IMMUNOSTIMULATION BY CPG OLIGONUCLEOTIDE-VIRUS COMPLEXES

CROSS-REFERENCES TO RELATED APPLICATIONS

[001] This application claims priority to U.S. Provisional Application No. 60/765,225 (Attorney Docket No. 021935-000900US) filed February 3, 2006 and is herein incorporated by reference for all purposes.

FIELD

[002] The present disclosure relates to immunogenic compositions. More particularly, the disclosure relates to complexes formed between viruses and oligonucleotides with enhanced immunogenic properties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[003] Aspects of this disclosure were made by individuals employed by and with the support of the United States Government pursuant to National Institutes of Health (National Institutes of Allergy and Infectious Diseases) Grant AI58734. Accordingly, the United States government has certain rights in the invention.

BACKGROUND

[004] Historically, vaccine development has been a largely empirical enterprise. This has been true for both immunogens, the component of a vaccine to which an immune response is induced, and for adjuvants, additional material administered with vaccines to enhance the immune response to the immunogen.

[005] Vaccines for eliciting an immune response against pathogens, such as viral pathogens have traditionally been based on isolated (recombinant or purified) pathogen proteins, pathogen strains with reduced virulence (attenuated strains) or chemically inactivated pathogens. Each of these approaches presents advantages and drawbacks. Isolated protein vaccines lack any capacity to cause infection. However, because an effective immune response often involves antibodies and T cell responses against epitopes on multiple pathogen proteins, such isolated protein vaccines have tended to provide suboptimal immunity to most pathogens. .

(006] Attenuated pathogen vaccines, typically based on strains with one or more mutations that affects pathogen replication or environmental sensitivity have proven

effective for some pathogens. However, because these strains remain viable, they may be subject to reversion or recombination and reactivation in some circumstances, and such vaccines have been shown to cause disease in immunocompromised individuals.

[007] Inactivated or killed pathogens have in some cases proven useful as vaccines. Once inactivated, the pathogen cannot cause disease, while possessing a broader antigenic spectrum than purified individual antigen alone. However, the chemical treatments necessary to ensure complete inactivation of the pathogen have frequently resulted in modifications to the antigenic epitopes, resulting in abrogation or reduction of the desired pathogen specific immune response.

[008] One approach to increasing the immune response to suboptimal vaccines has been to administer the antigen in combination with an adjuvant that non-specifically stimulates the immune system. Recently, various ligands of Toll-like receptors (TLRs) have been suggested as adjuvants to increase the potency of vaccines. One class of TLR ligands that has received much attention is immunostimulatory CpG oligonucleotides. Oligonucleotides including an internal unmethylated CpG oligonucleotide have been shown to enhance antigen specific immune responses when administered in combination with or in close temporal proximity to an antigen.

[009] The present disclosure addresses the need for improved immunogenic compositions, particularly for use as vaccines against human and animal pathogens, and provides a novel approach to enhancing pathogen specific immune responses using immunostimulatory oligonucleotides.

SUMMARY

[010] This disclosure concerns methods for eliciting immune responses with compositions such as immunogenic molecular complexes that include an inactivated virus coated with ssDNA or ssRNA oligonucleotides. The immunogenic complexes include at least one antigen (or antigenic epitope), which can be an antigen of the inactivated virus, or a heterologous antigen. Optionally, the complexes include one or more additional immunostimulatory moieties. The immunogenic complexes are administered to a subject to elicit an immune response, such as a protective immune response, and are therefqre useful as vaccine compositions.

[Oil] The foregoing and other objects, features, and advantages of the invention -will •"••■• become more apparent from the following detailed description, which proceeds with reference to the accompanying figures. - ^•

BRIEF DESCRIPTION OF THE DRAWINGS

[012] FIG. 1 is an image of an SDS-polyacrylamide gel illuminated by UV- backshadowing to visualize virus-ODN complexes.

[013] FIG. 2 (left panel) is an image of an SDS-polyacrylamide gel illuminated by UV- backshadowing to visualize virus-ODN complexes. The right panel is a line graph illustrating quantification of ODN binding to the virus particle.

[014] FIG. 3 illustrates the calculation of binding capacity of PS-ODN on SIV particles as molar ratio

[015] FIG. 4 left and right panels are line graphs illustrating binding affinity of PS-ODN toward SIV particle. The left panel indicates the ratio between free- and bound-ODN. The right panel is a Scatchard plot analysis.

[016] FIG. 5 is a line graph illustrating the length dependency of PS-ODN binding to inactivated SIV.

[017] FIG. 6 left and right panels are images of a coomassie blue stained gel and a southwestern analysis illustrating binding of biotinylated PS-ODN to SIV particles.

[018] FIG. 7 is a set of images depicting a coomassie blue stained gel (left panel); and south-western analyses demonstrating binding of PS- and PO-ODN to purified SIV subunit protein (center and right panels, respectively).

[019] FIG. 8 is an image of a south-western analysis demonstrating that PS-ODN binds to a protein with a size of around 16 kDa in intact SIV particles.

[020] FIG. 9 is an image of SDS-PAGE analysis illustrating purification of an SIV protein with 'a molecular weight of approximately 16kDa by binding to PS-ODN. :

[021] FIG. 10 provides the amino acid sequences of four independent segments of the pi 7 protein of SIV.

[022.] ^' FIG. 1 1 is a pair of images illustrating a coomassie blue stained SDS- . polyacrylamide gel (left panel) and a south-western analysis (right panel) showing expression and binding of recombinant HIV pi 7 protein to PS-ODN.

[023] FIG. 12 is a sequence comparison between SIV and HIV pi 7 proteins.

[024] FIG. 13 is a pair of images showing a coomassie blue stained SDS-polyacrylamide gel (left panel) and a south-western analysis (right panel) illustrating localization of the pi 7 domain involved in PS-ODN binding by deletion analysis.

[025] FIG. 14 is a pair of images showing a coomassie blue stained gel and southwestern analysis showing binding by PS-ODN to a chimeric TNFα protein with the pi 7 binding sequence (amino acids 21-40).

[026] FIG. 15 is a bar graph showing relative binding of PS-ODN to chimeric TNFα proteins with deletions in the pi 7 binding sequence.

[027J FIG. 16 is a bar graph showing relative binding of PS-ODN to pi 7 with deletions in the pi 7 binding sequence.

[028] FIG. 17 schematically illustrates the use of PS-ODN to attach immunogenic moieties to the surface of viral particles.

[029] FIG. 18 is a pair of images of SDS polyacrylamide gels illustrating attachment of the KLH polypeptide to an SIV particle via a PS-ODN.

[030] FIG. 19 is an image of an SDS-polyacrylamide gel illustrating attachment of streptatvidin to an SIV particle via a PS-ODN.

[031] FIG. 20 is a pair of images of SDS polyacrylamide gels illustrating attachment of a C3d peptide to an SIV particle via a PS-ODN.

[032] FIG. 21 is a pair of line graphs illustrating induction of CD80. and CD86 expression in dendritic cells contacted with an immunogenic complex including cholera toxin attached to an inactivated SIV particle via a PS-ODN linker. ^: • - .- •• i ^' • >:. ^' ■ ■ ^' • ■ _:

[033] FIG. 22 is a bar graph illustrating production of CD86 by antigen specific T cells in response to in vitro exposure to SIV and SIV immunogenic molecular complexes. ^{• ■}

[034] FIG. 23 is a bar graph illustrating expression of IFN-alpha by peripheral blood mononuclear cells in response to in vitro exposure to SIV and SIV immunogenic molecular complexes.

1035] FIG. 24 is a line graph showing inhibition of HIV replication by PS-ODN.

DETAILED DESCRIPTION

[036] The present disclosure concerns compositions and methods for their use for eliciting an immune response against an antigen. The disclosed compositions are highly immunogenic and can favorably be used as vaccines to generate a protective immune response against a variety of pathogens, including viral pathogens, as well as tumors. The compositions include an inactivated virus or viral particle complexed with a phosphorothioate modified oligonucleotide. Optionally, a heterologous antigen is included in the viral particle. The immunogenic complex is processed by antigen- presenting cells (APC) such that the antigen (whether a viral antigen inherent in the viral particle or an associated heterologous antigen) and the oligonucleotide are delivered to the same APC, enhancing the immune response as compared to delivery of the viral particle alone. The resulting virus-ODN complexes are highly immunogenic, even in the absence of other adjuvants.

[037] The properties of certain oligonucleotides (for example, ssDNA oligonucleotides including at least one unmethylated CpG) as adjuvants are well known in the art, and it has previously been widely speculated that administration of vaccine compositions in combination with immunostimulatory ODN adjuvants would increase the strength and efficacy of the elicited immune response. However, chemical conjugation of ODNs to viral particles using conventional methods has resulted in modification of the viral antigenic proteins, which reduces or abolishes their value as immunogens.

