Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
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  • A61P31/18—Antivirals for RNA viruses for HIV
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/525—Virus
  • A61K2039/5256—Virus expressing foreign proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
  • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/20011—Rhabdoviridae
  • C12N2760/20041—Use of virus, viral particle or viral elements as a vector
  • C12N2760/20043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• the present invention relates to the fields of molecular biology and virology, and to a method of treating an HTV-l infection and, more particularly, to the induction of both humoral and cellular immunity against HIV-1.
• HTV-1-seropositive New anti-retro viral strategies against human HTV-l result in a dramatic decrease in mortality among infected humans in developed countries, but the development of a successful vaccine to prevent infection is still the major goal to halt the HIV-1 pandemic.
• a human being is infected with HTV-l every 10 seconds on average, and in the heavily affected countries in Africa, such as Zambia and Kenya, nearly 40% of young adults are HTV-1-seropositive. (1).
• HJV vaccine strategies are being investigated, including recombinant proteins (Goebel, F.D., et al., European Multinational IMMUNO AIDS Vaccine Study Group Aids, 5:643-50, 1999; Quinnan, Q.Y., Jr., et al., AZDS Research & Human Retroviruses, 15:561-70, 1999; VanCott, T.C., et al., J.
• the ability of recombinant non-segmented negative-stranded RNA viruses expressing an immunodeficiency virus gene(s) as an immunodeficiency virus vaccine is disclosed.
• an immunodeficiency virus vaccine e.g., HTV-l vaccine
• the HIV-1 envelope protein is stably and functionally expressed and induces a strong humoral response directed against the HIV-1 envelope protein after a single boost with recombinant HIV-1 protein boost (gpl20) in mice.
• high neutralization titers against HIV-1 are detected m the mouse sera.
• the present invention fulfills this long sought need and further relates to recombinant RV vaccines expressing HTV-l envelope proteins to induce HTV-l -specific CTLs. Specifically, a single inoculation of the HTV-l virus vaccines of the present invention induce a solid and long-lasting memory CTL response specific for HTV-l proteins. These recombinant viruses are non-pathogenic for a wide range of animal species when administered orally or intramuscularly.
• the coding region of the HTV-l gpl60 (strains NL4-3 and 89.6) is cloned between the RV glycoprotein (G) and polymerase (L) proteins under the control of a RV transcription Stop/Start signal, the resulting recombinant RVs expressed HIV-1 gpl60 along with the other RV proteins.
• boost vaccine vector is "boost virus”.
• boost virus is "boost vaccine vector”.
• the present invention is directed to recombinant non-segmented negative-stranded
• RNA virus vectors expressing an immunodeficiency virus genes as a live-viral vaccine e.g., HTV-l vaccine
• the invention relates to recombinant Rhabdoviruses which express gene products of a human immunodeficiency virus and to immunogenic compositions which induce an immunological response against immunodeficiency virus infections when administered to a host.
• live-viral vaccines are non-pathogenic for a wide range of animal species when administrated orally or intramuscularly and induce protective immune responses such as neutralizing antibody response and long lasting cellular (such as cytotoxic T lymphocyte (CTL)) responses against the immunodeficiency viruses.
• CTL cytotoxic T lymphocyte
• the invention is a recombinant non-segmented negative-stranded
• RNA virus vector having: (a) a modified negative-stranded RNA virus genome that is modified to have one or more new restriction sites, or not to have one or more genes otherwise present in the genome; (b) a new transcription unit that is inserted into the modified negative-stranded RNA virus genome to express heterologous nucleic acid sequences; and (c) a heterologous viral nucleic acid sequence that is inserted into the new transcription unit, where the recombinant non-segmented negative-stranded RNA virus vector is replication competent, and the heterologous viral nucleic acid sequence encodes an antigenic polypeptide.
• the recombinant non-segmented negative-stranded RNA virus vector that is used as a live-viral vaccine is a recombinant Rhabdovirus vector.
• This vector includes (a) a modified Rhabdovirus genome; (b) a new transcription unit inserted into the Rhabdovirus genome to express heterologous nucleic acid sequences; and (c) a heterologous viral nucleic acid sequence that is inserted into the new transcription unit, where the recombinant Rhabdovirus vector is replication competent, and the heterologous viral nucleic acid sequence encodes an antigenic polypeptide.
