WO2010025016A2

WO2010025016A2 - Enhancement of transgene expression from virus-based vaccine vectors using suppressors of the type 1 interferon response

Info

Publication number: WO2010025016A2
Application number: PCT/US2009/052969
Authority: WO
Inventors: Jerald C. Sadoff; John Fulkerson; Michele Stone
Original assignee: Aeras Global Tb Vaccine Foundation
Priority date: 2008-08-06
Filing date: 2009-08-06
Publication date: 2010-03-04
Also published as: WO2010025016A3

Abstract

Baculovirus vectors with improved ability to express transgenes are provided. The baculovirus vectors are genetically engineered to contain and express nucleic acid sequences that encode one or more proteins that interfere with mammalian host cell type I interferon (IFN) responses. Thus, the expression of transgenes encoded by the baculovirus vector is not inhibited by a type I DFN response, and the transgenes are freely expressed in mammalian host cells. Alternatively, baculovirus vectors with nucleotide sequences encoding one or more genes of interest (e.g. tuberculosis or malarial antigens) are co-administered with one or more proteins that interfere with mammalian host cell type I IFN responses; or with viral vectors (e.g. adenovectors) that express one or more proteins that interfere with mammalian host cell type I IFN responses.

Description

ENHANCEMENT OF TRANSGENE EXPRESSION FROM VIRUS-BASED VACCINE VECTORS USING SUPPRESSORS OF THE TYPE 1 INTERFERON

RESPONSE

DESCRIPTION

BACKGROUND OF THE INVENTION

Field of the Invention

The invention generally relates to baculovirus vectors with improved ability to express transgenes. In an embodiment, baculovirus vectors are genetically engineered to contain and express nucleic acid sequences that encode one or more proteins that interfere with mammalian host cell type I interferon (IFN) responses, hi another embodiments, recombinant baculovirus vectors which express antigens of interest, including for example, tuberculosis or malaria antigens, also include nucleic acid sequences that encode one or more proteins that interfere with mammalian host cell type I IFN responses; or the recombinant baculovirus vectors are provided to subjects in combination with other vectors, e.g. recombinant adenovirus vectors or recombinant BCG, which include nucleic acids which encode and express one or more proteins that interfere with mammalian host cell type I IFN responses; or the recombinant baculovirus vectors are provided to a subject in combination with one or more proteins that interfere with mammalian type I IFN responses.

Background of the Invention A number of viral based vectors have been used to successfully transfect mammalian cells. Among those are adenovirus, adenovirus-associated virus (AAV), papovaviruses, and vacciniavirus. These viral based vectors have several disadvantages, however. Adenovirus vectors have been well studied and used in a number of gene therapy trials as well as in vaccine clinical trials; although, recent negative clinical trail outcomes may restrict their use in the United States (Gene Therapy, 7:110, 2000, Nature Biotechnology 26, 3-4, 2008).

There also have been clinical trials using adeno-associated virus (AAV); however, problems with this system have also arisen. AAV data have been published showing that the vector can produce abnormal T-cell responses (J. Clin. Ivest. 117, 3958-3970, 2007). The major problems demonstrated in the above viral based vectors appear to be non-selective cytotoxicity (particularly in the liver) and pre-existing immune responses against the viruses. The cytolytic T cell response induced against adenovirus derived peptides has been shown to mediate the destruction of the vector transduced cells and has been associated with local tissue damage and inflammation (Hum. Gene Ther. 6,

1265-1274,1995; J. Immunol. 155, 2564-2570, 1995). There is also a possibility of recombination with endogenous infecting adenovirus, particularly at high input doses, which posses further safety concerns. Adenoviruses will recombine with pre-existing material; a potential drawback where endogenous adenovirus is wide spread in the human population. Safety concerns have also been associated with the use of Herpes Simplex Virus. For example, lytic replication of the virus in the human brain has been linked to encephalitis (MoI. Biotechnol. 2, 179-195, 1994).

An alternative vector, which has been shown to infect mammalian cells, is the baculovirus (Trends Biotechnol., 20, 173-180, 2002). Baculovirus is a rod virus and therefore the amount of genetic material inserted into recombinant baculovirus is not limited, as in the capsid based viral systems. hi contrast to other human viral vectors (e.g. adevnovirus based vectors) baculovirus will not express its own genes from insect specific promoters in human cells (Virology 125: 107-117, 1983). Thus, baculovirus will not provoke an immune response as a consequence of viral gene expression of virally encoded genes, which is highly advantageous compared to other human viral vectors. Because baculovirus does not infect mammals, mammalian cells will have no pre-existing immunity to the baculovirus system. However, with the use of either a mammalian promoter (e.g. CAG) or a viral internal ribosome entry site (IRES)(e.g. EMCV IRES) upstream of a marker or therapeutic gene, expression of baculovirus encoded transgenes can be achieved in mammalian cells. Infection with baculovirus will not produce endogenous human viruses, as has been seen with the adenovirus vectors.

Another advantage of the baculovirus system is that it can be grown in a serum free culture media in large quantities, which removes the potential hazards of serum contamination of the therapeutic agent with viral and prion agents. Vaccine candidates using the baculovirus systems appear to have clear advantages over most other viral vaccine systems. Unfortunately, baculovirus based discharge of passenger DNA (i.e. transgenes) and other molecules for the expression of foreign proteins in mammalian cells will result in a type I interferon (IFN) response (J. Immunol., 178, 2361-2369, 2007). This IFN response limits the expression of foreign proteins by means of protein kinase R (PKR) and 2'-5' oligoadenylate-synthetase (2'-5' OAS). Activated PKR blocks translation by phosphorylating the α-subunit of eukaryotic initiation factor eIF2. On the other hand, 2-5A synthetases produce short, 2'-5' OAS associated oligoadenylates which activate RNase L, a single-stranded specific endoribonuclease that digests mRNA and ribosomal RNA. These mechanisms are likely to destroy or inhibit the transcription and translation of passenger nucleic acids encoded by a baculo virus system.

