WO2008109686A2

WO2008109686A2 - Non- infectious recombinant virus-like particles and their pharmaceutical applications

Info

Publication number: WO2008109686A2
Application number: PCT/US2008/055936
Authority: WO
Inventors: Igor Sivov; Dmitry Kulish
Original assignee: Neurok Pharma Llc
Priority date: 2007-03-05
Filing date: 2008-03-05
Publication date: 2008-09-12
Also published as: WO2008109686A3; WO2008109686A8

Abstract

The invention relates to novel virus-like particles (VLPs), including those based on Hepatitis C virus (HCV), as well as methods for their recombinant production and their use for stimulation of immunity and treatment of human and animal diseases. More specifically, the invention relates to novel synthetic constructs allowing the production of the novel recombinant non-infectious VLP ISVAC built of structural proteins of Hepatitis C virus (HCV), which VLP is efficiently produced in cultured BHK21 cells using the replication machinery of foot-and-mouth disease virus (FMDV). The pharmaceutical VLP preparations of the invention are highly efficient inducers of (i) endogenous Type I interferons (TYPE-I-IFN) such as interferon-q together with the general pool of interferons and (ii) general humoral immune response through stimulation of B cells (B-lymphocytes). VLPs of the invention are useful in treatment of human and animal diseases that can be treated by induction of interferons and/or B cell stimulation, including, e.g., hepatitis C, hepatitis B, FMD and certain malignancies, as well as asthmatic disorders, and allergies. VLPs of the invention are also useful as adjuvants that stimulate patient immune response to antigens and as viral diagnostic tools.

Description

Agent's file reference: 2207942- WOl

NON-INFECTIOUS RECOMBINANT HEPATITIS C VIRUS-LIKE PARTICLES

AND THEIR PHARMACEUTICAL APPLICATIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims priority under 35 U.S. C. § 119 to Russian patent application Serial No. 2007108037, filed March 5, 2007, and Russian patent application Serial No. 2007138092, filed October 16, 2007, the entire contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

[002] This invention belongs to the fields of biotechnology and medicine and relates to novel virus-like particles (VLPs), including those based on Hepatitis C virus (HCV), as well as methods for their recombinant production and their use for stimulation of immunity and treatment of human and animal diseases. More specifically, the present invention relates to novel synthetic constructs allowing the production of the novel recombinant non-infectious VLP ISVAC built of structural proteins of Hepatitis C virus (HCV), which VLP is efficiently produced in cultured BHK21 cells using the replication machinery of foot-and-mouth disease virus (FMDV). The pharmaceutical VLP preparations of the invention are highly efficient inducers of (i) endogenous Type I interferons (TYPE-I-IFN) such as interferon-α, together with the general pool of interferons and (ii) general humoral immune response through stimulation of B cells (B-lymphocytes). VLPs of the invention are useful in treatment of human and animal diseases that can be treated by induction of interferons and/or B cell stimulation, including, e.g., hepatitis C, hepatitis B, FMD and certain malignancies, as well as asthmatic disorders, and allergies. VLPs of the invention are also useful as adjuvants that stimulate patient immune response to antigens and as viral diagnostic tools.

BACKGROUND

[003] Virus-like particles (VLPs) (also called "pseudoviral particles") are biological nanoparticles built of viral structural proteins, which particles lack the viral genome and, therefore, are non-replicating and non-infectious. VLPs are generally composed of one or Agent's file reference: 2207942- WOl

more viral structural proteins, such as capsid, coat, shell, surface and/or envelope proteins, or particle-forming biological assemblies derived from these proteins. VLPs can generally assemble/form spontaneously upon recombinant expression of the viral structural proteins in an appropriate expression system. Designing and producing VLPs is a wide area of modern biological science that spans through different viral species, methods of production and conceptual applications. VLPs are useful tools that enable numerous distinct technologies from pharmaceutics to microelectronics (see, e.g., U.S. Patent Publication No. 20060216702). Three principal applications of VLPs are vaccination, immunotherapy and drug/gene delivery.

[004] VLPs built of hepatitis C virus (HCV) structural proteins attracted significant attention due to medical importance of Hepatitis C disease. HCV is recognized as the causative agent for most cases of non-A and non-B hepatitis, with an estimated worldwide prevalence of 170 million cases (Choo et al, Science, 1989, 244:359-362; Purcell, FEMS Microbiol. Rev., 1994, 14:181-192). Four million individuals may be infected in the United States alone (Alter and Mast, Gastroenterol. Clin. North Am., 1994, 23:437-455). Upon first exposure to HCV, only about 10% or less of infected individuals develop acute clinical hepatitis, while others appear to resolve the infection spontaneously. In most instances, however, the virus establishes a chronic infection that persists for decades, leading in about 50% of all cases to chronic hepatitis, which can, in turn, develop into liver cirrhosis and/or hepatocellular carcinoma (Iwarson, FEMS Microbiol. Rev., 1994, 14:201-204).

[005] HCV is a (+) strand enveloped RNA virus, i.e., its genome is represented by a coding single stranded RNA (cRNA) which is packaged with the structural proteins in a viral particle surrounded by a host cell-derived membrane. For review see Bartenschlager and Lohmann, J. Gen. Virol., 2000, 81:1631-1648. The HCV genome has a length of approximately 9.6 kb and its single, long open reading frame (ORF) encodes an approximately 3000 amino acid polyprotein that is proteolytically cleaved into a set of distinct products {see Rice, In: Virology, Fields et al. eds., Lippincott-Raven, 1996, Vol. 1, pp. 931-960; Clarke, J. Gen. Virol., 1997, 78:2397). The HCV ORF is flanked at the 5' and 3' ends by non-translated regions (NTRs or UTRs). Translation of the ORF is directed via an approximately 340 nucleotide (nt) long 5' NTR functioning as an internal ribosome entry site (IRES) and permitting the direct binding of ribosomes in close proximity to the start codon of the ORF (Tsukiyama-Kohara et al, J. Virol., 1992, 66:1476-1483; Wang et al, J. Virol., 1993, Agent's file reference: 2207942- WOl

67:3338-3344). The HCV polyprotein is cleaved co- and post-translationally by cellular and viral proteinases into ten different products, with the structural proteins located in the N-terminal one-third and the non-structural (NS) proteins (i.e., proteins which are not expected to be constituents of the virus particle) in the remainder (reviewed in Bartenschlager and Lohmann, supra; Bartenschlager, J. Viral Hepatitis, 1999, 6: 165-181; Reed and Rice, In: Hepatitis C Virus, Reesink ed., Basel: Karger, 1998, pp. 1-37). The first cleavage product of the polyprotein is a highly basic core protein (C), which is the major constituent of the nucleocapsid (Yasui et al, J. Virol., 1998, 72:6048-605). Envelope proteins El and E2 are highly glycosylated type 1 transmembrane proteins, forming two types of stable heterodimeric complexes (Deleersnyder et al, J. Virol., 1997, 71:697-704). Protein p7, located at the C-terminus of E2, is a highly hydrophobic polypeptide of unknown function. Most of the nonstructural proteins NS2-5B are required for replication of the viral RNA (Lohmann et al, Science, 1999, 285:110-113). The HCV virion was shown to be built of at least two layers: inner capsid scaffolded by protein C, and outer supercapsid formed by lipids stabilized by transmembrane proteins El and E2 (Kaito et al, 2006, Int. J. MoI. Med. 18(4):673-8; Nielsen et al, 2006, J. Virol. 80(5):2418-28).

[006] Attempts of obtaining HCV VLPs started immediately after deciphering

HCV genome in hopes of producing an HCV vaccine. The simplest approach to producing HCV VLP consists in self-assembly of a core (C) protein into a stable scaffold (Kunkel, M. et al, J Virol., 2001, 75(5):2119-29). However, these particles have not found much application because their structure is believed to be substantially different from the structure of wild-type HCV virion, and, therefore, physiologically irrelevant. To be physiologically relevant, HCV VLPs should, similarly to the wild type virion, have a two-layer composition built on three structural proteins C, El, El.

[007] The first HCV VLPs built on polyprotein C-E1-E2-P7 processing products and having shape and size similar to wild-type HCV, were obtained in insect cells infected with HCV genome inserted into baculovirus (Baumert et al, 1998, J. Virol. 72(5):3827-36; U.S. Patent No. 6,387,662). This type of particle was thoroughly studied over the years, but these studies yielded little practical application due to the fact that their assembly in the insect cellular environment likely resulted in a VLP composition that was different from the composition and structure of the viral particles generated in mammalian cells. Agent's file reference: 2207942- WOl

[008] Several technologies have been also developed for the production of recombinant HCV VLPs in mammalian cells (see, e.g., U.S. Patents Nos. 6,930,095 and 7,049,428). These methods provided physiologically relevant VLPs but were highly inefficient from the production standpoint. In order to increase the efficiency of HCV VLP production, various cell cultures have been tried, including BHK21 cells. Several such attempts have been reported (Blanchard et al.,. 2002, J. Virol. 76(8):4073-9; Ezelle et al., 2002, J. Virol. 76(23): 12325-34; Sivov et al., 2003, Dokl Biochem Biophys. 392:288-91) with little practical application due to remaining problems with the level of expression of HCV structural proteins resulting in low concentration of VLPs in cell culture. Low VLP yields in such systems are believed to be due to viral protein toxicity for host cells and formation of different lipoprotein assemblies in serum-based cultures which hinder further purification. For example, in Sivov et al., 2003, Dokl Biochem Biophys. 392:288-91, an expression vector encoding HCV structural proteins flanked by HCV untranslated regions (UTRs) and containing HCV internal ribosome entry site (IRES) provided a very low yield of VLPs upon transfection into BHK21 cells.

[009] PCT Publication No. WO 2004/061110 discloses generation of

"semi-recombinant" HCV VLPs in BHK21 cells by the technique of complementation. In this technique, the construct expressing non-structural HCV proteins is inserted into BHK21 host cell genome and the cell is infected with HCV particles incapable of replication. Such particles are known as "defective interfering particles" and are abundantly present in most HCV viral cultures and may be separated by gradient centrifugation. However, this approach also failed to provide sufficient increase in HCV VLP yield and is unacceptable for pharmaceutical applications, because it requires working with industrial volumes of cell culture infected by wild-type HCV that creates a significant biohazard.

[010] Several other variants of HCV VLPs have been obtained, such as those produced in yeast cells (U.S. Patent No. 7,048,930), large particles produced in different cultures (U.S. Patent No. 6,849,429) and hybrid particles that included elements of Hepatitis B virus (U.S. Patent No. 6,740,323). In addition to recombinant VLPs of HCV , prior art contains numerous references to recombinant VLPs of many other viruses including Hepatitis B, rotavirus, Foot-and-Mouth Disease Virus (FMDV), human papillomaviruses (HPV), retroviruses, alphaviruses, and even bacteriophages (Touze and Coursaget, Nucleic Acids Res., Agent's file reference: 2207942- WOl

Vol. 26, No. 5, 1998, pp. 1317-1324; Singh, Drug , Development Research, Vol. 67, Iss. 1,

2006, pp. 23-41; U.S. Patent No. 6,376,236; U.S. Patent Publications Nos. 20060251623 and 20060121468). For example, Yang et al. (J Virol. 2004, 78(20): 11152-60; see also U.S. Patent No. 6,420,160) disclose HPV VLPs capable of refolding in vitro and of inducing type I interferons (TYPE-I-IFN) such as interferon-alpha (IFN-α).

[011] Clearly, the need for novel technologies for production of non- infectious recombinant HCV VLPs remains urgent because any practical application of existing technologies is blocked by low yields and the fact that most current purification schemes use density gradient centrifugation which is very inefficient and expensive prohibiting industrial scale-up. The final consideration explaining the need for inventing new techniques for producing HCV VLPs is rooted in the fact that each HCV VLP produced in any principally novel system has a different composition and therefore may exert new unexpected properties that may be useful in treatment and prophylaxis of diseases related to HCV infection.

[012] Problems of HCV treatment and prophylaxis remain unsolved also because the high error rate of the viral RNA dependent RNA polymerase (RdRp) results in the rapid generation of HCV virus variants - a selection mechanism that allows some HCV variants to avoid anti-viral immune responses. Even within a single patient HCV does not exist as a single entity but rather as a collection of microvariants of a predominant "master sequence", a phenomenon that has been referred to as quasispecies (reviewed in Bukh et al., Semin. Liv. Dis., 1995, 15:41-63; Bukh et al, Clin. Exp. Rheumatol., 1995, 13(suppl.):S3-S7; Holland et al., Curr. Topics Microbiol. Immunol., 176:1-20). The master sequence, as well as the consensus sequence of the quasispecies sequence population have been found to change sequentially during the infection. Using comparative sequence analyses of HCV genomes isolated over intervals of 8 or 13 years, a mutation rate of 1.44xlO³ or 1.92xlO³ base substitutions per site per year was found, respectively (Ogata et al. , Proc. Natl. Acad. Sci. USA, 1991, 88:3392-3396; Okamoto et al, Virology, 1992, 190:894-899).

[013] To date, there is no single commercial HCV vaccine available on the market and the current news of development of such vaccines is disappointing (Dolan, Forbes,

2007, 178 (11) 50-51); Van Brunt, J., Signals Magazine, electronic publication http://www.signalsmag.com/, 05/30/2007). Agent's file reference: 2207942- WOl

[014] All modern approaches to HCV treatment, including WHO golden standard, are based on using recombinant interferon- alpha (IFN-α) that serves as an inducer of massive antiviral response that otherwise does not happen due to the mentioned mutation accumulation in HCV (Shepherd et al, Health Technol Assess. 2007 Mar; 11(11): 1-224; Younossi et al, Hepatology 2007 Mar; 45(3):806-16). Interferons are a group of vertebrate glycoproteins and proteins which are produced by the cells of the immune system and have antiviral, antiproliferative, and immunomodulatory activities. For review see Interferon: In Vivo and Clinical Studies, Volume 4, Eds: N. B. Finter and R. K. Oldham, Academic Press, New York, 1985. Interferons belong to the large class of glycoproteins known as cytokines. According to their receptor affinity, interferons are classified as either Type I or Type II. Type-I-interferons (TYPE-I-IFN) are induced by viral nucleic acids, while Type-II-interferons are produced as lymphokines through induction by antigens and mitogens. Historically, interferons were also classified into types on the basis of their antigenic specificities. In this older but widespread classification, the designations alpha (α), beta (β), and gamma (γ) have been used to correspond to previous designations of leukocyte, fibroblast, and immune interferons, respectively. Alpha interferons (IFN-α) and beta interferons (IFN-β) correspond to what is called type I interferons (TYPE-I-IFN); gamma interferon (IFN-γ) corresponds to what is called type II interferons (TYPE-II-IFN). See Journal of Interferon Research, 1 pp. vi (1980).