• the modified Rhabdovirus genome is, for example, modified rabies virus genome or a modified vesicular stomatitis virus genome.
• the modifications in the Rhabdovirus genome include creation of new restriction sites and/or deletion of one or more genes such as the native G (glycoprotein) gene of the Rhabdovirus, ⁇ gene of rabies virus, etc.
• the modified Rhabdovirus genome has a further modification to have a glycoprotein from another class of virus in place of the native glycoprotein.
• the glycoprotein from another class of virus is vesicular stomatitis virus glycoprotein.
• the modified rabies virus genome has a third modification to have contiguity of structural genes different from that in the rhabodvirus genome after the second modification.
• heterologous viral nucleic acid refers to the viral nucleic acid that encodes the antigenic polypeptide that induces immune response.
• a full- length HTV envelope protein, HIV gpl60, HTV gag, HTV g ⁇ l20, and full-length STV envelope protein are some of the antigenic polypeptides that are expressed in the recombinant viral vectors of the present invention.
• heterologous viral nucleic acid as used herein does not include the native gene sequences of the one or more classes of Rhabdoviruses in a recombinant Rhabdovirus such as, for example, VSV G gene in the recombinant RV.
• the sequence of the cytoplasmic domain of Rhabdovirus G gene is fused to other sequences before cloning into the modified Rhabdovirus genome.
• One such example is a chimeric VSV/RV glycoprotein where the fusion protein has VSV ectodomain and transmembrane domain, and RV cytoplasmic domain.
• Another such example is a chimeric HTV-l/RV glycoprotein where the fusion protein has HIV-1 gpl60 ectodomain and transmembrane domain, and RV cytoplasmic domain.
• the heterologous viral nucleic acid is fused to the sequence of the cytoplasmic domain of the G gene of the modified Rhabdovirus genome to produce a chimeric protein such that the resulting chimeric protein has a fusion between the transmembrane domain of the heterologous protein and cytoplasmic domain of the glycoprotein.
• the glycoprotein gene of the recombinant Rhabdovirus is deleted and the heterologous viral nucleic acid is fused to the sequence of the cytoplasmic domain of the G gene of the modified Rhabdovirus genome to produce a chimeric protein which functionally substitutes for the recombinant Rhabdoviruses glycoprotein gene.
• a recombinant Rhabdovirus that expresses a functional HIV envelope protein is provided.
• the recombinant Rhabdovirus is replication- competent.
• the Rhabdovirus can be a recombinant rabies virus or a recombinant vesicular stomatitis virus.
• the HIV envelope protein expressed from the recombinant Rhabdovirus is from any HIV-1 isolate.
• a recombinant ⁇ gene deficient Rhabdovirus having a heterologous nucleic acid segment encoding an immunodeficiency virus envelope protein or a subunit thereof is provided.
• the recombinant ⁇ gene deficient Rhabdovirus is a rabies virus and the immunodeficiency virus envelope protein, or a subunit thereof, is from a human immunodeficiency virus or from a simian immunodeficiency virus.
• the subunit or a fragment of the immunodeficiency envelope protein includes fragments having only a part of the contiguous amino acids of the envelope protein.
• subunits or fragments include, for example, HIV gpl20, HTV gp41, HIV gp40, the envelop proteins expressed by HTV NL4 - 3 and HIV 89 . 6 , and the subunits of other immunodeficiency viruses.
• a method of inducing an immunological response in a mammal includes the steps of: (a) delivering to a tissue of the mammal a recombinant Rhabdovirus vector that expresses a functional immunodeficiency virus envelope protein, or a subunit thereof, effective to induce an immunological response to the envelope protein; (b) expressing the envelope protein, or the subunit thereof, in vivo; (c) boosting the animal by delivering an effective dose of an isolated immunodeficiency virus envelope protein, or a subunit thereof, in an adjuvant or by delivering an effective dose of a boost vaccine vector; and (d) inducing a neutralizing antibody response and/or long lasting cellular immune response thereto to protect the mammal from an immunodeficiency virus.
• the recombinant Rhabdovirus has a rabies virus genome.
• the rabies virus genome In the method where the rabies virus genome is used, it is deficient in ⁇ gene.
• rabies virus genome is also deficient in a rabies virus glycoprotein gene or rabies virus genome has glycoprotein gene from another class of Rhabdovirus in place of the rabies virus glycoprotein.
