048] FIG. 8 shows the results of an antibody ELISA assay of serum from mice vaccinated 3 X (once every 2 weeks) with 100 meg of DNA plasmids by intramuscular injection. A HIV-1 Gag plasmid was used as the antigen. Plasmids encoding SP-D-CD40L, SP-D-CD27L, SP-D- 4IBBL, and SP-D-BAFF, IL-12, and IL-I5 were tested.

[0049] FIGS. 9A and 9B show the results of an antibody ELISA assay of serum from mice vaccinated 3 X (once every 2 weeks) with 100 meg of DNA plasmids by intramuscular injection. A HIV-1 Envelope membrane-bound gpl40 plasmid was used as antigen. Mice were vaccinated with IL-21 or IL-12 alone or in combination with various soluble trimeric TNFSF ligands. Samples were tested at 1 :480 and 1 : 1920 serum dilutions.

[0050] FIGS. 10A and 10B show the results of an antibody ELISA assay of serum from mice vaccinated 3 X (once every 2 weeks) with 100 meg of DNA plasmids by intramuscular injection. A HIV-1 Envelope membrane-bound gpl40 plasmid was used as antigen. Mice were vaccinated with IL-12 alone or in combination with various soluble trimeric TNFSF ligands. Samples were tested at 1 :480 and 1 : 1920 serum dilutions.

[0051] FIG. 11 shows neutralization titers for mouse serum samples graphed by vaccine group. Mice received plasmids encoding gpl40 and IL-12 plus either an empty vector, SP-D- BAFF, or SP-D-APRIL plasmids. All negative titers (Tier 1 titers <3 times the titer that neutralized the control SVA-MLV virus) were given a value of 0. [0052] FIG. 12 shows neutralization titers for mouse serum samples graphed by vaccine group. Mouse serum from mice that received plasmids encoding gpl40 and IL-12 plus either an empty vector, SP-D-BAFF plasmid, or SP-D-APRIL plasmid were assayed for neutralization against homologous virus 96ZM651, the source of the gpl40 gene encoded in the DNA vaccine. All negative titers (96ZM651 virus titers <3 times the titer that neutralized the control SVA- MLV virus) were given a value of 0.

[0053] FIGS. 13A and 13B show a graph of the IgG2a antibody titer of each mouse sample vs. the neutralization titer of the same sample (Fig. 13 A) and a graph of the total IgG antibody titer (IgGl + IgG2a) of each mouse sample vs. the neutralization titer of the same sample. (Fig. 13B). Only positive neutralization samples were graphed (Titer value >3 times neutralization titer for control virus SVA-MLV).

DETAILED DESCRIPTION

[0054] Disclosed herein is a composition and method that solves the problem of the difficulty of DNA vaccines to induce high amounts of antibody and to induce neutralizing antibodies. This is particularly important to generate antibodies, particularly as neutralizing antibodies, which has not yet been possible against particular viruses such as HIV-1 using traditional vaccine approaches. Neutralizing antibodies are antibodies that bind to the virus particle and neutralize infectivity. Neutralizing antibodies may stop infectivity by blocking endocytosis of the virus, preventing uncoating of the genome, causing aggregation of the virus particles, interfering with binding of the virus to receptors, or causing lysis of the enveloped virus when membranes are interrupted. This method will be useful in the development of new vaccines for the prevention or treatment of diseases in humans and animals. This method can be used to increase the amount of antibody production in response to any vaccine, including but not limited to those including, DNA vaccine, RNA vaccine, viral vaccine, and protein vaccines. This method can also be used to generate neutralizing antibodies in response to any vaccine, including but not limited to DNA vaccines, RNA vaccines, viral vaccines, and protein vaccines.

[0055] The composition and methods disclosed herein are different from the current technology because the adjuvants disclosed herein can markedly increase antibody production from DNA vaccines and increase neutralizing antibody production beyond the amount previously possible. Some researchers have discovered methods to increase antibody titers from DNA prime/protein boost vaccines. The priming step (immunization) is with the DNA that encodes the antigen and the boosting step is with the protein form of the antigen. The concern with this approach is that the protein boost will increase non-neutralizing antibodies to Envelope. Non-neutralizing antibodies bind to Env but the binding does not interfere with the ability of the virus to infect another cell. Neutralizing antibodies bind to Env in a manner that does interfere with the ability of the virus to infect another cell.

[0056] Disclosed herein is a useful method to increase antibody and neutralizing antibody levels in situations where it is preferable to use DNA or other gene-based vaccines or therapies. Few ways of increasing antibody production from DNA vaccines are known. The ability to generate high titer antibodies from DNA vaccines is particularly relevant for vaccines, such as an HIV vaccine, that require either glycosylated and/or membrane-bound antigens that are difficult or expensive to produce as protein-based vaccines.

[0057] The composition and methods disclosed herein use DNA vaccination and are expected to present membrane -bound glycosylated trimers of Envelope. Therefore, the composition, kits, and methods are expected to produce higher titers of neutralizing antibody compared to the DNA prime/protein boost approach. Envelope is a glycoprotein that is the only viral protein present on the HIV-1 virion. The precursor of Envelope is gpl60. The host cell cleaves gpl60 into gpl20 and gp41. The portion of gpl20 and gp41 that is outside of the membrane is known as gpl40. In various embodiments, the antigen present in the vaccine may include gpl40 or gpl60. In other embodiments, other antigens may be the antigen present in the vaccine.