[038] The compositions (immunogenic compositions) disclosed herein are based on the surprising discovery that phosphorothioate-modified oligodeoxyribonucleotides (PS- ODNs) spontaneously form stable complexes when mixed with retroviruses, such as HIV and SIV, inactivated with 2, 2'-dithiodipyridine (aldrithriol-2; AT-2). When mixed together PS-ODNs bind to the surface of the viral particle to generate "an adjuvant coat." This adjuvant coat substantially enhances the immunogenicity of the inactivated virus. • ■■ ^' ■ ^■ Prior attempts using inactivated virions and immunostimulatory ODNs have failed to

produce the immunogenic molecular complexes disclosed herein. This failure has apparently been due to the use of harsh inactivating agents that modify critical proteins involved in ODN binding to the virion, or to the use of viral particles lacking the appropriate binding protein. This disclosure provides methods suitable for the production of antigenically intact inactivated virions that lack the ability to replicate and thereby produce an infection, but that retain the capacity to form complexes upon contact with PS- ODNs. Because PS-ODNs bind to the inactivated virions spontaneously, without the use of chemical, cross-linking agents, the functional envelope glycoproteins and antigenic epitopes are preserved intact, retaining the capacity to elicit a strong and specific immune response to viral (and/or other) antigens of the immunogenic molecular complex.

[039] Importantly, the compositions and methods disclosed herein provide a generalizable approach to vaccine design and development by enabling a common platform technology with important potential advantages and widespread applications.

[040] An aspect of this disclosure relates to immunogenic molecular complexes made up of an inactivated virus or viral particle and phosphorothioate modified oligonucleotides (PS-ODNs). The virus is inactivated using a reagent, such as 2, 2'-dithiodipyridine (aldrithiol-2, AT-2) that preserves the structure and function of viral envelope proteins while eliminating potential infectivity by the virus. Preserved envelope glycoproteins facilitate uptake by antigen presenting cells, resulting in increased antigen presentation compared to virions without functional envelope glycoproteins. In exemplary embodiments, the inactivated virus is a retrovirus, such as HIV, SIV or SHIV.

[041] In certain embodiments, the inactivated virus or virus particle includes at least one immunogenic viral antigen. For example, the antigen can be a retroviral antigen such as an antigen derived from the viral envelope glycoproteins (such as SU and TM), viral gag proteins (such as CA, MA or NC, among others), the polymerase protein (RT), or accessory proteins (tat, rev, nef, vif, vpr, vpx, vpu). Alternatively, the inactivated virus includes at least one heterologous antigen that is not naturally expressed by the virus. For example, such an antigen can be an antigen of another virus, or a non-viral pathogen or of a tumor. Thus, the immunogenic molecular complexes disclosed herein serve as an antigen presentation platform for the delivery of a broad ^■ ■spectrum of antigens to elicit an immune response in a subject.

[042] In some embodiments, the oligonucleotide associated with the inactivated virus is typically a ligand of a Toll-like receptor (TLR) ₉ such as TLR9. For example, the oligonucleotide can be a synthetic ssDNA oligonucleotide, such as an oligonucleotide with an unmethylated CpG dinucleotide sequence. Such oligonucleotides are known in the art to have immunostimulatory properties. Immunostimulatory ODN with phosphorothioate modified backbones spontaneously associate with the inactivated virus to form stable immunogenic molecular complexes. Typically, the ODNs are at least nine nucleotides in length. For example, the immunogenic molecular complexes favorably include an immunostimulatory ODN with the generic formula 5'-XiCGX ₂ -3' ₃ wherein the central CG dinucleotide is unmethylated. Oligonucleotides with the formulas 5'-RRCGYY-3', 5'- RYCGY Y-3', 5'-RRCGYYCG-3', 5'-RYCGYYCG-3', with unmethylated CpG dinucleotides are all examples of immunostimulatory ODNs. In one particular example, the ODN complexed with the inactivated virus has the nucleotide sequence 5'- TGACCGTGAACGTTCGAGATGA-3'. In other examples, the PS-ODNs are ssRNA oligonucleotides.

[043] Optionally, the immunogenic molecular complexes also include one or more additional immunostimulatory moieties, such as additional antigens or additional adjuvants. In certain examples, the immunogenic molecular complexes include additional adjuvants that are ligands of TLRs (for example TLRs other than TLR9), such as an R848- like molecule, a lipid A molecule, a peptide, (such as the C3d peptide) or a polypeptide (such as keyhole limpet hemocyanin or streptavidin). Such additional adjuvants can be complexed to the inactivated virus using a PS-ODN to effect an association between the additional immunostimulatory moiety and the inactivated virus. In such a complex, the PS-ODN utilized as a linker can optionally be selected to possess immunostimulatory properties. For example, an additional TLR ligand can be chemically conjugated to the PS-ODN and complexed with the inactivated virus. In some instances, hybrid DNA-RNA ODN molecules that can bind to and activate both TLR9 and TLR7 are complexed with the inactivated virus.

[044] In some cases the immunogenic molecular complexes are formulated in pharmaceutical compositions or medicaments for administration to a subject, such as a ■ human subject. Typically, the pharmaceutical compositions (for example immunogenic compositions) include the immunogenic molecular complex and a pharmaceutically

acceptable carrier. Ih some cases, the pharmaceutical composition also includes an adjuvant (that is, an additional adjuvant, which is not a component of the immunogenic molecular complex).

[045] The disclosure also concerns methods of making immunogenic molecular complexes that include an inactivated virus and a PS-ODN, such as a phosphorothioate modified immunostimulatory oligonucleotide, such as an immunostimulatory CpG ODN). In an embodiment, the virus is inactivated with an agent that eliminates infectivity of the virus without destroying the structure or function of the viral envelope proteins, such as AT-2. In embodiments, a suspension of inactivated virus is contacted with a PS-ODN. Under appropriate conditions the inactivated virus and the PS-ODN spontaneously form immunogenic molecular complexes.

[046] Another aspect of the disclosure relates to methods of eliciting an immune response by administering to a subject an immunogenic molecular complex including an inactivated virus and a PS-ODN

Terms

[047] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19- 854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[048] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Additionally, . . numerical limitations given with respect to concentrations or levels of a substance, such as a growth factor, are intended to be approximate. Thus, where a concentration is indicated

to be at, least (for example) 200 pg, it is intended that the concentration be understood to be at least approximately (or "about" or "~") 200 pg.

[049] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." The abbreviation, ""e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[050] In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

[051] An "immunogenic molecular complex" is an assembly of naturally occurring and/or synthetic biological molecules that is capable of eliciting an immune response in an immunocompetent subject. In the context of this disclosure, an immunogenic molecular complex includes at least one antigen (or antigenic epitope) that elicits a specific immune response, for example antigen specific antibodies and/or antigen specific T cells. The immunogenic molecular complex also includes one or more molecules that increase the immune response (as compared to the response elicited by antigen alone). The increase can be qualitative (for example, by the inclusion of additional antigens and/or antigenic epitopes) such that an immune response is generated to two or more different antigens (or epitopes on a single antigen) or such that a specific response involving different components of the immune system (such as B cells, T cells, cytokines and/or antibodies) is generated. The increase can alternatively or additionally be quantitative, such that a stronger (e.g., as measured by increased antibody titer or by increased interferon production by antigen specific T cells) immune response is generated. For example, such molecules include adjuvants that increase the immune response in an antigen non-specific manner. Exemplary adjuvants that serve as constituents of an immunogenic molecular complex include Toll-like receptor (TLR) ligands, such as ssRNA and/or ssDNA oligonucleotides, which are ligands of TLR8 and TLR9, respectively.

[052] The phrase "complexed" or "complexed with" or "complexed to" indicates that the . referenced constituent(s) are associated or assembled into a macromolecular complex.

[053] A "vaccine composition" or "vaccine" is a composition of matter suitable for administration to a human or animal subject that is capable of eliciting a specific immune

response, e.g., against a pathogen. Thus, a vaccine is an immunogenic composition. A vaccine composition includes one or more antigens or antigenic epitopes. The antigen can be in the context of an isolated protein or peptide fragment of a protein, or can be a partially purified preparation derived from a pathogen. Alternatively, the antigen can be in the context of a whole live or inactivated pathogen. In the context of this disclosure, vaccines include immunogenic molecular complexes that contain an inactivated virus and one or more phosphorothioate modified oligonucleotides (PS-ODN).

[054] An "antigen" is a compound, composition, or substance that can stimulate the production of antibodies and/or a T cell response in an animal. Such compositions can be injected or absorbed into an animal. The term "antigen" includes all related antigenic epitopes. An "antigenic polypeptide" is a polypeptide to which an immune response, such as a T cell response and/or an antibody response, can be stimulated. "Epitope" or "antigenic determinant" refers to a site on an antigen to which B and/or T cells bind. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of an antigenic polypeptide. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and multidimensional nuclear magnetic resonance spectroscopy. In one embodiment, T cells respond to the epitope when the epitope is presented in conjunction with an MHC molecule.

1055] An antigenic polypeptide can include a virus-specific antigen, an organism-specific antigen or a disease-specific antigen. These terms are not mutually exclusive. For example, a virus-specific antigen can also be a disease-specific antigen. A virus-specific antigen is an antigenic epitope encoded by the viral genome. A disease-specific antigen is expressed coincidentally with a disease process. A disease-specific antigen can also be an antigen expressed due to a disease, condition or disorder that is not the result of infection by a pathogen, such as an antigen expressed by a tumob.- Thus, a tumor-specific, antigen is- ■ also a disease specific antigen. A disease-specific antigen may be an antigen recognized by T cells or B cells. For purposes of this disclosure, an organism-specific antigen

includes an antigen of a unicellular or multicellular organism, such as a bacteria or a eukaryotic cell or organism. An organism-specific antigen also includes a virus-specific antigen unless otherwise provided.