[015] TYPE-I-IFNs are "pivotal cytokines bridging together two aspects of host defense: innate and humoral immune systems" (Takaoka, A., and Yanai, H. 2006. Cell. Microbiol. 8(6):907-22); they possess three major biological activities: antiviral activity, antitumor activity and immunomodulatory activity. IFN-α is a hallmark representative of TYPE-I-IFN family. The first type of interferon to be identified and commercialized, IFN-α remains the most widely used interferon for clinical applications. In addition to IFN-α , TYPE-I-IFN family includes beta interferon (IFN-β) and several other minor species that bring the size of the family to more then 20 members. Another widespread interferon family is Type-II-interferon that is represented by interferon-γ, usually induced together with the rest of interferon family, but believed to be metabolically positioned downstream to TYPE-I-IFN action.

[016] Because of their crucial role, TYPE-I-IFNs are widely used in clinical practice where they are mostly represented by recombinant interferon-α-2 and recombinant Agent's file reference: 2207942- WOl

interferon-β that were approved for use in the treatment of a variety of tumors and viral diseases; most notably serve as the golden standard for treating Hepatitis C and multiple sclerosis. Despite their widespread use and obvious utility, recombinant interferons have two drawbacks that are believed to be linked to their exogenous nature. First, it is low efficacy (e.g. only 20-50% in HCV patients), and, second, significant side effects result from interferon administration. Major critique of using recombinant interferons comes from simple premise that bringing to patient body an enormous amount of only one member of the 20+-member family of interferons results in immediate shocks and disbalances. Furthermore, recombinant interferons are certainly not physiological enough in their post-translational modifications that results in lower relative activity.

[017] Modern HCV treatments usually represent a combination of recombinant

IFN-α with some general antiviral agent (e.g., ribavirin) (Shepherd J., et al, Health Technol Assess., 2007, 11(11): 1-224). This approach allows some increase in efficiency, but still remains unsatisfactory as it does not decrease side effects. Currently, several inhibitors of HCV enzymes are expected to enter the market thus creating a "second generation" of HCV therapeutics (Van Brunt, J., Signals Magazine, electronic publication http://www.signalsmag.com/, 05/30/2007; Toniutto, P., et al. Curr Opin Investig Drugs., 2007, 8(2): 150-8; Johnson, C.L., J Biol Chem., 2007, 282(14): 10792-803). Most probably, these novel therapeutics will improve treatment efficiency but will remain susceptible to HCV adaptive mutations in a manner similar to HIV adaptive mutations to enzyme inhibitors that proved to be a grave problem for HIV treatment.

[018] It is believed that a balanced induction of the entire pool of endogenous interferon family members would produce effective anti-viral response with little side effects. Hence, the identification of non-pathogenic inducers of a broad pools of endogenous interferons remains an important task (Komorizono et al, Anticancer Res. 2002, 22(6B):3573-8; Takaoka and Hideyuki, Cellular Microbiology (2006), v.8, No. 6, pp. 907-922; Pohl et al, Cellular Microbiology 2007, v. 9, No. 2, pp. 279-289; Stetso and Medzhitov, Immunity, 2006, v. 25, 373-381; Gordon & Minks, 1981, Microbiol. Rev. 45(2):244-66; Werner & Jolles, 1996, Eur. J. Biochem. 242(1): 1-19; De Clercq, 2006, J. Infect. Dis. 194 Suppl l:S19-26; U.S. Patent Publication No. 20060035335). It has proven to be a complex task because the induction of TYPE-I-IFNs is adjusted to detection of alien (e.g. , viral) or defective Agent's file reference: 2207942- WOl

nucleic acids (reviewed in Stetson and Medzhitov, 2006, Immunity. 25(3):373-81). After much trial and error, two major classes of interferon-α inducers have been identified: (i) long stretches of nucleic acids, most notably represented by "poly I:C" synthetic dsRNA polymer (Talmadge et al, 1985, Cancer Res. 45:1058; De Clercq, 2006, J. Infect. Dis. 194 Suppl 1:S 19-26), and (ii) CpG motif-containing oligodesoxinucleotides (CPG-ODNs) that serve as agonists for Toll-like receptors (TLR) (Kerkmann et al, 2005, J. Biol. Chem. 280(9):8086-93; Krieg, Annu. Rev. Immunol. 2002. v. 20, pp. 709-760; Krieg, Proc Am Thorac. Soc. 2007 JuI; 4(3):289-94; U.S. Patent Nos. 6,949,520 and 7,223,741; U.S. Patent Publication No. 20070078104). Studies of poly I:C and CPG-ODNs resulted in the development of a unified two-part model of interferon-α induction (reviewed in Stetson and Medzhitov, 2006, Immunity. 25(3):373-81) where cytosolic receptors {e.g., RIG-I and MDA5) sense intra-cellular DNAs such as poly I:C, and endosomal toll-receptors (TLRs) sense extra-cellular viral-motif RNAs associated with nanoparticles (Kerkmann et al, 2005, J. Biol. Chem. 280(9):8086-93; Kerkmann et al , 2006, Oligonucleotides. 16(4):313-22). Both types of receptors are expressed in specialized subset of blood cells called plasmacytoid dendritic cells (PDC) that were shown to serve as dominant producers of IFN-α (reviewed in Stetson and Medzhitov, 2006, Immunity. 25(3):373-81). On the clinical side, while both poly I:C and CPG-ODNs demonstrate significant interferon induction , they appeared not applicable due to toxicity {e.g., their potential accumulation in the body and fever- like side effects of their administration), viral relapse, difficulty and high cost of their chemical synthesis, and other problems (Ewel et al, 1992, Cane. Res. 52:3005; Krieg, 2007, Proc. Am. Thorac. Soc. 4(3):289-94, Krieg, 2007, J. Clin. Invest. 117(5): 1184-94). Therefore, the need for new types of TYPE-I-IFN inducers (which are clinically relevant) remains highly important.

[019] While interferons induce innate branch of human immunity, the second branch of immunity, called adaptive immunity, includes humoral immunity, which is commanded by specialized subset of cells called B cells (B-lymphocytes). Stimulation (or induction) of B cells results in multiple important immune reactions (Dδrner and Radbruch, 2007, Immunity 27(3):384-92; Mizoguchi and Bhan, 2006, J. Immunol. 176(2):705-10) including proliferation of antibody-producing cells. Thus stimulation of B cells is as important for treatment of diseases as induction of interferons. B cell stimulation is expected to be particularly crucial for treating immune system deficiencies (including asthmatic disorders and allergies), as well as for obtaining novel adjuvants that enhance patient response to vaccination Agent's file reference: 2207942- WOl

(U.S. Patent No. 7,223,741; U.S. Patent Publication No. 20070078104; Mizoguchi and Bhan, 2006, J. Immunol. 176(2):705-10). B cell stimulation is achieved by many different mitogens (Werner and Jolles, 1996, Eur. J. Biochem. 242(1):1-19; Dδrner & Radbruch, 2007, Immunity 27(3):384-92). It has been also reported that B cells and interferons can be induced by the same compounds (Mizoguchi & Bhan, 2006, J. Immunol. 176(2):705-10). For example, it was shown that, similarly to PDCs, B cells express TLRs and are stimulated by CpG ODNs (Krieg, A.M, et al, 1995. Nature. 374(6522):546-9). Interestingly, the same CpG ODNs that directly stimulate B cells, must be attached to a nanoparticle to stimulate PDCs to induce interferon (Kerkmann et al, 2005, J. Biol. Chem. 280(9): 8086-93).

SUMMARY OF THE INVENTION

[020] As follows from the above overview of the prior art, there is a great need in the art to develop-new treatments for Hepatitis C virus (HCV) infection and related diseases as well as new methods for obtaining non-infectious recombinant virus-like particles (VLPs) which can be produced in industrially meaningful quantities and acceptable host cells and are capable of efficiently inducing immunity, including inducing endogenous interferons and/or B cells, in animals and humans in vivo.

[021] The present invention satisfies these and other relevant needs by providing an industrially acceptable, pharmaceutically relevant and efficient method for producing novel non- infectious recombinant VLPs. VLPs of the invention can be derived from any virus, but are preferably derived from enveloped RNA viruses, such as, for example, members of the Sindbis-like superfamily (Togaviridae, Bromovirus, Cucumovirus, Tobavirus, Ilarvirus, Tobravirus, Potexvirus) and Flavivirus-like superfamily (Flaviviridae, Pestivirus), including, for example, yellow fever virus, dengue viruses, West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Murray Valley encephalitis virus, Rocio virus, tick-borne encephalitis viruses, Hepatitis C virus, Hepatitis E virus, Hepatitis G virus, hog cholera and bovine diarrhea viruses, and border disease virus of sheep. In a specific embodiment, the VLP of the invention is ISVAC derived from HCV subtype Ib.

[022] VLPs of the invention are characterized by a surprising ability to efficiently induce and/or enhance the production of interferons, including IFN-α, in human Agent's file reference: 2207942- WOl

blood and humans in vivo and can be therefore used to efficiently treat diseases treatable by the increased production of interferons (e.g., infections and malignancies). As shown herein, VLPs of the invention also strongly stimulate B cells and can be therefore used to efficiently treat relevant diseases that include immune system deficiencies, asthmatic disorders, and allergies. In addition, due to their interferon- and B cell- stimulating activity, VLPs of the invention are useful as adjuvants that stimulate patient response to antigens. Finally, VLPs of the invention are shown to be immunoprecipitated by virus-specific antibodies, which makes them useful as viral diagnostic tools.

[023] The present invention further provides pharmaceutical compositions comprising said VLPs and their use for diagnostics, prophylaxis and treatment of different diseases.

[024] Thus, in the first embodiment, this invention provides industrially acceptable, pharmaceutically relevant and efficient method for production of recombinant VLPs in an appropriate cell culture. In a preferred embodiment, this invention provides a method for production of HCV subtype Ib VLP called ISVAC in BHK21 cell line. In conjunction with the method for VLP production, the invention provides constructs utilized in this method as well as techniques for host cell cultivation and VLP isolation.

[025] There are two distinct constructs utilized in this invention. Together, these constructs form a binary expression system. The first construct (referred to as "replicon-producing"; schematically shown in Figure IA) encodes non-structural proteins of a first RNA virus which, upon expression in a host cell, assemble into a replication machinery ("replicon") of the first virus capable of directing the production of the structural proteins of a second virus. The second construct (referred to as "VLP-producing"; schematically shown in Figure IB) encodes structural proteins of the second virus that, upon expression in a host cell, assemble into a VLP. Importantly, in the "VLP-producing" construct, cDNA encoding structural proteins of the second virus is flanked by fragments of the UTR of the first virus which are recognized by the replication machinery of the first virus. The presence of the UTRs of the first virus in the "VLP-producing" construct results in the replication of mRNA produced by the "VLP-producing" construct under the control of the replication machinery encoded by the "replicon-producing" construct. The first and the second viruses may be Agent's file reference: 2207942- WOl

identical but in the most practical applications they will be different. According to the present invention, the first virus is an RNA virus, while the second virus can be any virus.

[026] The binary expression system of the invention is well suited for increasing efficiency of producing VLPs of many different viruses. In a specific embodiment, the binary expression system of the invention solves the problem of low efficiency of HCV VLP production that plagued the field for decades (see the discussion in the Background section, above). As HCV polyprotein expression appears to be toxic for cells (Sivov et ah, 2003, Dokl Biochem Biophys. 392:288-91), the present inventors hypothesized that it is necessary to accelerate the expression of the HCV polyprotein before said toxicity starts hurting cell processes. This acceleration is achieved herein by combining the increased transcription of cDNA and increased replication of mRNA encoding HCV structural proteins. The increased transcription is achieved by the use of a strong promoter {e.g. , CMV promoter). The increased replication is achieved by flanking the cDNA encoding the HCV polyprotein (in the "VLP-producing" construct) with UTRs recognized by the replication machinery of a heterologous virus {e.g., FMDV) encoded by the "replicon-producing" construct.

[027] Thus, according to the method of the present invention, HCV subtype Ib

VLP ISVAC is generated in BHK21 host cells using a binary plasmid system consisting of (i) a "replicon-producing" construct encoding and expressing the non-structural proteins of Foot-and-Mouth Disease virus (FMDV) under the control of 5 ' - and 3 ' -UTR of FMDV and (ii) a "VLP-producing" construct encoding and expressing the structural proteins of HCV under the control of 5'- and 3'-UTR of FMDV. The FMDV replication machinery encoded by the "replicon-producing" construct directs synthesis of the structural proteins of HCV (encoded by the "VLP-producing" construct) which then assemble into a non-infectious VLP.

[028] In one aspect, the invention provides a "replicon-producing" construct

(cDNA) which expresses in a host cell culture a set of non-structural proteins of a first RNA virus that further assemble into the replication machinery of the first virus. This "replicon-producing" construct comprises the following elements (schematically shown in Figure IA):

1) a promoter providing transcription in a host cell of mRNA encoding polyprotein of the non- structural proteins of the first virus; Agent's file reference: 2207942- WOl

2) 5 ' Untranslated Region (5"UTR) of the first virus, wherein such 5"UTR provides replication of the transcribed mRNA by the replication machinery of the virus;

3) Internal Ribosome Entry Site (IRES) providing translation of the polyprotein of the non- structural proteins of the first virus in the host cell;

4) a sequence encoding the polyprotein of the non-structural proteins of the first virus, wherein such polyprotein is processed into the replication machinery of this virus, and

5) 3"UTR of the first virus, wherein such 3"UTR provides replication of the transcribed mRNA by the replication machinery of this virus.

[029] As used herein, the terms "5 'UTR" and "3 'UTR" relate not to the position of these UTRs in the parent virus, but to their position in the mRNA transcribed from the DNA constructs of the invention. Also, as used herein such 5' and/or 3' UTR may be only a part of a larger UTR of the parent virus. It is preferable that the 3"UTR in the "replicon-producing" construct is stronger than the 5"UTR because the former drives the first round of replication of the transcribed mRNA. In a preferred embodiment, the "replicon-producing" construct contains weaker UTRs than the UTRs placed into the "VLP -producing" construct, to ensure the prevalent replication of the mRNA encoding a polyprotein of the VLP structural proteins. In a preferred embodiment, the IRES used in the "replicon-producing" construct is weaker than the IRES used in the "VLP-producing" construct, to allow for prevalent translation of the target polyprotein of structural VLP proteins.

[030] When the "replicon-producing" plasmid enters the host cell, it is expressed in three stages: first, transcription is guided by the promoter thus producing the mRNA replicon flanked by UTRs; second, non-structural proteins of the first virus are translated from the mRNA under the control of the IRES and then assemble into the replication machinery of the first virus; third, this replication machinery replicates the replicon as guided by the flanking UTRs. This chain of events results in fast accumulation of the replicon and replication machinery of the first virus in the host cell.

[031] The "replicon-producing" construct of the invention can be derived from many different RNA viruses, including, without limitation, picornaviruses (e.g., Foot-and-Mouth Disease Virus [FMDV]), alphaviruses and flaviviruses. In a preferred Agent's file reference: 2207942- WOl

embodiment, the "replicon-producing" construct is derived from FMDV. An important feature of the FMDV replicon utilized in this invention is its efficient propagation in BHK21 cell culture (see Escarmis et al, J Virol., 1998, v. 72, No. 12, p. 10171-10179).

