VIRAL ADJUVANTS

FIELD OF THE INVENTION

The present invention refers to a new product and to its use as an adjuvant for inducing an immune response against an antigen in a subject. In particular, the present invention refers to the use of insect virus as adjuvants promoting potent humoral and adaptive responses against co-administered antigens.

BACKGROUND OF THE INVENTION Vaccines have proven to be successful, highly acceptable methods for the prevention of infectious diseases. They are cost effective, and do not induce antibiotic resistance to the target pathogen or affect normal flora present in the host. In many cases, such as when inducing anti-viral immunity, vaccines can prevent a disease for which there are no viable curative or ameliorative treatments available. Vaccines function by triggering the immune system to mount a response to an agent, or antigen, typically an infectious organism or a portion thereof that is introduced into the body in a non-infectious or non-pathogenic form. Once the immune system has been "primed" or sensitized to the organism, later exposure of the immune system to this organism as an infectious pathogen results in a rapid and robust immune response that destroys the pathogen before it can multiply and infect enough cells in the host organism to cause disease symptoms.

The agent, or antigen, used to prime the immune system can be the entire organism in a less infectious state, known as an attenuated organism, or in some cases, components of the organism such as carbohydrates, proteins or peptides representing various structural components of the organism.

In many cases, it is necessary to enhance the immune response to the antigens present in a vaccine in order to stimulate the immune system to a sufficient extent to make a vaccine effective, i.e., to confer immunity. Many protein and most peptide and carbohydrate antigens, administered alone, do not elicit a sufficient antibody response to confer immunity. Such antigens need to be presented to the immune system in such a way that they will be recognized as foreign and will elicit an immune response. To this end, additives (adjuvants) have been devised which immobilize antigens and stimulate the immune response.

Adjuvants can be found in a group of structurally heterogeneous compounds (Gupta et al., 1993, Vaccine, 1:293-306). Classically recognized examples of adjuvants include oil emulsions (e.g., Freund's adjuvant), saponins, aluminum or calcium salts (e.g., alum), nonionic block polymer surfactants, lipopolysaccharides (LPS), mycobacteria, tetanus toxoid, etc. Theoretically, each molecule or substance that is able to favour or amplify a particular situation in the cascade of immunological events, ultimately leading to a more pronounced immunological response can be deemed as an adjuvant.

In principle, through the use of adjuvants in vaccine formulations, one can (1) direct and optimize immune responses that are appropriate or desirable for the vaccine; (2) enable mucosal delivery of vaccines, i.e., administration that results in contact of the vaccine with a mucosal surface such as buccal or gastric or lung epithelium and the associated lymphoid tissue; (3) promote cell-mediated immune responses; (4) enhance the immunogenicity of weaker immunogens, such as highly purified or recombinant antigens; (5) reduce the amount of antigen or the frequency of immunization required to provide protective immunity; and (6) improve the efficacy of vaccines in individuals with reduced or weakened immune responses, such as newborns, the aged, and immuno-compromised vaccine recipients.

Although little is known about their mode of action, it is currently believed that adjuvants augment immune responses by any of several means. Some assist in the presentation of antigen to antigen processing cells (APC). Oil-in-water emulsions, water-in-oil emulsions, liposomes and microbeads each assist in presenting antigen to APC. Small antigens or haptens are often linked to larger, immunogenic proteins or polysaccharides to facilitate recognition by the APC. Certain adjuvants have a depot effect holding antigen in place until the body has an opportunity to mount an immune response. Adjuvants may also increase the biological or immunologic half-life of antigens, mimic microbial structures leading to improved recognition of microbially- derived antigens by the pathogen recognition receptors (PRRs) which are localized on accessory cells from the innate immune system, mimic danger-inducing signals from stressed or damaged cells which serve to initiate an immune response, induce the production of immunomodulatory cytokines or bias the immune response towards a specific subset of the immune system (e.g., generating ThI- or Th2-polarized response). By illustrative way, mechanisms of adjuvant action are reviewed in WO 03/009812.

Recent observations strongly suggest that endogenously produced cytokines act as essential communication signals elicited by traditional adjuvants. The redundancy of the cytokine network makes it difficult to ascribe the activity of a particular adjuvant to one or more cytokines. Cytokines crucial for immunogenicity may include the proinflammatory (Type 1) substances: interferon (IFN), tumor necrosis factor (TNF) - alpha, interleukin (IL)-I, IL-6, IL-12, IL-15 and IL-18, which influence antigen presentation. Others may act more downstream during clonal expansion and differentiation of T and B cells, with IL-2, IL-4 and IFN-gamma as prototypes (Brewer et al., 1996, Eur. J. Immunol., 26:2062-2066; Smith et al., 1998, Immunology, 93:556562). Adjuvants that enhance immune responses through the induction of IFN- gamma and delayed type hypersensitivity also elicit the production of IgG subclasses that are the most active in complement-mediated lysis and in antibody-dependent cell- mediated-cytotoxicity effector mechanisms (e.g., IgG2a in mice and IgGl in humans) (Allison, Dev. Biol. Stand., 1998, 92:311; Unkeless, Annu. Rev. Immunol., 1988, 6:251-81; Phillips et al., Vaccine, 1992, 10:151-8).

Adjuvants may perform more than one function. As different adjuvants may have diverse mechanisms of action, their being chosen for use with a particular vaccine may be based on the route of administration to be employed, the type of immune responses desired (e.g., antibody-mediated, cell-mediated, mucosal, etc.), and the particular inadequacy of the primary antigen.

The benefit of incorporating adjuvants into vaccine formulations to enhance immunogenicity must be weighed against the risk that these agents will induce adverse local and/or systemic reactions. Local adverse reactions include local inflammation at the injection site and, rarely, the induction of granuloma or sterile abscess formation. Systemic reactions to adjuvants observed in laboratory animals include malaise, fever, adjuvant arthritis, and anterior uveitis (Allison et al. MoI. Immunol., 1991, 28:279-84; Waters et al., Infect. Immun., 1986, 51:816-25). Such reactions often are caused by the interaction of the adjuvant and the antigen itself, or may be due to the type of response to a particular antigen the adjuvant produces, or the cytokine profile the adjuvant induces. Thus, many potent immuno-adjuvants, such as Freund's Complete or Freund's Incomplete Adjuvant, are toxic and are therefore useful only for research purposes in experimental aimals, not animal or human vaccinations. Due to the limited range of

immuno-adjuvants available there is therefore a need in the art for new and improved immuno-adjuvants.

Inventors have now, surprisingly, found that certain insect viruses, e.g., baculoviruses, can be used as an adjuvant to promote and enhance antigen specific immunological responses in a subject.

Insect viruses are viruses which infect insect cells such as baculoviruses, granuloviruses, ascoviruses, iridoviruses, parvoviruses, polydnaviruses, poxviruses, reoviruses, rodaviruses, picorna-like viruses, tetraviruses, etc.

Baculoviruses (BVs) [(BV) baculovirus, in singular] are a family of large rod- shaped viruses with double- stranded DNA that are pathogenic for insects. They naturally infect only arthropods and have been isolated mostly from Lepidoptera but also from Hymenoptera, Diptera and Crustacea. BVs have very species-specific tropisms among the invertebrates with over 600 host species having been described.

Immature (larval) forms of moth species are the most common hosts, but BVs have also been found infecting mosquitoes, including adults. However, BVs cannot replicate in mammalian or other vertebrate animal cells and do not cause visible cytopathic effect even at high titers.

BVs are regarded therefore as safe and selective bioinsecticides that have been used worldwide against many insect pests. BV expression vectors such as Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) are also widely used as eukaryotic expression vectors for proteins requiring post-translational modifications such as glycosylation, proteolytic cleavage and fatty acylation. BVs have additionally been wide used in research and industry and for their dissemination into the environment.

SUMMARY OF THE INVENTION

Inventors have, surprisingly, shown that baculoviruses (BVs) have strong adjuvant properties in mice promoting potent humoral and CD8 ⁺ T-cell (CTL) adaptive responses against co-administered antigens. BVs also induce in vivo maturation of dendritic cells (DC) and production of inflammatory cytokines. The adjuvant effect of BVs was shown to be mediated by IFNα/β production and to play a major role in the strong immunogenicity of virus-like particles produced in baculovirus-insect cell expression system.