[056] An "adjuvant" is a composition that enhances immunogenicity (that is, the capacity of an antigen to elicit an immune response in an animal) in a non-antigen specific manner. Exemplary adjuvants include compositions such as such as suspensions of minerals (alum, aluminum hydroxide, or phosphate) onto which antigen is adsorbed; or water-in-oil emulsion in which antigen solution is emulsified in oil (Freund incomplete adjuvant, montanide-IS A), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance immunogenicity, for example, by inhibiting degradation of the antigen or promoting the localization of macrophages to the site of antigen injection.

[057] In the context of an immunogenic molecular complex of this disclosure, an adjuvant is typically a ligand of a Toll-like receptor (TLR). Exemplary TLR ligands are immunostimulatory oligonucleotides, including ssRNA and ssDNA oligonucleotides. In certain embodiments, the oligonucleotide includes an unrnethylated CpG. Typically, the TLR ligands that are oligonucleotides are at least nine nucleotides in length. Alternative TLR ligands that act as adjuvants include, for example, flagellins, R848, R848-like molecules (such as SM360320), and lipid A or similar molecules. In one example, the adjuvant is cholera toxin.

[058] An "inactivated" virus is a virus that has been treated so that it is incapable of replication and/or infection. An inactivated virus is incapable of causing disease, even in immunocompromised subjects. A "viral particle" is a macromolecular structure containing, at a minimum, an assembly of viral envelope and matrix proteins within a lipid membrane. A viral particle optionally contains additional components, such as a nucleocapsid core containing nucleic acids (including viral nucleic acids). A viral particle that includes both the replicative and genetic components of a virus is also termed a "virion." Viral particles and virions containing the replicative and genetic components of the virus can be capable of infection (and/or can be inactivated to eliminate infectivity). Thus these terms are not mutually exclusive, and in some specific cases the terms "virus," "virion" and "viral particle" can be used synonymously;. The term "inactivated" can be applied to any virus, virion or viral particle that contains biological rnacromolecules . -

corresponding to the replicative and genetic components of an intact virus, but that is incapable of causing infection.

[059] A "retroviral particle" is a viral particle that contains the envelope (and optionally, additional structural and/or functional components) of a retrovirus, such as HIV ₅ SIV or SHIV.

[060] The term "envelope protein" is used herein to refer generically to any viral protein that is integrated into and/or associated with the lipid membrane of a viral particle or virion. The term envelope protein is used in contradistinction to the phrase "inner core protein," which refers to the capsid (nucleocapsid) protein, and typically the replicative enzymes (for example, protease, transcriptase (e.g. , reverse transcriptase) and integrase proteins) associated with the viral genome. For example, in reference to the simian and human immunodeficiency viruses (SIV and HIV, respectively), the term envelope protein includes the envelope glycoproteins, gpl60 (gpl20 and gp41). In the context of this disclosure, the matrix protein (pi 7) is also included in the genus of envelope proteins. The matrix protein pi 7 is derived by cleavage of the gag protein and is localized adjacent to the inner leaflet of the lipid envelope of the viral particle.

[061] An "immunostimulatory" moiety or compound elicits or enhances an immune response when administered to a subject.

[062] An "immunostimulatory oligonucleotide" or ISS-ODN is a synthetic oligonucleotide that enhances an immune response, such as an antigen specific immune response. Exemplary ISS-ODNs are ligands of Toll-like receptors (TLRs). For example, ssRNA ISS-ODNs bind to TLR8, whereas ssDNA ISS-ODNs (such as DNA oligonucleotides with unmethylated CpG residues) bind to TLR9. Binding of such ISS- ODNs to a TLR expressed by certain immune system cells (including, for example, dendritic cells) increases expression of immunostimulatory cytokines, such as interferons, that induce proliferation and/or activation of antigen specific effector cells. In the context of the present disclosure ISS-ODNs typically include one or more phosphorothioate modified nucleotides. Exemplary ISS-ODNs are described iaU.S. Patent Nos, 6,194,348;. 6,207,646; 6,239,116; 6,406,705; 6,426,334; 6,429,199 ^" ; '6,514,948; 6,562,798; and ^' • ^{' • " •} • ^• 6,589,940, which are incorporated herein by reference.

[063] . The term "CpG" refers to a sequence of two nucleotides (a dinucleotide sequence) in which cytosine (C) precedes guanine (G) in a 5' to 3' direction. The "p" indicates that the C and G nucleotides are connected by a phosphodiester bond. In the context of an ISS-ODN, the cytosine is typically unmethylated.

[064] An "immune response" is an organism's (such as a mammal's) reaction to substances that are treated as foreign. Typically, the reaction to foreign substances, such as pathogens {e.g., viruses) involves the function of a variety of cell types, including B and T lymphocytes (B cells and T cells), Natural Killer cells, macrophages dendritic cells, among others. An immune response can be antigen specific (for example, an adaptive immune response) or antigen non-specific (for example, an innate immune response). An adaptive antigen specific immune response can involve the production of antigen specific antibodies and/or the production of antigen specific T cells, including antigen specific CD4+ cells (e.g., helper T cells) and CD8+ T cells (e.g., cytotoxic T cells) and immunoregulatory T cells. A "protective" immune response is an immune response that serves to prevent or reduce morbidity or mortality due to a disease, such as a disease caused by infection by a pathogen or abnormal cellular process (such as cancer). A protective immune response can be measured, for example, by the inhibition of viral replication or plaque formation in a plaque reduction assay or ELISA-neutralization assay, or by measuring resistance to viral challenge in vivo.

[065] Thus, to "elicit" (or "eliciting") or to "induce" (or "inducing") an immune response indicates that a stimulus, when administered to a subject, produces a reaction by the subject's immune system characterized by the proliferation, differentiation or activation of one or more type of immune effector cells, such as B cells, plasma cells, T cells and/or NK cells and their associated effector or immunoregulatory activities.

[066] The phrase "antigen presentation platform" refers to a generalizable (antigen independent) system for the presentation of antigens to a subject to elicit an immune response. In the context of this disclosure, the antigen presentation platform is an immunogenic molecular complex including an inactivated virus and an adjuvant. The presented antigen can be a viral antigen (for example, a viral antigen integral to the inactivated virus, or a heterologous viral antigen derived from a source other than the inactivated, virus), and antigen of another pathogenic organism (such as a bacterial antigen) or a tumor antigen.

[067] "AIdrithiol-2" or "AT-2" is a chemical compound with the formula Ci ₀ H ₈ N ₂ S ₂ . Aldrithiol-2 is also referred to as 2, 2'-dipyridyl disulfide or 2 ₃ 2'-dithiopyridine, which is available, for example from Aldrich Chemical Co. AT-2 covalently modifies proteins by mediating the covalent cross linking of free sulfhydryl residues on unpaired cysteines though a disulfide exchange reaction. In retroviruses, the cysteines of proteins on the interior of virions are typically found as free sulfhydryls, while the cysteines of proteins on the virion surface are typically found in disulfide linkages. Therefore, treatment of retroviral virions with AT-2 preferentially modifies cysteine residues in the internal proteins, including the strictly conserved zinc-finger motifs that are present in all retroviral nucleocapsid proteins, without affecting cysteine residues that are involved in disulfide linkages, such as those in the viral. envelope glycoproteins on the virion surface. Thus, AT-2 inactivates virus by modifying internal viral proteins, including destroying the structural integrity and ability to bind zinc of the zinc finger (motifs in retroviral nucleocapsid proteins) without appreciably affecting the conformation or function of proteins not containing free sulfhydryl moieties, such as the envelope proteins of the virus.

[068] In the context of this disclosure, the phrase "spontaneously formed" with respect to a molecular complex indicates that two or more constituents of the complex associate upon contact to form the complex without requirement for any additional constituent, agent or catalyst. Thus, the term spontaneously formed is intended to indicate that a complex is formed without the action of any chemical agent, such as a cross-linking agent, oxidizing agent, reducing agent, or the like.

[069] A "pharmaceutical composition" or "medicament" is a composition including at least one physiologically (for example, immunologically) active constituent in a formulation that is acceptable for in vivo administration to a subject mammal. Typically, the formulation includes a pharmaceutically acceptable carrier. Numerous examples of pharmaceutically acceptable formulations, preparation and carriers are provided in Remington's Pharmaceuticals Sciences, 19 ^th Ed., Mack Publishing Company, Easton, Pennsylvania (1995) (ISBN 0-912734-04-3).

[070] An "immunologically effective amount" is a quantity of a composition used to elicit an immune response in a subject. In the context of a vaccine administration, the , desired result is typically a protective pathogen-specific immune response. However,,to. , obtain protective immunity against a pathogen in an immunocompetent subject, multiple

administrations of the vaccine composition are commonly required. Thus, in the context of this disclosure, the term immunologically effective amount encompasses a fractional dose that contributes in combination with previous or subsequent administrations to attaining a protective immune response.

[071] A "subject" is a living, multicellular vertebrate organism. In the context of this disclosure the term subject includes both human and veterinary subjects for example, mammals, birds and primates. An "immunocompetent" subject is a subject that is capable of producing an immune response that is characterized as normal by medical practitioners (of either human or veterinary medicine).

Immunogenic molecular complexes

[072] This disclosure concerns immunogenic molecular complexes formed by the spontaneous association of an inactivated virus with immunostimulatory molecules that are capable of enhancing an immune response to antigens included in (or on) the inactivated viral particle. In brief, viruses, including retroviruses, such as HIV, SIV and SHIV are chemically inactivated by covalently modifying virion internal proteins to eliminate infectivity, using a reagent that preserves the structural and functional integrity of viral envelope proteins.