[032] In a more specific embodiment, this invention provides a

"replicon-producing" construct (cDNA) comprising the following elements (schematically shown in Figure 2A):

1) CMV promoter-enhancer that consists of SEQ ID NO: 1 ;

2) FMDV UTR fragment that consists of SEQ ID NO: 2;

3) IRES which is a hybrid between yellow fever virus (YFV) IRES and human immunodeficiency virus (HIV) long terminal repeat (LTR) and consists of SEQ ID NO: 3;

4) a cDNA encoding a polyprotein of FMDV non-structural proteins, and

5) FMDV UTR fragment that consists of SEQ ID NO: 5.

[033] In a preferred embodiment, the cDNA encodes for an FMDV polyprotein of FMDV non-structural proteins «2B-2C-3A-3B1/3B2/3B3-3C-3D» and consists of SEQ ID NO: 4.

[034] In a specific embodiment, the 3' end of cDNA coding for FMDV polyprotein of non-structural proteins is fused to a cDNA encoding a "reporter" protein to facilitate control of expression of the "replicon-producing" cDNA. In a preferred embodiment, this reporter protein is green fluorescent protein (GFP). However, many other reporters may be also used.

[035] In a preferred embodiment, this invention provides a

"replicon-producing" construct comprising SEQ ID NO: 6.

[036] It will be evident to those skilled in the art that, to obtain a functional expression construct, the "replicon-producing" cDNA containing the above elements can be inserted into many different DNA vectors including, without limitation, commercially Agent's file reference: 2207942- WOl

available vectors of series pUC (Promega, USA), pET (Clonetech, USA), pCDNA, and pDEST (Invitrogen, USA).

[037] In the most specific embodiment, the invention provides a

"replicon-producing" plasmid pDKIS-0107 of 10925 b.p. which consists of SEQ ID NO: 8. pDKIS-0107 efficiently expresses FMDV non-structural proteins in BHK21 cells. pDKIS-0107 is composed of two fused fragments:

1) DKIS-FUN: Clal-EcoRI fragment of 5757 b.p. that consists of SEQ ID NO: 7, wherein EcoRI end is blunted by Klenow, which CIaI- EcoRI fragment includes FMDV 5^{^}UTR having SEQ ID NO: 2, YFV-HIV hybrid IRES having SEQ ID NO: 3, cDNA having SEQ ID NO: 4 that encodes FMDV non-structural polyprotein, cDNA that encodes GFP, and FMDV 3^{^}UTR having SEQ ID NO: 5, and

2) CIaI- EcoRI fragment of 5168 b.p., wherein EcoRI end is blunted by Klenow, of recombinant expression vector pCI Neo (Promega, USA), that includes CMV promoter-enhancer and genes needed for replication in E.coli and antibiotic (ampicillin) resistance.

[038] In another aspect, this invention provides a "VLP-producing" construct that expresses in a host cell culture a set of structural proteins of a second virus, which proteins assemble into a VLP of the second virus, given that the "replication-producing" cDNA discussed above is simultaneously expressed in the same cell culture. The "VLP-producing" construct of the invention comprises the following elements (schematically shown in Figure IB):

1) a promoter providing transcription in a host cell of mRNA encoding polyprotein of the structural proteins of the second virus;

2) 5' Untranslated Region (5^{^}UTR) of the first virus, wherein such 5^VUTR provides replication of the transcribed mRNA by the replication machinery of the first virus;

[039] Internal Ribosome Entry Site (IRES) providing translation of the polyprotein of the structural proteins of the second virus in the host cell; in a preferred embodiment, the IRES used in the "VLP-producing" construct is stronger than the IRES used in the "replicon-producing" construct, to allow for prevalent translation of the target polyprotein of structural VLP proteins; Agent's file reference: 2207942- WOl

1) a sequence encoding the polyprotein of structural proteins of the second virus, wherein such polyprotein is processed and assembled into a VLP of the second virus, and

2) 3"UTR of the first virus, wherein such 3"UTR provides replication of the transcribed mRNA by the replication machinery of the first virus.

[040] The "VLP-producing" construct of the invention is expressed in three stages: first, transcription is guided by the promoter thus producing the mRNA replicon of the second virus flanked by UTRs derived from the first virus; second, UTRs of the first virus guide robust replication of the replicon of the second virus by the replication machinery of the first virus (said replication machinery being expressed in the same host cell); third, the polyprotein of structural proteins of the second virus is translated from the mRNA under the control of the IRES and then processed into individual proteins that further assemble into a VLP. Sequences encoding structural proteins derived from many different viruses are suitable for inclusion in the "VLP-producing" plasmids of the invention. Preferably, sequences encoding structural proteins are derived from enveloped RNA viruses, such as members of the Sindbis-like superfamily (Togaviridae, Bromovirus, Cucumovirus, Tobavirus, Ilarvirus, Tobravirus, Potexvirus) and Flavivirus-like superfamily (Flaviviridae, Pestivirus), including, for example, yellow fever virus, dengue viruses, West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Murray Valley encephalitis virus, Rocio virus, tick-borne encephalitis viruses, Hepatitis C virus, Hepatitis E virus, Hepatitis G virus, hog cholera and bovine diarrhea viruses, and border disease virus of sheep. In a preferred embodiment, sequences encoding structural proteins are derived from a hepatotropic RNA virus, most preferably Hepatitis C virus (HCV).

[041] In one specific embodiment, this invention provides a "VLP-producing" construct comprising the following elements (schematically shown in Figure 2B):

1) CMV promoter-enhancer that consists of SEQ ID NO: 1

2) FMDV UTR fragment that consists of SEQ ID NO: 2 or SEQ ID NO:

13;

3) HCV IRES;

4) a cDNA encoding a polyprotein of structural proteins of HCV;

5) FMDV UTR fragment consisting of SEQ ID NO: 4. Agent's file reference: 2207942- WOl

[042] In a preferred embodiment, the cDNA coding for IRES and polyprotein of

HCV structural proteins consists of SEQ ID NO: 9, and is built by fusion of a sequence encoding IRES of HCV type Ib to a cDNA encoding the «C-El-E2-p7» polyprotein of HCV type Ib.

[043] In a specific embodiment, the 3' end of the cDNA coding for HCV polyprotein is fused to a cDNA encoding a "reporter" protein to facilitate control of expression of the "VLP-producing" cDNA. In a preferred embodiment, this reporter protein is alpha-peptide of beta-galactosidase (β-gal_αpep). However, many other reporters may be also used.

[044] In a preferred embodiment, this invention provides a "VLP-producing" construct comprising SEQ ID NO: 10.

[045] It will be evident to those skilled in the art that, to obtain a functional expression construct, the "VLP-producing" cDNA containing the above elements can be inserted into many different DNA vectors including, without limitation, commercially available vectors of series pUC (Promega, USA), pET (Clonetech USA), pCDNA, and pDEST (Invitrogen, USA).

[046] In the most specific embodiment, the invention provides a

"VLP-producing" plasmid pISVAC-0905 of 8901 b.p. which consists of SEQ ID NO: 12. pISVAC-0905 efficiently expresses HCV subtype Ib structural proteins in BHK21 cells, given that the "replicon-producing" cDNA" or, more specifically, recombinant expression vector pDKIS-0107 is being simultaneously expressed in the same host cell. pISVAC-0905 is composed of two fused fragments:

1) ISVAC-FUN: Hindlll-Pstl fragment of 4879 b.p. that consists of SEQ ID NO: 11, which includes sequentially fused sequences of FMDV 5^VUTR having SEQ ID NO: 2, SEQ ID NO: 9 that represents a fusion of HCV type Ib IRES and cDNA encoding HCV structural proteins polyprotein, cDNA coding for the alpha-peptide of beta-galactosidase reporter protein, and FMDV 3^VUTR having SEQ ID NO: 5, and

2) Hindlll-Pstl fragment of 4022 b.p. of recombinant expression vector pcDNA3.1 (Invitrogen, USA), that includes CMV promoter-enhancer, Agent's file reference: 2207942- WOl

and genes needed for replication in E.coli and antibiotic (ampicillin) resistance.

[047] In another aspect, the present invention provides a method for production of a recombinant non-replicating VLP of a second virus, wherein said method comprises:

1) co-expressing in a susceptible host cell (i) a "replicon-producing" construct which expresses a replication machinery of a first RNA virus and (ii) a "VLP-producing" construct which expresses structural proteins of the second virus under the control of the replication machinery of the first virus, under the conditions wherein said structural proteins of the second virus are capable of assembling into the recombinant VLP, and

2) isolating said VLP from the cells.

[048] According to the present invention, co-expression of the

"replicon-producing" and "VLP-producing" constructs can be achieved, for example, using one of the following methods:

1) by both constructs being integrated into a host cell genome; such integration may be easily achieved by anybody skilled in the art by introducing selective markers into the integrating fragment; furthermore, additional promoter and effector DNA elements may be introduced to silence and control transcription of integrated constructs;

2) by one construct being integrated into the host cell genome while another being transfected into the host cell in the form of a recombinant expression vector (e.g. , pDKIS-0107 or pISVAC-0905) or in the form of a cDNA-containing restriction fragment of the expression vector or in the form of a cDNA-containing PCR fragment amplified from the expression vector;

3) by co-transfection without integration of both constructs (e.g. , pDKIS-0107 and pISVAC-0905) or corresponding cDNA-containing restriction fragments or corresponding cDNA-containing PCR fragments.

[049] A preferred embodiment of this invention uses the third technique, i.e. co-expression of the expression vectors without integration, due to its relative simplicity and practicality. Transfection of a host cell by constructs of the invention can be performed by any method known in the art, including, without limitation, lipofection and electroporation. One specific embodiment of the invention uses a method of co-transfection by TAT-transduction Agent's file reference: 2207942- WOl

(e.g. , as described in Jong-Sub Yoon et al. J. Microb. 328, 42(4), 2004; Carsten R. et al, J. Biol. Chem. 2003, v.278, no. 13, pp. 11411-11418).

[050] According to the present invention, host cells for VLP production can be selected from various mammalian cultured cell lines. In a preferred embodiment, the host cell line is a cell line that survives without serum, such as, e.g., BHK21 cells. Serum-free host cell cultures are important to ensure minimal contamination with low density lipoproteins that dramatically hinder further VLP purification. The most preferred host cells of the invention are BHK21 cells.

[051] In another aspect, this invention provides incubation of the host cells, prior to cDNA expression, under conditions that facilitate growth of the endoplasmic reticulum in said cells. Such induction is expected to facilitate HCV (and other enveloped viruses) VLP assembly because HCV virions are shown to mature in endoplasmatic reticulum (see, e.g., Roingeard et al, 2004, Biol Cell. 96(2):103-8). Stimulation of the growth of endoplasmic reticulum can be achieved, for example, by induction of cytochrome p450 by, e.g., phenobarbital CYP2B6 (see Bar-nun et al, Proc. Natl. Acad. Sci USA, 1980, 77(2): 965-969), barbiturates, rifampicin, zixorin, etc., or by other well-known methods.

[052] In a separate embodiment, the invention provides a method for isolating

VLPs produced according to the above methods from the host cell, said method comprising the following steps, which are performed sequentially:

1) adding to the transfected VLP-producing cell culture polyethylene glycol (PEG) 3000-8000 to concentration 13-15% w/w, supplied by appropriate salt and buffer (e.g., PBS);

2) incubating the resulting solution (e.g., overnight);

3) adding a non-ionic detergent (e.g., Triton-XIOO) to the solution;

4) collecting VLP-containing supernatant by either centrifugation or filtration;

5) adding to the resulting VLP-containing supernatant PEG 3000-8000 to concentration 20-40% w/w;

6) incubating the resulting solution (e.g., overnight); Agent's file reference: 2207942- WOl

7) collecting the VLP-containing precipitate by either centrifugation or filtration (preferred method is centrifugation at 5000-1000 g for 10-40 mins), and

8) dissolving the collected VLPs in an appropriate solvent (e.g., a pharmaceutically acceptable solvent such as PBS).

[053] In a specific embodiment, the above method includes an additional set of steps 5-8 (i.e., VLP purification/concentration step of precipitation by PEG 3000-8000 to concentration 20-40% w/w after incubation).

[054] While overnight incubation is usually preferred, it will be evident to those skilled in the art that upon robust precipitation resulting from PEG addition, incubation time may be shortened to several hours and even 20 minutes.

[055] In one embodiment, VLPs are additionally purified using a method comprising the following steps:

1) adding to the VLP solution prepared according to steps 1-6, above, PEG 3000-8000 to concentration higher than 30% w/w;

2) incubating the resulting solution for an appropriate period of time (e.g. , overnight or several hours or 20 minutes in case of robust precipitation);

3) collecting VLP-containing precipitate by either centrifugation or filtration (preferred method is centrifugation at 5000-1000xg for 10-40 min), and

4) dissolving the collected VLPs in an appropriate solvent (e.g., a pharmaceutically acceptable solvent such as PBS).

[056] As disclosed herein, PEG precipitation may serve as intermittent or final step of VLP isolation. As noted in the Background section, above, most reports of VLP production propose isolation of VLPs by gradient centrifugation (e.g., in sucrose or CsCl gradient, see, e.g., U.S. Patent No. 6,387,662), which is very inefficient. The present invention surprisingly demonstrates that repetitive PEG precipitations lead to retaining and amplifying useful properties of VLPs (such as the ability to induce interferon-α in animal blood or its components) and generate higher total VLP yields and more concentrated VLP preparations (e.g., preparations having at least 10¹⁰ particles/ml, preferably at least 10¹² particles/ml) Agent's file reference: 2207942- WOl

making them useful in pharmaceutical applications. After PEG precipitations, VLPs are obtained in highly concentrated wet paste that conveniently allows further solution in any appropriate buffer in any appropriate concentration. In a specific embodiment, the buffer is pharmaceutically acceptable such as PBS.

[057] The present invention also provides a novel method for improving the stability of immuno-stimulating activity of the VLPs of the invention by limiting their oxidation in the final purified preparations and, optionally, in the course of their purification. The VLP oxidation can be limited by any methods known in the art, including, without limitation, the addition of a reducing agent (such as, e.g., a disulfide bond reducing reagent [e.g., dithiothreitol (DTT), tris(2-carboxyethyl)phosphine HCl (TCEP), β-mercaptoethanol (ME), glutathione (GSH), cysteine, etc.), storage in the absence of air access, degassing, sonication, etc. In the absence of such oxidation prevention, the VLPs of the invention should be preferably used within 24 hours of their purification.

[058] In another aspect, the invention provides an additional step of purification of HCV VLPs, that is batch affinity chromatography on lactoferrin immobilized on an appropriate sorbent (e.g., magnetic particles), to which HCV VLPs bind under physiological conditions (e.g., in PBS). According to one embodiment, after elution with an appropriate salt (e.g., IM KCl), the preparation of HCV VLPs can be transferred to the appropriate (e.g., pharmaceutically acceptable) diluent by dialysis or precipitation with PEG 3000-8000 at concentration 20-40% w/w, or by any other known method.