There is a need to provide improved baculoviral vectors to mediate successful expression of transgenes in mammalian host cells.

SUMMARY OF THE INVENTION

The invention provides baculovirus expression vectors genetically engineered to contain and express nucleic acid sequences that encode one or more proteins that interfere with mammalian host cell type I interferon (IFN) responses, hi one embodiment of the invention, such baculoviruses are also genetically engineered to encode a transgene of interest. Upon administration of such a baculoviral vector to a mammalian host, expression of the one or more proteins that interfere with mammalian host cell type I IFN results in suppression of the host type I IFN response, and successful expression of the transgene in the host occurs unimpeded. It is an object of this invention to provide baculovirus vectors comprising, nucleic acid sequences that encode and express one or more proteins that modulate a Type 1 Interferon (IFN) response; and nucleic acid sequences that encode and express one or more transgenes of interest. In one embodiment, the one or more proteins that modulate a Type 1 IFN response are selected from the group consisting non-structural protein 1 (NSP- 1 ) from rotavirus, NS 1 protein from influenza virus, C 12R protein from ectromlia virus. In another embodiment, the one or more transgenes of interest include one or more transgenes that encode antigens, for example, at least one tuberculosis or malaria antigen.

The invention also provides a vaccine composition for administration to a mammal. The vaccine compositions comprises i. at least one baculovirus vector comprising, nucleic acid sequences that encode and express one or more proteins that modulate a Type 1

Interferon (IFN) response; and nucleic acid sequences that encode and express one or more transgenes of interest; and ii. a physiological acceptable carrier. The one or more proteins that modulate a Type 1 EFN response are selected from the group consisting non-structural protein 1 (NSP-I) from rotavirus, NSl protein from influenza virus, C12R protein from ectromlia virus. The one or more transgenes of interest include one or more transgenes that encode antigens. The antigens include at least one tuberculosis or malaria antigen. Pn some embodiments, the mammal is a human.

The invention also provides a method of eliciting an immune response to one or more antigens in a mammal. The method comprises the step of administering to said mammal a composition comprising: i. at least one baculovirus vector comprising,nucleic acid sequences that encode and express one or more proteins that modulate a Type 1 Interferon (IFN) response; and nucleic acid sequences that encode and express said one or more antigens; and ii. a physiological acceptable carrier. The composition is administered in an amount sufficient to elicit an immune response to said one or more antigens, hi one embodiment, the one or more proteins that modulate a Type 1 IFN response are selected from the group consisting non-structural protein 1 (NSP-I) from rotavirus, NSl protein from influenza virus, C12R protein from ectromlia virus. The one or more antigens may include at least one tuberculosis or malaria antigen. The mammal may be a human.

The invention also provides a method of eliciting an immune response in a mammal, the method comprising the steps of: administering to a subject a recombinant baculovirus vector genetically engineered to contain and express nucleotides coding for one or more genes of interest; and administering to said subject either one or more proteins that modulate a Type 1 Interferon (EFN) response, or a viral vector which expresses nucleic acid sequences that encode one or more proteins that modulate a Type 1 Interferon (EFN) response. Ln one embodiment, the one or more genes of interest are nucleic acids which encode for one or more tuberculosis and malarial antigens, hi another embodiment, one or more proteins that modulate a Type 1 EFN response are administered in the second administering step. Ln one embodiment, a viral vector which expresses nucleic acid sequences that encode one or more proteins that modulate a Type 1 EFN response is administered in the second administering step. The viral vector may be a recombinant adenoviral vector. The invention also provides a method of eliciting an immune response in a mammal, the method comprising the steps of: administering to a subject a recombinant baculovirus vector which expresses nucleic acid sequences that encode one or more proteins that modulate a Type 1 Interferon (IFN) response; and administering to said subject either one or more antigen proteins, or a viral vector which expresses nucleic acid sequences that encode one or more antigen proteins.

The invention also provides a method of suppressing a Type 1 Interferon (IFN) response in a mammal in need thereof. The method comprises the step of administering to said mammal a composition comprising i. at least one baculovirus vector comprising, nucleic acid sequences that encode and express one or more proteins that modulate a Type 1 Interferon (IFN) response. The composition is administered in an amount sufficient to suppress said Type 1 Interferon (EFN) response in said mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure IA-C. A, amino acid sequence of Cl 2R protein of ectromelia virus; B, amino acid sequence of NSPl protein of rotavirus; amino acid sequence of NSl of influenza virus.