[0058] Disclosed are compositions and methods utilizing cytokines such as IL-21 or IL-12, or combinations of these cytokines and soluble trimeric TNFSF ligands, to enhance antibody production and neutralizing antibody levels from DNA vaccines. The compositions and methods disclosed herein may be utilized in a DNA or gene-based vaccine that encodes an antigen such as HIV-1 Envelope and one or more of IL-21, IL-12, or soluble trimeric TNFSF ligand. Viral vector vaccines may also be used.

[0059] In HIV-1 infection, neutralizing antibodies have not controlled the infection. The human monoclonal antibodies 2G12, 2F5, and 4E10 represent broad neutralizing antibodies against HIV-1. (Wei, X. et al., Antibody neutralization and escape by HIV-1. Nature, 422: 307- 312 (2003)). These antibodies are extremely rare and it is crucial to find the right adjuvants to elicit neutralizing antibodies with candidate immunogen vaccines.

[0060] The tumor necrosis factor superfamily ligand (TNFSF ligand) contains many immunologically active proteins that have the potential to act as potent molecular adjuvants. One of the most immunologically important TNF-superfamily proteins is CD40 ligand (CD40L), which is important for activating dendritic cells to induce CD 8+ T-cell memory responses (Stone, G.W. et al., Macaque Multimeric Soluble CD40 Ligand and GITR Ligand Constructs Are Immunostimulatory Molecules In Vitro. Clinical and Vaccine Immunology. 1223-1230 (2006)). Multimeric soluble forms of TNF-superfamily molecules have been shown to induce potent immune responses in mice. Disclosed herein is the use of Surfactant Protein D (SP-D) to multimerize a variety of TNF-superfamily molecules. (Stone, G.W. et al., Macaque Multimeric Soluble CD40 Ligand and GITR Ligand Constructs Are Immunostimulatory Molecules In Vitro. Clinical and Vaccine Immunology. 1223-1230 (2006)). SP-D is a water soluble protein that spontaneously forms 4-trimer multimers as well as higher order "pinwheel" structures. For example, SP-D-CD40L includes 4 trimers of CD40L linked by SP-D. (Fig. 1).

[0061] Disclosed herein are costimulatory SP-D-TNFSFL that act as adjuvants in combination with IL-12 or IL-21 to induce strong neutralizing antibodies to membrane bound GP140 antigen in a mouse model. The antibodies can be tested for their ability to neutralize HIV-1 infection in human CD4 T cells.

[0062] Soluble multimeric forms of the TNFSF proteins CD40 ligand (CD40L) or glucocorticoid-induced tumor necrosis factor receptor ligand (GITRL) have been shown to act as adjuvants for HIV-1 DNA vaccines in mice. These constructs combine the extracellular domain of murine CD40L or GITRL with the body of the spontaneously multimerizing Surfactant Protein-D (SP-D). (Fig. 1).

[0063] A DNA vaccine vector expressing membrane -bound HIV-1 gpl40 and adjuvant plasmids expressing at least one of IL-12, IL-21, and various soluble multimeric TNFSF ligands (SP-D-CD40L, SP-D-CD27L, SP-D-4-1BBL, SP-D-BAFF, SP-D-APRIL, and SP-D-GITRL) in mice models is disclosed herein. Membrane-bound gpl40 (mgpl40) Envelope can form stable trimers on the transfected cell membrane and induce neutralizing antibodies in vaccinated animals. [0064] DNA vectors, either individually or in particular combinations, increase antibody production for vaccines expressing membrane bound HIV-1 gpl40 protein. These DNA vectors may be referred to as DNA vaccine vectors and are capable of increasing antibody production from DNA vaccines or other gene -based vaccines. Alternatively, viral vector vaccines may be used. Vaccines can be generated against a variety of infectious agents and other diseases. These antibody-based therapeutics can include, but are not limited to, cancer vaccines targeting antibodies to cancer cell associated membrane-surface antigens and vaccines targeting HIV-1 infection.

[0065] Soluble trimeric TNFSF ligands such as SP-D-CD40L, SP-D-CD27L, SP-D-GITRL, SP-D-LIGHT, SP-D-41BBL, and SP-D-RANKL are able to induce proliferation of T cells.

[0066] The below described preferred embodiments illustrate adaptations of these compositions, kits, and methods. Nonetheless, from the description of these embodiments, other aspects of the invention can be made and/or practiced based on the description provided below.

Biological Methods

[0067] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises such as Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, (1992) (with periodic updates). Immunology techniques are generally known in the art and are described in detail in methodology treatises such as Current Protocols in Immunology, ed. Coligan et al, Greene Publishing and Wiley-Interscience, New York, (1992) (with periodic updates), Advances in Immunology, volume 93, ed. Frederick W. Alt, Academic Press, Burlington, MA (2007); Making and Using Antibodies: A Practical Handbook, eds. Gary C. Howard and Matthew R. Kaser, CRC Press, Boca Raton, Fl (2006); Medical Immunology, 6th ed., edited by Gabriel Virella, Informa Healthcare Press, London, England (2007); and Harlow and Lane ANTIBODIES: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988).