[059] In a preferred embodiment, the invention provides a method of making

HCV VLP ISVAC (see schematic representation in Figure 3) that comprises the following steps performed sequentially:

1) incubating BHK21 cells under conditions that facilitate growth of the endoplasmic reticulum (e.g., with barbiturate to induce cytochrome p450);

2) co-expressing in the BHK21 cells expression plasmids pDKIS-0107 (SEQ ID NO: 8) and pISVAC-0905 (SEQ ID NO: 12) (e.g., by co-transfection of said plasmids into BHK21 cells by the method of TAT-transduction), and Agent's file reference: 2207942- WOl

3) isolation of HCV VLP ISVAC from the cells by differential PEG precipitation followed by batch chromatography on lactoferrin, and completed by PEG re-precipitation.

[060] In a specific embodiment, step 3 comprises the following steps performed sequentially:

1) adding to the cell culture polyethylene glycol (PEG) 3000-8000 to concentration 13-15% w/w, supplied by appropriate salt and buffer;

2) incubating the resulting solution;

3) adding a non-ionic detergent to the solution;

6) incubating the resulting solution;

7) collecting VLP-containing precipitate by either centrifugation or filtration, and

8) dissolving the collected VLPs in an appropriate solvent.

[061] The novel PEG precipitation method of the present invention provides a versatile tool for handling recombinant VLPs allowing VLP preparations to be drastically concentrated (e.g., to particle concentrations higher than 10¹⁰ particles/ml, preferably higher than 10¹² particles/ml), transferred into another buffer, or additionally purified to remove VLPs that were damaged (e.g., oxidized or proteolyzed) during storage.

[062] In conjunction with the novel constructs and methods of the invention, provided herein are-non-infectious recombinant VLPs obtained using these constructs and methods and pharmaceutical compositions comprising such VLPs in a pharmaceutically acceptable carrier or excipient. In a specific embodiment, the invention provides a non-infectious HCV subtype Ib VLP ISVAC which is produced using the pDKIS-0106 and pISVAC-0905 constructs disclosed above. Agent's file reference: 2207942- WOl

[063] Also provided herein are methods of using the VLPs and VLP-containing pharmaceutical compositions of the invention for prophylaxis, treatment and prevention of human and animal diseases.

[064] Thus, in a specific embodiment, the present inventions provides that the

VLPs and VLP-containing pharmaceutical compositions of the invention can be used to increase endogenous interferon production in an animal blood or its components (such as, e.g., serum, peripheral blood mononuclear cells (PBMC), plasmacytoid dendritic cells (PDCs), leukocyte-trombocute layer, buffy coat fraction, trombocytes, and any combination thereof), upon direct administration. The induced interferons include TYPE-I-IFNs (e.g., IFN-α, IFN-β) as well as TYPE-II-IFN. The animal can be human or any other animal, including, e.g., domestic species of cattle, swine, birds, and insects.

[065] In a specific embodiment, the invention provides that the HCV subtype Ib

VLP ISVAC induced by direct treatment in the whole human blood more then 2 ng/ml of serum interferon- α-2b as measured by ELISA, and more then 10000 IU/ml of general interferon activity as measured by the interferon bioassay (i.e., protection of fibroblasts against vesicular stomatitis virus [VSV]).

[066] In another specific embodiment, rectal administration of HCV subtype Ib

VLP ISVAC to healthy humans resulted in a brief but strong increase of interferon in the blood with peak in 2-3 hours after administration and full clearance in 6-9 hours. The peak corresponded to 300 pg/ml of interferon-α-2b (measured by ELISA) and 7500 IU/ml of general interferon activity (measured by the interferon bioassay). In addition, the subjects' blood after ISVAC administration was found to be "primed" for amplified interferon induction upon viral infection. The subjects were clinically followed-on for 6 months after the experiment; neither subject reported any side effects of ISVAC administration.

[067] To achieve an increase in the blood interferon production, the VLPs of the invention can be administered by any known route of administration. Preferably, the route of administration is rectal. Rectal administrability of the VLP compositions of the present invention constitutes a significant advantage of the invention as it insures a better patient

99 Agent's file reference: 2207942- WOl

compliance (as compared to parenteral and other invasive routes of administration) and a simpler administration, production, formulation, and storage process.

[068] When used to increase interferon production, the VLPs and

VLP-containing pharmaceutical compositions of the invention can be used in combination with recombinant interferons (e.g., recombinant interferon-α or recombinant interferon-β or interferon inducers such as CpG motif-containing oligodesoxinucleotides (CPG-ODNs) or poly I:C synthetic dsRNA polymer.

[069] Due to their ability to induce interferons in blood and blood components, the VLPs of the invention can be also used for clearing conserved blood from various infections susceptible to treatment with interferons. Thus, in a specific embodiment, the present invention provides a method for clearing a blood sample from an infection susceptible to treatment with interferons, comprising adding to said blood sample a VLP-containing pharmaceutical composition of the invention and incubating the resulting mixture.

[070] As disclosed herein, due to their ability to increase interferon production, the VLPs and VLP-containing pharmaceutical compositions of the invention can be used to treat any diseases treatable by an increased interferon (e.g., TYPE-I-IFN) production, including, without limitation, various cancers, infections (e.g., viral, fungal, bacterial or parasitic infection), asthmatic disorders, allergic reactions, and other conditions. Examples of encompassed cancers include, without limitation, acute leukocytic leukemia, hairy cell leukemia, chronic myelogenous leukemia, multiple myeloma, reticulosarcoma, thrombocytosis, cutaneous T-cell leukemia, follicular lymphoma, malignant melanoma, squamous cell carcinoma, AIDS-related Kaposi's sarcoma, renal cell carcinoma, prostate carcinoma, bladder cell carcinoma, cervical dysplasia, colon carcinoma, kidney carcinoma, ovarian cancer, malignant melanoma, including post-operational prophylactic of malignant propagation, basal cell carcinoma, squamous cell carcinoma. Infections include, without limitation, hepatitis A, acute hepatitis B, acute hepatitis B+D, chronic hepatitis B, chronic hepatitis B+D, chronic hepatitis C, herpes virus infection, including herpes virus-associated stomatitis and gingivitis, poliomyelitis, Foot-and-Mouth Disease (FMD), papylloma virus infection, laryngeal papillomatosis, recurrent respiratory papillomatosis, infections by such viruses as, e.g., Hepatitis E virus, Hepatitis F virus, Hepatitis G virus, Human Agent's file reference: 2207942- WOl

Immunodeficiency Virus (HIV), cytomegalovirus (CMV), measles virus, West Nile fever virus, Epstein-Barr virus, Swine plague virus, Cattle plague virus, Encephalomyelitis virus, Reovirus, Yellow Fever Virus (YFV), Newcastle virus (NCV), and Polyhedrosis virus, as well as infectious diseases such as, e.g., mycosis, condylomatosis, encephalitis and meningoencephalitis, viral conjunctivitis, keratoconjunctivitis, keratitis, sepsis, including post-operational sepsis, pneumonia, meningitis, respiratory virus infection, influenza, including avian influenza, pyelonefritis, chlamydia infection, ureaplasmosis, toxoplasmosis, mycoplasmosis, gardnerellosis, trochomoniasis, bacterial vaginosis, and vaginal candidosis. Other encompassed diseases include, without limitation, rheumatoid arthritis, multiple sclerosis, cervical erosion, cervicitis, vulvovaginitis, bartolinitis, adneksitis, prostatitis, uretritis, balanitis, balanopostitis, cervical endometriosis, hemorrhagic fever, secondary immunodeficiency syndrom, allergic conjunctivitis, and bronchial asthma.

[071] In another embodiment, the present invention provides that the VLPs and

VLP-containing pharmaceutical compositions of the invention can be used to stimulate B-cells in an animal. The animal can be human or any other warm-blooded animal. In a specific embodiment, the HCV subtype Ib ISVAC VLP of the invention is shown to dramatically stimulate B cells in mice. To achieve stimulation of B cells, the VLPs of the invention can be administered by any known route of administration. As specified above, a preferred route of administration is rectal. As disclosed herein, due to their ability to stimulate B cells, the VLPs and VLP-containing pharmaceutical compositions of the invention can be used to treat any diseases treatable by such stimulation, including, without limitation, diseases related to an immune system deficiency such as cancer or infection (e.g., viral, fungal, bacterial or parasitic infection), as well as asthmatic disorders, and allergies. In a specific embodiment, a VLP-containing pharmaceutical composition of the invention is administered to a patient in conjunction with a particular allergen as a type of desensitization therapy to treat or prevent the occurrence of an asthmatic disorder or an allergic reaction associated with an asthmatic disorder.

[072] In an additional aspect, the ability of the VLPs and VLP-containing pharmaceutical compositions of the invention to induce interferons and/or stimulate B cells allow their use as adjuvants to stimulate patients' immune response to various antigens. Thus, in a specific embodiment, the invention provides a method for enhancing the immunogenicity Agent's file reference: 2207942- WOl

of an antigen, comprising administering to an animal the antigen and an adjuvant, wherein said adjuvant comprises a VLP-containing pharmaceutical composition of the invention.

[073] The VLPs and VLP-containing pharmaceutical compositions of the invention can be also used directly as anti-viral vaccines to prevent or alleviate infection by the virus corresponding to the virus from which the VLP structural proteins are derived (e.g., HCV subtype Ib VLP ISVAC can be used as HCV vaccine).

[074] When administered to human and animal patients with the goal of both inducing interferon and stimulating B cells, various administration regimens can be used. A preferred treatment regimen of the invention involves a twice- weekly administration of 0.1-1 mg of VLPs per 100 kg of the patient weight.

[075] The final aspect of the invention provides using VLPs of the invention for diagnosing an infection by a virus from which the VLP structural proteins are derived. Thus, in a specific embodiment, the invention provides a method for detecting a virus infection in an animal comprising adding to a sample from said animal a VLP preparation corresponding to said virus and detecting antibodies interacting with the VLP, wherein the presence of antibodies interacting with the VLP is indicative of the viral infection. The diagnostic test can be, for example, ELISA of the VLPs with the patient's blood. Indeed, as demonstrated herein, HCV subtype Ib VLP ISVAC can be reproducibly immunoprecipitated by the blood serum of HCV-infected individuals.

BRIEF DESCRIPTION OF THE DRAWINGS

[076] Figure 1 is a schematic representation of a "replicon-producing" construct (A) and a "VLP-producing" construct (B) of the invention.

[077] Figure 2 is a schematic representation of a "replicon-producing" construct (A) and a "VLP-producing" construct (B) designed for expression of HCV VLP under the control of FMDV replication machinery. Agent's file reference: 2207942- WOl

[078] Figure 3 is a schematic representation of the steps involved in HCV VLP

ISVAC production and purification.

[079] Figure 4 are graphs showing efficient induction of interferon-α-2b

(IFN-α-2b) in whole human blood by HCV VLP ISVAC. Donor blood was incubated in the presence of 10% v/v and 3% v/v of intact and boiled (negative control) ISVAC at 37°C for 20 hours. Newcastle disease virus (NDV) was used as positive control. IFN-α-2b induction was measured by (A) IFN-α-2b ELISA showing material presence of the protein in pg/ml and (B) interferon bioassay (protection of fibroblasts against vesicular stomatitis virus [VSV]) showing general interferon activity in international units (IU)/ml.

[080] Figure 5 are graphs showing efficient induction of IFN-α-2b in the blood of four (4) healthy human volunteers upon rectal administration of 1 ml of 2 mg/ml ISVAC dissolved in 5 ml PBS. (A): IFN-α-2b concentration in volunteer serum determined by ELISA (bars) and interferon bioassay (squares, D). (B): response to infection by Newcastle disease virus (NDV) (positive control) of blood of volunteers administered with intact ISVAC (diamonds, ♦) and boiled ISVAC (negative control) (circles, •).

[081] Figure 6 is the graph showing efficient induction of antibody secreting B cells in mice spleen upon administration of HCV VLP ISVAC. 1 and 10 μ 1 of ISVAC dissolved in 200 μ 1 of PBS, 200 μ g of polyacrylate 80 kDa (positive control) in 200 μ 1 of PBS, and 200 μ 1 PBS (negative control) were administered subcutaneously to 3 groups of 10 mice each. Number of antibody secreting cells (cell number per spleen) induced in four (4) days after immunization with sheep erythrocytes is shown.

DEFINITIONS

[082] As used herein, the terms "virus-like particle" (VLP), "pseudovirion" and

"pseudoviral particle" refer to a non-replicating viral particle derived from a virus. VLPs lack the viral genome and, therefore, are non- infectious in a clinical sense, i.e. they may be able to enter a cell, but are incapable of starting viral reproduction. VLPs are generally composed of one or more viral proteins, such as, but not limited to, those proteins referred to as capsid, coat, Agent's file reference: 2207942- WOl

shell, surface and/or envelope proteins, or particle-forming polypeptides derived from these proteins. For example, the non-replicating ISVAC VLPs of the present invention are composed of all structural proteins of HCV subtype Ib (i.e., C, El, E2, and p7) but lack all of HCV non-structural proteins which are responsible for the viral replication. VLPs can assemble/form spontaneously upon recombinant expression of the viral structural proteins in an appropriate expression system (e.g., the BHK21 expression system as disclosed herein). The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electrophoresis, immunoblotting, electron microscopy, density gradient centrifugation, X-ray crystallography, and the like. See, e.g., Baker et al, Biophys. J. (1991) 60:1445-1456; Hagensee et al, J. Virol. (1994) 68:4503-4505.

[083] The terms "viral structural proteins", "virus structural proteins," and

"structural proteins of the virus" are used interchangeably, and are terms of art. These terms refer to proteins encoded by viral genome and participating in building a capsid, virion or any other structural component of the virus. It will be evident to those skilled in the art that a wide variety of sequences which encode structural proteins of viruses, in addition to those discussed above, can be utilized in the present invention, and are therefore deemed to fall within the scope of the phrase "viral structural proteins".

[084] The terms "viral non-structural proteins", "virus non-structural proteins," and "nonstructural proteins of the virus" are used interchangeably, and are terms of art. These terms refer to proteins encoded by viral genome but not participating in building a capsid, virion or any other structural component of the virus. Many non-structural proteins of RNA viruses are involved in viral replication and assemble into "replication machinery" sufficient for independent replication of the viral nucleic acid genome or of any nucleic acid fragment carrying origin of replication and necessary signals for viral replication. Such fragment is called "replicon". As "replication machineries" of different viruses may be different in protein composition and assembly, the term "replication machinery" is defined by functional, not structural, features. It will be evident to those skilled in the art that a wide variety of sequences which encode non-structural proteins of different viruses, in addition to those discussed herein, can be utilized in the present invention. Agent's file reference: 2207942- WOl

[085] The term "replicon" refers to a self-replicating fragment of a nucleic acid that contains an origin of replication, signal sequences, as well as a sufficient set of genes encoding non-structural proteins that assemble into the replication machinery that drives and performs replication of this replicon guided by said signal sequences. In a broader sense, the term "replicon" is often used synonymously to the term "replication machinery" and relates not only to a nucleic acid but also to the replication machinery itself that drives self -replication of a viral nucleic acid in a host cell.

[086] The term "internal ribosome entry site" or "IRES" defines a special region of some viral and cellular RNA molecules that is capable of directing cap-independent binding of the ribosome and ribosomal assembly thus initiating and guiding translation of these RNAs.