Thus, in one aspect, the present invention refers to a product comprising: a) an antigen; and b) an adjuvant, wherein said adjuvant comprises the nucleic acid of an insect virus. The components (antigen and adjuvant) of said product may be contained in the same composition or, alternatively, separately, in different compositions. Advantageously, the nucleic acid of an insect virus of said product is included, obtained or encapsidated into a viral or non-viral vehicle or vector, which constitutes a further aspect of this invention. In another aspect the invention refers to a product comprising, separately: a) an antigen, and b) an enhancer agent of the immune response against said antigen, wherein said enhancer agent comprises an the nucleic acid of an insect virus, as a combination for its simultaneous or successive administration in a subject, for inducing an immune response against said antigen in said subject.

In another aspect the invention refers to a product as previously described as a medicament. In a particular embodiment, such medicament is a medicament for inducing an immune response against said antigen in said subject.

In another aspect, the invention refers to a pharmaceutical composition comprising a product as previously defined and a pharmaceutically acceptable excipient or vehicle.

In another aspect, the invention refers to the use of the nucleic acid of an insect virus as enhancer agent of the immune response against an antigen, in combination with said antigen, for the manufacture of a pharmaceutical composition, for inducing an immune response against said antigen in said subject or for preventing and/or treating an infectious disease, or for preventing and/or treating a cancer.

In another aspect, the invention refers to an adjuvant composition comprising, at least, the nucleic acid of an insect virus and, alternatively, one o more additional adjuvants and/or pharmaceutically acceptable vehicles.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows that BVs strongly enhance humoral and CTL responses against co-administered antigens. Figure Ia shows the kinetics of the OVA-specific antibody

(Ab) response induced by immunization with OVA protein in combination with BVs. C57BL/6 mice received a single intravenous (i.v.) or subcutaneous (s.c.) injection of OVA protein (10 μg) either alone or with 10 ⁶ pfu of BVs. Control mice received BVs alone (10 ⁶ pfu). Mice were bled at different times after injection and individual sera were tested for OVA protein specific IgG by ELISA. Figure Ib shows the effect of the dose of BVs in the induction of OVA-specific IgG. Mice received a single i.v. injection of OVA protein (10 μg) either alone or in combination with different doses of BVs (10 ⁶ , 10 ⁵ , 10 ⁴ or 10 ³ pfu). Results are expressed as mean ± SEM for 4-5 mice. Data are representative of two independent experiments. Figure Ic shows the induction of OV A ₂ 57-264- specific CD8 ⁺ T-cells by immunization with BOVAp and BVs. C57BL/6 mice were left untreated or were immunized by a single i.v. injection of BOVAp (10 ⁹ beads) either alone or in combination with BVs (10 ⁶ pfu). Control group of mice received BVs alone (10 ⁶ pfu). At different times after injection, O V A257-264- specific CTLs (OVATetr ⁺ ) in spleen were quantified by tetramer staining. The frequency of OVATetr ^"1" after i.v. injection of BOVAp and PoIyLC (25 μg) is shown for comparison. Results are expressed as mean ± SEM for 5-7 mice. (d,e) C57BL/6 mice were left untreated or were immunized by i.v. route with BOVAp (10 ⁹ beads) either alone or in combination with different doses of BV (10 ⁶ , 10 ⁵ , 10 ⁴ and 10 ³ pfu). Control groups of mice received BVs alone at the same doses. The specific CTL response was analyzed at day 7 by Figure Id represents an in vivo killing assay and Figure Ie shows tetramer staining. Dots represent individual mice. All results are representative of at least two independent experiments.

Figure 2 shows that inactivation of BVs abrogates its adjuvant property. In Figure 2a it is shown that C57BL/6 mice were left untreated or were immunized by i.v. route with BOVAp (10 ⁹ beads) alone or in combination with supernatant (100 μL) from Sf9 cell culture (72 hours) infected or not with BVs [SN (BV-infected Sf9) and SN (non infected Sf9), respectively]. The titer of BVs in the supersnatant from BV-infected Sf9 cells was 10 ⁷ pfu/mL. As control, mice were also immunized with supernatant without BOVAp. Figure 2b shows the effect of the inactivation of BVs on its capacity to promote CD8 ⁺ T cell priming. Mice were immunized i.v. with BOVAp alone or in combination with either BVs untreated (BV) or treated with UV-light (BV-UV), BEI (BV-BEI), Triton (BV-Triton) or Benzonase (BV-Benzo), as described in Material and

Methods (Example 1). Each mouse received a volume of treated BVs equivalent to the volume of untreated BVs containing 10 ⁶ pfu. As control, a group of mice was immunized with BOVAp and Benzonase (Benzo). Figure 2c shows the adjuvant properties of BV-DNA. C57BL/6 mice were left untreated or were immunized i.v. with BOVAp alone or in combination with BV-DNA (10 μg) either free (BV-DNA) or mixed with 30 μL of DOTAP. As controls, mice were immunized with BOVAp and CpG-ODN (10 μg), either free or mixed with DOTAP (30 μL) or with BOVAp + DOTAP (30 μL). (a,b,c) CTL responses were analyzed by in vivo killing assay at day 7 after injection. Dots represent individual mice. Data are representative of two independent experiments.

Figure 3 shows that IFNα/β mediates the adjuvant property of BVs. In Figure 3a in vivo production of type I IFN in response to BVs it is shown. Sera from C57BL/6 mice were collected at different time points after injection of BVs and titrated for IFNα and IFNβ by ELISA. Different doses of BV were tested (10 ⁶ , 10 ⁵ , 10 ⁴ and 10 ³ pfu). In Figure 3b level of IFNα detected in the sera of C57BL/6 mice 8 hours after the injection of BVs either untreated (BV) or treated with UV light (BV-UV), BEI (BV- BEI) or Triton (BV-Triton) it is represented. Each mouse received a volume of treated BV equivalent to the volume of untreated BV containing 10 ⁶ pfu. Figure 3c shows the effect of the absence of type I IFN receptor on the level of seric IFNα and IFNβ induced by BV injection. 129 Sv and IFNARko mice were injected with 10 ⁶ pfu of BV and their sera were titrated for IFNα and IFNβ by ELISA. (a,b,c) Results are expressed as mean ± SEM for 3 mice per group. In Figure 3d it is shown that 129 Sv and IFNARko mice received a single i.v. injection of OVA protein (10 μg) either alone or in combination with BV (10 ⁶ or 10 ⁵ pfu). Mice were bled at different times after injection and individual sera were tested for OVA protein specific IgG by ELISA. Results are expressed as mean ± SEM for 4 mice. Figure 3e shows that 129 Sv and IFNARko mice were left untreated or were immunized by i.v. route with BOVAp (10 ⁹ beads) either alone or in combination with 10 ⁶ or 10 ⁵ pfu of BVs. The specific CTL response was analyzed at day 7 by tetramer staining, ELISPOT and by in vivo killing assay (upper, medium and lower panel, respectively). (f,g) IFNARko mice are able to mount a normal humoral and CD8 T-cell response under appropriate conditions. In Figure 3f 129 Sv and IFNARko mice received a single s.c. injection of OVA protein (10 μg) either alone

or in combination with alum. Fifteen days after injection, anti-OVA specific IgG were titrated by ELISA. In Figure 3g 129 Sv and IFNARko mice were left untreated or were i.v. immunized with BOVAp (10 ⁹ beads) either alone or in combination with 100 μg of anti-CD40 mAb. The specific CTL response was analyzed at day 7 by in vivo killing assay. (f,g) Results are expressed as mean ± SEM for 4-5 mice. All data are representative of at least two independent experiments.

Figure 4 shows that in vivo maturation of pDC and cDC by BV is dependent of IFNα/β. In Figure 4a C57BL/6 mice received an i.v. injection of PBS or BV (10 ⁶ , 10 ⁵ , 10 ⁴ or 10 ³ pfu) and 15 hours later, splenic pDC and cDC were magnetically sorted using anti-CDllc and anti-mpdca-1 coated magnetic beads. Phenotypic activation markers (CD40, CD80, CD86, K ^b and I-A ^b ) were analyzed on CDll ^int B220 ⁺ (pDC) and on CDll ^hlgh B220 ^" populations (cDC). Figure 4b shows that the inactivation of BV abrogated in vivo maturation of pDC and cDC. C57BL/6 mice were injected i.v. with PBS or BV, either untreated (BV, 10 ⁶ pfu) or treated with UV light (BV-UV), BEI (BV- BEI) or Triton (BV-Triton). Phenotypic activation markers (CD40 and CD86) were analyzed on magnetically sorted splenic cDC and pDC 15 hours after injection. In Figure 4c sorted splenic DC (pDC and cDC) from 129 Sv and IFNARko mice treated with PBS, LPS (25 μg) or BV at two different doses (10 ⁶ and 10 ⁵ pfu) were analyzed for the expression of CD40 and CD86 markers 15 hours after treatment (b and c). Geometric Mean Fluorescence ± SEM for 3 mice per group is shown. All data are representative of at least two independent experiments.