[0068] Nucleic acid molecules encoding an antigen, IL-12, IL-21, or TNFSF ligand as described herein may be in the form of RNA (e.g., mRNA, microRNA, siRNA, shRNA or synthetic chemically modified RNA) or in the form of DNA (e.g., cDNA, genomic DNA, and synthetic DNA). The DNA may be double-stranded or single-stranded, and if single-stranded, may be the coding (sense) strand or non-coding (anti-sense) strand. In one embodiment, a nucleic acid can be an RNA molecule isolated or amplified from immortalized or primary tumor cell lines.

[0069] Any suitable biological sample can be tested. Examples of biological samples include blood, saliva, serum, plasma. The steps of the method can be performed using any suitable protocol or assay. Examples of suitable assays include enzyme-linked immunosorbent assays (ELISAs), Western blots, flow cytometry assays, immunofluorescence assays, qPCR, microarray analysis, etc.

[0070] In an embodiment, an antibody (e.g., monoclonal, polyclonal, Fab fragment, etc.) specific for a given protein may be used. In some embodiments, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the kits, assays and methods described herein. A kit may contain antibodies and other components, packaging, instructions, or other material to aid in the use of the kit.

[0071] Described herein are kits for preparation of a vaccine formulation. A typical kit for may include at least one molecule selected from the group consisting of IL-12, IL-21 , and a TNFSF ligand. A kit may include a well plate to carry the mixture of the different reagents, as well as one or more washing buffers. Optionally, kits may also contain one or more of the following: containers which include positive controls, containers which include negative controls, photographs or images of representative examples of positive results and photographs or images of representative examples of negative results.

Effective Doses

[0072] The compositions described above are preferably administered to a mammal (e.g., non-human primate, bovine, canine, rodent, human) in an effective amount, that is, an amount capable of producing a desirable result in a treated subject (e.g., delaying or preventing onset of a disease or disorder in the subject). Toxicity and therapeutic efficacy of the compositions utilized in methods described herein can be determined by standard pharmaceutical procedures. As is well known in the medical and veterinary arts, dosage for any one animal depends on many factors, including the subject's size, body surface area, age, the particular composition to be administered, time and route of administration, general health, and other drugs being administered concurrently.

[0073] Typically, the subject is one who will receive a vaccine, or for whom vaccine administration is being considered.

[0074] The amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the pathology of the disease. A composition as described herein is typically administered at a dosage that increases production of neutralizing antibodies to a given antigen.

[0075] Therapeutic compositions described herein can be administered to a subject by any suitable delivery vehicle and route. Examples of delivery vehicles and means for delivering compositions include antibody (vaccine) delivery, gene therapy including viral vectors, liposomes, aptamers, and other biologies. The administration of a composition including a therapeutically effective amount of at least one of IL-12, IL-21, or a TNFSF ligand. The composition may be provided in a dosage form that is suitable for local or systemic administration (e.g., parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, intracranially). In various embodiments, the composition may be provided in a dosage form that is suitable for oral administration or intranasal administration. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins (2000) and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, , Marcel Dekker, New York (1988-1999)).

[0076] Compositions as described herein including small molecules may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra. [0077] Compositions for parenteral use may be provided in unit dosage forms (e.g., in single-dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. The composition may include suitable parenterally acceptable carriers and/or excipients. The active therapeutic agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing agents.

[0078] As indicated above, the pharmaceutical compositions described herein may be in a form suitable for sterile injection. To prepare such a composition, the suitable active therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1 ,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the compounds is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.

[0079] Materials for use in the preparation of microspheres and/or microcapsules are, e.g., biodegradable/bioerodible polymers such as polygalactia poly-(isobutyl cyanoacrylate), poly(2- hydroxyethyl-L-glutam- nine) and poly(lactic acid). Biocompatible carriers that may be used when formulating a controlled release parenteral formulation are carbohydrates (e.g., dextrans), proteins (e.g., albumin), lipoproteins, or antibodies. Materials for use in implants can be nonbiodegradable (e.g., polydimethyl siloxane) or biodegradable (e.g., poly(caprolactone), poly(lactic acid), poly(gly colic acid) or poly(ortho esters) or combinations thereof).

[0080] At least two of IL-12, IL-21 , and a TNFSF ligand may be mixed together in a single composition, or may be administered separately. An antigen and at least one molecule selected from the group consisting of IL-12, IL-21 , and a TNFSF ligand may be encoded on a single plasmid or vector. Alternatively, each of the antigen and at least one molecule selected from the group consisting of IL-12, IL-21 , and a TNFSF ligand may be encoded on a separate plasmid or vector. At least one of IL-12, IL-21 , and a TNFSF ligand may be administered in combination with any standard or newly emerging therapy (e.g., HIV infection therapy or cancer therapy). Such methods are known to the skilled artisan and described in Remington's Pharmaceutical Sciences by E. W. Martin.

[0081] Formulations for oral use include a liquid containing the active ingredient(s) (e.g., at least one of IL-12, IL-21 , and a TNFSF ligand) in a mixture with non-toxic pharmaceutically acceptable excipients.