[087] The terms "untranslated region (UTR)" and "non-translated region

(NTR)" are used interchangeably, and are terms of art (see Bukh et al, Proc. Nat. Acad. Sci. USA, 89, 4942-4946 (1992)). These terms refer to nucleic acid fragments that are positioned outside of translation reading frame at the ends of the viral replicon, a complement thereof {e.g. , a negative- sense RNA), and the corresponding DNA sequences of the positive-sense and the negative-sense RNA sequences. UTRs of different viruses may be very different but they always fulfill several crucial roles among which is driving viral replication due to recognition by viral replication machinery. Another possible role for UTRs may include serving as IRES or cis-element for viral processes.

[088] The term "polyprotein" refers to a polypeptide that is post-translationally cleaved to yield more than one polypeptide. This invention deals with both natural polyproteins of different viruses and with the artificially engineered ones.

[089] "Fusion PCR" is a technology for obtaining long fragments of DNA composed from pieces of different genes or nucleic acid fragments. For review, see Zhu et al, BioTechniques, 2007, 43(3):354-359. Briefly, to perform a fusion PCR of two pieces of nucleic acid, one uses a PCR oligonucleotide which on one end hybridizes with the first piece of nucleic acid while on the other end hybridizes with the second piece of nucleic acid. PCR of the first piece of nucleic acid with such oligonucleotide and another oligonucleotide flanking the first piece results in a fragment carrying at one of its ends the sequence that hybridizes to Agent's file reference: 2207942- WOl

the second piece of nucleic acid. PCR of the second piece of nucleic acid using as a primer the fragment obtained in the first PCR and another oligonucleotide flanking the second piece results in a DNA molecule consisting of the first and second nucleic acids fused together.

[090] As used herein, the term "viral RNA" refers to RNA from the viral genome, fragments thereof, transcripts thereof, and mutant sequences derived therefrom.

[091] The terms "vector", "construct", and "plasmid" are used interchangeably to refer to the vehicle by which a DNA or RNA sequence can be introduced into a host cell, so as to transfect the host and clone the vector or promote expression of the introduced sequence. Vectors include plasmids, cosmids, phages, viruses, etc. Vectors may further comprise selectable markers.

[092] As used herein, the term "isolated" means that the material being referred to has been removed from the environment in which it is naturally found, and is characterized to a sufficient degree to establish that it is present in a particular sample. Such characterization can be achieved by any standard technique, such as, e.g., sequencing, hybridization, immunoassay, functional assay, expression, size determination, or the like. Thus, a biological material can be "isolated" if it is free of native cellular components, i.e., components of the cells in which the material is found or produced in nature. A nucleic acid molecule excised from the chromosome that it is naturally a part of is considered to be isolated. Such a nucleic acid molecule may or may not remain joined to regulatory, or non-regulatory, or non-coding regions, or to other regions located upstream or downstream of the gene when found in the chromosome. Nucleic acid molecules that have been spliced into vectors such as plasmids, cosmids, artificial chromosomes, phages and the like are considered isolated.

[093] An isolated material may or may not be "purified". The term "purified" as used herein refers to a material (e.g. , a nucleic acid molecule or a protein) that has been isolated under conditions that detectably reduce or eliminate the presence of other contaminating materials. Contaminants may or may not include native materials from which the purified material has been obtained. A purified material preferably contains less than about 90%, less than about 75%, less than about 50%, less than about 25%, less than about 10%, less than about Agent's file reference: 2207942- WOl

5%, or less than about 2% by weight of other components with which it was originally associated.

[094] The term "about" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e. , the limitations of the measurement system. For example, "about" can mean within an acceptable standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and more preferably still up to ±1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term "about" is implicit and in this context means within an acceptable error range for the particular value.

[095] In the context of the present invention insofar as it relates to any of the disease conditions recited herein, the terms "treat", "treatment", and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition. Treatment may be effected prophylactically (e.g., prior to infection) or therapeutically (e.g., following infection). For example, in relation to infections, the term "treat" may mean (i) prevention of infection or re-infection, (ii) reduction or elimination of symptoms of an infection, (iii) substantial or complete elimination of the pathogen in question, etc. In relation to cancer, the term "treat" may mean to relieve or alleviate at least one symptom selected from the group consisting of tumor growth, metastasis, sensitivity of tumor cells to treatments such as chemotherapy, radiation therapy, thermo therapy, etc. Within the meaning of the present invention, the term "treat" also denotes to arrest, delay the onset (i.e., the period prior to clinical manifestation of a disease) and/or reduce the risk of developing or worsening a disease. The term "protect" is used herein to mean prevent, delay or treat, or all, as appropriate, development or continuance or aggravation of a disease in a subject. Within the meaning of the present invention, disease conditions include without limitation various cancers, infections (e.g., viral, fungal, bacterial or parasitic infection), asthmatic disorders, allergic reactions, and other conditions. Examples of encompassed cancers include, without limitation, acute leukocytic leukemia, hairy cell leukemia, chronic myelogenous leukemia, multiple myeloma, reticulosarcoma, thrombocytosis, cutaneous T-cell leukemia, Agent's file reference: 2207942- WOl

follicular lymphoma, malignant melanoma, squamous cell carcinoma, AIDS-related Kaposi's sarcoma, renal cell carcinoma, prostate carcinoma, bladder cell carcinoma, cervical dysplasia, colon carcinoma, kidney carcinoma, ovarian cancer, malignant melanoma, including post-operational prophylactic of malignant propagation, basal cell carcinoma, squamous cell carcinoma. Infections include, without limitation, hepatitis A, acute hepatitis B, acute hepatitis B+D, chronic hepatitis B, chronic hepatitis B+D, chronic hepatitis C, herpes virus infection, including herpes virus-associated stomatitis and gingivitis, poliomyelitis, Foot-and-Mouth Disease (FMD), papylloma virus infection, laryngeal papillomatosis, recurrent respiratory papillomatosis, infections by such viruses as, e.g., Hepatitis E virus, Hepatitis F virus, Hepatitis G virus, Human Immunodeficiency Virus (HIV), cytomegalovirus (CMV), measles virus, West Nile fever virus, Epstein-Barr virus, Swine plague virus, Cattle plague virus, Encephalomyelitis virus, Reovirus, Yellow Fever Virus (YFV), Newcastle virus (NCV), and Polyhedrosis virus, as well as infectious diseases such as, e.g., mycosis, condylomatosis, encephalitis and meningoencephalitis, viral conjunctivitis, keratoconjunctivitis, keratitis, sepsis, including post-operational sepsis, pneumonia, meningitis, respiratory virus infection, influenza, including avian influenza, pyelonefritis, chlamydia infection, ureaplasmosis, toxoplasmosis, mycoplasmosis, gardnerellosis, trochomoniasis, bacterial vaginosis, and vaginal candidosis. Other encompassed diseases include, without limitation, rheumatoid arthritis, multiple sclerosis, cervical erosion, cervicitis, vulvovaginitis, bartolinitis, adneksitis, prostatitis, uretritis, balanitis, balanopostitis, cervical endometriosis, hemorrhagic fever, secondary immunodeficiency syndrom, allergic conjunctivitis, and bronchial asthma.

[096] As used herein, the term "therapeutically effective" applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to an animal in need thereof. Within the context of the present invention, the term "therapeutically effective" refers to that quantity of a VLP or a pharmaceutical composition comprising such VLP which is sufficient (i) to increase the production of interferon (e.g., TYPE-I-IFN, and/or TYPE-II-IFN, and/or IFN-α, and/or IFN- β, and/or IFN-γ) in an animal (e.g., human) blood or its components, and/or (ii) to stimulate proliferation or any other property of B cells in an animal (e.g. , human), and/or (iii) to reduce or eliminate at least one symptom of an infection, cancer, asthma, allergy, or of another relevant disease as listed above. Note that when a combination of active ingredients is Agent's file reference: 2207942- WOl

administered, the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually.

[097] The phrase "pharmaceutically acceptable", as used in connection with compositions of the invention, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to an animal (e.g., a human). Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[098] As used herein, the term "blood components" encompasses, without limitation, serum, peripheral blood mononuclear cells (PBMC), plasmacytoid dendritic cells (PDCs), leukocyte-trombocute layer, buffy coat fraction, trombocytes, and any combination thereof.

DETAILED DESCRIPTION OF THE INVENTION

1.1. Preparation of "replicon-producing" and "VLP-producing" constructs

[099] Two plasmids necessary for production of VLPs of the invention may be prepared using standard methods of genetic engineering that are evident to those skilled in the art and disclosed in many basic manuals and textbooks. These plasmids are designated in this invention as "replicon-producing" and "VLP-producing". Both plasmids are produced by ligating two fragments of DNA, a "functional fragment" and a "core fragment". The "functional fragment" of the "replicon-producing" plasmid encodes viral non- structural proteins. The "functional fragment" of the "VLP-producing" plasmid encodes viral structural proteins. The "core fragment" of both plasmids is a vector that carries sets of sequences necessary for plasmid replication in E.coli and mammalian cells as well as other useful utilities. Such "core" elements are widely available in many commercially available vectors, such as, e.g., vectors of series pUC (Promega, USA), pET (Clonetech USA), pCDNA or pDEST(Invitrogen, USA). Ligating the first and the second fragments together provides operational plasmids used in this invention. Agent's file reference: 2207942- WOl

[0100] While the "core fragment" is available commercially, the "functional fragment" can be produced (e.g., by fusion PCR or ligation) using various viral and marker sequences available from naturally-occurring sources as well as from various depositories (e.g. the American Type Culture Collection, Rockville, MD). For example, viral strains utilized in the generation of ISVAC VLP particles of the invention are HCV subtype Ib (GenBank accession No. AB031663, strain VAT96), and FMDV «A» (GenBank accession No. X74812, strain A22550, and GenBank accession No. AY593833).

[0101] The "functional" fragment prepared by fusion PCR consists of several distinct DNA sequences picked from different sources. Design of primers for this fusion PCR must account for proper DNA orientation (i.e., 5" to 3" for coding thread) as well as for intact translation reading frame (i.e., nucleotide triplets, corresponding to amino acids) to prevent frameshifting, so that the resulting DNA construct is properly expressed in the context of the final plasmid and its product is properly processed in the context of the final cell culture.

1.2. Preparation of BHK21 host cells suitable for HCV VLP production

[0102] BHK21 cells utilized in this invention are widespread around the world and may be readily obtained from many different commercial and academic sources.

[0103] During the preparation stage, BHK21 cells can be grown according to the standard techniques known to those skilled in the art. However, in a preferred embodiment of this invention, cells were additionally stimulated to grow the endoplasmic reticulum. As it is known that HCV virions mature in the endoplasmic reticulum, the induction of the growth of the endoplasmic reticulum is used herein to further facilitate the assembly of HCV virions (see, e.g., Roingeard, P., et al., 2004 Biol Cell., 96(2): 103-8.). Stimulation of the growth of endoplasmic reticulum can be achieved, for example, by induction of cytochrome p450 by the addition of, e.g., phenobarbital CYP2B6 (see Bar-nun et al, Proc. Natl. Acad. Sci. USA, 1980, 77(2): 965-969), barbiturates, rifampicin, zixorin, etc. to cell culture media, or by other well-known methods. The accumulation of cytochrome p450 can be controlled by, e.g., spectrophotometry and optimally should reach OD₄₅₀>0.15. It will be evident to those skilled in the art that growth of the endoplasmic reticulum can be also controlled by microscopy as well as several other well-known methods. Agent's file reference: 2207942- WOl

1.3. Transfection of BHK21 cell culture with "replicon-producing" and "VLP-producing" constructs

[0104] Introduction of exogenous cDNA into cells with the goal of expressing said cDNA in said cells may be achieved by many different techniques. Two distinct approaches that are evident to those skilled in the art and should be discriminated as conceptually different are plasmid transfection and integration into cell genome. In the preferred embodiment of this invention, DNA plasmids are designed so that they do not integrate into BHK21 genome, however, it will be evident to those skilled in the art that one or both cDNAs may be readily modified for such integration by inserting appropriate DNA fragments coding for distinct selective markers (e.g., by introducing selective markers into the integrating fragment and/or additional promoter and effector DNA elements to silence and control transcription of integrated DNAs). cDNAs may also be transfected into cells in the form of plasmids or in the form of cDNA-containing restriction fragments of the corresponding expression construct, as well as cDNA-containing PCR fragments amplified from the corresponding expression construct.

[0105] A preferred embodiment of this invention uses co-expression of the expression constructs, due to its relative simplicity and practicality. Transfection of the cell by plasmids of the invention can be performed by any method known in the art. Co-transfection by TAT-transduction (Jong-Sub Yoon et al. J. Microb. 328, 42(4), (2004); Carsten R. et al., J. Biol. Chem. 2003, v.278, No. 13, pp. 11411-11418) was chosen as a preferred embodiment of this invention. However, it will be evident to those skilled in the art that the other transfection and transduction protocols (such as, e.g., lipofection or electroporation) can be used.

[0106] In the production of HCV ISVAC VLPs of the invention, BHK21 cells are co-transfected with "replicon-producing" plasmid pDKIS-0106 (which expresses a polyprotein of FMDV non-structural proteins fused to GFP: «2B-2C-3A-3B1/3B2/3B3-3C-3D-GFP») and "VLP-producing" plasmid pISVAC-0905 (which expresses a polyprotein of HCV-Ib structural proteins fused to β-gal_αpe_P: «C-El-E2-p7-β-gal_αpep»). Importantly, the pDKIS-0106 plasmid dramatically enhances the expression of the pISVAC-0905 plasmid. Agent's file reference: 2207942- WOl

1.4. Isolation of VLPs

[0107] In a preferred embodiment of this invention, cells expressing VLPs desintegrate spontaneously, otherwise cells should be disintegrated by the techniques familiar to those skilled in the art. The initial purification of VLPs can be achieved by a two-stage polyethyleneglycol (PEG) precipitation: first, a low concentration of PEG 3000-8000 (13-15% w/w) is used to precipitate cell debris, second, a high concentration of PEG 3000-8000 (20-40% w/w) is used to precipitate VLPs. It will be evident to those skilled in the art that there are many types of salts and buffers and their concentrations and treatment conditions that can be varied in this procedure.

[0108] VLP-containing precipitate obtained after PEG treatment can be dissolved in an appropriate (e.g., pharmaceutically acceptable) buffer such as PBS and can be stored at +4°C and used. In a preferred embodiment of this invention, additional purification of VLP is performed and includes batch chromatography of VLPs on the sorbent carrying lactoferrin. The most preferred embodiment employs lactoferrin magnetic particles (Promega, USA), to which VLPs bind in PBS at room temperature. It will be evident to those skilled in the art that lactoferrin may be immobilized on many other commercially available sorbents. After elution with an appropriate salt (e.g., IM KCl), the preparation of VLPs can be transferred to an appropriate (e.g., pharmaceutically acceptable) diluent by dialysis or re-precipitation with PEG 3000-8000 (20-40% w/w); or by any other known method. It will be evident to those skilled in the art that many diverse methods of purification can be used for further purification of VLPs.