[073] When such inactivated viral particles, such as inactivated retroviral particles, are contacted with oligonucleotides with phosphorothioate modified backbones .(PS-ODNs), the viral particles and the PS-ODNs spontaneously form stable complexes. Binding of the phosphorothioate modified oligonucleotides (PS-ODN) is mediated via a specific interaction between the oligonucleotide and retroviral protein. This interaction is independent of the nucleotide sequence of the oligonucleotide.

[074] In certain embodiments, the PS-ODN is an immunostimulatory ODN (ISS-ODN). Such ISS-ODN enhance the immunogenic properties of the complex by interacting with a Toll-like receptor (TLR) expressed by various cells of the immune system, such as antigen presenting cells, including, for example, dendritic cells. This interaction enhances the immune response to antigens delivered by the immunogenic complex by acting as an adjuvant that stimulates cells of the immune system, for example by enhancing expression of costimulatory molecules. In contrast to simply administering mixtures of antigens and- ^■

ISS-ODN as has previously been described, the administration of immunogenic complexes

including the antigen and ISS-ODN in a single molecular structure ensures that both the antigen and adjuvant are delivered to the same antigen presenting cells, maximizing the immune response generated against the antigen.

[0751 The core of the immunogenic molecular complexes disclosed herein is inactivated viruses. The inactivated viruses are produced using a reagent, such as aldrithiol-2 (AT-2) that selectively modifies zinc finger containing proteins critical for infectivity, but that does not appreciably affect viral envelope proteins. Accordingly, these inactivated viruses possess the structural and antigenic properties of infectious viral particles without posing a risk of infection (even in immunocompromised individuals).

[076] In some embodiments, where the immunogenic molecular complex is to be used to elicit an immune response against a viral antigen, it is convenient to use an inactivated viral particle corresponding to the virus to which an immune response is desired. For example, to elicit an immune response specific for a retrovirus, an inactivated retroviral particle is usually selected as the core of the immunogenic molecular complex. Accordingly, in certain embodiments an SIV, HIV or SHIV viral particle produced by inactivating an SIV virus, an HIV virus or an SHIV virus provides the core of the immunogenic molecular complex. In other embodiments, an alternative virus that can be rendered non-infectious by exposure to AT-2 is employed as the core of the immunogenic molecular complex.

[077] Typically, the selected virus possesses integral immunogenic proteins that serve as antigens to elicit a humoral or cytotoxic immune response (such as a protective immune response) in a subject. For example, to elicit an immune response against HIV ₅ an inactivated HIV particle is selected that includes one or more of viral glycoproteins, such as the Surface glycoprotein (SU or gpl20) or transmembrane glycoprotein (TM or gp41)), viral gag proteins, such as the capsid (CA), matrix (MA), nucleocapsid (NC),or others, or the polymerase protein or reverse transcriptase (RT).

[078] Optionally, one or more heterologous antigens are also incorporated into the immunogenic molecular complex. Additional antigens can be selected from other pathogenic organisms (including other viruses) or vaccines, such as tetanus toxoid; KLLH, and the like. Alternatively, the additional antigen can be derived from a cellular source, such as an antigen expressed by an HIV infected cell (such as tat, rev, nef, vit, vpu, vpr or

vpx) or an antigen produced by a tumor (for example, a tumor antigen). Viruses that express heterologous antigens can be produced using standard recombinant expression technologies, as described below.

[079] Contact of such inactivated virus particles with PS-ODN results in the spontaneous formation of a molecular complex in which a viral particle core is coated or covered with PS-ODN. The interaction between a PS-ODN and the viral particle core is mediated by a specific interaction between the phosphorothioate backbone and- a viral envelope protein, such as the pi 7 matrix protein of an immunodeficiency virus (such as SIV, HIV or SHIV). Conserved amino acids in the N-terminal region (for example, amino acids between residues 21 and 32 of the pi 7 protein) are involved in the binding interaction between the viral particle and the PS-ODN.

[080] Thus, viral particles suitable for use in immunogenic molecular complexes can be produced from any virus that expresses amino acids 21-32 of SIV or HIV (or SHIV) pi 7. The binding effect is maintained when these amino acids are present in a chimeric (for example a fusion) protein, so long as the relevant amino acids are accessible on the surface of the protein, and available for binding in the context of the viral envelope. Accordingly immunogenic molecular complexes can be produced from viruses other than SIV ₅ HIV or SHIV by engineering an envelope protein of the virus to include this PS-ODN binding site.

[081] Essentially any oligonucleotide that includes one or more phosphorothioate backbone modification can be used to produce the immunogenic molecular complexes disclosed herein. Typically, the PS-ODN is a synthetic oligonucleotide produced using standard synthetic procedures. PS-ODN (including ISS-ODN) are available as from commercial sources (e.g., Coley Pharmaceuticals, Dynavax), or can be synthesized using known chemistries and a commercially available apparatus such as the MerMade Synthesizers available from Bioautomation, Inc.

[082] In exemplary immunogenic complexes, the inactivated virus or viral particle is combined with a phosphorothioate modified immunostimulatory ODN. ISS-ODNs are well known in the art. ISS-ODN bind to and activate Toll-like receptors (TLRs) expressed by a variety of cells, including antigen presenting cells, of the immune system. Activation of TLRs, which is not dependent on the identify of any particular antigen, contributes to an

enhanced immune response, both by increasing the magnitude of the immune response, and in some cases, by involving different cellular components of the immune response. ISS-ODNs that are ligands of TLRs include, for example, ssDNA oligonucleotides (particularly ssDNA oligonucleotides that include one or more unmethylated CpG dinucleotide) and ssRNA oligonucleotides. Single-stranded DNA and RNA oligonucleotides bind to TLR9 and TLR7/8 (in humans), respectively.

[083] Exemplary ISS-ODN are well known in the art, and methods for their production and use as adjuvants, as well as numerous exemplary nucleotide sequences of ISS-ODNs are disclosed in U.S. Patent Nos. 6,194,398; 6,207,646; 6,239,116; 6,406,705; 6,426,334; 6,429,199; and 6,514,948, each of which is incorporated herein in its entirety. In addition, ODNs including chemically modified bases, such as halogen-modified bases, as disclosed in U.S. Patent No. 6,562,798, which is incorporated herein by reference, can also be used in the context of immunogenic molecular complexes. For example, U.S. Patent No. 6,589,940 describes the production and use as adjuvants of phosphorothioate modified ISS-ODN. U.S. Patent No. 6,589,940 is incorporated herein by reference.

[084] For example, ssDNA ODNs that include an unmethylated CpG can be used as PS- ODN in the context of the immunogenic molecular complexes described herein. In exemplary embodiments, the PS-ODN is an oligonucleotide with the generic formula 5'- X ₁ CGX ₂ -3', wherein the central CG dinucleotide is unmethylated. Alternatively, the oligonucleotides can be selected from PS-ODN with the formulas 5'-RRCGYY-3', 5'- RTCGYY-3', 5'-RRCGYYCG-3', 5'-RTCGYYCG-3', with unmethylated CpG dinucleotides. In these examples, X represents any nucleotide, R represents a purine (A,G) and Y represents a pyrimidine (C,T). In one particular example, the PS-ODN complexed with the inactivated virus has the nucleotide sequence 5'-TGACCGTGAACGTTCGAGATGA-S'.

[085] ssRNA-ODNs are ligands of TLR7/8 in humans and in some other mammals, such as macaques (Wille-Reece et al., Proc. Natl. Acad. ScI USA, 102:15190-15194, 2005). Such ssRNA-ODNs with phosphorothioate modified backbones (regardless of sequence) also spontaneously bind to inactivated virus. Any phosphorothioate modified ssRNA oligonucleotide is immuηostimulatory in the context of the complexes disclosed herein; In one exemplary embodiment, the ssRNA ODN included in the immunogenic molecular complex is an oligonucleotide with the sequence 5'-GCCCGUCUGUUGUGUGACUC-S'.

In addition, chimeric phosphorothioate modified oligonucleotides, including both DNA and RNA nucleotides can be used in the complexes disclosed herein.

[086] Binding of a PS-ODN is independent of the sequence of the oligonucleotide. PS- ODNs with essentially any sequence bind to pi 7, so long as the ODN is at least about nine nucleotides in length and has at least one phosphorothioate modification of its backbone. Typically, a phosphorothioate modified RNA or DNA ODN is selected that is at least 9, and generally no longer than about 60 nucleotides in length. Longer ODNs can of course be used, however, increasing the length of the ODN increases the cost without increasing the immunostimulatory effect of the ODN. Typically, an ODN of at least 10, or at least 12, or at least 15, or at least 18 nucleotides is used to produce an immunogenic molecular complex. Most frequently, the ODN is no longer than about 60, or about 50 or about 40 nucleotides in length. Although binding to pi 7 is sequence independent, the strength of binding increases with the number of phosphorothioate modifications. Accordingly, the ODN is typically selected to have multiple phosphorothioate modifications. In some cases most, or indeed all of the residues are phosphorothioate modified.

[087] In applications where additional immunostimulatory moieties are attached to the oligonucleotide for inclusion the immunogenic molecular complex, longer ODNs can be used to facilitate the conjugation (attachment) procedure, while ensuring that sufficient length remains to effect association of the ODN with the inactivated virus. Within these parameters, selection of the PS-ODN can be made on the basis of convenience and/or availability.