[0082] Antigen or antigens to which an immune response is mounted as a result of vaccination may be from any pathogen. In one embodiment, the antigen may be derived from, but not limited to, pathogenic bacterial, fungal, or viral organisms, Streptococcus species, Candida species, Brucella species, Salmonella species, Shigella species, Pseudomonas species, Bordetella species, Clostridium species, Norwalk virus, Bacillus anthracis, Mycobacterium tuberculosis, human immunodeficiency virus (HIV), Chlamydia species, human Papillomaviruses, Influenza virus, Paramyxovirus species, Herpes virus, Cytomegalovirus, Varicella-Zoster virus, Epstein-Barr virus, Hepatitis viruses, Plasmodium species, Trichomonas species, sexually transmitted disease agents, viral encephalitis agents, protozoan disease agents, fungal disease agents, bacterial disease agents, cancer cells, or mixtures thereof.

[0083] A subject may be treated for an infectious pathogen or cancer. Examples of infectious pathogens include viruses such as, but not limited to, influenza, HIV, dengue virus, rotavirus, HPV, HBV, HCV, CMV, HSV, HZV, and EBV, pathogenic agents including the causative agents of Malaria, Plasmodium(p) falciparum, P. malariae, P. ovale, P. vivax and P. knowlesi; the causative agent of Leishmania (L), L. major, L. tropica, L. aethiopica, L. mexicana, L. donovani, L. infantum syn. L. chagas; and pathogenic bacteria including Bacillus anthracis, Bordetella pertussis, Streptococcus pneumonia, and meningococcus.

[0084] The vaccine may be used against any cancer or with any other therapy or intervention for cancer. Examples of cancers include HPV-induced cervical cancers (e.g. , E7/E7 tumor associated antigens (TAA)), human melanoma (e.g., TRP-1 , TRP-2, gp-100, MAGE-1 , MAGE-3 and / or p53), and prostate cancer (e.g., TSA). Similarly for lung tumors, breast tumors, and leukemia, any suitable tumor associated antigen can be used, and many have been described. Many such TAA are common between various cancers (e.g., CEA, MUC-1 , Her2, CD20). EXAMPLES

[0085] The present invention is further illustrated by the following specific examples. The examples are provided for illustration only and should not be construed as limiting the scope of the invention in any way.

Example 1

Vaccination of Animals

[0086] Antigen plasmid p96ZM651gpl40-CD5-opt encodes a membrane-bound HIV-1 gpl40 protein (mgpl40) (AIDS Research Reagent Program). Various soluble 4-trimer adjuvant constructs of TNFSF ligands were tested for their immune response in combination with a plasmid expressing murine IL-12. BALB/c mice were vaccinated intramuscularly (i.m.) every two weeks X3 with 80 μg of antigen plasmid and 20 μg pIL-12 plus 20 μg of various TNFSF ligand plasmids. Spleen cells and serum were harvested two weeks later for ELISA and ELISpot. The antibody response for IgGl and IgG2a was detected by an ELISA assay to detect 96ZM651 gpl20 protein.

Example 2

Neutralization assay

[0087] Neutralization was determined by using the PhenoSense assay system. In this system, pseudovirus was harvested 48 h after cotransfecting HEK293 cells with pCXAS-Env libraries plus an HIV genomic vector that contains a firefly luciferase indicator gene. Two pseudoviruses were tested: autologous clade C virus 96ZM651 and a clade C Tier 1 (easy to neutralize) virus MW965.26. Pseudoviruses were then incubated with heat-inactivated mouse sera at graded dilutions for 1 h at 37°C. U87 cells expressing CD4 and the CCR5 and CXCR4 coreceptors were inoculated with virus-antibody mixtures in the absence of added cations. Virus infectivity was determined 72 h postinoculation by measuring the amount of luciferase activity expressed in infected cells. Neutralizing activity was calculated as the percent inhibition of viral replication (luciferase activity) at each antibody dilution compared with a mouse serum-free control as follows: % inhibition = [1 - (luciferase + immune sera)/(luciferase - immune sera)] X 100. Murine leukemia virus (SVA-MLV) was also included in each assay to rule out nonspecific neutralizing activities.

Example 3

[0088] The antigen plasmid used was p96ZM65 lgpl40-CD5-opt. The adjuvants used in the studies disclosed herein are SP-D-CD40L, SP-D-CD27L, SP-D-GITRL, SP-D-APRIL, SP-D- BAFF, SP-D-41BBL, IL-12, and IL-21. All genes were cloned into the pcDNA3.1(+) or pVAX- 1 vector, except IL-12 that was cloned into the pORF vector.

[0089] Antigen plasmid p96ZM651gpl40-CD5-opt encodes the membrane-bound HIV-1 gpl40 protein (AIDS Research Reagent Program). Various soluble 4-trimer adjuvant constructs of TNFSF ligands were tested for an immune response in combination with a plasmid expressing murine IL-12 or IL-21.

[0090] BALB/c mice were vaccinated i.m. every two weeks X 3 with 80 μg of antigen plasmid (gpl40-CD5-opt) and 20 μg pIL-12 or IL-21 plus 20 μg of various TNFSF ligand plasmids. Spleen cells and serum were harvested two weeks later to be analyzed by ELISA and ELISpot. IL-12 is comprised of a p35 and p40 subunit. IL-12 was encoded on the pORF-mlL- 12 plasmid. Both p35 and p40 are expressed under the control of the same promotor.

[0091] The inclusion of IL-12 plus TNFSF ligands in a membrane-associated gpl40 vaccine can induce both humoral and cellular immune responses against gpl40. Therefore IL-12 in combination with at least one TNFSF ligand is useful as an adjuvant for HIV vaccination.