[0109] The resulting quality and concentration of VLP preparations can be tested by various methods known to those skilled in the art such as, e.g., SDS-PAGE, immunoblotting, immunoprecipitation, electron microscopy (e.g., negative contrasting), light microscopy (e.g., by analysis of dispersed particles in a dark field), etc. In the Examples section, below, the quality and concentration of the final preparation of HCV ISVAC VLPs was determined by immunoprecipitation with anti-HCV antibodies (see Example 2) and by testing the VLP ability to induce interferon production in whole human blood serum (i.e., by functional testing, see Example 3.1). Agent's file reference: 2207942- WOl

1.5. Improving VLP stability by preventing VLP oxidation

[Ol 10] The present invention provides a novel method for improving the stability of immuno-stimulating activity of the VLPs of the invention by limiting their oxidation in the final purified preparations and, optionally, in the course of their purification. It is demonstrated in Example 3.2, below, that a mere incubation of ISVAC solution in the presence of air dramatically decreases its interferon-inducing ability. The VLP oxidation can be limited by any methods known in the art, including, without limitation, the addition of a reducing agent (such as, e.g., a disulfide bond reducing reagent [e.g., dithiothreitol (DTT), tris(2-carboxyethyl)phosphine HCl (TCEP), β-mercaptoethanol (ME), glutathione (GSH), cysteine, etc., storage in the absence of air access, degassing, sonication, etc. In the absence of such oxidation prevention, the VLPs of the invention should be preferably used within 24 hours of their purification.

ILl. ELISA of VLPs

[0111] ELISA is an important method of the control of authenticity of VLPs.

Two approaches may be used here: 1) immunoprecipitation with blood of patients infected by the virus corresponding to the VLP and hence carrying antibodies to this virus; 2) immunoprecipitation with isolated antibodies for the virus or its component. In a specific embodiment of this invention, ELISA is used to precipitate HCV VLPs with anti-HCV antibodies contained in the blood of HCV-infected patients (see Example 2, below).

II.2. Using VLPs as diagnostics

[0112] As demonstrated in Example 2, below, ISVAC is reproductively precipitated by the blood of HCV-infected patients, therefore, ISVAC and similar pseudovirions may be used as diagnostics of related viruses, more precisely as an antigen component of the relevant designated ELISA kit. Agent's file reference: 2207942- WOl

111.1. Using VLPs of the invention for inducing interferon production in human or animal blood and its components

[0113] As disclosed herein, VLPs of the invention, including HCV VLP ISVAC, possess unique and unexpected ability to induce and/or enhance the production of interferons (including IFN-α) in human blood and its components (e.g., serum, peripheral blood mononuclear cells (PBMC), plasmacytoid dendritic cells (PDCs), leukocyte-trombocute layer, buffy coat fraction, or any combination thereof). As demonstrated in Example 3.1, below, the HCV subtype Ib ISVAC VLP of the invention produced a dose-dependent induction on interferons in whole human blood that reached 2000 pg/ml of interferon-α-2b (as determined by ELISA) and general interferon activity of 12500 IU/ml (as determined by bioassay) (see Figures 4A-B).

[0114] The ability of HCV VLP ISVAC and similar VLP preparations of the invention to induce and/or increase the production of TYPE-I-IFN in human blood and its components has an important pharmaceutical application. Blood banks of the entire world are plagued by the problem of conserved blood contamination by different viruses. Treating such conserved blood with VLPs of the invention will induce TYPE-I-IFN that, in turn, will jump-start antiviral defense mechanisms that will either clear out or dramatically suppress the contaminating viruses. The treatment of blood or blood components using VLPs of the invention can be performed simply by a single or repetitive addition of such VLPs to a blood sample followed by incubation (e.g., for 6 hours). Virus inactivation in the sample can be monitored using various well-known functional assays such as, e.g., ELISA, PCR, or viral propagation on plates.

111.2. Using VLPs for inducing interferon production in human and animal patients

[0115] One of the unique properties of the VLPs of the present invention, including HCV VLP ISVAC, is their ability to induce the production of interferons, including interferon-α, in human and animal patients. Thus, as specified in the Example 3.3 below, rectal administration of ISVAC to four healthy human subjects resulted in interferon rising and peaking in 3 hours after administration at approximately 7500 IU/ml (as determined by the bioassay) and 500 pg/ml of IFN-α-2b (as determined by ELISA) (see Figure 5A). As further Agent's file reference: 2207942- WOl

specified in Example 3.3, below, the ISVAC-induced interferon enhanced the ability of patient blood to respond to attack of a live HCV virus (see Figure 5B), thus demonstrating "priming" phenomenon that supports the physiological relevance of the interferon induced by ISVAC.

[0116] The ability of the VLPs of the invention to induce the production of interferons in human and animal patients can be tested, for example, by administration of the appropriate amount of the VLP to the patient followed by collection of the patient's blood at specific time points and detection of interferon by such methods as, e.g., ELISA or interferon bioassay. These two independent techniques supplement each other by providing different angles on interferon presence: 1) interferon-α ELISA monitors physical presence of a representative member of IFN-α subfamily (e.g., IFN-α-2b as disclosed in Example 3), and 2) interferon bioassay measures protection of fibroblasts from vesicular stomatitis virus (VSV) infection (e.g., as described in Meager, 2002, J. Immunol. Methods. 261:21-36).

IV.1. Using VLPs for stimulating B cells in human or animal patients

[0117] One of the unique properties of the VLPs of the present invention is their ability to stimulate B cells in human and animal patients. Thus, as specified in the Example 4, below, intracutaneous administration of ISVAC to mice dramatically and in dose-dependent manner stimulated formation of antibody secreting cells to the level exceeding that for known strong mitogens and adjuvants (see Figure IB).

[0118] The ability of the VLPs of the invention to stimulate B cells in human and animal patients can be tested, for example, by administration of the appropriate amount of the VLP to the patient followed by such methods as 3H-thymidine incorporation assay (Krieg, A.M, et al, 1995, Nature. 374(6522):546-9) and/or Jerne-Nordin local hemolysis assay (Jerne et al, 191 A, Transplant. Rev. 18:130-91).

V.I. Using VLP preparations for treating diseases in human and animal patients

[0119] The ability of the VLPs of the invention to induce interferons and stimulate B cells in human and animal patients has important therapeutic consequences. Agent's file reference: 2207942- WOl

Interferon induction is an important component in fighting viral infections, cancers and many other diseases. As disclosed herein, due to their ability to increase interferon (e.g., TYPE-I-IFN) production, the VLPs and VLP-containing pharmaceutical compositions of the invention can be used to treat any disease treatable by an increased interferon production, including, without limitation, various cancers, infections (e.g., viral, fungal, bacterial or parasitic infection), asthmatic disorders, allergic reactions, and other conditions. Examples of encompassed cancers include, without limitation, acute leukocytic leukemia, hairy cell leukemia, chronic myelogenous leukemia, multiple myeloma, reticulosarcoma, thrombocytosis, cutaneous T-cell leukemia, follicular lymphoma, malignant melanoma, squamous cell carcinoma, AIDS-related Kaposi's sarcoma, renal cell carcinoma, prostate carcinoma, bladder cell carcinoma, cervical dysplasia, colon carcinoma, kidney carcinoma, ovarian cancer, malignant melanoma, including post-operational prophylactic of malignant propagation, basal cell carcinoma, squamous cell carcinoma. Infections include, without limitation, hepatitis A, acute hepatitis B, acute hepatitis B+D, chronic hepatitis B, chronic hepatitis B+D, chronic hepatitis C, herpes virus infection, including herpes virus-associated stomatitis and gingivitis, poliomyelitis, Foot-and-Mouth Disease (FMD), papylloma virus infection, laryngeal papillomatosis, recurrent respiratory papillomatosis, infections by such viruses as, e.g., Hepatitis E virus, Hepatitis F virus, Hepatitis G virus, Human Immunodeficiency Virus (HIV), cytomegalovirus (CMV), measles virus, West Nile fever virus, Epstein-Barr virus, Swine plague virus, Cattle plague virus, Encephalomyelitis virus, Reovirus, Yellow Fever Virus (YFV), Newcastle virus (NCV), and Polyhedrosis virus, as well as infectious diseases such as, e.g., mycosis, condylomatosis, encephalitis and meningoencephalitis, viral conjunctivitis, keratoconjunctivitis, keratitis, sepsis, including post-operational sepsis, pneumonia, meningitis, respiratory virus infection, influenza, including avian influenza, pyelonefritis, chlamydia infection, ureaplasmosis, toxoplasmosis, mycoplasmosis, gardnerellosis, trochomoniasis, bacterial vaginosis, and vaginal candidosis. Other encompassed diseases include, without limitation, rheumatoid arthritis, multiple sclerosis, cervical erosion, cervicitis, vulvovaginitis, bartolinitis, adneksitis, prostatitis, uretritis, balanitis, balanopostitis, cervical endometriosis, hemorrhagic fever, secondary immunodeficiency syndrom, allergic conjunctivitis, and bronchial asthma.

[0120] Stimulation of B cells is an important component in fighting immune system deficiency (e.g., a tumor or cancer or a viral, fungal, bacterial or parasitic infection), Agent's file reference: 2207942- WOl

asthmatic disorders, and allergic reactions. As disclosed herein, due to their ability to stimulate B cells, the VLPs and VLP-containing pharmaceutical compositions of the invention can be used to treat any disease treatable by such stimulation.

[0121] The VLPs of the invention can be produced in unit dosage forms for administration by rectal, oral, parenteral, transmucosal, intranasal, vaginal, transdermal, or topical (e.g., by application to skin wounds) routes. Parenteral routes include intravenous, intra-arteriole, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, intrathecal, and intracranial administration. A preferred route of administration according to the present invention is rectal. Being non-invasive, this route of administration insures better patient compliance. It is also preferred because of the simpler production and handling of the pharmaceutical compositions.

[0122] For administration to human and animal patients, VLPs of the present invention can be formulated in pharmaceutical compositions in combination with one or more pharmaceutically acceptable carriers and excipients such as, e.g., lubricants, diluents, flavorants, colorants, buffers, and disintegrants. Suitable pharmaceutically acceptable carriers include any and all conventional solvents (such as, e.g., water, physiological solution, glycerol, ethanol), emulgators, buffers, conservants, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like with which the compound is administered. Examples of such useful substances can be found in "Remington's Pharmaceutical Sciences" by E. W. Martin. The term "pharmaceutically acceptable carrier" refers to a carrier that does not cause an allergic reaction or other untoward effect in patients to whom it is administered. As noted above, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans. Specific, suitable pharmaceutically acceptable carriers include, but are not limited to, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers can further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of one or more of the active components of the composition. The use of such media and agents for pharmaceutically active substances is well known in the art and Agent's file reference: 2207942- WOl

suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in compositions of the present invention is contemplated.

[0123] In addition to the VLPs, the pharmaceutical compositions of the invention can also include one or more other biologically active substances. Such substances include but are not limited to vitamins, oils and other immunostimulators. In one embodiment, said pharmaceutical compositions may be administered to a patient in conjunction with a particular allergen as a type of desensitization therapy to treat or prevent the occurrence of an asthmatic disorder or an allergic reaction associated with an asthmatic disorder.

[0124] The formulation and dose for therapeutic administration of the VLP preparations of the invention will depend on the severity of the disease condition being treated, whether other drugs are being administered, whether other actions are taken (such as diet modification), the weight, age, and sex of the subject, and other criteria. The skilled medical practitioner will be able to select the appropriate formulation and dose in view of these criteria and based on the results of published clinical trials. The dosage and administration regimen for the VLP preparations of the invention can be further adjusted for an individual patient by monitoring the level of TYPE-I-IFN (e.g., IFN-α or IFN-γ) in patient's blood or blood serum (e.g., by ELISA, virus suppression, or any other suitable method).

[0125] In one embodiment, suitable dose ranges of the VLP preparations are from about 0.1 mg to about 1 mg per 100 kg of body weight taken twice weekly in the course of 10 weeks.

VLl. Using VLP preparations as adjuvants to enhance patient response to various antigens

[0126] It was discussed earlier that there is a major need for improved and more powerful pharmaceutically acceptable adjuvants because recombinant or synthetic antigens used in modern day vaccines are generally far less immunogenic than older style live or killed whole organism vaccines (reviewed in Petrovsky, N., and Aguilar, J.C., 2004, Immunol Cell Agent's file reference: 2207942- WOl

Biol. 82(5):488-96). With few exceptions, alum remains the sole adjuvant approved for human use in the majority of countries worldwide.

[0127] Most adjuvants are distinguished by their ability to stimulate B cells

(Kwissa M, et al, 2007, Expert Rev Vaccines. 6(5):673-84; Aguilar, J.C., and Rodriguez, E.G., 2007, Vaccine, 25(19):3752-62); actually it is one of their crucial functions. Since ISVAC is found to strongly and directly stimulate both B cells and IFN-I-TYPE, it most probably will be very useful as strong adjuvant inducing ThI immunity. Such application may be performed simply by adding ISVAC to an antigen or vaccine pharmaceutical composition. Also, it may be done by separate administration of ISVAC, that is simultaneous or prior to vaccination.

VILl. General Techniques

[0128] In accordance with the present invention, there can be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989 (herein "Sambrook et al, 1989"); DNA Cloning: A Practical Approach, Volumes I and II (Glover ed. 1985); Oligonucleotide Synthesis (Gait ed. 1984); Nucleic Acid Hybridization (Hames and Higgins eds. 1985); Transcription And Translation (Hames and Higgins eds. 1984); Animal Cell Culture (Freshney ed. 1986); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); Ausubel et al eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc. 1994; among others.

EXAMPLES

[0129] The present invention is also described and demonstrated by way of the following examples. However, the use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiments described herein. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the invention in spirit or in scope. The invention is Agent's file reference: 2207942- WOl

therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which those claims are entitled.

Example 1 : Production of HCV VLP ISVAC

1.1. Design and production of expression vectors

[0130] Two expression vectors were used for production of ISVAC: pDKIS-0107, which encodes non-structural proteins of FMDV and hence is referred to as "replicon-producing", and pISVAC-0905, which encodes structural proteins of HCV subtype Ib and hence is referred to as "VLP -producing". Both vectors were built from two fragments: 1) a "functional" fragment which contains a cDNA encoding viral proteins preceded by an IRES and flanked by UTRs (also schematically shown in Figures 2A-B), and 2) a "core" fragment which contains standard expression vector elements such as DNA sequences for replication in E.coli and sequences for expression in BHK21 cells.