[088] As indicated above, the adjuvant coat can be made up of phosphorothioate modified RNA or DNA oligonucleotides. Typically, the inactivated virus core is coated with between about 1000 and 5000 molecules of an ssDNA-ODN, with an approximate modal ratio of 1500 molecules of a phosphorothioate modified DNA-ODN per inactivated viral particle. An inactivated virus is typically coated with between about 200 and 500 molecules of an ssRNA-ODN, with an approximate modal ratio of approximately 250 molecules of a phosphorothioate modified RNA-ODN. In some cases, the bound ODN share an identical nucleotide sequence. In other cases, ODNs with different sequences, are attached to the immunogenic molecular complex. In some cases the inactivated virus is coated with a combination of ssDNA- and ssRNA-ODN. Optionally, the oligonucleotides

coating the inactivated virus can include chimeric phosphorothioate modified oligonucleotides that include both DNA and RNA nucleotides,

[089] In some embodiments, the immunogenic molecular complex includes one or more additional immunostimulatory moieties. The additional immunostimulatory moiety (or moieties) can be an antigen (for example an antigen that is not normally an integral part of the viral particle, such as tat, rev, nef, vit, vpu, vpr or vpx, a tumor antigen, or a pathogen derived antigen such as tetanus toxoid) as indicated above. Alternatively, the additional immunostimulatory moiety is a compound or biological molecule that stimulates the immune system in an antigen non-specific manner. Such immunostimulatory moieties can include other TLR ligands, such as TLR5 ligands (such as flagellins), R848, R848-like molecules (such as SM360320), lipid A or similar molecules, other adjuvants (such as cholera toxin and heat labile toxin), and immunostimulatory peptides, such as the C3d peptide. Alternatively, the immunostimulatory moiety can be a polypeptide, such as keyhole limpet hemocyanin (KLH) or streptavidin. Optionally, combinations of such moieties are included in an immunogenic molecular complex (alternatively, immunogenic molecular complexes with different additional immunostimulatory moieties are administered in combination). Such additional immunostimulatory moieties further enhance the immune response elicited upon administration of the immunogenic molecular complex.

Formation of virus-ODN complexes

[090] Immunogenic molecular complexes are produced by contacting virus inactivated with an agent, such as AT-2, that renders the virus non-infectious without impairing structure, assembly or antigenicity of viral envelope proteins. The virus can be a retrovirus that naturally expresses a protein to which PS-ODNs bind, such as the pi 7 matrix protein of immunodeficiency viruses. Alternatively, the virus can be other than a retrovirus, so long as it is expresses (naturally expresses from an endogenous gene or is engineered so that it expresses from a heterologous gene) a polypeptide that includes an amino acid sequence to which a PS-ODN binds. For example, a virus other than a retrovirus can be engineered according to recombinant DNA technologies well known to those of skill in the art to express a chimeric (fusion) protein that includes amino acids selected from an HIV or SIV pi 7 protein. For example, the chimeric protein can be engineered to include amino acids 21-32, or any additional subsequence encompassing

amino acids 21-32, such as amino acids 1-40 of an HIV or SIV pl7 protein, without impairing the structure or function of the modified viral protein.

[091] The virus is produced according to any methods known in the art for the production of the specific virus selected. Appropriate methods for virus production are well known to those of skill in the art. For example, methods for growing animal viruses can be found, for example, in DNA Viruses: A Practical Approach, Alan J. Cann (ed.) Oxford University Press, 2000; Robinson and Cranage (eds.) Vaccine Protocols (Methods in Molecular Medicine) Humana Press, 2003, and references cited therein.

[092] For example, for the production of HIV, cells capable of supporting the growth of virus, such as H9 cells chronically infected with virus, are cultured in a suitable liquid medium. The H9 T-lyrnphoid cell line can be cultured in RPMI 1640 supplemented with 5% fetal bovine serum, 2 mM glutamine (optionally, with the addition to the medium of antibiotics, such as penicillin (100U) and/or streptomycin (100 μg/ml)) at approximately 37 ⁰ C. Alternatively, HIV can be produced by infection of primary cells (for example, peripheral blood mononuclear cells, PBMC) or other lymphoid cell lines. If the virus is to be grown for administration to a human subject, the virus is typically cultured in serum free medium and manufactured according to cGMP procedures approved by the FDA (e.g., under 21 Code of Federal Regulations, parts 210 and 211, available on the world wide web at fda.gov/cder/dmpq). In one exemplary protocol, the virus is grown in AIM V medium supplemented with GMP qualified screened human serum. With time cGMP compliant procedures may change. Any methods disclosed herein can be adapted in accordance with new cGMP requirements as mandated by the FDA. SIV can produced under similar culture conditions, for example using a suitable cell line, such as H9, HuT 78, CEM X 174, SUP-Tl cells engineered to express CCR5, or other suitable cell lines.

[093] When the antigen of interest is derived from the virus (that is, the virus to be inactivated and used as the core of the immunogenic molecular complex), essentially any cells suitable for viral replication and packaging can be used. When the antigen is a heterologous antigen derived from a source other than the virus that serves as the core of the immunogenic molecular complex, the cell is selected (br modified) to express the heterologous antigen (for. example an antigen of another virus, a non- viral pathogen or a tumor antigen). Cellular membrane proteins are incorporated within the viral membrane as a function of virus packaging in cells. Thus, heterologous proteins incorporated into the

cell membrane of the host cell utilized to produce virus are. exposed, on the surface of the .. - viral particle. By engineering the host cell to express the desired antigen (or antigenic epitope) as a membrane protein, infectious virus (and inactivated viral particles) can be produced with essentially any antigen. Similarly, by modifying the host cell to express membrane bound costimulatory molecules and/or cytokines, viral particles can be produced that incorporate such additional immunostimulatory moieties.

[094] Detailed protocols for numerous such procedures are described in, for example, Sambrook et al., Molecular Cloning - A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001 ; and Ausubel et ■ al., Current Protocols in Molecular Biology (supplemented through 2004) John Wiley & Sons, New York. In addition to the above references, protocols for in vitro amplification techniques, useful in the production of recombinant nucleic acids (including expression vectors), such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), Q3-replicase amplification, and other RNA polymerase mediated techniques (e.g., NASBA), useful e.g., for amplifying cDNA probes of the invention, are found in U.S. Patent No. 4,683,202; and in Innis et al. (eds), PCR Protocols A Guide to Methods and Applications Academic Press Inc. San Diego, CA, 1990; Bartlett and Stirling (Eds), PCR Protocols (Methods in Molecular Biology) , Humana Press, Totowa, NJ, 2003.

[095] Cells can be grown to a suitable density at essentially any scale from 96 well plates to commercial tissue culture reactors reaching a scale in excess of 100 1. An appropriate scale can be selected by the practitioner based on the particular application for which the virus is desired. Virus is recovered from the supernatant (culture medium) and if desired concentrated by sucrose gradient. Viral stocks can then be stored at -20 ⁰ C.

[096] Virus is titered (if desired) by any procedure known in the art. For example virus can be titered by inoculating cells, such as H9 cells, AA2 cells or human PBMC, at a concentration of 2 x 10 ⁶ cells per 1 ml volume, with serial dilutions of supernatant or viral stock, and incubated overnight (e.g., 14-20 hours). The cells are washed, then seeded at 10 ⁵ cells in 250 μl in 96 well culture plates in multiple replicates. Cells are cultured in RPMI 1640 supplemented as ^• described above, and approximately 1/3. to! ¹ A of the meϋiuiri is replaced twice weekly. On day 10 post inoculation, supernatants are harvested and analyzed for p24 content as an index of productive infection, for example, using a capture enzyme-linked immunosorbent assay (ELISA).

[097] . To inactiyate the virus, a stock solution of inactivating agent is added directly to a suspension of virus. The inactivating agent is selected to specifically target viral proteins that contain zinc finger motifs. The cysteine residues involved in coordinating zinc ions, which are essential for protein function, and hence viral infectivity, are selectively modified whereas cysteine residues involved in disulfide linkages, such as viral envelope proteins, are not affected by the chemical treatment. One exemplary inactivating agent is aldrithiol-2 (AT-2; 2,2'-dithiopyridine, 2,2'-dipyridyl disulfide) other suitable agents can be found, for example, in U.S. Patent Nos. 6,001,555 and 6,989,263, which are incorporated herein in their entirety.

[098] For example, a stock solution of AT-2 (for example 100 mM AT-2 in dimethyl sulfoxide, DMSO) is added to viral supernatants or viral stocks to achieve a final concentration of at least about 250 μM. Typically, the final concentration of AT-2 is in a range between about 250 μM and about 5 mM. For example, in one protocol virus is treated with 1 mM (final concentration) AT-2. In some instances (e.g., for certain strains of virus) lower or higher concentrations can be used to iully eliminate infectivity. For specific viral strains, higher or lower concentrations can be determined empirically by those of skill in the art. The virus is treated with AT-2 for at least 1 hour at 37 ⁰ C, and more typically overnight (18 hr) at 4 ⁰ C. At the conclusion of the inactivating treatment, inactivated virus is purified and AT-2 is removed by either sucrose gradient purification and ultracentrifugation (large scale) or double ultracentrifugation pelleting (small scale). Purification is typically performed at 4 ⁰ C. Once inactivated, the viral particles are kept on ice and used within approximately 2 hours.