Example 4

[0092] An anti-mouse CD3 antibody costimulation assay for CD4+ T cell proliferation in vitro was performed.

[0093] Referring to Fig. 2, SP-D-TNFSFL induced in vitro costimulation. Supernatant from AD293 cells transfected with SP-D-CD40L (**), SP-D-CD27L (**), SP-D-GITRL (***), SP-D- LIGHT (***), SP-D-41BBL (***), SP-D-RANKL (**) induced proliferation of CD4+ T cells significantly compare to pCDNA3.1 alone. However, supernatants from SP-D-APRIL or SP-D- BAFF, which mainly induce B cell proliferation, did not induce CD4+ T cell proliferation. Example 5

[0094] BALB/c mice were vaccinated intramuscularly at Days 1, 14 and 28 with gpl40- CD5-opt + IL-12 or IL-21 with or without different TNFSF ligands and sacrificed after 2 weeks. (Fig. 3).

[0095] A combination study of mgpl40-CD5-opt + IL-12 or IL-21 with or without various SP-D-TNFSFLs was performed. The combination of mgpl40 + IL-12 or IL-21 + SP-D- TNFSFL induced strong antibody responses. Antibody secretion against the Env protein (5 μg/ml) was determined by ELISA. The number of mice used was n=4/group.

[0096] There was an IgGl and IgG2a antibody response to Env protein induced by gpl40- CD5-opt + IL-12 or IL-21 with or without SP-D-TNFSFL. (Fig. 4).

[0097] At a 1 :480 serum dilution, IL-21 alone induced a very strong IgGl response, whereas IL-21 + SP-D-BAFF drastically reduced Env specific IgGl antibody response. (Fig. 4A). However, mgpl40 + IL-12 induced moderate Env specific IgGl, but in combination with SP-D- CD27L or SP-D-BAFF, a very strong IgGl response was induced, even at the 1 : 1920 serum dilution. (Figs. 4A and 4B). SP-D-41BBL and SP-D-CD40L showed a moderate response (only at the 1 :480 serum dilution) compared to mgpl40 + IL-12. (Fig. 4A). The mgpl40 vaccine alone did not induce any IgGl response. (Figs. 4A and 4B). Serum from mgpl40 + IL-21 mice showed strong induction of IgGl response (Thl mediated), whereas a combination containing SP-D-BAFF suppressed the IgGl response induced by mgpl40 + IL-21. (Figs. 4A and 4B).

[0098] The same ELISA plate was analyzed for an IgG2a response. The IgG2a + IgGl response drastically increased by the combination of mgpl40 + IL-12 + SP-D-CD27L or SP-D- BAFF (Figs. 4C and 4D), while mgpl40 + IL-12 + SP-D-CD40L and SP-D-41BBL showed a moderate increase at 1 :480 dilution. (Fig. 4C). There was not much change in mgpl40 + IL-21 induced IgG2a + IgGl response. (Figs. 4C and 4D).

[0099] The mice vaccinated with mgpl40 + IL-12 + SP-D-TNFSFL strongly induced both cellular and humoral responses. Previous results had suggested that IL-12 does not induce antibody responses in mice models. The combination of mgpl40 + IL-12 induced a moderate antibody response. But the combination of SP-D-CD27L and SP-D-BAFF enhanced strong IgGl and IgG2a response (both Thl and Th2 response). (Figs. 4C and 4D).

[0100] SP-D-CD27L induced high titers of IgG2a and IgGl anti-gpl40 antibodies and SP- D-BAFF plus pIL-12 induced significantly higher IgGl titers (1 :480) when compared to gpl40 plasmid plus pIL-12 alone (p<0.05). (Figs. 4A-4D). Overall, anti-gpl40 titers of greater than 1 : 1920 could routinely be induced in mice vaccinated with pIL-12 plus various TNFSF ligands.

Example 6

[0101] BALB/c mice were vaccinated intramuscularly at Days 1, 14 and 28 with gpl40- CD5-opt + IL-12 without SP-D-TNFSFL or with various SP-D-TNFSFL and sacrificed after 2 weeks. (Fig. 5).

[0102] Antibody secretion against the Env protein (5 μg/ml) was determined by ELISA. Antibody (IgG2a and IgGl) responses induced by gpl40-CD5-opt + IL-12 with or without SP- D-TNFSFL were measured. The combination of mgpl40 + IL-12 + SP-D-TNFSFL induced antibody responses. (Figs. 6C and 6D). The number of mice used was n=10/group.

[0103] The combination of mgpl40 + IL-12 induced a moderate Env specific IgG2a response, whereas a combination of mgpl40 + IL-12 + SP-D-CD27L induced a very strong IgG2a response specific to Env protein even at the 1 : 1920 serum dilution. (Figs. 6A and 6B). The combination of SP-D-APRIL and SP-D-GITRL did not induce any IgG2a response, even relative to mgpl40 + IL-12 alone. (Figs. 6A and 6B).

[0104] The same IgG2a ELISA plate was used to look for IgGl response. (Figs. 6C and 6D). The IgG2a + IgGl response was drastically increased by the combination of mgpl40 + IL- 12 + SP-D-CD27L, and moderately by SP-D-BAFF, even at the 1 : 1920 dilution. (Figs. 6C and 6D). Though SP-D-APRIL and SP-D-GITRL showed some response at 1 :480, it was not seen at 1 : 1920. (Figs. 6C and 6D).