[0131] The "functional" fragments of the final expression vectors were produced by fusion PCR followed by restriction and blunting of EcoRI sticky end. The DNA promoter used in both plasmids was the CMV promoter embedded into commercial expression vectors. Because of that, fusion PCR was used to prepare "truncated" versions of constructs (SEQ ID NO: 7 and SEQ ID NO: 11, for pDKIS-0107 and pISVAC-0905, respectively) that do not contain CMV promoter but contain all other elements of constructs as follows:

1) FRAGMENT DKIS-FUN (SEQ ID NO: 7: 5757 b.p. flanked by Clal-EcoRI restriction sites):

2) FMDV 5' UTR of SEQ ID NO: 2 (GenBank accession No. AY593833 or X74812; strain A22550)

3) hybrid YFV-HIV IRES of SEQ ID NO: 3 ;

4) cDNA coding for FMDV non-structural proteins polyprotein of SEQ ID NO: 4 (GenBank accession No. AY593833 or X74812);

5) cDNA coding for GFP from the cloning vector pEGFP-N3 (Invitrogen, USA) (GenBank accession No. U57609);

6) FMDV 3' UTR of SEQ ID NO: 5 (GenBank accession No. AY593833 or X74812). Agent's file reference: 2207942- WOl

7) FRAGMENT ISVAC-FUN of SEQ ID NO : 11 (flanked by Hindlll-Pstl restriction sites):

8) FMDV 5' UTR of SEQ ID NO: 2 (GenBank accession No. AY593833 or X74812);

9) SEQ ID NO: 9 that represents a fusion of HCV type Ib IRES and cDNA encoding HCV structural proteins polyprotein (GenBank accession No. AB031663; strain VAT96) having the structure «C-El-E2-p7»;

10) cDNA coding for beta-galactosidase alpha-peptide (β-gal_αpep) from the expression vector pUC19 (Invitrogen, USA) (GenBank accession No. EF153731.1);

11) FMDV 3' UTR of SEQ ID NO: 5 (GenBank accession No. AY593833 or X74812).

[0132] Hybrid YFV-HIV IRES of DKIS-FUN was prepared by oligonucleotide synthesis and inserted into the construct by fusion PCR. Genes coding for GFP and β-gal_αpep were prepared by PCR and inserted into the construct by fusion PCR.

[0133] The "core" fragments of final expression vectors were produced by restriction and blunting of EcoRI sticky end, designed to produce the following DNA fragments (disclosed as parts of SEQ ID NO: 8 and SEQ ID NO: 12, for pDKIS-0107 and pISVAC-0905, respectively):

1) FRAGMENT DKIS-CORE: Clal-EcoRI fragment of 5168 b.p. prepared by single-hit CIaI- EcoRI DNA restriction, wherein EcoRI end is blunted by Klenow, of recombinant expression vector pCI Neo (Promega, USA; GenBank accession No. U47120), that includes CMV promoter-enhancer, and genes needed for replication in E.coli and antibiotic (ampicillin) resistance;

2) FRAGMENT ISVAC-CORE: Hindlll-Pstl fragment of 4022 b.p. prepared by single-hit Hindlll-Pstl DNA restriction of recombinant expression vector pcDNA3.1 (Invitrogen, USA; GenBank accession No. EF550208), that includes CMV promoter-enhancer, and genes needed for replication in E.coli and antibiotic (ampicillin) resistance.

[0134] Ligation of fragments DKIS-FUN and DKIS-CORE produced the plasmid pDKIS-0107 of 10925 b.p. Ligation of fragments ISVAC-FUN and ISVAC-CORE produced the plasmid pISVAC-0905 of 8901 b.p. Both plasmids were amplified in E.coli and used for TAT-transduction into BHK21 cells for ISVAC production (Example 1.2). Agent's file reference: 2207942- WOl

[0135] Oligonucleotides for PCR were designed by computer programs such as

Oligo 5.0 and Blast, and commercially synthesized at Syntol LLC (Moscow, Russia). Fusion PCR primers had size around 70-100 nt, regular PCR primers - 20-35 nt.

[0136] Restriction, ligation and blunt end filling were performed by enzymes from Promega (USA) according to the manufacturer's recommendations.

1.2. Preparation of BHK21 culture and Tat-transduction

[0137] BHK21 cells were obtained from Leytran LLC (Moscow, Russia). Cell culture was grown in commercial media RPMI1640 (Gibco, USA) with 10% FCS (Fetal Calf Serum, Gibco, USA) for 24 hours. Then cells were transferred to DMEM media supplied with 10% FCS and 0.1% potassium phenobarbital (Sigma, USA) and 1 mM hemin (Sigma, USA); and incubated for 60 hours in total 10 liters of culture until reaching concentration of 10 cells/ml as defined by microscopy. Concentration of cytochrome was controlled by spectrophotometry. After the incubation, cells were thoroughly washed from the media and incubated for 2 hours in 200 ml of fresh DMEM media without serum but supplied with 0.1% potassium phenobarbital, 1 mM hemin, and 50 mM lisophostphatidilcholine (all reagents from Sigma, USA). This batch of competent cell culture was used for further transfection.

[0138] Tat-transfection was performed by standard protocol (see Jong-Sub Yoon et al., 2004, J. Microb. 328, 42(4); Carsten R. et al, 2003, J. Biol. Chem. 2003, v.278, No. 13, pp. 11411-11418). HIV-I Tat-protein peptide for transduction (YGRKKRRQRRR; SEQ ID NO: 14) was produced at Syntole LLC (Moscow, Russia) by standard peptide synthesis technique. 0.1 μM of Tat-peptide dissolved in 4 ml of deionized water was added to samples of 100 μg of each expression vector prepared as discussed above in section 1.1 and resuspended after alcohol precipitation in 4 ml of fresh DMEM media without serum. To form desoxynucleoprotein for transfection, the mixture was incubated at room temperature for 30 minutes. Formation of the desoxynucleoprotein was controlled by staining DNA with ethidium bromide. The resulting mixture containing the transfection desoxynucleoprotein was added to 200 ml batch of competent BHK21 cells prepared as discussed above and used in the further stages of ISVAC production (Example 1.3). Agent's file reference: 2207942- WOl

1.3. Expression, isolation and harvesting of ISVAC

[0139] The mixture of competent cells with desoxynucleorpotein (prepared as discussed above), was incubated for 36-72 hours at 37⁰C. After incubation, Triton-XlOO was added to the final concentration of 0.2 % and PEG 6000 (Sigma, USA) was added to 15% w/w. The efficiency of transfection and further protein expression and processing was controlled by measuring production of beta-galactosidase alpha-peptide (β-gal_αpep) encoded by pISVAC-0905 by X-GaI complementation.

[0140] The mixture with Triton-X100 and PEG was incubated for 2 hours at 37⁰C.

After that, it was centrifuged at 5000xg for 20 minutes at room temperature: ISVAC-containing supernatant was collected. 1/2 volume of 1.5M NaCl supplied with 60% PEG-6000 was added to the supernatant and the resulting mixture was incubated for 12-18 hours at +4⁰C. The ISVAC-containing precipitate was obtained by centrifugation at 5000xg for 30 minutes at room temperature. The precipitate was dissolved in 100 ml PBS (50 mM, pH 7.4) supplemented with 25 mM NaCl, 1 mM MgSO₄, and 0.1 mM CaC^. Approximately 10 g of precipitate were obtained from 10¹⁰ BHK21 cells suspended in 200 ml of transfection mixture.

[0141] Further purification of ISVAC was achieved by batch chromatography on lactoferrin magnetic sorbent prepared by immobilization of lactoferrin on magnetic beads (Promega, USA). 4 mg of avidin-lactoferrin conjugate was immobilized on 20 ml of the biotinylated magnetic beads. 100 ml ISVAC prepared as described above was mixed with 25 ml of such sorbent. The mixture was gently shaken for 5 minutes at room temperature and then the beads were separated magnetically. Then the beads were washed two times by 20 ml of PBS (50 mM, pH 7.4) and shaken for another 5 minutes at room temperature. ISVAC VLPs were eluted with 1.5 M NaCl, PBS (50 mM, pH 7.4) at room temperature. VLPs were re-precipitated by 35% PEG-6000 and resuspended in PBS (50 mM, pH 7.4). Approximately 3 g of precipitate were obtained at this stage from 10¹⁰ BHK21 cells suspended in 200 ml of transfection mixture. The final ISVAC preparation was used within 24 hours of purification and was characterized by immunoblotting, immunoprecipitation (Example 2), and functional assays of inducing interferon in human blood (Example 3) and stimulating B cells in mice (Example 4). Agent's file reference: 2207942- WOl

Example 2: ISVAC immunoprecipitation with anti-HCV antibodies

[0142] Blood of HC V- infected patients randomly mixed together with the group of non-infected patients was tested by standard HCV ELISA kit (Orion Trading Company, Canada). In a parallel experiment, the same samples were tested by the same kit under the same conditions, however, the HCV antigen was replaced with ISVAC in the amount 50 μl per well of standard 96- well plate. Out of 295 blood samples tested, The Orion kit provided 181 "positive" results and 114 "negative" ones. ISVAC provided 165 "positive" results and 130 "negative" ones, the latter including all 114 negatives from the kit. Hence, ISVAC immunological behavior is very similar to that of HCV and ISVAC may be used as a diagnostics for the presence of antibodies to HCV.

Example 3: Testing of the ability of ISVAC to induce endogenous interferons

3.1 Using ISVAC for IFN-oc induction in human blood

[0143] ISVAC PBS solution prepared according to Example 1.3 was used within

24 hours for direct treatment of blood obtained from 4 (four) healthy anonymous donors of different age, gender, and complexion. For each experiment 250 μl of blood was mixed with 250 μl of RPMI-1640 medium (Sigma, USA), supplemented with 50 μl of PBS containing 3% v/v or 10% v/v of ISVAC or relevant controls (3% v/v and 10% v/v of boiled ISVAC as negative controls and Newcastle disease virus (NDV) as a positive control) and incubated at 37°C for 20 hours. Serum was decanted and used for interferon bioassay or for ELISA - 100 μl per well in both cases. To prepare the "boiled" ISVAC negative control sample, one of intact ISVAC tubes was submerged to boiling water for 20 minutes. As shown in Figures 4A-B, incubation of human blood with 3% v/v and 10% v/v ISVAC provided dose-dependent induction on interferons that reached a very high level: 2000 pg/ml of IFN-α-2b by ELISA and general interferon activity of 12500 IU/ml by bioassay.

[0144] To investigate the relative strength of ISVAC-driven interferon induction, it was compared with interferon production in response to Newcastle disease virus (NDV) which is a well-known inducer of IFN- α in human blood and phytohemagglutinin (PHA) which is a well-known inducer of IFN-γ. Boiled ISVAC was used as a negative control. In the Agent's file reference: 2207942- WOl

bioassay, 2xlO⁶ cells of diploid propagated culture of human embryo fibroblasts diluted in DMEM medium supplied with 5% fetal calf serum were used per 96-well plate. 100 μl of tested blood samples were added per fibroblast-containing well and incubated overnight at 37°C. After incubation fluid was removed and monolayers were washed with PBS. Plates were then infected with VSV. Samples were titrated in parallel with NDV that was used as positive control and recombinant interferon standard that was used as a reference. All cell materials: VSV, NDV, fibroblasts, were obtained from Gamaleya Institute of Microbiology (Moscow, Russia). Assays were standardized against international reference standards thus providing activity measurements in international units of interferon activity (IU) per ml of tested blood.

[0145] Under the above experimental conditions, NDV provided 32000 IU/ml while PHA provided 3500 IU/ml that correlates with the ability of IFN-α and IFN-γ to confer viral infection resistance to cells. 12500 IU/ml induced by 10% ISVAC (see Figure 4B) demonstrated that ISVAC-driven induction went well beyond IFN-γ and almost reached the level of IFN-α induced by the aggressive live virus NDV. ELISA of IFN-α- 2b confirmed that high amount of IFN-α was induced (see Figure 4A). The final total production of TYPE-I-IFN in this experiment is higher then the quantities obtained here for IFN-α-2b because many other TYPE-I-IFN subtypes are also induced. As VLPs by their nature are rather complicated mixtures, it is always necessary to provide a control that proves that the investigated activity resides within VLPs rather with some impurity (Vanlandschoot et al, 2007). Since in the presence of boiled ISVAC no interferon induction was detected (see Figures 4A-B), ISVAC interferon-inducing activity is clearly linked to VLP structure rather then to any potential contaminants.

3.2. Investigation of the effect of oxidation on ISVAC activity

[0146] It was noticed that the interferon-inducing activity of ISVAC prepared according to Example 1.3 significantly decreases within 24 hours of the preparation if ISVAC solution is stored in partially filled eppendorf. We hypothesized that such decrease in activity is due to oxidation, and prepared two samples in closed 1.6 ml eppendorf tubes: 1) one containing 0.8 ml of ISVAC solution in PBS and air and 2) one containing 1.6 ml of ISVAC solution in PBS, wherein the buffer had been thoroughly degassed and the air was almost Agent's file reference: 2207942- WOl

totally absent in the eppendorf. After 1 day of incubation at +4oC, ISVAC activity was assessed by its ability to induce interferon in human blood (as disclosed in Example 3.1). The interferon-inducing activity of the degassed (and protected from air) sample corresponded to 2000 pg/ml by ELISA discussed in example 3.1, while the activity of the sample exposed to the air was at least 10 times lower, indicating that the activity of ISVAC prepared by PEG precipitation can be destroyed by oxidation.

[0147] Thus, in order to improve stability of immuno-stimulating activity of

ISVAC VLPs, it is important to limit oxidation of the final ISVAC purified preparations and, optionally, in the course of their purification. The VLP oxidation can be limited by any methods known in the art, including, without limitation, the addition of a reducing agent (such as, e.g., a disulfide bond reducing reagent [e.g., dithiothreitol (DTT), tris(2-carboxyethyl)phosphine HCl (TCEP), β-mercaptoethanol (ME), glutathione (GSH), cysteine, etc., storage in the absence of air access, degassing, sonication, etc. In the absence of such oxidation prevention, the VLPs of the invention should be preferably used within 24 hours of their purification.

3.3 Using ISVAC for TYPE-I-IFN induction in human volunteers

[0148] A six- month program of full-scale rodent toxicology studies according to international standards and requirements of Russian Ministry of Health was carried out. Acute toxicity studies in mice and rats showed that LD₅₀ is not achieved in either rectal or intramuscular ISVAC administration at the levels exceeding recommended human dosages 1400-fold. Chronic toxicity studies were performed in two settings: 2-month rectal in rats and 1 -months rectal in rabbits. Neither study revealed any internal or external pathology. Specific studies of mutagenecity, embryotoxicity, teratogenecity, and allergenecity in rodents failed to demonstrate any toxicity of ISVAC. Additionally, ISVAC was shown not to influence growth of the culture of human stem cells, that is believed to be a strong indication of low toxicity of a given compound in humans. Based on these results, ISVAC was determined to be safe allowing to proceed with experiments in human volunteers. The experiments in human volunteers were performed in full compliance with international legal and ethical standards and monitored by the Ethical Committee of Neurok Pharma LLC. Agent's file reference: 2207942- WOl

[0149] 1 ml of 2 mg/ml ISVAC dissolved in 5 ml PBS prepared as disclosed in

Example 1.3 was rectally administered within 24 hours of final purification by clyster to 4 (four) healthy volunteers at 9AM of a given day. Volunteers then provided blood samples for interferon detection throughout the day. Each of these volunteers displayed interferon rising and peaking in 3 hours after administration at approximately 7500 IU/ml by the bioassay and 500 pg/ml of IFN-α-2b by ELISA (Figure 5A). Then interferon level fell down to the baseline at 6-9 hours after administration. In a negative control studies the same volunteers received administration of ISVAC deactivated by boiling and no one produced any serum interferon.