DETAILED DESCRIPTION Successful viral pathogens have evolved mechanisms that enable them to establish infection by blocking autocrine and paracrine responses of EFNs. Recent studies have indicated that the rotavirus nonstructural protein NSPl interacts with ERF3 and that this interaction results in the proteasome-mediated degradation of ERJF3, which in turn suppresses ENF-β production The NSl protein of influenza has been shown to have several effects on the type I EFN pathway. The activity of the carboxy-terminal domain of the NSl protein is able to inhibit the host mRNA's processing mechanisms. Second, it facilitates the preferential translation of viral mRNA by direct interaction with the cellular translation initiation factor eEF4GI. Third, by binding to dsRNA and interaction with putative cellular kinase (s), the NSl protein is able to prevent the activation of EFN-inducible dsRNA activated kinase (PKR), 2', 5'-oligoadenylate synthetase system, and cytokine transcription factors such as NF-KB or ERF 3 and c- Jun/ATF2. As a result, the NSl protein inhibits the expression of FNF-α and ENF-genes, delays the development of apoptosis in the infected cells and prevents the formation of the antiviral state in neighboring cells. The C12R protein binds to INF-α/β thereby modulating the immune response. Therefore, by co-administering such a protein, together with a gene of interest that is desired to be expressed in a mammalian host cell, transcription and translation of the gene of interest can occur without IFN suppression. In one embodiment, this is accomplished by constructing a baculovirus that harbors both a passenger gene or genes of interest (e.g. a gene encoding an antigen) and an immune response modulator (e.g. one or more proteins that interfere with host cell type I IFN responses) a superior viral vector from which significant amounts of transgene expression are observed can be generated. Such a vector may be used, for example, as a vaccine vector for overexpression of antigens, or for many other purposes, discussed in detail below.

In some embodiments of the invention, a single baculovirus encodes and delivers to a host cell both one or more IFN repressor genes and one or more transgenes of interest. In other embodiments, the IFN repressor proteins and the genes of interest are encoded by separate baculoviral vectors, which may be administered together. Alternatively, a baculoviral vector encoding an IFN repressor protein or a gene of interest (e.g. a tuberculosis or malaria antigen) may be administered with a different type of vector (e.g. an adenoviral or other vector) which encodes either the IFN repressor protein or the gene of interest, which, e.g. encodes a tuberculosis if malarial antigen. That is, the repressor protein may be encoded by another type of vector, and the IFN repressor may be encoded by a baculoviral vector and vice versa, hi addition, in some embodiments, one or more repressor proteins are administered to host cells as fully translated proteins, rather than being delivered as gene sequences that must be transcribed and translated. This would be part of a vaccine compositions or regimen where the repressor protein(s) are provided in concert with a baculovirus which expresses a gene of interest such a s a tuberculosis or malaria antigen, or both. hi all cases, the expression, from a vector, of one or more transgenes is increased when the IFN repressing proteins are present, compared to when the transgenes are present in a host cell in which the IFN repressing proteins are not present. For example, the increase in expression will maybe at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 905, 100% or even higher, i.e. the increase may be several fold higher. Those of skill in the art are familiar with the measurement of protein expression, e.g. by quantitating mRNA (directly or indirectly), by quantitating protein (e.g. using antibodies), or by measuring activity, etc. Examples of proteins capable of modulating the type I interferon (IFN) pathway which may be used in the practice of the invention (i.e. which may be cloned into a baculovirus) include, but are not limited to C12R protein of ectromelia virus, the NSPl protein of rotavirus, and NSl of influenza. The amino acid sequences of these three proteins are depicted in Figures IA, IB and 1C, respectively. Those of skill in the art will recognize that the precise sequences presented in Figure 1 need not be employed, but sequences with high (e.g. at least about 70, 80, 85, 90, 95 or higher) identity may be used, as may fragments (e.g. carboxyl and/or amino terminal truncated proteins) that still retain full or sufficient activity (e.g. at least 25, preferably 50, or even 60, 70, 80, 90% or higher activity ) of the full length protein. Those of skill in the are will recognize that, due to the redundant nature of the genetic code, many nucleic acid sequences may be utilized to encode such proteins, and the use of any or all such suitable sequences is contemplated. hi addition, other suitable IFN modulating proteins include but are not limited to:

Ebola VP35 (The Ebola virus VP35 protein functions as a type I IFN antagonist. CF Basler, X Wang, E Mϋhlberger, V Volchkov, Proceedings of the National Academy of Sciences, 2000. National Acad Sciences); Vaccinia Bl 8R (Waibler et al. Journal of Virology. 2009 Feb;83(4): 1563-71); rabies phosphoprotein P (Krzysztof Brzόzka, et al. Journal of Virology, March 2006, p. 2675-2683, Vol. 80, No. 6); lymphocytic choriomeningitis virus (LCMV) nucleoprotein (Martinez-Sobrido Luis et al. Journal of Virology 2006;80(18):9192-9); and Hepatitis C virus (HCV) protease NS3/4A (Xiao-Dong Li, et al. Proc Natl Acad Sci U S A. 2005 December 6; 102(49): 17717-17722). hi addition, Weber and Haller (Biochemie 89, 2007, 836-842) describe other examples of suitable proteins such as the E3L protein of poxviruses, the sigma3 protein of reoviruses, the USl 1 protein of herpes simplex virus, and murine cytomegalovirus proteins ml 42 and ml 43.

The construction of recombinant baculovirus is well documented; and in the practice of this invention the recombinant baculovirus which expresses one or more of the type I JJ⁷N response repressors and the gene or genes of interest (e.g. tuberculosis, malaria, human immunodeficiency virus, dengue fever antigens, etc.). For example, EP0340359, discloses a method of obtaining a recombinant baculovirus incorporating a foreign gene/s through the use of a transfer vector. A recombinant baculovirus incorporating the foreign gene(s) is derived from the transfer vector by co-transfecting insect cells susceptible to baculovirus infection with wild type baculovirus and a transfer vector, m addition, United States patent No. 6,126,944 to Pellett et al., the complete contents of which is herein incorporated by reference, describes the construction of a baculovirus transfer vector for expression of foreign genes which are juxtaposed with the baculovirus polyhedrin gene at the translation initiation site, without the addition of further nucleotides to the initiation site. Those of skill in the art will recognize that many types of baculo virus may be employed in the practice of the invention and all such are encompassed by the present invention.