Example 7

[0105] Referring to Figs. 7A-7C, IFN-γ, IL-2, and IL-4 secretion of Env specific splenocytes was induced by the combination of mgpl40 + IL-12 + SP-D-TNFSFL. IFN-γ, IL-2, and IL-4 secretion by T cells were measured by ELISpot.

[0106] IFN-γ secretion of Env specific spleen cells were enhanced by SP-D-CD27L, SP-D- APRIL and SP-D-GITRL (**) when combined with gpl40 + IL-12 compared to gpl40 + IL-12 alone. *p<0.05, **p<0.01, ***P<0.001. (Fig. 7 A). [0107] IL-2 secretion by Env specific spleen cells was increased by SP-D-CD27L, SP-D- APRIL and SP-D-GITRL (**) when combined with gpl40 + IL-12 compared to gpl40 + IL-12 alone. *p<0.05, **p<0.01, ***P<0.001. (Fig. 7B).

[0108] IL-4 secretion by spleen cells was strongly enhanced by SP-D-CD27L, SP-D-BAFF, SP-D- APRIL and SP-D-GITRL when combined with gpl40 + IL-12 compared to gpl40 + IL-12 alone. *p<0.05, **p<0.01, ***P<0.001. (Fig. 7C).

[0109] Combinations of p96ZM651gpl40-CD5-opt with pIL-12 and TNFSF ligand adjuvant plasmids induced strong immune responses. The combination induces both humoral and cellular responses in mice models. Vaccination with SP-D-CD27L, SP-D-GITRL (p<0.01), or SP-D- APRIL plasmids with an IL-12 plasmid and gpl40 plasmid synergistically enhanced IFN-γ and IL-2 ELISpot responses compared to gpl40 plasmid plus pIL-12 alone.

Example 8

[0110] Referring to Fig. 8, an antibody ELISA assay of serum from mice vaccinated 3 X (once every 2 weeks) with 100 meg of DNA plasmids by intramuscular injection was performed. A HIV-1 Gag plasmid was used as the antigen. Plasmids encoding as soluble trimers of a TNFSF ligand, produced by fusion with Surfactant Protein D (SP-D), were tested (including SP-D- CD40L, SP-D-CD27L, SP-D-4IBBL, and SP-D-BAFF). Also tested were plasmids encoding cytokines IL-12 (Invivogen pORF-mIL-I2) and IL-I5. Only IL-12 was able to induce significant (p<0.05) titers at a serum dilution of 1 : 1920.

[0111] A DNA expression plasmid encoding the mouse cytokine IL-12 can increase antibody production from a HIV-1 Gag antigen DNA vaccine (Fig. 8). Another laboratory did not observe increased antibody with a mouse IL-12 plasmid. The plasmid utilized by the other laboratory expressed the two subunits from different promoters. The plasmid used in this study encodes the two subunits of IL-12 linked by a flexible linker.

Example 9

[0112] Referring to Figs. 9A and 9B, an antibody ELISA assay of serum from mice vaccinated 3 X (once every 2 weeks) with 100 meg of DNA plasmids by intramuscular injection was performed. A HIV-1 Envelope membrane-bound gpl40 plasmid was used as the antigen. [0113] The combination of the pORF-mIL-12 with soluble trimeric TNFSF ligands (TNFSF ligands) was tested. A distinct cytokine IL-21 was also tested. This distinct cytokine IL-21 was known to be capable of enhancing immune responses. A codon-optimized IL-21 based on publicly available sequence data was generated. Increased antibody responses relative to IL-12 using either IL-21 or combinations of IL-12 and soluble trimeric TNFSF ligands were obtained (Figs. 9 A and 9B). IL-21 or IL-12 in combination with SP-D-BAFF or SP-D-CD27L were particularly effective at 1 :480 and 1 : 1920 serum dilutions.

Example 10

[0114] Referring to Figs. 10A and 10B, an antibody ELISA assay of serum from mice vaccinated 3 X (once every 2 weeks) with 100 meg of DNA plasmids by intramuscular injection was performed. Mice were vaccinated with either IL-12 alone or in combination with a variety of soluble trimeric TNFSF ligands. A HIV-1 Envelope membrane -bound gpl40 plasmid was used as the antigen.

[0115] IL-12 in combination with SP-D-TNFSFL adjuvants induced higher titer antibody. IL-12 + SP-D-CD27L was a particularly effective combination with high titers, even at 1 : 1920 dilution. (Figs. 10A and 10B). Ten mice per group experiment were used in this experiment. Therefore, IL-12 + TNFSFL plasmids are able to enhance a HIV-1 Env gpl40 DNA vaccine. (Figs. 1 OA and 10B).