Figure 5 shows the humoral responses induced by immunization with OVA protein and alum either alone or in combination with BVs. C57BL/6 mice received a single s.c. injection of OVA protein (10 or 0.5 μg), and alum (1 mg) with or without BVs (10 ⁶ pfu). Control group received only OVA. Mice were bled at different times after injection and individual sera were tested for OVA protein specific IgG by ELISA. Data are representative of two independent experiments.

Figure 6 represents the percentage of O V A ₂₅₇ - ₂₆₄ - specific lysis determined by in vivo killing assay. C57BL/6 mice were left untreated or were immunized by a single i.v. injection of BOVAp (10 ⁹ beads) either alone or in combination with BV (10 ⁶ pfu). Control mice remained untreated or were injected with BV (10 ⁶ pfu). At day 7, naϊve and primed mice were injected i.v. with a mix (1:1) of OVA ₂₅₇ - ₂₆₄ peptide-loaded CFSE ^hlgh and unloaded CFSE ^low splenocytes. The number of CFSE-positive cells

remaining in the spleen after 20 hours was determined by FACS analysis. Plots are representative of data obtained from more than 30 mice.

Figure 7 represents the percentage of O V A ₂₅₇ - ₂₆₄ - specific lysis determined by in vivo killing assay 80 days after injection. C57BL/6 mice were left untreated or were immunized by a single i.v. injection of BOVAp (10 ⁹ beads) either alone or in combination with BVs (10 ⁶ pfu). As control, a group of mice was only injected with BV (10 ⁶ pfu). At days 7 and 80, naϊve and primed mice were injected i.v. with a mix (1:1) of OVA ₂ 57-264 peptide-loaded CFSE ^hlgh and unloaded CFSE ^low splenocytes. The number of CFSE-positive cells remaining in the spleen after 20 hours was determined by FACS analysis. Data are representative of two independent experiments.

Figure 8 represents the percentage of OVATetr ^"1" cells among CD8 ⁺ T-cells after i.v. or s.c. injection of BOVAp and BV. C57BL/6 mice were left untreated or were immunized by a single i.v. or s.c. injection of BOVAp (10 ⁹ beads) either alone or in combination with BVs (10 ⁶ pfu). Seven days after injection, OVATetr ^"1" were quantified in spleen by tetramer staining. Data are representative of two independent experiments.

Figure 9 shows that immunization with BOVAp together with BV induces a strong type 1 CTL response. C57BL/6 mice were left untreated or were immunized by i.v. route with BOVAp (10 ⁹ beads) either alone or in combination with different doses of BV (10 ⁶ , 10 ⁵ , 10 ⁴ or 10 ³ pfu). As control, groups of mice received only BV at the same doses. The specific CD8 ⁺ T-cell response was analyzed at day 7 by measuring the frequency of IFN-γ and IL-4 spot-forming cells (SFC) by ELISPOT. Dots represent individual mice. All results are representative of at least two independent experiments.

Figure 10 shows the CTL response induced by BVs and Beads coated with different peptides. Latex beads were coated with the following H-2 ^b -epitopes containing peptides: E7 ₄₉ _ ₅₇ from Human Papillomavirus E7 protein, HY _738-746 from the Minor Histocompatibility (H) male specific (Y) antigen (H-Y), LCMV ₃₃ _ ₄ i from Lymphocytic choriomeningitis virus (LCMV) glycoprotein and PBl ₇₀₃ - ₇₁₁ from the PBl subunit of the Influenza virus RNA polymerase. Female C57BL/6 mice were left untreated or were immunized by a single i.v. injection of the corresponding beads (10 ⁹ beads) either alone or in combination with BV (10 ⁶ pfu). As control, a group of mice was only injected with BV (10 ⁶ pfu). Seven days later, naϊve and primed mice were injected i.v. with a mix (1:1) of homologue peptide-loaded CFSE ^hlgh and unloaded CFSE ^low splenocytes. The

number of CFSE-positive cells remaining in the spleen after 20 hours was determined by FACS analysis. Data are representative of two independent experiments.

Figure 11 shows that in vivo production of inflammatory cytokines in response to BV is dependent on IFNα/β. 129 Sv and IFNARko mice were injected with BVs either untreated (10 ⁶ or 10 ⁵ pfu) or UV-treated (10 ⁶ pfu) and sera harvested at various time points were titrated for IL-12p40, IL-12p70, IL-6, IFN-γ, MIG and IP-IO by ELISA. Results are expressed as mean ± SEM for 5 mice per group and are representative of two independent experiments.

DETAILED DESCRIPTION OF THE INVENTION

In an aspect, the invention refers to a product, hereinafter referred to as the product of the invention (1), said product comprising: a) an antigen; and b) an adjuvant, wherein said adjuvant comprises the nucleic acid of an insect virus.

As used herein "antigen" refers, in general, to any product capable binding to an antibody or to a T-cell receptor when it is presented by major histocompatibility complex (MHC) molecules, and includes entire organisms or portions or components thereof, e.g., carbohydrates, proteins or peptides representing various structural components of said organisms. As such, an antigen is any substance capable of being recognized by the immune system of a subject and/or capable of inducing in a subject a humoral immune response or a cellular immune response which leads to the activation of lymphocytes T and/or B when it is introduced into a subject, what may require, sometimes, that the antigen contains or is joined to an epitope of Th cells. An antigen may contain one or more epitopes (e.g., epitopes B and T). The product of the invention (1) may contain just one antigen or, alternatively, two or more different antigens.

In a particular embodiment of the present invention, said antigen is a viral antigen, a bacterial antigen, a fungal antigen, a protozoa antigen, a nematode antigen, a tumour antigen, an allergen, etc. Illustrative non limitative examples of said antigens can be found in WOO 1/026682.

Thus, illustrative, non limitative, examples of bacterial antigens are, for example, Bordetella antigens, Mycobacterium tuberculosis antigens, porin antigens from Bactericides which is associated with ulcerative colitis and inflammatory bowel

disease, Helicobacter pylori antigens, Streptococcus antigens associated with dental caries, antigens derived from Campylobacter jejuni which is associated with diarrheal disease, the P-glycoprotein cell surface antigen which is correlated with multidrug resistance in mammalian species, a pilus antigen present in adhesion-forming bacteria, Moraxella catarrhalis outer membrane vesicle antigens associated with pulmonary disease, etc.

Illustrative, non limitative, examples of viral antigens include rotavirus antigens, human immunodeficiency virus (HIV) antigens, HIV type 2 (HIV-2) antigens, simian immunodeficiency virus (SIV) antigens, non-A, non-B hepatitis virus antigens, delta antigen of hepatitis D virus, influenza virus antigens, foot-and-mouth disease virus (FMDV) antigens, poliovirus antigens, human rhinovirus (HRV) antigens, human papillomavirus (HPV) antigens, hepatitis C virus (HCV) antigens, hepatitis B core antigens, Epstein Barr virus-related antigens, hepatitis V virus C33 antigen, cytomegalovirus (CMV) antigens, herpes simplex virus antigens, HTLV-I and HTLV-II antigens, etc.

Illustrative, non limitative, examples of protozoa and nematodes antigens include Plasmodium vivax which causes malaria, Leishmania antigens which are associated with Leishmaniasis, the antigens of the nematode parasite Dirofilaria immitis, antigens oϊAnaplasma marginale which causes bovine anaplasmosis. Illustrative, non limitative, examples of tumour antigens are, for example, the esophageal cancer associated antigen, the mammary- specific protein (mammaglobin) which is associated with breast cancer, the prostate mucin antigen which is associated with prostate adenocarcinomas, human prostate specific antigen (PSA), the SF-25 antigen of colon adenocarcinoma, urinary tumor associated antigens, melanogenic antigen, the MART-I melanoma antigen, human tumor-associated antigen (PRAT), TRP-2 protein tumor antigen, the human tumor-associated antigen (TUAN), the tumor specific T antigen which is associated with virally-induced tumors, etc.