Generally, the genes that are placed into the baculovirus via genetic engineering are under control of an expression sequence such as a promoter, internal ribosomal entry site

(IRES), various enhancer sequences, etc.. Such sequences and promoters may be naturally within the baculovirus (i.e. native to the baculovirus), and the sequences of interest placed at a location such that their expression is driven by the wildtype baculoviral sequences. Alternatively, promoters from organisms other than baculovirus may be cloned into the baculovirus, together with the gene(s) of interest. Exemplary promoters that may so utilized in the practice of the invention include but are not limited to various vira, prokaryotic or eukaryotic promoters, e.g. cytomegalovirus (CMV) promoters, cauliflower mosiac virus promoter, influenza and HIV viral promoters, heat shock promoters (e.g. hspόO promoter) and other promoters from M. tuberculosis, etc. Of these, both constitutive and inducible promoters may be utilized.

The ease of construction, and capacity to accept large foreign DNA- fragments (>20 kbp), allows the development of baculoviruses having enlarged or targeted cell tropism along with more stable, temporal and cell type-specific control of transgene expression. In one embodiment, the baculoviral expression vectors of the invention are genetically engineered to encode and deliver both the IFN inhibiting factors and one or more other genes of interest i.e. passenger genes or transgenes. The passenger genes are typically heterologous transgenes ("foreign" genes) that originate from another organism, such another virus, a bacteria or other pathogen, and may be from any organism. "Passenger gene" is intended to refer not only to entire "genes" but to any sequence that encodes a peptide, polypeptide, protein, or nucleic acid of interest, i.e. an entire "gene" per se may not be included, but rather the portion of a gene that encodes a polypeptide or peptide of interest e.g. an antigenic peptide. Further, various other constructions may be encoded by passenger genes, e.g. chimeric proteins, or various mutant (either naturally occurring or genetically engineered) forms of an amino acid sequence. In addition, totally artificial amino acid sequences that do not appear in nature may also be encoded. The baculoviral expression vector is genetically engineered to contain one or more of such "passenger genes", and may also encode multiple copies of individual passenger genes. The recombinant baculoviral expression vector functions as a vector to carry the passenger gene(s) and/or genes encoding the suppression factors into host cells that are invaded by the baculovirus, where the gene products are expressed, i.e. the gene sequences are expressible and transcription and/or translation of the gene products occurs within the host cell that is invaded by the bacterium. The sequences encoding the passenger genes and the genes encoding the suppression factors are operatively (operably) linked to expression control sequences, particularly expression control sequences that allow expression within the eukaryotic host cell. In some embodiments, if multiple passenger genes are encoded, each will have its own expression control system. In other embodiments, one expression control system will serve to drive expression of more than one passenger gene, e.g. as a single transcript with a plurality of gene sequences. Similarly, if multiple suppression factors are encoded in a single baculovirus, the transcription of each may be separately controlled, or multiple sequences may be under the control of one expression control sequence.

In particular, such passenger genes may encode one or more peptides or proteins that are antigens, and to which it is desired to elicit an immune response. Those of skill in the art will recognize that a wide variety of such antigens exists, including but not limited to those associated with infectious agents such as various viruses, bacteria, and fungi, etc. The viral pathogens, from which the viral antigens are derived, include, but are not limited to, Orthomyxoviruses, such as influenza virus (Taxonomy BD: 59771; Retroviruses, such as RSV, HTLV-I (Taxonomy ID: 39015), and HTLV-II (Taxonomy ID: 11909),

Papillomaviridae such as HPV (Taxonomy ID: 337043), Herpesviruses such as EBV Taxonomy ID: 10295); CMV (Taxonomy ID: 10358) or herpes simplex virus (ATCC #: VR-1487); Lentiviruses, such as HIV-I (Taxonomy ID: 12721) and HIV-2 Taxonomy ID: 11709); Rhabdoviruses, such as rabies; Picornoviruses, such as Poliovirus (Taxonomy ID: 12080); Poxviruses, such as vaccinia (Taxonomy ID: 10245); Rotavirus (Taxonomy ID:

10912); and Parvoviruses, such as adeno-associated virus 1 (Taxonomy DD: 85106).

Examples of viral antigens can be found in the group including but not limited to the human immunodeficiency virus antigens Nef (National Institute of Allergy and Infectious Disease HIV Repository Cat. # 183; Genbank accession # AF238278), Gag, Env (National Institute of Allergy and Infectious Disease HIV Repository Cat. # 2433; Genbank accession

# U39362), Tat (National Institute of Allergy and Infectious Disease HIV Repository Cat. # 827; Genbank accession # M13137), mutant derivatives of Tat, such as Tat-31-45 (Agwale et al., Proc. Natl. Acad. Sci. USA 99:10037; 2002), Rev (National Institute of Allergy and Infectious Disease HIV Repository Cat. # 2088; Genbank accession # L14572), and Pol (National Institute of Allergy and Infectious Disease HIV Repository Cat. # 238; Genbank accession # AJ237568) and T and B cell epitopes of gpl20 (Hanke and McMichael, AIDS Immunol Lett, 66:177; 1999); (Hanke, et al, Vaccine, 17:589; 1999); (Palker et al, J.