Example 11

[0116] The combination IL-12 plus SP-D-BAFF or SP-D- APRIL increases the proportion of animals with positive Tier 1 virus neutralizing antibody titers compared to IL-12 adjuvant alone. (Table 1). Mice were vaccinated with plasmids encoding HIV-1 Env gpl40, IL-12 and either an empty vector, SP-D-BAFF, or SP-D- APRIL. Mouse serum from each vaccine group was tested for neutralizing titers against the Tier 1 HIV-1 virus MW965.26 or against a non-HIV control virus (SVA-MLV). Positive titers were defined as values >20 as well as >3 times the control virus neutralization titer. Table 1

Example 12

[0117] Referring to Fig. 11, the combination of adjuvants IL-12 plus SP-D-BAFF or SP-D- APRIL enhances the mean neutralization titers against Tier 1 viruses. The neutralization titers for each mouse serum sample were graphed by vaccine group. Mice received plasmids encoding gpl40 and IL-12 plus either an empty vector, SP-D-BAFF, or SP-D-APRIL plasmids. All negative titers (Tier 1 titers <3 times the titer that neutralized the control SVA-MLV virus) were given a value of 0.

[0118] Referring to Fig. 12, IL-12 plus SP-D-BAFF enhances the proportion of animals producing neutralizing antibody titers against virus 96ZM651 compared to IL-12 alone. The mouse serum tested in Fig. 11 was also assayed for neutralization against homologous virus 96ZM651, the source of the gpl40 gene encoded in the DNA vaccine. Neutralization titers for mouse serum samples were graphed by vaccine group. All negative titers (96ZM651 virus titers <3 times the titer that neutralized the control SVA-MLV virus) were given a value of 0.

[0119] Referring to Figs. 13A and 13B, Tier 1 virus neutralization titer correlates with IgG2a antibody titer but not total IgG titer, suggesting that the Thl biased immune response induced by SP-D-BAFF and SP-D-APRIL enhances neutralization titers. Only positive neutralization samples were graphed (Titer value >3 times neutralization titer for control virus SVA-MLV). The IgG2a antibody titer of each mouse sample was graphed vs. the neutralization titer of the same sample. (Fig. 13 A). There was a significant positive correlation. The total IgG antibody titer (IgGl + IgG2a) of each mouse sample was graphed vs. the neutralization titer of the same sample. (Fig. 13B). IgG2a is induced by a Thl immune response, suggesting that the Thl response mediated by the vaccine correlates with increased neutralization. [0120] ELISA data for DNA vaccination with adjuvants IL-12 or IL-21 shows that IL-12 and IL-21 can enhance antibody titers. This is also the case when these cytokines are combined with SP-D-TNFSFL constructs such as SP-D-BAFF and SP-D-APRIL. The neutralization data provides evidence that SP-D-BAFF and SP-D-APRIL enhance neutralization titer of the antibodies produced. (Figs. 1 1 , 12, 13A, and 13B). IL-12 alone is unable to induce neutralizing antibodies, but when combined with SP-D-BAFF, there is a significant increase in neutralization titers, despite no significant increase in IgG titers. (Figs. 1 1 and 12). Neutralization of HIV-1 virus is critical for protection from infection and a vaccine that can induce high neutralization titers is a major goal of HIV vaccine research.

[0121] Combinations of IL-12 and either SP-D-BAFF or SP-D-APRIL increased the proportion of mice with measurable neutralizing activity compared to IL-12 alone, suggesting that SP-D-BAFF and SP-D-APRIL are specifically enhancing neutralizing activity. (Fig. 12 and Table 1). A significant correlation between IgG2a titers and neutralization titer suggests that SP- D-BAFF and SP-D-APRIL are increasing neutralization by increasing IgG2a (and a Thl immune response) in the vaccinated animals. (Fig. 13 A) This correlation with IgG2a titers may also reflect underlying changes in B cell maturation and somatic hypermutation induced by the SP-D constructs.

[0122] When neutralization titer values were compared between groups, SP-D-BAFF enhanced mean titer values compared to IL-12 alone. (Fig. 1 1). This increase was observed for both HIV-1 viruses tested (autologous virus and a Tier 1 virus), highlighting the ability of SP-D- BAFF to increase neutralization titers in the context of multiple viral strains. These data support the hypothesis that SP-D-BAFF is able to enhance neutralization of HIV-1 virus in vaccinated animals, and that the incorporation of SP-D-BAFF in a DNA based HIV-1 vaccine will lead to better protection from HIV-1 infection via virus neutralization.

[0123] The antibodies may be tested for neutralizing ability against HIV-1 infection in CD4 T-cells.

Other Embodiments

[0124] Any improvement may be made in part or all of the compositions, kits, and method steps. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended to illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. Any statement herein as to the nature or benefits of the invention or of the preferred embodiments is not intended to be limiting, and the appended claims should not be deemed to be limited by such statements. More generally, no language in the specification should be construed as indicating any non-claimed element as being essential to the practice of the invention. This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contraindicated by context.

REFERENCES

[0125] Advances in Immunology, volume 93, ed. Frederick W. Alt, Academic Press, Burlington, MA (2007)

[0126] Crouch, E. D., et al., Recombinant pulmonary surfactant protein D. Post- translational modification and molecular assembly. J Biol Chem. 269: 15808-15813 (1994).

[0127] Current Protocols in Immunology, ed. Coligan et al., Greene Publishing and Wiley- Interscience, New York, (1992) (with periodic updates).

[0128] Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, (1992) (with periodic updates).

[0129] Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, Marcel Dekker, New York (1988-1999)).

[0130] Harlow and Lane, ANTIBODIES: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988).

[0131] Huster, K.M., et al., Selective expression of 11-7 receptor on memory T cells identifies early CD40L-dependent generation of distinct CD8+ memory T cell subsets. PNAS. 101 :5610-5615 (2004).