Illustrative, non limitative, examples of allergens include the vespid antigen 5 which is used to treat patients with vespid venom allergy, the CRX JII Cryptomeria japonica major pollen allergens, ryegrass pollen allergens LoI p Ib. 1 and LoI p Ib. 2, allergens of alder pollen, hazel pollen and birch pollen, the house dust mite Dermatophagoides farinae Derf I and Derf II allergens, and D. pteronssinus Der p I and

Der p VII allergens, cat allergen (FeI d I), cockroach (CR) allergens, peanut allergen (Ara h II), etc.

Other antigens which are associated with the development of diseases are also included within the scope of the invention. These include, but are not limited to, the exemplary GAGE tumor rejection antigen precursor which is associated with cancer development, the antigens extracted from mammalian malpighian epithelia (e.g., esophagus and epidermis) and associated with rheumatoid arthritis, the Rh blood group antigens, antigens indicative of the presence and progression of atherosclerotic plaque, the IgG Fc-binding protein antigen associated with autoimmune diseases such as ulcerative colitis, Crohn's disease, rheumatoid arthritis, and systemic lupus, the Sm-D antigen associated with system lupus erythematosus (SLE), monocyte antigens, the antigen associated with autoimmune inner ease Meniere's disease, the mesothelin differentiation-associated antigen which is implicated in mesotheliomas and ovarian cancers, the osteogenic and fibroblastic antigen (OFA) associated with bone-related diseases, the mast cell function-associated antigen (MAFA) which is associated with inflammatory and allergic reactions, etc. Also included within the invention's scope are cytokines antigens such as the exemplary interleukin-lcX, interleukin-1, interleukin-2, interferon-a, interferon-Y, and tumor necrosis factor, interleukin-6, the TGF- superfamily which includes the TGF- family, the inhibin family, the DPP/VG1 family, and Mullerian Inhibiting Substance Family, interleukin-11, leukemia inhibitory factor, oncostatin M, and ciliary neurotrophic factor, interleukin-12, etc.

The product of the invention (1) further comprises, in addition to an antigen, an adjuvant, wherein said adjuvant comprises the nucleic acid of an insect virus. As it is shown in the Example accompanying the present description, insect viruses can exert a strong influence on mammalian humoral and cellular responses against co-administered antigens through DC maturation and production of inflammatory mediators.

The term "insect virus" as used herein refers to viruses which infect insect cells and are pathogenic for insects; although said insect virus can infect mammal and other vertebrate animal cells, they are not pathogenic for them. Illustrative, non limitative, examples of insect viruses included within the scope of the present invention include, DNA viruses such as baculoviruses (e.g., nuclear polyhedrosis viruses (NPV)), granuloviruses (GV), ascoviruses, iridoviruses, parvoviruses, polydnaviruses, poxviruses, etc., and RNA viruses such as reoviruses (cytoplasmic polyhedrosis

viruses), rodaviruses, picorna-like viruses, tetraviruses, etc. In a particular embodiment, said insect virus is a baculovirus (BV).

Additionally, data herein included show that the nucleic acid of said insect virus also produces said adjuvant effect. The term "nucleic acid" as used herein refers to a complex, high-molecular-weight biochemical macromolecule composed of nucleotide chains that convey genetic information. The most common nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Effectively, data herein included show that DNA from BVs (BV-DNA) also produces said adjuvant effect. In fact, inventors have demonstrated that inactivation of BVs by, for example, treatment with aziridine binary ethylenimine (BEI), a potent alkylating agent that selectively reacts with nucleic acids but not with proteins, abolished the adjuvant properties of BVs. Thus, the nucleic acid of an insect virus may be responsible for its adjuvant properties. Further, it appears that the nucleic acid of the insect virus should be present in an infectious form so that it could exert its adjuvant properties; thus, in a particular embodiment, the adjuvant of the product of the invention (1) comprises full-length nucleic acid of an insect virus.

Inventors have additionally shown that, advantageously, said isolated nucleic acid from said insect virus should be delivered by an appropriate vehicle for preventing its degradation or damage. Thus, the nucleic acid of the insect virus which is present in the product of the invention (1) is, advantageously, included, contained or encapsidated within an appropriate vehicle or vector in order to prevent its degradation or damage. Said vehicle or vector may be a viral vehicle or vector, such as, for example, the virion per se (i.e., the mature insect virus particle consisting of the viral nucleic acid core and protein coat, and in some types an outer lipid envelope), a viral-like particle (VLP) (i.e., an empty viral particle comprising viral structural protein(s)) containing the nucleic acid of said insect virus, said VLP can derive from the same insect virus or from other insect or non-insect virus (e.g., parvovirus, etc.), etc.; alternatively, said vehicle or vector may be a non-viral vehicle or vector, such as, for example, a liposome, a polymer, or a mixture thereof, etc., to render a liposome/nucleic acid complex (lipoplex), a polymer/nucleic acid complex (polyplex), a liposome/polymer/nucleic acid complex (lipopolyplex), etc. According to the invention, virtually any suitable vehicle or vector which prevents, or cooperates in preventing, said viral nucleic acid from degradation or damage can be used; nevertheless, in a particular embodiment, said vehicle or vector is

a viral vector such as the virion of the insect virus, whereas in another particular embodiment, said vehicle or vector is a non-viral vehicle or vector, such as a cationic liposome which facilitates the uptake of DNA and also prolongs its life span in vivo.

Therefore, in a particular embodiment, the adjuvant which is present in the product of the invention (1) comprises a vehicle or vector containing the nucleic acid of an insect virus, said vehicle or vector being a viral vehicle or vector, such as, for example, the virion per se of the insect virus, a VLP containing the nucleic acid of said insect virus, said VLP deriving from the same insect virus or from other insect or non- insect virus, etc.; or alternatively, said a non- viral vehicle or vector, such as, for example, a liposome, a polymer, or a mixture thereof, etc., to render a liposome/nucleic acid complex (lipoplex), a polymer/nucleic acid complex (polyplex), a liposome/polymer/nucleic acid complex (lipopolyplex), etc. In a preferred embodiment, the adjuvant which is present in the product of the invention (1) comprises a vehicle or vector containing the nucleic acid of an insect virus, said vehicle or vector being the virion of the insect virus, e.g., a baculovirus, or a cationic liposome containing the nucleic acid of an insect virus, e.g., the nucleic acid (DNA) of a baculovirus.

Therefore, in another aspect, the invention relates to a vehicle or vector comprising a nucleic acid from an insect virus with the proviso that said vehicle or vector is not the virion per se of an insect virus. Said vehicle or vector may be a viral vehicle or vector, such as, for example, a VLP containing the nucleic acid of said insect virus, etc.; or, alternatively, a non-viral vehicle or vector, such as, for example, a liposome, a polymer, or a mixture thereof, etc., to render a liposome/nucleic acid complex (lipoplex), a polymer/nucleic acid complex (polyplex), a liposome/polymer/nucleic acid complex (lipopolyplex), etc. The liposomes and polymers useful for generating non-viral vehicles or vectors are known by the skilled person in the art.

In another particular embodiment, the product of the invention (1) may contain, in addition to said adjuvant containing the nucleic acid of an insect virus, one or more additional adjuvants. Said additional adjuvants may also cooperate in enhancing the immune response to the antigen in a subject. Virtually any adjuvant, non-incompatible with the adjuvant containing the nucleic acid of the in sect virus, may be used, such as, but not limited to, those that contain an emulsion system and a synthetic resin material that is capable of complexing with antigens, copolymers of

polyoxyethylene/polyoxypropylene block copolymers, lH-imidazo[4,5-C-quinolin]-4- amine and its derivatives, mutant Escherichia coli heat-labile enterotoxin holotoxin, formyl methionyl peptide (fMLP), ADP-ribosylating exotoxin which is particularly suitable for transcutaneous administration, interleukin-12, polydimethylsiloxane and a complex emulsifier, hemozoin or P-hematin, Saccharomyces cerevisiae glucan, zinc hydroxide/calcium hydroxide gel, lecithin, and polyalphaolefin, polyoxyethylene sorbitan monoesters (PS) which are useful for topical administration of antigens via mucosal membranes, and transdermal liposomes. Furthermore, methods of using the crystalline bacterial surface layers (SL) as adjuvants by conjugating antigens to SL are also known in the art (Jahn- Schmid et al. (1997) International Immunology 9:1867- 1874).