• Boosting the animal can be done by delivering an effective dose of a boost vaccine vector instead of the isolated immunodeficiency virus envelope protein.
• an immunogenic composition having any of the above mentioned recombinant Rabdo viruses along with an adjuvant is provided.
• a method of inducing an immunological response in a mammal which includes the steps of: (a) delivering to a tissue of the mammal a non-segmented negative-stranded RNA virus that expresses a functional immunodeficiency virus envelope protein, or a subunit thereof, effective to induce an immunological response to the envelope protein; (b) expressing the envelope protein, or the subunit thereof, in vivo; (c) boosting the animal by delivering an effective dose of an isolated immunodeficiency virus envelope protein, or a subunit thereof, in an adjuvant or by delivering an effective dose of a boost vaccine vector; and (d) inducing a neutralizing antibody response and/or long lasting cellular immune response thereto to protect the mammal from an immunodeficiency virus.
• the method where the non-segmented negative-stranded RNA virus is used includes a Rabies virus or a Vesicular Stomatitis virus.
• a non-segmented negative-stranded RNA virus that expresses a functional immunodeficiency virus envelope protein, or subunit thereof is administered to the mammal.
• This RNA virus will express the functional immunodeficiency virus envelope protein, or subunit thereof.
• An effective dose of an isolated immunodeficiency virus envelope protein, or subunit thereof, in an adjuvant or an effective dose of a boost vaccine vector is delivered to the mammal, thereby inducing a neutralizing antibody response and/or long lasting cellular immune response to the functional immunodeficiency virus envelope protein, or subunit thereof.
• the immunodeficiency virus is any HTV-l virus.
• the non-segmented negative-stranded RNA virus is a Rhabdovirus.
• the long-lasting cellular response is a cross-reactive CTL response wherein the cross-reactive CTLs are directed against envelope proteins, or subunits thereof, from different immunodeficiency virus strains. It is another object of the invention to present a method of protecting a mammal from an immunodeficiency virus infection.
• a non-segmented negative-stranded RNA virus that expresses a functional immunodeficiency virus envelope protein, or subunit thereof is administered to the mammal.
• This RNA virus will express the functional immunodeficiency virus envelope protein, or subunit thereof, thereby thereby inducing a neutralizing antibody response and/or long lasting cellular immune response to the functional immunodeficiency virus envelope protein, or subunit thereof.
• the immunodeficiency virus is any HIV-1 virus.
• the non-segmented negative-stranded RNA virus is a Rhabdovirus.
• the long-lasting CTL response is a cross-reactive CTL response wherein the cross-reactive CTLs are directed against envelope proteins, or subunits thereof, from different immunodeficiency virus strains.
• Figure 1 Schematically shows a method for the construction of recombinant RV genomes.
• Figure 2 A graph showing One-step growth curves of BSR cells that were infected with the recombinant RVs (SBN, SBN-89.6, and SBN-NL4-3)
• FIG. 1 Western blot analysis of recombinant rabies viruses (RVs) expressing HTV- 1 gpl60.
• Figure 4 A composite photograph showing Sup-Tl cells after these cells were infected (using a MOI of 1) with SBN, SBN-89.6, or SBN-NL4-3.
• Figure 5. A graph showing ELISA reactivity of mouse sera against HTV-l gpl20.
• FIG. 7 Schematic representation of a method for the construction of RV-based expression vectors with foreign viral glycoproteins.
• Figure 8 Schematic representation of a method for the construction of full- length and RV-glycoprotein deleted RVs expressing HTV-l gpl60.
• FIG. 9 CTLs from HTV-l gpl60 immunized mice induce long-lasting HTV-l gpl60-specific CTLs.
• Groups of three 6- to 8-week-old female BALB/c mice (Harlan Sprague) are inoculated i.p. with 2xl0 7 foci-forming units of recombinant RV expressing HTV-1 NL4 - 3 envelope protein.
• 105 to 135 days after the single inoculation spleens are aseptically removed and single cells suspensions are prepared (infra). Stimulator cells are prepared (infra), then added back to the effector cell population at a ratio of 3:1.
• Cytolytic activity of cultured CTLs is determined by measurement of the percent Cr released (infra).