[099] Typically, the immunogenic molecular complexes disclosed herein include an inactivated virus particle core. Nonetheless, equivalent immunogenic molecular complexes can be produced using microvesicles produced from uninfected cells that have been engineered to express retroviral pi 7 and an antigen of interest according to recombinant DNA and protein expression procedures. Microvesicles are spontaneously budded from a broad variety of different cell types, which can be transformed with one or more expression vectors encoding HIV or SIV (or SHIV) pi 7 and an antigen of interest under the regulatory control of a strong constitutive or inducible promoter. : ^* . ^* ; r : - : ^•*

[010OJ The inactivated virus (or microvesicle) is contacted with phosphorothioate modified ODN, typically in a suspension in buffer (such as phosphate buffered saline or

• ^{• • ■ ■ • • ■} 23

PBS). PS-ODN (for example, immunostimulatory ssDNA oligonucleotide) is combined with inactivated viral particles at a weight per weight (w/w) ration of 2:100 PS-ODN to virus particle. The mixture is incubated at room temperature (between about 18 and 25 ⁰ C) for a period sufficient to achieve saturation binding, for example, approximately ten minutes. At saturation, binding of PS-ODN to inactivated virus reaches a molar ration of approximately 1500 PS-ODN per viral particle (with a range of approximately 1000- 5000), effectively coating the viral particle with PS-ODN. In the event that removal of free ODN is desired, the mixture is separated by centrifugation at 13,000 rpm for 30 minutes at 4 ⁰ C. After discarding the supernatant, the coated viral pellet is rinsed and resuspended in an appropriate buffer.

[0101] Inactivated virus can be coated with phosphorothioate modified ssRNA oligonucleotides in the same manner as described above with respect to phosphorothioate modified ssDNA oligonucleotides.

[0102] In certain embodiments, one or more additional immunostimulatory moieties are attached to the PS-ODN, thereby incorporating it into the immunogenic molecular complex. In such embodiments, the PS-ODN serves as a linker, attaching the additional immunostimulatory moiety to the inactivated virus. For example, other TLR ligands, or other immune modulators (such as cholera toxin and heat labile toxin) can be incorporated into the immunogenic molecular complexes to optimize quantitative and qualitative features of the immune response induced by administration of the immunogenic molecular complex to a subject (for example, as a vaccine). TLR ligands or other desirable imunomodulators that do not spontaneously associate with the inactivated viral particle can be incorporated into the complex by covalently linking them to synthetic oligonucleotides that do spontaneously associate (such as a phosphorothioate modified immunostimulatory ssDNA or ssRNA oligonucleotide). In addition to DNA containing unmethylated CpG motifs that stimulate dendritic cells and B cells via TLR9, examples of additional TLR ligands or other immune modulators that may be usefully incorporated into particulate immunogen/adjuvant complexes to induce or enhance particular types of immune responses, include: include TLR2 ligands, such as bacterial lipoproteins; TLR3 ligands, such as double stranded RNA (e.g., poly IC) that induce interferon responses; TLR4 ligands, such as LPS; TLR5 ligands, such as bacterial flagellin proteins; TLR7 ligands, such as single stranded viral RNA; TLR8 ligands like imiquimod and R848 and

related molecules, as vyell as the complement cleavage product C3d. The distinct immunomodulatory properties of these different molecules may be usefully exploited to direct the immune responses elicited by an immunogenic composition, such as a vaccine, in various defined directions, depending on the application, For example, TLR7 and TLR8 ligands are helpful in stimulating strong, Tm directed cellular immune response important for host defense against intracellular pathogens, particularly relevant to chronic viral infections, whereas C3d enhances antibody response, which may be helpful in the induction of protective antibodies by prophylactic vaccines.

[0103] Such additional immunostimulatory moieties can be conjugated to PS-ODNs using known chemistries to attach various reactive groups on the immunostimulatory moiety to the ODN via, for example, the thiol group of the phosphorothioate modified ODN. For example, immunostimulatory molecules that contain accessible amine groups (such as peptides or polypeptides) can be treated with sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SSMCC) and then reacted with a PS-ODN. Alternatively, immunostimulatory molecules with accessible amine groups can be treated with succinimidyl 4-hydrazinonicotinate acetone hydrazone (SANH) and then reacted with a 5' or 3' terminus aldehyde modified PS-ODN. Alternatively, the additional immunostimulatory moieties can be conjugated to other polysulfated linkers such as heparin (sulfated), dextran sodium sulfate, chondroitin sulfate, and the like, for attachment to virus particles.

An antigen presentation platform

[0104] Based on the ability to complex both antigens and additional immunostimulatory moieties to an inactivated virus coated with PS-ODN, the immunogenic molecular complexes disclosed herein provide a broad based, generalizable antigen delivery platform that can be used for the development of immunogenic compositions, including vaccines designed to elicit, enhance or modulate an antigen specific immune response.

[0105] The selected antigen, whether a viral antigen, an antigen of non-viral pathogen or a cellular antigen, such as a tumor antigen, can be incorporated into an immunogenic molecular complex either be expressing it in the cells in which virus is produced,' or "by attaching it to the surface of an inactivated viral particle via a PS-ODN. Essentially any antigen of interest can be incorporated into an immunogenic molecular complex, ^"

providing a highly immunogenic particulate antigen delivery system that includes both antigenic and non-specific immune modulators (such as adjuvants).

[0106] The antigen delivery platform effectively delivers both the antigen(s) and the adjuvant(s) to the same antigen presenting cells to custom tailor the immune response based on a rational vaccine design incorporating selection of optimal antigenic components and delivering them in a manner calculated to elicit and bias the immune response in a way that is predicted to provide effective protection against the particular pathogen (or non-pathogenic disease). Thus, where an antibody response is desired, adjuvants and/or immunostimulatory moieties (for example, cytokines and costimulatory molecules) predicted to enhance proliferation and differentiation of B cells, and maturation and activation of antigen specific plasma cells, are incorporated into the immunogenic molecular complex. Similarly, where a T cell response (or particular type of T cell response, such as a THl or TH2 response) is desired, the immunogenic molecular complexes are engineered to incorporate TLR ligands and/or other immunostimulatory moieties that enhance and modulate the response to desired T cell response. In addition, immunomodulatory molecules such as mitogens (for example, ConA), intermediates of intracellular signaling pathways (for example, diacylglycerol), and inhibitors (for example, siRNA molecules) of inhibitory signaling pathways such as STATl, STAT3, and SMAD pathways can also be incorporated into the antigen presentation platforms disclosed herein.

Immunogenic Compositions and Methods

[0107] Immunogenic molecular complexes as described above containing an inactivated virus and PS-ODNs can be administered to a subject to elicit an immune response, including the production of antigen specific antibodies, antigen specific T cells and the like. Most commonly, the immunogenic molecular complexes are administered to elicit a prophylactic immune response against a pathogenic organism (such as a virus) to which the subject has not yet been exposed. For example, the immunogenic molecular complexes disclosed herein can be administered as part of a localized or wide-spread vaccination effort (for example, to protect against infection by HIV). .

[0108] In such methods, an immunogenically effective amount of an immunogenic molecular complex is administered to a subject to prevent, inhibit or to treat a condition,

symptom or disease, such as a disease resulting from exposure to a pathogenic organism, such as a virus.

[0109] Accordingly, a pharmaceutical composition or medicament (particularly an immunogenic composition) containing an immunogenic molecular complex that includes an inactivated virus complexed to PS-ODNs (for example, immunostimulatpry ssDNA or ssRNA ODNs) is a feature of this disclosure. As discussed above, the immunogenic molecular complex includes at least one antigen (typically an antigen to which an immune response is desired): The antigen can be an integral component of the virus that constitutes a component of the immunogenic molecular complex, or the antigen can be a heterologous antigen derived from a different virus, a non- viral pathogen, or even a tumor.

[0110] The quantity of an immunogenic molecular complex included in the vaccine composition is sufficient to elicit an immune response when administered to a subject. For example, when administered to a subject in one or more doses, a vaccine composition containing an immunogenic molecular complex favorably elicits a protective immune response against a pathogen. A dose of the vaccine composition can include at least about 0.1% wt/wt of an immunogenic molecular complex to about 99% wt/wt immunogenic molecular complex , with the balance of the vaccine composition is made up of pharmaceutically acceptable constituents, such as a pharmaceutically acceptable carrier and/or pharmaceutically acceptable diluent. Guidelines regarding vaccine formulation can be found, e.g., in U.S. Patent Nos. 6,890,542, and 6,651,655. In one specific, non-limiting example the vaccine composition (medicament) includes at least about 1%, such as about 5%, about 10%, about 20%, about 30% or about 50% wt/wt immunogenic molecular complex. Alternatively, the dosage can be provided in terms of protein content or concentration. For example, a dose can include from approximately 0.1 μg, such as at least about 0.5 μg protein. For example, a dose can include about 1 μg of an immunogenic molecular complex up to about 100 μg, or more of an immunogenic molecular complex. As will be apparent to one of ordinary skill in the art, the quantity of pathogen present in the vaccine formulation depends on whether the composition is a liquid or a solid. The amount of immunogenic molecular complex in a solid composition can exceed that tolerable in a liquid composition. - "• v ^{> ■ • ■ •• • • ■ ■■'■ ■}

[0111] Typically, preparation of a vaccine composition (medicament) entails preparing a pharmaceutical composition that is essentially free of pyrogens, as well as any other

impurities that could be harmful to humans or animals. Typically, the pharmaceutical composition contains appropriate salts and buffers to render the components of the composition stable. Such components can be supplied in lyophilized form, or can be included in a diluent used for reconstitution of a lyophilized form into a liquid form suitable for administration. Alternatively, where the immunogenic molecular complex is prepared for administration in a solid state (e.g. , as a powder or pellet), a suitable solid carrier is included in the formulation.