The components (antigen and adjuvant) of the product of the invention (1) may be contained in the same composition or, alternatively, they may be, separately, in different compositions. Therefore, in an embodiment, the invention refers to a product comprising, separately: (a) an antigen, and, (b) an adjuvant, wherein said adjuvant comprises the nucleic acid of an insect virus.

As mentioned above, an insect virus and its nucleic acid can be used as adjuvant for enhancing the immune response against an antigen in a subject. Therefore, in another aspect, the invention refers to a product, hereinafter referred to as product of the invention (2), comprising, separately: a) an antigen, and b) an enhancer agent of the immune response against said antigen, said enhancer agent comprising the nucleic acid of an insect virus, as a combination for its simultaneous or successive administration in a subject, for inducing an immune response against said antigen in said subject.

The term "subject", as used in this description, refers to a member of a mammal species, and includes, but is not limited to, domestic animals, rodent, primates and humans; the subject is preferably a human being, male or female, of any age or race. The particulars of the antigen which may be present in the product of the invention (2) are the same than the antigen of the product of the invention (1). Also, the product of the invention (2) may contain just one antigen or, alternatively, two or more different antigens.

The particulars of the insect virus and its nucleic acid which may be present in the product of the invention (2) are the same than those of the product of the invention (1). Thus, both DNA insect viruses such as baculoviruses, granuloviruses, ascoviruses, irido viruses, parvoviruses, polydnaviruses, poxviruses, etc., and RNA insect viruses such as reoviruses, rodaviruses, picorna-like viruses, tetraviruses, etc., can be used; the nucleic acid from said insect virus should be delivered by an appropriate viral or non- viral vehicle or vector for preventing its degradation or damage. Therefore, in a particular embodiment, the enhancer agent which is present in the product of the invention (2) comprises a vehicle or vector containing the nucleic acid of an insect virus, said vehicle or vector being a viral vehicle or vector, such as, for example, the virion per se of the insect virus, a VLP containing the nucleic acid of said insect virus, said VLP deriving from the same insect virus or from other insect or non-insect virus, etc.; or alternatively, said a non- viral vehicle or vector, such as, for example, a liposome, a polymer, or a mixture thereof, etc., to render a liposome/nucleic acid complex (lipoplex), a polymer/nucleic acid complex (polyplex), a liposome/polymer/nucleic acid complex (lipopolyplex), etc. In a preferred embodiment, the enhancer agent which is present in the product of the invention (2) comprises a vehicle or vector containing the nucleic acid of an insect virus, said vehicle or vector being the virion of the insect virus, e.g., a baculovirus, or a cationic liposome containing the nucleic acid of an insect virus, e.g., the nucleic acid (DNA) of a baculovirus.

The product of the invention (2) can be used for the simultaneous or successive administration in a subject of an antigen and an enhancer agent of the immune response against said antigen, for inducing an immune response against said antigen in said subject. Thus in a particular embodiment, the antigen and the enhancer agent is administered simultaenously to a subject, whereas, in another particular embodiment, the antigen and the enhancer agent are administered successively (sequentially in the time), at any order.

For its administration to a subject, both products provided by this invention [i.e., the product of the invention (1) and the product of the invention (2)], can be administered by any suitable administration route which results in an enhanced immune response against the antigen used, to which end said product will be formulated in the dosage form suited to the chosen administration route. In a particular embodiment, the administration of the product provided by this invention is carried out orally or

parenterally, for example, intravenous, subcutaneously, etc. Further, in a particular embodiment of the invention, said antigen is lyophilized or in a form of presentation suitable for its administration in a suitable administration form for its, for instance, oral or parenteral administration. In a preferred embodiment, said antigen is in a form of presentation suitable for its intravenous or subcutaneous administration.

In a particular embodiment, the ratio antigen:adjuvant (or enhancer agent) can vary from 10 to 1,000 micrograms of antigen to 10 ³ to 10 ⁸ plaque forming units of insect virus. In another particular embodiment, said ratio antigen:adjuvant (or enhancer agent) ranges from 10 to 1,000 micrograms of antigen to 0.1 to 100 micrograms of viral nucleic acid.

In another aspect, the invention refers to a product, such as any one of the previously described products [products of the invention (1) and (2)], as a medicament. In a particular embodiment, such medicament is a medicament for inducing an immune response against said antigen in a subject. In another aspect, the invention refers to a pharmaceutical composition comprising a product as previously defined [i.e., the product of the invention (1) and the product of the invention (2)] and a pharmaceutically acceptable excipient or vehicle. The excipients, carriers and auxiliary substances must be pharmaceutically and pharmacologically tolerable, so that they can be combined with other components of the formulation or preparation and do not cause adverse effects in the treated organism. The pharmaceutical compositions or formulations include those which are suitable for oral or parenteral (including subcutaneous, intradermal, intramuscular or intravenous) administration, although the best administration route depends on the condition of the patient and the nature of the compound to be administered. The formulations can be in the form of single doses. The formulations are prepared according to methods known in the pharmacology field. The active substance amounts to administer may vary according to the particularities of the therapy.

In another aspect, the invention refers to the use of the nucleic acid of an insect virus as an enhancer agent of the immune response against an antigen, in combination with said antigen, for the manufacture of a pharmaceutical composition for inducing an immune response against said antigen in said subject. In another particular embodiment, said pharmaceutical composition can be used for preventing and/or treating an infectious disease or for preventing and/or treating a cancer.

In another aspect, the invention refers to an adjuvant composition comprising, at least, the nucleic acid of an insect virus and, alternatively, one o more additional adjuvants and/or pharmaceutically acceptable vehicles. The pharmaceutically acceptable adjuvants and vehicles which can be used in said adjuvant compositions are those adjuvants and vehicles known by the persons skilled in the art and normally used in the manufacture of adjuvants compositions.

In a particular embodiment, said adjuvant composition is prepared in form of an aqueous solution or suspension in a pharmaceutically acceptable diluent, such as saline solution, phosphate-buffered saline solution (PBS), or any other pharmaceutically acceptable diluent.

The following example illustrates the invention and should not be considered limiting the scope thereof.

EXAMPLE 1 Baculo virus has strong adjuvant properties in mice promoting potent humoral and CD8 ⁺ T-cell (CTL) adaptive responses against co-administered antigens

I. MATERIALS AND METHODS

Mice. C57BL/6 and 129 Sv mice were obtained from CER Janvier (Le Genset

St. Isle, France) and Charles Rivers (Arbresle, France), respectively. 129 mice deficient for type I IFN receptor (IFNARko) were purchased from the specific pathogen-free (SPF) unit at the Pasteur Institute. Animals were kept in the Pasteur Institute animal facilities under SPF conditions. Experiments involving animals were conducted according to the institutional guidelines for animal care.

Baculovirus and viral DNA. Purified baculovirus Autographa californica nuclear polyhedrosis virus was obtained from Agate Bioservice SARL (Boisset et Gaujac, France). Briefly, BV was propagated in Spodoptera frugiperda (Sf9) cells (1 x 10 ⁸ ) in Sf-900 II SFM serum- free insect cell culture medium (Gibco Invitrogene, Cergy Pontois Cedex, France) (MOI: 0.5) and 72 hours later, the supernatant culture (120 mL) was harvested and cell debris was removed by centrifugation at 5,000 rpm for 15 min at 4°C. The virus was pelleted by centrifugation at 57,000 g for 60 min at 4°C, and

resuspended in 1 niL of PBS under sterile conditions. The infectious titer was determined by standard plaque assay. The virus stock was free of endotoxin (< 0.01 endotoxin units/mL) as shown by Limulus Amebocyte Ly sate test (QCL-1000) (BioWhittaker-Cambrex, Emerainville, France). The inactivation of BV by UV-light exposition was performed using an