Immunol., 142:3612 3619; 1989) chimeric derivatives of HIV-I Env and gpl20, such as but not restricted to fusion between gρl20 and CD4 (Fouts et al., J. Virol. 2000, 74:11427-11436; 2000); truncated or modified derivatives of HIV-I env, such as but not restricted to gpl40 (Stamatos et al, J Virol, 72:9656-9667; 1998) or HIV-I Env and/or gpl40 or derivatives thereof (Binley, et al, J Virol, 76:2606-2616; 2002); (Sanders, et al, J

Virol, 74:5091-5100 (2000); (Binley, et al J Virol, 74:627-643; 2000), the hepatitis B surface antigen (Genbank accession # AF043578); (Wu et al, Proc. Natl. Acad. Sci., USA, 86:4726 4730; 1989); rotavirus antigens, such as VP4 (Genbank accession # AJ293721); (Mackow et al, Proc. Natl. Acad. Sci., USA, 87:518 522; 1990) and VP7 (GenBank accession # AY003871); (Green et al, J. Virol, 62:1819 1823; 1988), influenza virus antigens such as hemagglutinin or (GenBank accession # AJ404627); (Pertmer and Robinson, Virology, 257:406; 1999); nucleoprotein (GenBank accession # AJ289872); (Lin et al, Proc. Natl. Acad. Sci., 97: 9654-9658; 2000) herpes simplex virus antigens such as thymidine kinase (Genbank accession # AB047378; (Whitley et al, In: New Generation Vaccines, pages 825-854).

The bacterial pathogens, from which the bacterial antigens are derived, include but are not limited to: Mycobacterium spp., Helicobacter pylori, Salmonella spp., Shigella spp., E. coli, Rickettsia spp., Listeria spp., Legionella pneumoniae, Pseudomonas spp., Vibrio spp., Bacillus anthracis and Borellia burgdorferi, hi particular, Mycobacterium tuberculosis antigens of interest include but are not limited to Rv0079, RvO 101, RvO 125, RvO 170,

RvO198c, RvO211, RvO227c, RvO243, RvO251c, RvO282, RvO283, RvO284, RvO285, RvO286, RvO287, Rv0288, RvO289, Rv0290, RvO29, Rv0350, Rv0351, Rv0383c, RvO384c, Rv0450c, RvO467, RvO468, Rv0503c, RvO569, RvO572c, RvO574c, Rv0588, RvO628c, Rv0685, RvO754, RvO798c, RvO824c, RvO847, RvO867c, Rv0885, RvI 006, RvI 009, Rvl057, RvlO94, RvI 124, RvI 130, RvI 131, RvI 169c, RvI 174c, RvI 182, RvI 186c,

RvI 187, RvI 188, RvI 196, Rvl221, Rvl347c, Rvl348, Rvl349, Rvl411c, Rvl436, Rvl461, Rvl462, Rvl464, Rvl465, Rvl466, Rvl477, Rvl478, Rvl594, Rvl636, Rvl733c, Rvl734c, Rvl735c, Rvl736c, Rvl737c, Rvl738, Rvl793, Rvl812c, Rvl813c, Rvl876, Rvl884c, Rvl886c, Rvl908c, Rvl926c, Rvl980c, Rvl986, Rvl996, Rvl997, Rvl998c, Rv2004c, Rv2005c, Rv2006, Rv2007c, Rv2008c, Rv201 Ic, Rv2028c, Rv2029c, Rv2030c, Rv2031c, Rv2032, Rv2110c, Rv2123, Rv2140c, Rv2182c, Rv2224c, Rv2244, Rv2245, Rv2246, Rv2251, Rv2377c, Rv2378c, Rv2380c, Rv2381c, Rv2382c, Rv2383c, Rv2386c,

Rv2389c, Rv2428, Rv2429, Rv2430c, Rv2450c, Rv2457c, Rv2466c, Rv2510c, Rv2515c, Rv2516c, Rv2557, Rv2590, Rv2620c, Rv2621c, Rv2622, Rv2623, Rv2625c, Rv2626c, Rv2627c, Rv2628, Rv2629, Rv2657c, Rv2659c, Rv2660, Rv2710, Rv2744c, Rv2780, Rv2833c, Rv2856, Rv2869c, Rv2875, Rv2930, Rv2999, Rv3126c, Rv3127, Rv3129, Rv3130c, Rv3131, Rv3132c, Rv3133c, Rv3134c, Rv3139, Rv3140, Rv3173c, Rv3229c,

Rv3250c, Rv3251c, Rv3283, Rv3290c, Rv3347c, Rv3372, Rv3406, Rv3516, Rv3546, Rv3570c, Rv3593, Rv3597c, Rv3616c, Rv3619c, Rv3660c, Rv3763, Rv3804c, Rv3812, Rv3833, Rv3839, Rv3840, Rv3841, Rv3871, Rv3873, Rv3874, Rv3875, Rv3876, Rv3878, and Rv3879c.(See also United States patent application 11/945, 680 to Shafferraan et al, publication # 20090136534, the complete contents of which are herein incorporated by reference.)