[0150] An important test for physiological relevance of induced interferon is its ability to "prime" blood for response to a viral infection. The term "priming" refers to the long-standing observation of an amplifying effect of type-I-IFN signaling on further response to viral induction. To test the "priming" effect of ISVAC, each blood sample obtained from volunteers after ISVAC administration was treated by NDV and subjected to the bioassay. As shown in Figure 5B, NDV induced significantly higher levels of interferon in blood obtained after ISVAC administration then in the intact blood thus proving that ISVAC-induced interferon engages in "positive feedback" circle similarly to the native interferon.

[0151] Neither volunteer receiving ISVAC and having increased serum interferon concentration reported any side effects. This finding is surprising because it is now generally believed that any interferon induction in human body should bring side effects, most prominently, the flu-like syndrome (Krieg, A.M. 2007. Proc. Am. Thorac. Soc. 4(3):289-94; Krieg, A.M. 2007. J. Clin. Invest. 117(5): 1184-94). Perfect tolerability of ISVAC may be explained by its most physiological manner of interferon induction. Indeed, ISVAC significantly differs in structure from all presently used interferon inducers (poly I:C, CpG ODNs and recombinant interferons) and therefore may induce interferon via a more physiologically relevant set of receptors and other biological stimuli.

Example 4: Using ISVAC for B cell stimulation in mice

[0152] Experiment was performed in mice CBAxC57Bl/6)F_! (provided by

Institute of Immunology of Russian Academy of Sciences, Moscow, Russia), each weighting 19+0.4 g. 4 groups of 10 mice each were subcutaneously administered with negative control of Agent's file reference: 2207942- WOl

200 μl PBS, positive control of 200 μg of polyacrylate (average molecular weight 80 kDa), or 10 μl or 1 μl of ISVAC (prepared according to Example 1.3 and used within 24 hours) dissolved in 200 μl PBS. Three hours after administration each mouse was administered 5x10 sheep erythrocytes. Number of antibody- secreting cells formed per spleen was measured after 4 days by standard technique of Jerne-Nordin local hemolysis plaque forming assay (Jerne et al, 1974, Transplant. Rev. 18:130-91). Simultaneously the number of mononuclears per spleen was measured to control directness of ISVAC action towards B cells. Figure 6 shows that, as compared to the negative control (PBS), ISVAC dramatically and in dose-dependent manner stimulated formation of antibody secreting cells to the level clearly exceeding that for polyacrylate which is known as a strong mitogen and adjuvant (Hilgers et al, 2000). The number of mononuclears increased by 10% in 1 μl ISVAC group and by 28% in 10 μl ISVAC group, which is much lower than the induction of antibody-secreting cells indicating that mononuclears cannot be responsible for the major part of B cell stimulation that must be exerted directly by ISVAC.

[0153] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Claims

Agent's file reference: 2207942- WOlCLAIMS:

1. An isolated cDNA that encodes a replication machinery of a first RNA virus and comprises the following fragments:

1) a promoter providing transcription in a host cell of mRNA encoding polyprotein of the non- structural proteins of the virus;

2) 5' Untranslated Region (5"UTR) of the virus, wherein such 5"UTR provides replication of the transcribed mRNA by the replication machinery of the virus;

3) Internal Ribosome Entry Site (IRES) providing translation of the polyprotein of the non- structural proteins of the virus in the host cell;

4) a sequence encoding the polyprotein of the non-structural proteins of the virus, wherein such polyprotein is processed into the replication machinery of this virus, and

5) 3"UTR of the virus, wherein such 3"UTR provides replication of the transcribed mRNA by the replication machinery of the virus.

2. The isolated cDNA according to claim 1, wherein the virus is Foot-and- Mouth Disease Virus (FMDV).

3. The isolated cDNA according to claim 1 having a structure shown in Figure IA.

4. The isolated cDNA according to claim 2, which comprises the following fragments:

1) CMV promoter-enhancer that consists of SEQ ID NO: 1;

2) FMDV UTR fragment that consists of SEQ ID NO: 2;

4) a cDNA encoding the polyprotein of FMDV non- structural proteins, and

5) FMDV UTR fragment that consists of SEQ ID NO: 5.

5. The isolated cDNA according to claim 4, wherein the cDNA encoding FMDV polyprotein consists of SEQ ID NO: 4. Agent's file reference: 2207942- WOl

6. The isolated cDNA according to claim 4, wherein the cDNA encoding the polyprotein of FMDV non- structural proteins is fused at the 3' end to a cDNA encoding a reporter protein.

7. The isolated cDNA according to claim 6, wherein the reporter protein is green fluorescent protein (GFP).

8. The isolated cDNA according to claim 7 which comprises SEQ ID NO: 6.

9. The isolated cDNA of claim 7 having the structure shown in Figure 2A.

10. A recombinant expression vector comprising the isolated cDNA of any one of claims 1-9, wherein said vector directs expression of the non-structural proteins of the first virus in an appropriate host cell resulting in the assembly of said non-structural proteins into the replication machinery of said virus.

11. The recombinant expression vector according to claim 10 which consists of SEQ ID NO: 8 (pDKIS-0107).

12. An isolated cDNA that encodes control elements of a first RNA virus and structural proteins of a second virus and which comprises the following fragments:

2) 5' Untranslated Region (5"UTR) of the first virus, wherein such 5"UTR provides replication of the transcribed mRNA by the replication machinery of the first virus;

3) Internal Ribosome Entry Site (IRES) providing translation of the polyprotein of the structural proteins of the second virus in the host cell;

4) a sequence encoding the polyprotein of structural proteins of the second virus, wherein such polyprotein is processed and assembled into a virus-like particle (VLP) of the second virus, and

5) 3"UTR of the first virus, wherein such 3"UTR provides replication of the transcribed mRNA by the replication machinery of the first virus.

13. The isolated cDNA according to claim 12, wherein the first virus is Foot-and-Mouth Disease Virus (FMDV). Agent's file reference: 2207942- WOl

14. The isolated cDNA according to claim 12, wherein the second virus is a hepatotropic RNA virus.

15. The isolated cDNA according to claim 14, wherein the second virus is Hepatitis C Virus (HCV).

16. The isolated cDNA according to claim 12 having a structure shown in Figure IB.

17. The isolated cDNA according to claim 12, which comprises the following fragments:

1) CMV promoter-enhancer that consists of SEQ ID NO: 1

2) FMDV UTR fragment that consists of SEQ ID NO: 2 or SEQ ID NO:

13;

3) HCV IRES;

4) a cDNA encoding a polyprotein of structural proteins of HCV, and

5) FMDV UTR fragment consisting of SEQ ID NO: 4.

18. The isolated cDNA according to claim 17, wherein the cDNA encoding the polyprotein of structural proteins of HCV consists of SEQ ID NO: 9.

19. The isolated cDNA according to claim 17, wherein the cDNA encoding the polyprotein of structural proteins of HCV is fused at the 3' end to a cDNA encoding a reporter protein.

20. The isolated cDNA according to claim 19, wherein the reporter protein is alpha-peptide of beta-galactosidase (β-gal_αpep).

21. The isolated cDNA according to claim 20 which comprises SEQ ID NO: 10.

22. The isolated cDNA of claim 20 having the structure shown in Figure 2B.

23. A recombinant expression vector comprising the isolated cDNA of any one of claims 12-22, wherein said vector directs expression of the structural proteins of the second virus under the control of the replication machinery of the first virus in an appropriate host cell Agent's file reference: 2207942- WOl

resulting in the assembly of said structural proteins into a virus-like particle (VLP) of the second virus.

24. The recombinant expression vector according to claim 23 which consists of SEQ ID NO: 12 (pISVAC-0905).

25. A method of making a recombinant virus-like particle (VLP) of a second virus, wherein said method comprises:

1) co-expressing in a susceptible host cell (i) the cDNA of claim 1 which expresses a replication machinery of a first RNA virus and (ii) the cDNA of claim 12 which expresses structural proteins of the second virus under the control of the replication machinery of the first virus, under the conditions wherein said structural proteins of the second virus are capable of assembling into the recombinant VLP, and

2) isolating said VLP from the cells

26. The method according to claim 25, wherein the cDNAs of the first and second virus are integrated into the host cell genome.

27. The method according to claim 25, wherein one of the cDNAs is integrated into the host cell genome and the other cDNA is expressed from a recombinant expression vector.

28. The method according to claim 25, wherein the cDNAs of the first and second virus are co-transfected into the host cell in the form of recombinant expression vectors.

29. The method according to claim 28, wherein the recombinant expression vectors are pDKIS-0107 (SEQ ID NO: 8) and pISVAC-0905 (SEQ ID NO: 12).

30. The method according to claim 25, wherein the host cell is a mammalian cell.

31. The method according to claim 30, wherein the host cell is BHK21.

32. The method according to claim 25, wherein, prior to cDNA expression, the host cell is incubated under conditions that facilitate growth of the endoplasmic reticulum in said cell.

33. The method according to claim 32, wherein the host cell is incubated in the presence of an inducer of cytochrome p450. Agent's file reference: 2207942- WOl

34. The method according to claim 25, wherein the second virus is a hepatotropic RNA virus.

35. The method according to claim 34, wherein the second virus is Hepatitis C virus (HCV).

36. The method according to claim 25, wherein the stage of isolating VLPs comprises the following steps, which are performed sequentially:

1) adding to the host cell culture polyethylene glycol (PEG) 3000-8000 to concentration 13-15% w/w, supplied by appropriate salt and buffer;

2) incubating the resulting solution;

3) adding a non-ionic detergent to the solution;

6) incubating the resulting solution;

8) dissolving the collected VLPs in an appropriate solvent.

37. The method according to claim 36, wherein the non-ionic detergent is Triton-X100.

38. The method according to claim 36, wherein the incubation of step 2 is an overnight incubation.

39. The method according to claim 36, wherein the final VLP solution is prepared under conditions where oxidation is prevented.

40. The method according to claim 39, wherein the final VLP solution comprises a reducing agent.

41. The method according to claim 36, wherein steps 5-8 are repeated. Agent's file reference: 2207942- WOl

42. The method according to claim 36, further comprising the following steps, which are performed sequentially:

1) adding to the VLP solution of step 8, PEG 3000-8000 to concentration higher than 30% w/w;

2) incubating the resulting solution for an appropriate period of time;

3) collecting VLP-containing precipitate by either centrifugation or filtration, and

4) dissolving the collected VLPs in an appropriate solvent.

43. The method according to claim 36, further comprising purifying VLPs using batch chromatography on lactoferrin immobilized on an appropriate sorbent.

44. A recombinant VLP of a virus obtained according to the method of any one of claims 25-43.

45. The recombinant VLP of claim 44, wherein the virus is Hepatitis C virus.

46. A pharmaceutical composition comprising the recombinant VLP of claim 44 or claim 45 and a pharmaceutically acceptable carrier or excipient.

47. The pharmaceutical composition of claim 46, wherein the VLP concentration is at least 10¹⁰ particles/ml.

48. A method of making a recombinant virus-like particle (VLP) ISVAC of Hepatitis C virus subtype Ib, wherein said method comprises the following steps performed sequentially:

1) incubating BHK21 cells under conditions that facilitate growth of the endoplasmic reticulum;

2) co-expressing in the BHK21 cells expression plasmids pDKIS-0107 (SEQ ID NO: 8) and pISVAC-0905 (SEQ ID NO: 12);

3) adding to the cell culture polyethylene glycol (PEG) 3000-8000 to concentration 13-15% w/w, supplied by appropriate salt and buffer;

4) incubating the resulting solution;

5) adding a non-ionic detergent to the solution;

6) collecting VLP-containing supernatant by either centrifugation or filtration; Agent's file reference: 2207942- WOl

7) adding to the resulting VLP-containing supernatant PEG 3000-8000 to concentration 20-40% w/w;

8) incubating the resulting solution;

9) collecting VLP-containing precipitate by either centrifugation or filtration, and

10) dissolving the collected VLPs in an appropriate solvent.

49. A recombinant VLP ISVAC of Hepatitis C virus subtype Ib obtained according to the method of claim 48.

50. A pharmaceutical composition comprising the HCV subtype Ib VLP ISVAC of claim 49 and a pharmaceutically acceptable carrier or excipient.

51. The pharmaceutical composition of claim 50, wherein said composition is formulated for rectal administration.

52. A method for increasing interferon (IFN) production in an animal blood or its components, comprising administering to the animal or the preparation of the animal blood or its components the pharmaceutical composition of claim 46 or claim 50.

53. The method of claim 52, wherein the animal is human.

54. The method of claim 52, wherein the interferon (IFN) is a TYPE-I-IFN.

55. The method of claim 52, wherein the interferon (IFN) is IFN-α or IFN-β.

56. The method of claim 52, wherein the pharmaceutical composition of claim 46 or claim 50 is administered together with another interferon-inducing agent.

57. The method of claim 56, wherein the additional interferon-inducing agent is selected from the group consisting of recombinant interferons, CpG motif-containing oligodesoxinucleotides (CPG-ODNs), and poly I:C synthetic dsRNA polymer.

58. A method for enhancing the immunogenicity of an antigen, comprising administering to an animal the antigen and an adjuvant, wherein said adjuvant comprises the pharmaceutical composition of claim 46 or claim 50.

59. The method of claim 58, wherein the animal is human. Agent's file reference: 2207942- WOl

60. A method for clearing a blood sample from an infection susceptible to treatment with interferons, comprising adding to said blood sample the pharmaceutical composition of claim 46 or claim 50 and incubating the resulting mixture.

61. A method for stimulating B cells in an animal, comprising administering to the animal the pharmaceutical composition of claim 46 or claim 50.

62. The method of claim 61, wherein the animal is human.

63. A method of treating a disease treatable by increasing interferon production and/or by B cell stimulation in an animal, comprising administering to the animal a therapeutically effective amount of the pharmaceutical composition of claim 46 or claim 50.

64. The method of claim 63, wherein the animal is human.

65. The method of claim 63, wherein the animal is selected from the group consisting of cattle, swine, birds, and insects.

66. The method of claim 63, wherein the disease is selected from cancer, infection, asthmatic disorder, and allergic reaction.

67. The method of claim 66, wherein the infection is selected from viral, fungal, bacterial, and parasitic infection.

68. A use of the pharmaceutical composition of claim 46 or claim 50 to treat a disease treatable by increasing interferon production and/or by B cell stimulation in an animal.

69. A method for detecting a virus infection in an animal comprising adding to a sample from said animal the VLP preparation of claim 44 corresponding to said virus and detecting antibodies interacting with the VLP, wherein the presence of antibodies interacting with the VLP is indicative of the viral infection.

70. The method of claim 69, wherein antibodies interacting with the VLP are detected by ELISA.

71. A method for improving the stability of immuno- stimulating activity of the VLP preparation of claim 44 or claim 49 comprising limiting VLP oxidation in the VLP preparation, and, optionally, in the course of VLP purification. Agent's file reference: 2207942- WOl

72. The method of claim 71, wherein the VLP oxidation is limited by a method selected from the group consisting of the addition of a reducing agent, storage in the absence of air access, degassing, and sonication.

73. A use of the VLP-containing pharmaceutical composition of claim 46 to prevent or alleviate an infection by a virus from which VLP structural proteins are derived.