Ultraviolet Crosslinker (Amersham Life Science, Saclay, France). Briefly, 300 μL of purified BV (1 x 10 ⁹ pfu/mL) was spread out in a thin layer on a well from a 24- well sterile plate and the plate was located into the crosslinker at a distance of 10 cm from the UV-light source and on ice. The sample received a total of 2 x 10 ⁴ mJ/cm ² . BV was also inactivated by Binary ethylenimine (BEI) and Triton treatment as described in (Rueda, P. et al. (2000) Effect of different baculovirus inactivation procedures on the integrity and immunogenicity of porcine parvovirus-like particles. Vaccine 19, 726-34). Briefly, BEI was freshly prepared by cyclation of 0.2 M 2-bromoethylamine hydrobromide (Merck, Barcelona, Spain) in 0.4 M NaOH at 37°C for 2 hours. One hundred fifty μL of BV (1 x 10 ⁹ pfu/mL) were incubated with 10 mM BEI for 48 hours at 37°C. The residual BEI was hydrolyzed with 15 mM sodium thiosulfate. For triton treatment, 150 μL of BV (1 x 10 ⁹ pfu/mL) were incubated for 30 min at 25°C in the presence of 1% Triton and 0.3% tri-n-butyl-phosphate and dialyzed overnight against PBS. For the Benzonase treatment, 150 μL of BV (1 x 10 ⁹ pfu/mL) were diluted with PBS to 500 μL and incubated with Benzonase (90 units/mL) (Novagen, Merck Eurolab, Fontenay sous Bois, France) and Mg ₂ Cl (2 mM) for 2 h at 37°C. The enzyme was inactivated with 150 mM ClNa.

DNA from BV (BV-DNA) was isolated from the purified virions by treatment with proteinase K (Sigma- Aldrich, Saint Quentin Fallavier, France) and 10% sodium dodecyl sulfate in sterile PBS for 2 h at 55 ⁰ C. RNAs were removed by incubation with Rnase A for 1 h at 37 ⁰ C. Viral DNA was purified by phenol-chloroform-isoamyl alcohol extraction, precipitated at 12,000 g, and resuspended in sterile endotoxin-free water. The resultant DNA exhibited a single DNA band by electrophoresis and neither protein nor chromosomal DNA of insect cells was detected.

PPV-VLPs. The production of chimeric and non-recombinant PPV VLPs was previously described in (Sedlik, C, Saron, M., Sarraseca, J., Casal, I. & Leclerc, C. (1997) Recombinant parvovirus-like particles as an antigen carrier: a novel

nonreplicative exogenous antigen to elicit protective antiviral cytotoxic T cells. Proc Natl Acad Sci U S A 94, 7503-8). PPVOVA particles result from the assembly of VP2 proteins carrying the OVA ₂₅₇ - ₂₆₄ epitope. Wild-type PPV VLPs and chimeric PPVOVA VLPs were expressed in Sf9 cells using a baculovirus expression system. VLPs were precipitated with 20% ammonium sulfate followed by dialysis. The concentration of VLPs was determined by densitometry and by double-antibody sandwich ELISA. This VLP preparation will be referred as "standard" preparation. The LPS endotoxin content in the standard preparation was below 0.01 units/mL. The titer of BV in the VLP standard preparation measured by plaque assay varied from 10 ⁷ -10 ⁸ pfu/mL. For some experiments, VLPs was purified by size-exclusion chromatography (SEC) using a Sephacryl S-1000 SF column (Amersham Pharmacia Biotech, Barcelona, Spain) as described in (Rueda, P. et al. (2000) cited supra). In some experiments, BV present in VLPs was inactivated by treatment with BEI and Triton as described above (Boisgerault, F. et al. (2005) Cross-priming of T cell responses by synthetic microspheres carrying a CD8+ T cell epitope requires an adjuvant signal. J Immunol 174, 3432-9). No BV was detected in VLPs purified by SEC or treated with either BEI or Triton.

Immunizations. For analysis of antibody responses, mice received a single i.v. or s. c. injection of OVA protein (10 μg) (Calbiochem, France Biochem, Meudon, France) either alone or in combination with BV or alum (1 mg). LPS concentration in the OVA solution was 65 EU/mg of protein. For CTL priming, mice were i.v. or s.c immunized with 10 ⁹ synthetic latex beads (1 μm-diameter) (Polysciences, Warrinngton, PA) covalently linked (BOVAp) to the OVA257-264 synthetic peptide (Neosystem, Strasbourg, France) as previously described (Boisgerault, F. et al. (2005) Cross-priming of T cell responses by synthetic microspheres carrying a CD8+ T cell epitope requires an adjuvant signal. J Immunol 174, 3432-9) either alone or in combination with BV, polyLC (25 μg) (Invivogen, Toulouse, France), anti-mouse CD40 (100 μg) (clone 3/23, BD Biosciences, Le Pont de Claix, France), PPV VLPs (10 μg) or a mix containing 10 μg of either purified BV-DNA or CpG 2216 (5'-ggG GGA CGA TCG TCg ggg gg-3') (Genset, Paris, France) and 30 μL of a cationic liposome preparation (DOTAP, Roche Diagnostics, Meylan, France). In some experiments, mice were injected i.v. with 10 μg of PPV-OVA VLPs either alone or together with BV.

Antibodies and cytokine ELISA assays. Mice were bled at different times after injection and individual sera were tested for OVA protein- specific IgG by incubation of the sera in a plate (Nunc Maxisorp plates, Nunc, Strasbourg, France) coated with OVA protein (1 μg/well) in 0.1 M Na ₂ CO3 (pH = 9.5) and further incubation with anti-mouse IgG-HRP (Sigma- Aldrich). Titers are expressed as the highest serum dilution giving two times the absorbance mean of negative sera.

For in vivo analysis of cytokines and chemokines production, mice were injected i.v. with BV and sera were collected at the indicated time points after treatment. IFN-α, IFN-β, MIG and IP-IO levels were detected by using ELISA Kits (IFN-α and IFN-β: PBL Biomedical Laboratories, Piscataway, NJ, USA) ( MIG and IP-IO: R&D System, Lille, France). IL-12p40, IL-12p70, IL-6 and IFN-γ were measured by in house ELISA assays, as described in (Schlecht, G. et al. (2004) Murine plasmacytoid dendritic cells induce effector/memory CD8+ T-cell responses in vivo after viral stimulation. Blood 104, 1808-15). Purified monoclonal antibodies (mAbs) anti-IL-12p40 (C15.6), anti-IL- 12p70 (9A5), anti-IL-6 (MP5-20F3) and anti-IFN-γ (R4-6A2) were used for coating and biotinylated mAbs anti-IL-12p40/p70 (C17.8), anti-IL-6 (MP5-32C11) and anti-IFN-γ (XMGl.2) were used as secondary mAbs. All mAbs used in the in house ELISA assays were from BD Biosciences (Le Pont de Claix, France).

In vivo killing assay. Naϊve syngenic splenocytes were pulsed at 25 x 10 ⁶ cells/mL with the OVA257-264 peptide (10 μg/mL) for 30 min at 37 ⁰ C, washed extensively to remove free peptide and labeled at 5 x 10 ⁶ cells/mL with high concentration (1.25 μM) of CFSE (CFSE ^hlgh ) [5(6)-carboxyfluorescein diaceteted N- succinimidyl ester] (Molecular Probes, Leiden, The Netherlands). The control non- pulsed population was labeled with low concentration (0.125 μM) of CFSE (CFSE ^low ). CFSE ^hlgh and CFSE ^low cells were mixed in a 1:1 ration (5 x 10 ⁶ cells of each population) and injected i.v. into mice. The number of CFSE-positive cells remaining in the spleen after 20 h was determined by FACS analysis. Specific lysis was calculated as follow: % specific Lysis: 100-[100 x (%CFSE ^hlgh primed mice/%CFSE ^low primed mice) / (%CFSE ^hlgh naϊve mice/%CFSE ^low naϊve mice)].

ELISPOT. IFN-γ and IL-4 ELISPOT was performed as described in

(Boisgerault, F. et al. cited supra). Purified anti-IFN-γ mAb (R4-6A2) or anti-IL-4 mAb

(BVD4-1D11) were used for capture and biotinylated anti-IFN-γ (XMGl.2) or anti-IL-4

(BVD4-24G2) were used as secondary rriAbs. Frequency of IFN-γ or IL-4-producing cells was determined by counting the number of spots/well with a computer-assisted

ELISPOT image analyzer (Bioreader-3000, Bioreader, Karben, Germany). The results were expressed as the number of spot- forming cells (SFC) per million splenocytes. For each mouse, the number of OVA257-264 specific IFN-γ SFC was determined by calculating the difference between the number of spots generated in the absence and in the presence of the OVA257-264 peptide (1 μg/mL).