Examples of protective antigens of bacterial pathogens include the somatic antigens of enterotoxigenic E. coli, such as the CFA/I fimbrial antigen (Yamamoto et al, Infect. Immun., 50:925 928; 1985) and the nontoxic B subunit of the heat labile toxin ( et al, Infect. Immun., 40:888-893; 1983); pertactin of Bordetella pertussis (Roberts et al., Vacc,

10:43-48; 1992), adenylate cyclase hemolysin of B. pertussis (Guiso et al., Micro. Path., 11:423-431; 1991), fragment C of tetanus toxin of Clostridium tetani (Fairweather et al., Infect. Immun., 58:1323 1326; 1990), OspA oϊBorellia burgdorferi (Sikand et al, Pediatrics, 108:123-128; 2001); (Wallich et al, Infect Immun, 69:2130-2136; 2001), protective paracrystalline-surface-layer proteins of Rickettsia prowazekii and Rickettsia typhi

(Carl et al, Proc Natl Acad Sci U S A, 87:8237-8241; 1990), the listeriolysin (also known as "LIo" and "HIy") and/or the superoxide dismutase (also know as "SOD" and "p60") of Listeria monocytogenes (Hess, J., et al, Infect. Immun. 65:1286-92; 1997); Hess, J., et al, Proc. Natl. Acad. Sci. 93:1458-1463; 1996); (Bouwer et al, J. Exp. Med. 175:1467-71; 1992), the urease of Helicobacter pylori (Gomez-Duarte et al, Vaccine 16, 460-71; 1998);

(Corthesy-Theulaz, et al, Infection & Immunity 66, 581-6; 1998), and the Bacillus anthracis protective antigen and lethal factor receptor-binding domain (Price, et al, Infect. Immun. 69, 4509-4515; 2001).

The parasitic pathogens, from which the parasitic antigens are derived, include but are not limited to: Plasmodium spp., such as Plasmodium falciparum (ATCC#: 30145); Trypanosome spp., such as Trypanosoma cruzi (ATCC#: 50797); Giardia spp., such as Giardia intestinalis (ATCC#: 30888D); Boophilus spp., Babesia spp., such as Babesia microti (ATCC#: 30221); Entamoeba spp., such as Entamoeba histolytica (ATCC#: 30015); Eimeria spp., such as Eimeria maxima (ATCC# 40357); Leishmania spp. (Taxonomy ID: 38568); Schistosome spp., Brugia spp., Fascida spp., Dirofilaria spp., Wuchereria spp., and Onchocerca spp. (See also International patent application PCT/US09/30734 to Shaffermann et al., the complete contents of which is herein incorporated by reference.)

Examples of protective antigens of parasitic pathogens include the circumsporozoite antigens of Plasmodium spp. (Sadoff et al., Science, 240:336 337; 1988), such as the circumsporozoite antigen of P. berghei or the circumsporozoite antigen of P. falciparum; the merozoite surface antigen of Plasmodium spp. (Spetzler et al., hit. J. Pept. Prot. Res., 43:351-358; 1994); the galactose specific lectin of Entamoeba histolytica (Mann et al., Proc.

Natl. Acad. Sci., USA, 88:3248-3252; 1991), gp63 of Leishmania spp. (Russell et al, J. Immunol., 140:1274 1278; 1988); (Xu and Liew, Immunol., 84: 173-176; 1995), gp46 of Leishmania major (Handman et al., Vaccine, 18:3011-3017; 2000) paramyosin of Brugia malayi (Li et al., MoL Biochem. Parasitol., 49:315-323; 1991), the triose-phosphate isomerase of Schistosoma mansoni (Shoemaker et al., Proc. Natl. Acad. Sci., USA, 89:1842

1846; 1992); the secreted globin-like protein of Trichostrongylus colubriformis (Frenkel et al., MoI. Biochem. Parasitol., 50:27-36; 1992); the glutathione-S-transferase's of Frasciola hepatica (Hillyer et ah, Exp. Parasitol., 75:176-186; 1992), Schistosoma bovis and S. japonicum (Bashir et al., Trop. Geog. Med., 46:255-258; 1994); and KLH of Schistosoma bovis and S. japonicum (Bashir et al., supra, 1994).

Alternatively, it may be desired to elicit an immune response to antigens that are not associated with infectious agents, for example, antigens associated with cancer cells, Alzheimer's disease, Type 1 diabetes, heart disease, Crohn's disease, multiple sclerosis, etc. hi addition, the passenger genes that are carried by the baculovirus vector need not encode antigens, but may encode any peptide or protein of interest. For example, the methods of the invention can be used for the delivery of passenger molecules for correction of hereditary disorders, e.g. the vectors may be used for gene therapy. Such genes would include, for example, replacement of defective genes such as the cystic fibrosis transmembrane conductance regulator (CFTR) gene for cystic fibrosis; or the introduction of new genes such as the integrase antisense gene for the treatment of HIV; or genes to enhance Type I T cell responses such as interleukin-27 (IL-27); or genes to modulate the expression of certain receptors, metabolites or hormones such as cholesterol and cholesterol receptors or insulin and insulin receptors; or genes encoding products that can kill cancer cells such as tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL); or a naturally occurring protein osteoprotegerin (OPG) that inhibits bone resorption; or to efficiently express complete-length humanized antibodies, for example, humanized monoclonal antibody that acts on the HER2/neu (erbB2) receptor on cancer cells.