[0112] Aqueous compositions typically include an effective amount of the immunogenic molecular complex dispersed (for example, dissolved or suspended) in a pharmaceutically acceptable diluent or aqueous medium. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other undesirable reaction when administered to a human or animal subject. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents and the like. Optionally, a pharmaceutically acceptable carrier or diluent can include an antibacterial, antifungal or other preservative. The use of such media and agents for pharmaceutically active substances is well known in the art. Numerous pharmaceutically acceptable carriers are described in Remington's Pharmaceuticals Sciences, 19 ^th Ed., Mack Publishing Company, Easton, Pennsylvania (1995).

[0113] Except insofar as any conventional media or agent is incompatible with production of an immune response by an inactivated pathogen, its use in the immunogenic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions. For example, certain pharmaceutical compositions can include the inactivated pathogen in an aqueous diluent, mixed with a suitable surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and/or in oils. In some cases (for example, in liquid formulations), these preparations contain a preservative to prevent the growth of microorganisms.

[0114] The compositions can be administered for therapeutic (for example, prophylactic) treatment to elicit an immune response in a subject. Favorably, the immune response is a protective immune response that protects against subsequent exposure or challenge by an infectious organism that expresses the antigen (or protects against recurrence or expansion

or mediates reduction of a tumor). In therapeutic applications, a therapeutically effective (e.g., immunogenically effective) amount of the composition is administered to a subject. Single or multiple administrations of the immunogenic molecular complexes are administered depending on the dosage and frequency as required to elicit the desired immune response and as tolerated by the subject. In one embodiment, the dosage is administered once as a bolus, but in another embodiment it can be administered in multiple or repeated doses until a satisfactory response (for example, as measured by antibody titer, by antigen- _^ specific T cell response or the like) is achieved. Generally, the dose is sufficient to elicit an immune response without producing unacceptable toxicity to the subject.

[0115] A immunogenic molecular complex can be administered by any means known to one of skill in the art, such as by intramuscular, subcutaneous, or intravenous injection, but even oral, nasal, and transdermal routes are contemplated. In one embodiment, administration is by subcutaneous or intramuscular injection, for example in a liquid suspension. To extend the time during which the immunogenic molecular complex is available to stimulate a response, the complex can be provided as an oily injection, as a particulate system, or as an implant.

[0116] As an alternative to liquid formulations, the vaccine composition can be administered in solid form, e.g., as a powder, pellet or tablet. For example, the vaccine composition can be administered as a powder using a transdermal needleless injection device, such as the helium-powered POWDERJECT® injection device. This apparatus is a uses pressurized helium gas to propel a powder formulation of a vaccine composition, e.g., containing an inactivated pathogen, at high speed so that the vaccine particles perforated the stratum corneum and land in the epidermis.

[0117] Dosages of inactivated pathogen are administered that are sufficient to elicit an immune response, e.g., a protective immune response, in a subject. In some cases, the dose includes an amount in excess of the amount of a live virus utilized to elicit an immune response, because the inactivated vaccine is incapable of increasing in number after administration into the subject. For example, therdose can include at least about 100 nanograms (or 200 nanograms, or 500 nanograms, or 1 microgram) of immunogenic . molecular complex per dose to. about 25 mg {e.g., about 10 rng, or about 15 ing, or about

20 mg), or even more. Most commonly a dose includes between 10 and 100 μg p24 (CA) equivalents for HIV (or 10-100 μg p28) equivalents for SIV).

[0118] Guidelines regarding vaccine formulation can be. found, e.g. , in U.S. Patent Nos. 6,890,542, and 6,651,655. Doses can be calculated based on protein concentration (or infectious units, such as PFU, of infectious unit equivalents). The optimal dosage can be determined empirically, for example, in preclinical studies.in mice and non-human primates, followed by testing in humans in a Phase I clinical trial.

[0119] In some cases it is desirable to administer the immunogenic molecular complex in a sustained fashion. Controlled release parenteral formulations can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems, see, Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems. 2 ^nd Edition. CRC Press, Boca Raton, FL, 2005. Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles, Microcapsules contain the therapeutic protein as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 μm so that only nanoparticles are administered intravenously. Microparticles are typically around 100 μm in diameter and are administered subcutaneously or intramuscularly (see, Kreuter, Colloidal Drug Delivery Systems. J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342, 1994 ; Tice & Tabibi, . Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, 1991.

[0120] Polymers can be used for ion-controlled release. Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer, polaxamer 407 exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and..urease (Johnston et al., ^~ Pharrri... Res. 9:425-934, 1992; and Pec, J. Pharm. Sci. Tech. 81 :626-30, 1992). Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et a!.. Int. J. Pharm. 112:215-224. 1994V In yet another aspect, liposomes are used for

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controlled release as well as drug targeting of the lipid-capsulated drug (Betageri et aL, Liposome Drug Delivery Systems. CRC Press, Boca Raton, FL, 1993; ISBN 1-56-676030- 5). Numerous additional systems for controlled delivery of therapeutic proteins are known (e.g., U.S. Patent No. 5,055,303; U.S. Patent No. 5,188,837; U.S. Patent No. 4,235,871; O.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; U.S. Patent No. 4,957,735; and U.S. Patent No. 5,019,369; U.S. Patent No. 5,055,303; U.S. Patent No. 5,514,670; U.S. Patent No. 5,413,797; U.S. Patent No. 5,268,164; U.S. Patent No. 5,004,697; U.S. Patent No. 4,902,505; U.S. Patent No. 5,506,206; U.S. Patent No. 5,271,961; U.S. Patent No. 5,254,342; and U.S. Patent No. 5,534,496).

[0121] Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceuticals Sciences, 19 ^th Ed., Mack Publishing Company, Easton, Pennsylvania (1995) (ISBN 0-912734-04-3).

Examples Example 1 : Binding of oligonucleotides to viral particles is backbone specific but not sequence specific

[0122] To clarify the parameters of oligonucleotide binding to inactivated virus, oligonucleotides with different backbones were incubated with increasing amount of SIV (inactivated as described above) in suspension in PBS. AT-2 inactivated SIV particles were incubated for 10 minutes at room temperature in PBS with a 22-mer CpG-ODN (5'- TGACTGTGAACGTTCGAGATGA-S') that had a phosphodiester (PO), phosphorothioate (PS) or morpholino (MO) backbone (synthesized by TriLink, San Diego, CA). The SIV particles were then pelleted by centrifugation, and the supernatant was loaded on a Tricine SDS-PAGE 10-20% gradient gel (Invitrogen, Carlsbad, CA) to visualize ODN that, had not associated with SIV. UV shadowing of the gel showed that incubation of inactivated SIV with PS-ODN, but not PO-ODN or MO-ODN, resulted in depletion of ODN from the supernatant, indicating association of PS-ODN with AT-2 inactivated SIV (FIG.. 1). Thus, binding of oligonucleotides to inactivated SIV particles was backbone specific. Changing the CpG dinucleotides in the sequence to GpG did not alter the ability of the PS-ODN to bind SIV, demonstrating that binding of PS-ODN tσ virus particle was sequence independent. Time course experiments demonstrated that -

association of PS-ODN with AT-2 inactivated SIV occurred within one minute at room temperature or 4 ⁰ C.

Example 2: Binding capacity of PS-ODN on SIV particle

[0123] Excess PS-ODN was mixed with SIV particle suspended in buffer. To remove unbound ODN SIV particle was pelleted by centrifugation and was rinsed with buffer. The amount of ODN bound to SIV particle was estimated by ^" UV shadowing as described above using known amount of PS-ODNs as standard (FIG. 2). The weight/weight ratio of PS-ODN to SrV particle was approximately 2.5%.

Example 3: Rough calculation of binding capacity of PS-ODN on SIV particle as molar ratio

[0124] An apparent molecular weight of SIV particle was calculated as shown in FIG. 3 based on a viral particle size of 110 ran and assuming a spherical SIV particle). Weight /weight ratio of PS-ODN to SIV particle was transformed' to molar ratio using an estimated molecular weight of SIV of approximately 4.2 x 10 ^s g/mol. The resulting calculation yielded a molar ratio of approximately 1000-1,500 molecules of PS-ODN per virus particle.

Example 4: Binding affinity of PS-ODN toward SIV particle

[0125] To measure the binding affinity of PS-ODN toward SIV particle, increasing amounts of a Cy3-labeled PS-ODN (22 nucleotides in length) was incubated with lOμg (p28 CA equivalent) of SIV particle. After centrifugation to separate bound from unbound oligonucleotide, fluorescent intensity was measured in supernatants (free ODN) and in pellets (bound ODN) using a fluorometer (FIG. 4, left panel). FIG. 4, right panel illustrates a Scatchard plot analysis showing that the Kd of PS-ODN toward SIV particle is around 140 tiM.

Example 5: Binding of ssRNA oligonucleotides to inactivated SIV particle [0126] In experiments similar to those described in the previous examples, inactivated SIV particles were incubated with phosphorothioate modified ssRNA oligonucleotides. After separation by centrifugation, free and bound oligonucleotide was measured. ssRNA bound to SIV at a weight per weight (w/w) ratio of 0.5%, indicating a molar ratio of- ^' . ■ approximately 250 ssRNA-ODNs per SIV particle.