DC purification. DCs were isolated from spleen of untreated, BV- or LPS- injected mice as previously described (Sun, C. M., et al (2003) Ontogeny and innate properties of neonatal dendritic cells. Blood 102, 585-91). Briefly, spleens were harvested and treated for 15 min with 400 U/mL collagenase D and 50 μg/mL Dnase I (Roche Diagnostics, Meylan, France). Spleens were then dissociated and the single cell suspension was incubated with anti-CD llc-coated magnetic beads (N418) either alone or together with anti-mpdcal -coated magnetic beads (JF05-1C2.4.1). Beads were from Miltenyi Biotec (Bergisch-Gladbach, Germany). Purified anti-CD 16/32 (2.4G2) monoclonal antibody was also added (BD Biosciences). Cells were selected on an automated magnetic cell sorter (AutoMACS, Miltenyi Biotec) using posseld2 program. LPS used for in vivo maturation was Ultra-pure LPS from Invivogen (Toulouse, France).

Antigen presentation assay. AutoMACS sorted splenic CDlIc ⁺ DCs

(10 ⁵ /wells) from C57BL/6 mice and the OVA ₂₅ 7-264 CTL hybridoma B3Z (10 ⁵ /wells) were co-cultured (18 h) with different concentration of either PPV-OVA or PPV VLPs in complete medium consisting in RPMI- 1640 Glutamax-I supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 5 x 10 ^"5 mol/L 2-mercaptoethanol and 10% of heat- inactivated Fetal Calf Serum (FCS) (Invitrogene, Life Technologies, Cergy-Pontoise, France). Activation of B3Z cells was determined by measuring the

amount of IL-2 in the supernatant by CTL-L assay. Results are expressed as counts per minute of incorporated [ ³ H] Thymidine.

Flow cytometry analysis. To analyze DC maturation, cells were magnetically sorted 15 hours after injection and resuspended in PBS supplemented with 5% of FCS and 2.5 mM EDTA (Sigma-Aldrich) (staining buffer). In each experiments, cells were incubated with rat anti-CD 16/32 mAbs (2.4G2) to block nonspecific binding and labeled for 10 min in the dark with the following mAbs: anti-CD 1 Ic-APC (HL-3), anti- B220-PE (RA3-6B2) and biotinylated anti-CD40 (HM40-3), anti-CD86 (GLl), anti-K ^b (AF6, 88.5), anti-IA ^b (25-9-17), rat IgM (R4-22), rat IgG _2a (R35-95) and hamster IgG ₂ (B81-3). All mAbs were from BD Biosciences. Biotinylated mAbs were detected with streptavidin-FITC (BD Biosciences). To measure the in vivo frequency of OVA ₂ 5 ₇ _ ₂ 6 ₄ - specific CTLs (OVATetr ⁺ ), spleen cells from naϊve or primed mice were stained first with PE-conjugated H-2K ^b -OVA ₂₅₇ _ ₂₆₄ -tetramer (Beckman Coulter France, Roissy CDG cedex, France) and then with anti-CD3-APC (145-2C11) and anti-CD8-FITC (53-6.7). Results are expressed as percentage of OVATetr ^"1" cells among CD8 ⁺ T-cells. Events were acquired on a FACScalibur flow cytometer and analyzed using CellQuest Software (BD Biosciences).

II. RESULTS

BV strongly enhances humoral and CTL responses against co-administered antigens

In order to evaluate whether baculovirus could affect the induction of adaptive immune responses, inventors first analyzed antibody responses elicited by a single injection of OVA protein either alone or in combination with 10 ⁶ pfu of BV. Coinjection of BV together with OVA protein resulted in the priming of a potent and long lasting

OVA-specific humoral response (Fig. Ia). This adjuvant effect was observed with doses of BV ranging from 10 ⁶ to 10 ³ pfu (Fig. Ib). Interestingly, the adjuvant effect of BV was however not observed in mice immunized with OVA and alum (Figure 5).

To next evaluate whether BV can also promote CTL responses, we used latex beads coated with the OVA ₂ S _7-264 peptide (BOVAp) that can transfer in vivo this CTL epitope into the MHC-I pathway of DC but are devoid of stimulatory capacity and fail

to induce CTL responses unless an appropriate stimulatory signal is co-delivered. Mice were injected i.v. with 10 ⁹ BOVAp either alone or together with 10 ⁶ pfu of BV. Immunization with BOVAp together with BV induced a massive expansion of OVA ₂₅₇ - ₂₆₄ -specific CTLs (OVATetr ⁺ ) that represented 10% of total CD8 ⁺ T cells at day 7 after injection (Figure Ic). O V A257-264- specific CTLs generated in the presence of BV showed full in vivo lytic function, as shown by in vivo killing assay 7 days after immunization (Figure 6) that was still observed 80 days after injection (Figure 7). This adjuvant effect of BV was observed both after i.v. and s.c. immunization, although a lower frequency of OVATetr ^"1" CTLs was detected upon s.c. immunization (Figure 8). The potency of BV to promote CTL responses was dose-dependent as demonstrated either by in vivo killing assay (Figure Id) or tetramer staining (Figure Ie). Despite the low frequency of OVATetr ^"1" CTL, a measurable lytic activity was still detected upon injection of BOVAp and 10 ³ pfu of BV. Simultaneous administration of BOVAP and BV induced a vigorous type 1 CTL response, characterized by a high frequency of IFN- γ-producing cells that diminished with the dose of BV and a low frequency of IL-4- producing cells (Figure 9). The frequencies of IFN-γ and IL-4 producing cells detected in mice immunized with BOVAp and 10 ³ pfu of BV were equivalent to those found in naϊve animals. This ability of BV to strongly increase CTL response was not only restricted to beads coated with the OVA257-264 peptide but also to beads coated with three other peptides containing CTL epitopes (Figure 10). Altogether, these data show that BV can provide the signals required to promote specific humoral and CTL specific responses against co-injected antigens.

In order to exclude that the adjuvant effect of BV preparations was due to contaminants from insect cells, mice were immunized with BOVAp and supernatant from either BV-infected or uninfected Sf9 cells. Only immunization with BOVAp and supernatant from BV-infected cells was able to prime OVA ₂₅₇ _ ₂₆₄ -specific CTLs (Figure 2a). To mimick BV purification, supernatants from non-infected Sf9 cells (120 mL) were pelleted and resuspended in PBS (1 mL). No CTL response was detected after immunization with BOVAp and 200 μL of this suspension (data not shown).

Inactivation of BV abrogates its adjuvant property

To further confirm that BV was responsible for this strong stimulation of adaptive immune responses, BV preparations were subjected to three different protocols

for virus inactivation: UV-radiation, treatment with Triton or alkylation with aziridine binary ethylenimine (BEI) (Rueda, P. et al. (2000) Effect of different baculovirus inactivation procedures on the integrity and immunogenicity of porcine parvovirus-like particles. Vaccine 19, 726-34). Inactivation of BV by UV-radiation has been shown to damage viral DNA but also to induce the denaturation of the gp64 envelope glycoprotein, preventing target cell infection. The non-ionic detergent Triton X-IOO in combination with the solvent tri-n-butyl-phosphate disturbs the lipidic envelope of BV and other enveloped viruses without affecting proteic or nucleic components. BEI is a potent alkylating agent that selectively reacts with nucleic acids but not with proteins. A suspension of BV (10 ⁹ pfu/mL) was subjected to UV-radiation, Triton or BEI treatment and live BV was assessed by plaque assay. No infectious BV was detected after UV- light or BEI treatment and only a residual titer of 7 x 10 ³ pfu/mL was detected after Triton treatment. The capacity of BV to promote CTL responses was strongly impaired by these treatments leading to BV inactivation (Figure 2b). To assess if the adjuvant property of BV is due to contaminant soluble oligonucleotides, purified BV was treated with Benzonase that digests both DNA and RNA oligonucleotides. Due to the size of this enzyme (60 kD), it was expected that Benzonase will digest soluble oligonucleotides but not those inside the virion. Accordingly, we found that the virus titer of BV suspension was not strongly affected by Benzonase treatment (1 x 10 ⁹ and 1.6 x 10 ⁸ pfu/mL before and after treatment, respectively). BV digested with Benzonase showed the same adjuvanticity than untreated virus (Figure 2b). Altogether, these data indicate that a compound intrinsically associated to BV and not a soluble contaminant from the Sf9 cell culture is responsible for the adjuvant properties of the BV preparations. Moreover, the adjuvanticity of BV is dependent of its infective capacity since disruption of the viral envelope and/or damage of the viral DNA abolished the adjuvant properties of BV.