In addition, the passenger genes may encode inhibitory RNAs such as "small inhibitory" siRNAs. As is known in the art, such RNAs are complementary to an mRNA of interest and bind to and prevent translation of the mRNA, e.g. as a means of preventing the expression of a gene product. Similar methods can be used for delivery of passenger molecules to down regulate the immune system in order to prevent or control autoimmune diseases or other diseases of immune system. Examples include the prevention or treatment of diabetes mellitus, multiple sclerosis, lupus erythematosis and Crohn's disease and inflammatory joint and skin diseases. Other examples include fine tuning of immune responses that hamper specific immune responses such as down regulation of immune responses that divert the therapeutic immune responses to cancer and other diseases. For example, down regulation of Th2 responses when ThI responses are appropriate for prevention and treatment of cancer, Leishmaniasis, tuberculosis, and HIV. This can be achieved by means of the present technology through manipulation of the immunosuppressive nature of the immune system in combination with the ability to express the suitable cytokine milieu for stimulation of the proper immune response and inhibition of improper immune responses. hi a preferred embodiment, the present invention relates to a method for the introduction of IFN resistance genes into host cells, either in vitro or in vivo. Such a method would comprise introduction of the desired IFN resistance genes, along with sequences encoding a gene or nucleic acid sequence of interest, into a baculovirus based delivery system such that the IFN resistance proteins and nucleic acid sequences of interest are expressed upon administering the baculovirus to a host. Further, all genetic sequences may be either constitutively expressed or induced by environmental cues.

Gene sequences for cloning may be obtained by various known molecular biology techniques, e.g. using restriction enzymes, polymerase chain reactions, ligases, etc. Alternatively, gene sequences can be made synthetically using, for example, an Applied Biosystems ABF^M 3900 High-Throughput DNA Synthesizer (Foster City, CA 94404

U.S.A.) using procedures provided by the manufacturer. To synthesize large sequences i.e. greater than about 200 bp, a series of segments of the full-length sequence are generated by PCR and ligated together to form the full-length sequence using procedures well know in the art. However, smaller sequences, i.e. those smaller than about 200 bp, can be made synthetically in a single round.

The present invention also provides preparations for administering the recombinant baculo virus expression vectors of the invention. In particular, vaccine preparations and preparations for use in eliciting immune responses are provided. The preparations include at least one genetically engineered baculovirus as described herein, and a pharmacologically suitable carrier. The preparation of such compositions (e.g. for use as vaccines) is well known to those of skill in the art. Typically, such compositions are prepared either as liquid solutions or suspensions, however, solid forms such as tablets, pills, powders and the like are also contemplated. Solid forms suitable for solution in, or suspension in, liquids prior to administration may also be prepared. The preparation may also be emulsified. The active ingredients may be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredients. Suitable excipients are, for example, water, saline, dextrose, raffinose, glycerol, ethanol and the like, or combinations thereof, hi addition, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like. The vaccine preparations of the present invention may further comprise an adjuvant, suitable examples of which include but are not limited to Seppic, Quil A, Alhydrogel, etc.

If it is desired to administer an oral form of the composition, various thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders and the like may be added. The composition of the present invention may contain any such additional ingredients so as to provide the composition in a form suitable for administration. The final amount of recombinant baculovirus in the formulations may vary. However, in general, the amount in the formulations will be from about 1-99 percent. Further, the preparations of the present invention may contain a single type of recombinant baculo virus or more than one type of recombinant baculovirus.

In the case of vaccine preparations, the present invention also provides methods of eliciting an immune response to antigens encoded by the baculovirus vector, and methods of vaccinating a mammal against diseases or conditions associated with such antigens. By eliciting an immune response, we mean that administration of the vaccine preparation of the present invention causes the synthesis of specific antibodies (at a titer in the range of 1 to 1 x 10⁶, preferably 1 x 10³, more preferable in the range of about 1 x 10³ to about 1 x 10⁶, and most preferably greater than 1 x 10⁶) and/or cellular proliferation, as measured, e.g. by ³H thymidine incorporation. The methods involve administering a composition comprising a baculovirus strain of the invention in a pharmacologically acceptable carrier to a mammal. The vaccine preparations of the present invention may be administered by any of the many suitable means which are well known to those of skill in the art, including but not limited to by injection, orally, intranasally, by ingestion of a food product containing the recombinant baculovirus, etc. hi preferred embodiments, the mode of administration is oral, subcutaneous, intradermal or intramuscular. The targeted host is generally a mammal, and may be a human, although this need not always be the case, as veterinary applications are also contemplated.

EXAMPLES

EXAMPLE 1. Construction of Baculoviral Vectors which Express IFN Suppressing Proteins

Recombinant baculovirus encoding a fusion protein comprising M. tuberculosis antigens Ag85A, Ag85B, and RV3406 from is constructed. Baculoviruses have been shown to infect mammalian cells; therefore Chinese hamster overy (CHO), HeLa, and baby hamster kidney (BHK) cells grown in tissue culture flasks are transfected with pcDNA3.1 encoding NS 1 of influenza- A or NSP 1 of rotavirus under the control of the CMV promoter, or pcDNA3.1 alone. Zeomycin resistant stable transformants are expanded and seeded into 6-well tissue culture flasks in Dulbecco's Modified Eagle Medium (DMEM) and incubated to > 60% confluence. Test wells from each group are counted and cells in fresh, serum-free media are infected with recombinant Bacultovirus (rBaculovirus) at multiplicities of infection (MOIs) of 10, 100, 1000, and 5000 for a period of 1-2 hours. Culture media can optionally be supplemented with 10 mM sodium butyrate to maximize transgene expression. After removal of the virus, fresh medium is added and cultures are incubated at 37⁰C for 48 hours. Cultures are examined for antigen expression by immunoblotting. For Western blot analysis, cell extracts are resolved in denaturing polyacrylamide gels, and proteins are transferred to nitrocellulose membranes and immunoblotted by using standard methods and Ag85-specific antisera.