• Figure 10 CTLs from HIV-1 gpl60 immunized mice cross-kill target cells expressing heterologous HIV-1 envelope proteins. Groups of six 6- to 8-week-old female BALB/c mice are inoculated i.p. with 2x10 foci-forming units recombinant RV expressing HTV-l envelope protein from strains NL4-3 (A) or 89.6 (B). Three and four weeks after the single inoculation, spleens were aseptically removed and splenocytes were stimulated in-vitro with vaccinia virus expressing the homologous HIV-1 envelope protein (infra).
• Target cells are prepared by infection with vaccinia virus expressing HTV-l envelope proteins from strains NL4-3 (vCB41), 89.6 (vBD3), JR-FL (vCB28), or Ba-L (vCB43). Chromium release assays are completed (infra). The results are shown from two different, independent experiments.
• Cytolytic activity is mediated by CD8 + T-cells.
• Groups of three 6- to 8- week-old female BALB/c mice are inoculated i.p. with 2xl0 7 foci-forming units recombinant RV expressing HIV-1 envelope protein from the NL4-3 strain.
• Eighteen weeks after the single inoculation spleens are aseptically removed and splenocytes are stimulated in vitro with vaccinia virus expressing HTV-1 NL . 3 envelope protein (infra).
• CD8 + T-cells are depleted from the cell culture (CD8 " ) and enriched (CD8 + ) using Dynabeads Mouse CD8 (Lyt2), as described by the manufacturer. Chromium release assays are completed (infra) on cultures depleted (CD8 " ) or enriched (CD8 + ) of CD8 T-cells, or unprocessed cultures (CD8 + /CD8 " ). Target cells are prepared (infra) by infection with vaccinia virus expressing HIV-1 envelope proteins from NL4-3 (vCB41). Background levels were equal to, or below, 6% specific lysis. DETAILED DESCRIPTION OF THE INVENTION
• Rhabdoviruses such as Rabies virus and Vesicular Stomatitis virus are members of the family Rhabdoviridae.
• Rabies virus possesses a negative stranded RNA genome of approximately 12kb. The genome is modularly organized and similar to that of vesicular stomatitis virus (VSV).
• VSV vesicular stomatitis virus
• These Rhabdoviruses encode five structural proteins.
• the five open reading frames coding for the viral structural proteins are nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and polymerase (L).
• N nucleoprotein
• P phosphoprotein
• M matrix protein
• G glycoprotein
• L polymerase
• the nucleoprotein (N), the phosphoprotein (P), the viral polymerase (L), and the genomic RNA form a helical ribonucleoprotein complex (RNP).
• the RNP is surrounded by a host cell- derived envelope membrane which contains the matric protein (M) on the inner side of the membrane, and the transmembrane glycoprotein (G) which mediates binding of the virus to specific receptors on the cell membrane.
• Non-segmented negative-strand RNA viruses entirely from cDNA has been reported by the inventors. (Schnell et. al., EMBO, 13:4195-4203, 1994).
• the approach involved intracellular expression of anti-genomic RNA in cells also expressing the viral proteins required for formation of an active RNP complex, namely, the nucleoprotein (N), the phosphoprotein (P), and the viral polymerase (L).
• N nucleoprotein
• P phosphoprotein
• L viral polymerase
• Rhabdovirus vectors are generated and are used to express functional genes, including full-length HTV-l envelope proteins. From the recombinant Rhabdovirus vectors of the invention all the dominant epitopes for neutralizing antibodies, cytotoxic T-lymphocytes (CTL), and antibody-dependent cell cytotoxicity are expressed at one time.
• CTL cytotoxic T-lymphocytes
• the construction of different recombinant Rhabdovirus vectors expressing HIV or SIV or other viral genes is described in the following paragraphs. Recombinant Rhabdovirus expression vectors
• RNA vectors that express heterologous genes or gene sequences are constructed.
• an expression vector with its own glycoprotein is constructed.
• X can be cloned at different genome sites to regulate expression levels.
• an expression vector with a glycoprotein from another virus or another viral serotype is constructed (see Fig. 7 as an example for the RV vector with VSV glycoprotein).
• This vector is used as boost virus to induce a stronger immune response.
• X can be cloned at different genome sites to regulate expression levels.
• the present invention relates to constructs of recombinant RVs (rabies viruses) expressing HIV-1 gpl60, where the RV glycoprotein (G) is replaced with that of a chimeric vesicular stomatitis virus (VSV) G /RV- cytoplasmic domain (serotype Indiana or New Jersey).