Example 6: Length Parameter of PS-ODN

[0127] To determine the minimum nucleotide length required for optimal binding of PS- ODN to SIV particle, PS-ODN of different lengths were synthesized. To avoid any possibility of sequence-dependent variation in binding affinity. PS-ODNs consisting of various repeats of a trinucleotide unit (TCG) were examined in binding assay as described above. As illustrated in FIG. 5, optimal binding is obtained with PS-ODNs of at least nine nucleotide (3 TCG repeats) in length.

Example 7: Characterization of PS-ODN binding molecule(s) in SIV particle [0128] To characterize the moiety to which PS-ODN binds, SIV particles obtained from infected host cells and microvesicles derived from uninfected host cells were separated by SDS-PAGE (as described above) and transferred onto nitrocellulose membranes. This membrane was first incubated with biotinylated PS-ODN, and the bound PS-ODN was detected with HRP conjugated-streptavidin. Binding of PS-ODN was visualized using a chemiluminescence development assay. A representative south-western analysis is shown in FIG. 6. PS-ODN binding to a protein with a molecular weight of approximately 16-17 kDa was observed only in the SIV sample and not in the microvesicle sample, indicating that PS-ODN bound to a virus specific protein rather than a host cell protein.

Example 8: Binding of PS- and PO-ODN to purified SIV subunit proteins [0129] To determine the identity of the SIV protein to which PS-ODN were bound, five purified SIV subunit proteins, gpl20, p28, pl7, pl4 and p8 were tested for binding to PS- and PO-ODN by South-Western analysis (FIG. 7). Neither PS- nor PO-ODN bound to either p28 or pi 4. In contrast, purified p8 showed a tight binding to both type of ODNs. Both gpl20 and pl7 showed a high affinity toward PS-ODN but low affinity toward PO- ODN.

Example 9: Photo-labeling of PS-ODN binding protein in intact SIV particle

[0130] To determine the identity and specificity of PS-ODN binding in intact inactivated virus particles, SIV particles were incubated with photo-active PS-ODN. After photo- activation with UV exposure, photo-labeled protein with PS-ODN was detected by southwestern analysis using biotinylated ODN complementary to photo-labeled QDN as _. hybridization probe. A single protein with a size of 17kDa was specifically labeled with PS-ODN in intact SIV virions. (FIG. 8)

Example 10: Purification of PS-ODN binding protein from SIV particle [0131] To confirm the identify the PS-ODN binding protein in SIV particles, bound protein was purified using biotinylated PS-ODN. SIV particles were incubated with native- or biotinylated-PS-ODN. The incubation mix was then contacted with streptavidin-sepharose beads to capture the PS-ODN binding protein. After repeated washing of with buffer containing mild detergent to remove non-specifically bound proteins, the sepharose-bound proteins were analyzed by SDS-PAGE. A major protein with a molecular weight of around 16kDa was isolated (FIG. 9).

Example 11: Identification of pl7 as PS-ODN binding protein in SIV particle [0132] Protein isolated from SIV particles as described above was analyzed by mass spectroscopy to identify PS-ODN-binding protein. Four independent segments were identified as SIV pi 7 protein. The amino acid sequences of these proteins are provided in FIG. 10.

Example 12: Recombinant HIV pi 7 protein is able to bind PS-ODN [0133] To confirm that pl7 binds to PS-ODN, recombinant pl7 was generated using an E. coli protein expression system. Binding affinity of the expressed recombinant pi 7 protein toward PS-ODN was tested by south-western analysis as described above (FIG. 11). TNFo, was used as a negative binding control. PS-ODN was observed to bind specifically to expressed recombinant pl7 but not to TNFα. The binding occurred even after heat- denaturation of pl7, demonstrating that binding of PS-ODN to pl7 is a function of primary rather than tertiary structure of the protein.

Example 13: Amino acid sequence homology of pl7 derived from SIV and HIV [0134] PS-ODNs bind to SIV and HIV particles with comparable affinity. It was therefore predicted that the binding site of PS-ODN would be to an amino acid sequence that was conserved between SIV and HIV. Comparison of pl7 amino acid sequences derived from SIV and HIV (FIG. 12) revealed that the sequences of these proteins are substantially identical in their N-terminal halves, suggesting that PS-ODN binding is mediated by a sequence in this region of the protein.

Example 14: Deletion analysis of pl7

[0135] To locate the position of PS-ODN binding domain in pl7 several N-terminus deletion mutants were generated and their binding affinity toward PS-ODN was tested by

south-western analysis as described above (FIG. 13). Deletion of amino acids 1-20 from the N -terminus of pi 7 did not show any significant influence on PS-ODN binding. However, deletion of the next additional 20 amino acids (through amino acid position 40) resulted in complete loss of PS-ODN binding. This analysis indicated that PS-ODN binding is mediated by amino acids between residue 20 and 40 of pi 7.

Example 15: Confirmation of pl7 subfragment (21-40) as a binding site for PS-ODN [0136] To confirm that sequence specific binding by PS-ODNs was mediated by amino acids in the pl7 subfragment between residues 21 and 40, this subfragment was fused to the C-terminus of mouse TNFα, and the binding affinity of this chimeric protein and PS- ODN was analyzed by south-western analysis as described above. As shown in FIG. 14, the chimeric protein with amino acids 21-40 of SIV pl7 was bound by PS-ODN.

Example 16: N-terminus deletion analysis of pl7 PS-ODN binding subfragment (21-

40)

(0137] To identify specific amino acid residues involved in the pi 7 subfragment (amino acids 21-40) involved in PS-ODN binding, individual amino acids were sequentially deleted from the N-terminus of the TNFα-pl7 chimeric protein, and binding affinity of these deletion mutants toward PS-ODN was analyzed using Cy3 labeled PS-ODN. As shown in FIG. 15, deletion of each basic amino acid dropped the affinity toward PS-ODN and the deletion of amino acids 21-30 resulted in an almost complete loss of binding affinity toward PS-ODN.

Example 17: C-terminus deletion analysis of pl7 PS-ODN binding subfragment (21-

40)

[0138] A similar deletion analysis was performed starting from the C-terminus of the same pi 7 subfragment (that is, from amino acid position 40 with respect to pi 7). Deletion of first 6 amino acids from C-terminus did not result in any appreciable reduction of binding affinity. However, deletion of the last 4 amino acids including a single basic amino acid drastically reduced the affinity (FIG. 16). Based on deletion analysis of this pl7 subfragment, amino acid residues 21-32 from the N-terminal region of p>17 are . , involved in PS-ODN binding. Based on 3D structure of pi 7 PS-ODN binding segment (21-32) locates between 1 ^st and 2 ^nd α-helix structures and exists as free loop.

^{" ' ■ ' ■ ■} 35

Example 18: Attachment of immunostimulatory moieties to viruses via PS-ODN

[0139] Additional immunostimulatory moieties can be conjugated (for example, by chemical cross-linking) to PS-ODNs without disrupting binding of the PS-ODN to the viral particle as illustrated schematically in FIG. 17.

[0140] PS-ODN was conjugated to the polypeptide adjuvant KLH to demonstrate that large immunostimulatory moieties can be attached to inactivated virus via a PS-ODN linker. This conjugate was incubated with SIV particle, and bound and unbound conjugate were separated by centrifugation. The pellet was washed, and the amount of bound KLH conjugate was analyzed by SDS-PAGE as illustrated in FIG. 18. Using similar procedures SIV-streptavidin complexes were generated (FIG. 19).

[0141] Similarly a small molecule adjuvant, the C3d peptide was conjugated to PS-ODN. Binding of this ODN-peptide conjugate to SIV particle was analyzed as essentially as described above (FIG. 20).

Example 19: Virus-OND complexes with attached immunostimulatory moieties are immunologically active

[0142] Bone marrow-derived dendritic cells were incubated overnight with increasing concentrations of inactivated SIV particles (SIV), SIV particles non-covalently associated with immunostimulatory oligonucleotides (SIV+C274), or SIV particles non-covalently associated with C274-cholera toxin conjugates (SIV+C274-CT). CD80 and CD86 expression levels on the CDl Ic ⁺ dendritic cells were then assessed by flow cytometry. Results are shown as a mean fluorescence intensity ration (MFIR) where MFIR=fluorescence of treated cell/fluorescence of untreated cell. A substantial increase in expression of both CD80 and CD86 was observed in the cells treated with immunogenic complexes including the immunostimulatory moiety cholera toxin conjugated to a PS- ODN linker, indicating that the immunogenic complex induced maturation of the contacted dendritic cells (FIG. 21). Similarly, SIV immunogenic complexes induced CD86 and INF -alpha expression in peripheral blood mononuclear cells (FIGS. 22 and 23, respectively).

^: Example 20:

[0143] The ability of PS-ODN to inhibit HIV replication was assessed using in vitro replication assays based on published protocols (Kimpton and Emmerman, J. Virol.

^{• ■} - ^{■ ••} 36

66:2232-2239, 1992). P4R5 HeLa cells, engineered to express CD4, CxCR4, CCR5 and lacZ under the HIV-I LTR, were infected with tittered amounts of HIV NL4-3 (R4 strain). After two hours, PS-ODNs at various concentrations and of varying lengths consisting of triplet TCG repeats were added to the culture medium. The cells were incubated for two days, then fixed and stained for β-galactosidase production, β-galactosidase production is an indication of virus replication. The percent inhibition was determined in comparison to infected cells without PS-ODN treatment. Inhibition of viral replication by PS-ODN was dependent on dose and length as illustrated in FIG. 24.

[0144] In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.