Results show that NSl and the NSPl expressing cells produce easily detectable transgene fusion proteins in excess of that observed in the parental (pcDNA3.1 only) CHO, HeLa, or BHK cells. EXAMPLE 2. Construction and Testing of Baculoviral Vectors which Express IFN Suppressing Proteins and Transgenes

The next step is the encoding of NSl and a transgene encoding e.g. the hemagglutinin (HA) of avian influenza H5N1 in a bicistronic expression cassette in a recombinant baculovirus. Expression of the transgene is tested in cells infected with an analogous recombinant baculovirus construct expressing the same HA transgene but not the NSl protein. This study validates the approach of using a recombinant baculovirus encoding a suppressor of the type I interferon. Immunogenicity studies in mice also demonstrate the anticipated gain in type and magnitude of the immune response elicited by the recombinant baculo viruses expressing both the NSl suppressor of the type I interferon response and the hemagglutinin immunogen of interest. EXAMPLE 3. Vaccine Development

These observations have clear and significant implications for the use and development of recombinant baculovirus vaccines. Increased transgene expression upon administration of a baculoviral vector to a mammalian host as described herein leads to improved cellular and humoral immune responses to encoded antigens. This invention thus has a broad range of applications for recombinant baculovirus vaccines for the prevention and treatment of a wide variety of diseases.

While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.

Claims

CLAIMSWe claim:

1. A baculovirus vector comprising, nucleic acid sequences that encode and express one or more proteins that modulate a Type 1 Interferon (IFN) response; and nucleic acid sequences that encode and express one or more transgenes of interest.

2. The baculovirus vector of claim 1, wherein said one or more proteins that modulate a Type 1 IFN response are selected from the group consisting non-structural protein 1 (NSP- 1) from rotavirus, NSl protein from influenza virus, C12R protein from ectromlia virus.

3. The baculovirus vector of claim 1, wherein said one or more transgenes of interest include one or more transgenes that encode antigens.

4. The baculovirus vector of claim 3, wherein said antigens include at least one tuberculosis or malaria antigen.

5. A vaccine composition for administration to a mammal, comprising i. at least one baculovirus vector comprising, nucleic acid sequences that encode and express one or more proteins that modulate a Type 1 Interferon (IFN) response; and nucleic acid sequences that encode and express one or more transgenes of interest; and ii. a physiological acceptable carrier.

6. The vaccine composition of claim 5, wherein said one or more proteins that modulate a Type 1 IFN response are selected from the group consisting non-structural protein 1 (NSP- 1) from rotavirus, NSl protein from influenza virus, C12R protein from ectromlia virus.

7. The vaccine composition of claim 5, wherein said one or more transgenes of interest include one or more transgenes that encode antigens.

8. The vaccine composition of claim 7, wherein said antigens include at least one tuberculosis or malaria antigen.

9. The vaccine composition of claim 5, wherein said mammal is a human.

10. A method of eliciting an immune response to one or more antigens in a mammal, comprising the step of administering to said mammal a composition comprising i. at least one baculo virus vector comprising, nucleic acid sequences that encode and express one or more proteins that modulate a Type 1 Interferon (IFN) response; and nucleic acid sequences that encode and express said one or more antigens; and

11. a physiological acceptable carrier; in an amount sufficient to elicit an immune response to said one or more antigens.

11. The method of claim 10, wherein said one or more proteins that modulate a Type 1 IFN response are selected from the group consisting non-structural protein 1 (NSP-I) from rotavirus, NSl protein from influenza virus, C12R protein from ectromlia virus.

12. The method of claim 10, wherein said one or more antigens include at least one tuberculosis or malaria antigen.

13. The method of claim 10, wherein said mammal is a human.

14. A method of eliciting an immune response in a mammal, comprising the steps of: administering to a subject a recombinant baculo virus vector genetically engineered to contain and express nucleotides coding for one or more genes of interest; and administering to said subject either one or more proteins that modulate a Type 1 Interferon (BFN) response, or a viral vector which expresses nucleic acid sequences that encode one or more proteins that modulate a Type 1 Interferon (IFN) response.

15. The method of claim 14 wherein said one or more genes of interest are nucleic acids which encode for one or more tuberculosis and malarial antigens.

16. The method of claim 14 wherein said one or more proteins that modulate a Type 1 IFN response are administered in the second administering step.

17. The method of claim 14 wherein a viral vector which expresses nucleic acid sequences that encode one or more proteins that modulate a Type 1 IFN response is administered in the second administering step.

18. The method of claim 17 wherein the viral vector is a recombinant adenoviral vector.

19. A method of eliciting an immune response in a mammal, comprising the steps of: administering to a subject a recombinant baculovirus vector which expresses nucleic acid sequences that encode one or more proteins that modulate a Type 1 Interferon (FFN) response; and administering to said subject either one or more antigen proteins, or a viral vector which expresses nucleic acid sequences that encode one or more antigen proteins.

20. A method of suppressing a Type 1 Interferon (FFN) response in a mammal in need thereof, comprising the step of administering to said mammal a composition comprising i. at least one baculovirus vector comprising, nucleic acid sequences that encode and express one or more proteins that modulate a Type 1 interferon (FFN) response; in an amount sufficient to suppress said Type 1 Interferon (IFN) response in said mammal.