[0132] Making and Using Antibodies: A Practical Handbook, eds. Gary C. Howard and Matthew R. Kaser, CRC Press, Boca Raton, Fl (2006)

[0133] Medical Immunology, 6th ed., edited by Gabriel Virella, Informa Healthcare Press, London, England, (2007) [0134] Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001).

[0135] Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins (2000).

[0136] Stone, G.W. et al, Macaque Multimeric Soluble CD40 Ligand and GITR Ligand Constructs Are Immunostimulatory Molecules In Vitro. Clinical and Vaccine Immunology, 13(11): 1223-1230 (2006).

[0137] Wei, X. et al., Antibody neutralization and escape by HIV-1. Nature, 422:307-312 (2003).

Claims

What is claimed is:

1. A composition comprising at least one DNA vector encoding an antigen and at least one molecule selected from the group consisting of IL-12, IL-21, and a TNFSF ligand.

2. The composition of claim 1, wherein the antigen and at least one molecule selected from the group consisting of IL-12, IL-21, and the TNFSF ligand are encoded on different DNA vectors.

3. The composition of claim 1, wherein the antigen is HIV-1 gpl40.

4. The composition of claim 1, wherein the TNFSF ligand is selected from the group consisting of SP-D-CD40L, SP-D-CD27L, SP-D-4-1BBL, SP-D-BAFF, SP-D-APRIL, and SP- D-GITRL.

5. The composition of claim 4, wherein the TNFSF ligand is selected from the group consisting of SP-D-BAFF and SP-D-APRIL.

6. The composition of claim 1, wherein the at least one molecule is IL-12 and the TNFSF ligand.

7. The composition of claim 1, wherein the at least one molecule is IL-21 and the TNFSF ligand.

8. A method of increasing antibody production in response to a DNA vaccine comprising administering to a subject a composition comprising at least one DNA vector encoding an antigen and at least one molecule selected from the group consisting of IL-12, IL-21, and a TNFSF ligand.

9. The method of claim 8, wherein the antigen and at least one molecule selected from the group consisting of IL-12, IL-21, and the TNFSF ligand are encoded on different DNA vectors.

10. The method of claim 8, wherein the antibody production comprises production of neutralizing antibodies.

11. The method of claim 10, wherein the antibody production comprises neutralizing antibodies directed to HIV-1 gpl40.

12. The method of claim 8, wherein the TNFSF ligand is selected from the group consisting of SP-D-CD40L, SP-D-CD27L, SP-D-4-1BBL, SP-D-BAFF, SP-D-APRIL, and SP-D-GITRL.

13. The method of claim 12, wherein the TNFSF ligand is selected from the group consisting of SP-D-BAFF and SP-D-APRIL.

14. A vaccine formulation for preventing or treating a disease or condition in a subject, comprising at least one DNA vector encoding IL-12 and a TNFSF ligand.

15. The vaccine formulation of claim 14, further comprising at least one DNA vector encoding an antigen.

16. The vaccine formulation of claim 15, wherein the antigen, IL-12, and the TNFSF ligand are encoded on different DNA vectors.

17. The vaccine formulation of claim 15, wherein the antigen is HIV-1 gpl40.

18. The vaccine formulation of claim 14, wherein the disease or condition is HIV infection.

19. The vaccine formulation of claim 14, wherein the TNFSF ligand is selected from the group consisting of SP-D-CD40L, SP-D-CD27L, SP-D-4-1BBL, SP-D-BAFF, SP-D-APRIL, and SP-D-GITRL.

20. The vaccine formulation of claim 19, wherein the TNFSF ligand is selected from the group consisting of SP-D-BAFF and SP-D-APRIL.

21. A vaccine formulation for preventing or treating a disease or condition in a subject, comprising at least one DNA vector encoding IL-21 and a TNFSF ligand.

22. The vaccine formulation of claim 21 , further comprising at least one DNA vector encoding an antigen.

23. The vaccine formulation of claim 22, wherein the antigen, IL-21, and the TNFSF ligand are encoded on different DNA vectors.

24. The vaccine formulation of claim 22, wherein the antigen is HIV-1 gpl40.

25. The vaccine formulation of claim 21, wherein the disease or condition is HIV infection.

26. The vaccine formulation of claim 21, wherein the TNFSF ligand is selected from the group consisting of SP-D-CD40L, SP-D-CD27L, SP-D-4-1BBL, SP-D-BAFF, SP-D-APRIL, and SP-D-GITRL.

27. A kit for preparation of a vaccine formulation for preventing or treating a disease or condition in a subject, the kit comprising at least one DNA vector encoding at least one molecule selected from the group consisting of IL-12, IL-21, and a TNFSF ligand.

28. The kit of claim 27, wherein the at least one molecule selected from the group consisting of IL-12, IL-21, and a TNFSF ligand are encoded on different DNA vectors.

29. The kit of claim 27, wherein the disease or condition is HIV infection.

30. The kit of claim 27, wherein the TNFSF ligand is selected from the group consisting of SP-D-CD40L, SP-D-CD27L, SP-D-4-1BBL, SP-D-BAFF, SP-D-APRIL, and SP-D-GITRL.

31. The kit of claim 30, wherein the TNFSF ligand is selected from the group consisting SP-D-BAFF and SP-D-APRIL.