It has been reported that the baculoviral DNA (BV-DNA) contains CpG motifs. To examine the effect of the purified BV-DNA on the induction of CD8 ⁺ T-cell responses, mice were immunized with BOVAp together with BV-DNA. No CTL response was detected after priming of mice with BOVAp either with CpG-ODN or BV-DNA (Figure 2c). Inventors reasoned that a non-adequate uptake and/or a quick degradation of the DNA given as a soluble molecule could prevent the induction of an effective CTL response. Administration of DNA in cationic liposome facilitates the

uptake of DNA and also prolongs its life span in vivo. Thus, mice were immunized with BOVAp together with either BV-DNA or CpG-ODN mixed with the cationic liposomal preparation, DOTAP. A strong O V A ₂₅₇ - ₂₆₄ - specific CTL response was detected in both cases (Figure 2c) whereas no CTL response was detected upon immunization with BOVAp in combination with DOTAP alone (Figure 2c). No CTL response was detected after immunization with BOVAp and BV-DNA treated with Benzonase and mixed with DOTAP (data not shown). These data show that BV-DNA delivered by an appropriate vehicle is able to promote CTL responses, suggesting that the viral DNA may be responsible for the adjuvant properties of BV. However, we cannot totally exclude that other viral components, or even some insect components encapsulated inside the virion, could also play some role in the adjuvanticity of BV.

IFNα/β mediates the adjuvant property of BV

Type I Interferons (IFNs) are expressed by most, if not all cells, in response to virus infection and are an essential link between innate and adaptive immunity. Inventors thus wondered whether BV adjuvanticity was mediated by type I IFNs. Inventors first investigated the ability of BV to induce type I IFNs secretion in vivo. A dose dependent production of IFNβ and IFNα was detected in the serum of mice injected with 10 ⁶ , 10 ⁵ or 10 ⁴ pfu of BV (Figure 3a). Inactivation of BV totally abrogated the in vivo production of IFNα (Figure 3b). IFNα was neither detected after injection of 200 μL of supernatant from uninfected Sf9 cells, whereas high levels (17.2 ± 4.3 ng/mL) were detected upon injection of the same volume of a supernatant from BV- infected Sf9 cells (BV dose: 10 ⁶ pfu) (data not shown). These data indicate that BV rather than soluble contaminants from Sf9 cells, is responsible for the in vivo production of IFNα/β and that inactivation of BV by disrupting the viral envelope and/or damaging the viral DNA abolished its capacity to induce in vivo the secretion of IFNα/β.

To investigate the regulation by type I IFN receptor (IFNAR) of the BV-induced in vivo production of IFNα/β, WT (129Sv) mice and mice deficient for IFNAR (IFNARko) were injected with 10 ⁶ pfu of BV. Whereas, sustained levels of IFNβ were detected in the sera of WT mice between 2 to 8 hours after injection, a huge and quick increase of IFNβ followed by a drastic decrease was observed in IFNARko mice, with IFNβ levels almost undetectable 6 hours after injection (Figure 3c). The seric IFNβ

level in IFNARko mice peaked 2 hours after injection and was 5 fold-times higher than the maximum level detected in WT mice. Lack of IFNAR and, hence, lack of sequestration of secreted IFNβ, could be a likely explanation for this high level of IFNβ observed in IFNARko mice. By contrast, the IFNα production was drastically diminished in IFNARko mice as compared to WT mice, although low level of IFNα could still be detectable (Figure 3c). These results indicate that in vivo production of IFNα/β by BV is strongly regulated by IFNAR.

To determine whether BV adjuvanticity was dependent of type I IFN, inventors first studied the effect of IFNAR absence on the ability of BV to enhance humoral responses. WT and IFNARko mice were immunized with OVA protein alone or in combination with 10 ⁶ or 10 ⁵ pfu of BV. As in C57BL/6 mice, BV markedly enhanced the OVA- specific IgGs response in 129Sv mice but not IFNARko mice (Figure 3d). However, a small increase in antibody titers was still observed in IFNARko mice, indicating that BV can promote the induction of antibodies independently of IFNα/β. To assess the effect of IFNAR signaling on the adjuvant effect of BV on CTL responses, 129Sv and IFNARko mice were immunized with BOVAp alone or in combination with 10 ⁶ or 10 ⁵ pfu of BV and the specific CTL response was analyzed at day 7 by tetramer staining, ELISPOT and by in vivo killing assay. The enhancement of CD8 ⁺ T cell responses was partially dependent of type I IFN, since BV treatment of IFNARko mice resulted in some increase in the CTL response (Figure 3e). To check if the impaired immune response observed in IFNARko mice was due to the lack of IFNAR signaling and not to a general defect of this strain, WT and IFNARko mice were immunized with OVA protein mixed with alum or with BOVAp together with anti- CD40 monoclonal antibodies and the induction of specific antibodies and CTLs was analyzed. IFNARko mice were indeed able to mount humoral (Figure 3f) and CTL (as measured by in vivo killing assay) (Figure 3g) responses comparable to control mice.

To investigate whether BV could induce DC maturation, C57BL/6 mice were injected with PBS or BV and 15 hours later, phenotypic activation markers were analyzed on splenic CDll ^int B220 ⁺ and CDll ^hlgh B220 ^" DC subsets (plasmacytoid (pDC) and conventional (cDC), respectively). BV strongly activated pDC and cDC in vivo and the activation of both DC subsets was totally abrogated by BV inactivation and was strongly dependent of IFNAR signaling (Figure 4). Finally, inventors analyzed the ability of BV to induce in vivo the secretion of inflammatory mediators (Figure 11).

High levels of IL-12p40, IL-12p70, IL-6, IFN-γ, MIG and IP-IO were detected in the sera of mice injected with BV. Secretion of these inflammatory mediators was totally abrogated by UV-light BV inactivation (data not shown). Interestingly, in the absence of IFN α/β signaling, the BV-induced production of IL-6, IFN-γ, MIG and IP-IO was strongly impaired whereas the secretion of IL-12p40 and p70 was enhanced. It has indeed been reported that type I IFN can both positively and negatively regulate IL-12 production although this controversy still remains to be enlightened.

III. DISCUSSION

Collectively, these experiments demonstrate that non-pathogenic insect viruses can exert a strong influence on mammalian humoral and cellular responses against coadministered antigens through DC maturation and production of inflammatory mediators. These adjuvant properties are mainly mediated by IFNα/β, although mechanisms independent of IFNAR signaling are also involved. The adjuvant properties of BV are abolished by disruption of the viral envelope and/or damage of the viral DNA. It has been demonstrated that BV can efficiently transduce a wide range of mammalian cells but does not replicate in them. The data here presented show that purified BV-DNA in cationic liposome mimics the adjuvant BV effect strongly suggesting that internalization of viral DNA and release into TLR9-expressing cellular compartments are important clue for the stimulatory properties of BV. Importantly such adjuvant effects are observed with quantities of BV virions as minute (10 ⁴ -10 ³ pfu) as those potentially contaminating recombinant proteins produced in BV-expression systems, such as virus-like particles. Purification processes based on size discrimination are the most commonly used for recombinant VLPs purification. Thus, gradient centrifugation, microfiltration (0.45 μm) and ultrafiltration (300 kDa) are very usual protocols found in the downstream processing of different VLPs. However, none of these methods would sharply discriminate between VLPs and BV. The absence of pathogenicity due to BV, at least for mammalians, could have help to ignore the presence of BV in the BV-derived recombinant proteins. On the basis of the data presented here, some of the immunological properties described for proteins expressed in the BV-expression system might suspiciously be due to contaminant BV. Indeed, several BV-derived VLPs were

shown to induce in vivo the production of inflammatory cytokines as well as to activate macrophages and DC through mechanism dependent of the Myd88 pathway. Many of these stimulatory properties have been attributed to the particulate structure of the VLPs or to some structural components or even to internal contaminants that could be recognized as pathogen associated molecules by mammalian cells.