• RVs rabies viruses
• VSV chimeric vesicular stomatitis virus
• Rhabdoviruses have only a single surface protein on their virions, chimeric RV/VSV viruses are not neutralized by the humoral response against the RV G and therefore allow a second productive infection.
• RV/VSV recombinant chimeric RV/VSV
• repeated expression of the RV nucleoprotein which was previously shown to be an exogenous superantigen (Lafon, et al., Nature, 358, 507-10, 1992; Lafon, M. Research in Immunology, 144:209-13, 1993), might help to enhance the immune response against the HIV-1 envelope.
• RV Rabies Virus
• the cytoplasmic domain of the RV glycoprotein is fused to the foreign glycoproteins. It should be noted that all genes within the recombinant genome can be rearranged to attenuate the virus or to enhance transcription of the foreign gene.
• a recombinant RV with rearranged genome, VSV glycoprotein, and HTV-l gpl60 can be constructed to have: 3'-X-N-P-G(VSV serotype NJ)-M-L-5 ⁇
• a recombinant expression vector (either RVs or VSVs) having a foreign glycoprotein instead of their own is constructed for entry into specific host cells, i.e., to mimic the tropism of another virus (e.g., HTV-l, Hepatitis C) in order to induce a stronger immune response (Fig. 8).
• This construct can be represented as 3 - N-P-M-FflV-l-gpl60-L.
• these constructs can have, in addition, their own glycoproteins (e.g., 3 -N-P-M-HTV-l-gpl60-G-L).
• all genes within the recombinant genome can be rearranged to attenuate the virus or to enhance transcription of the foreign gene.
• Transgenic mice expressing human CD4 and CXCR4 are generated to analyze the in vivo induction of an immune response of the G-related RVs expressing HIV-1 gpl60/RVG.
• a recombinant expression vector (either RV or VSV) having a multiple antigens and multiple transcription stop/start signals is constructed.
• This construct is represented as 3 -N-P-M-G-X-Y-L-5' where X and Y are heterologous genes.
• X can be HTV-l gpl60 and Y can be HTV-l gag.
• An alternative construct can be 3 -N-Z-P-M-G-X-Y-L-5' where, for example, X can be HTV-l g ⁇ l60, Y can be HIV-1 gag and Z can be HTV-l tat.
• Rabies virus is a negative-stranded RNA virus of the Rhabdovirus family and it possesses a relatively simple, modular genome organization coding for five structural proteins (supra and Conzelmann, et al., Virology, 175:485-99, 1990).
• the present invention relates to an RV vaccine strain-based vector, which is non- pathogenic for a wide range of animal species when administrated orally or intramuscularly. This vector shows advantages over other viral vectors, for several reasons. First, its modular genome organization makes genetic modification easier than for the majority of more complex genomes of DNA and plus-stranded RNA viruses.
• Rhabdoviruses have a cytoplasmic replication cycle and there is no evidence for recombination and/or integration into the host cell genome.
• Rose, et al. Rhabdovirus genomes and their products, Plenum Publishing Corp., New York, 1997.
• RV In contrast to most other viral vectors only a negligible seropositivity exists in the human population to RV and immunization with a RV-based vector against HTV-l will not interfere with immunity against the vector itself.
• RV grows to high titers 10 foci forming units (FFU) in various cell-lines without killing the cells, which probably results in longer expression of HTV-l genes compared to a cytopathogenic vector.
• FFU foci forming units
• rabies virus vectors The following different recombinant rabies virus vectors are constructed.
• This vector also contains a Smal site upstream of the RV glycoprotein, which is used to delete the RV glycoprotein gene (G).
• the vector is called RV-SBN.
• RVs expressing HIV-lgp-160 ecto- and transmembrane domain fused to the RV G cytoplasmic domain are also constructed.
• the chimeric gpl60/RVG protein is expressed by RV and incorporated into RV virions.
• a recombinant virus displaying a foreign envelope protein on its surface will induce a strong immune response against this antigen.
• RV-SPBN Another RV vector is also generated which is identical to RV-SBN but has, in addition, a single Pad site downstream of RV G protein. This vector is used to functionally replace RV G with VSV G or other viral glycoproteins. This vector is called RV-SPBN and is used as a boost vaccine vector or a boost virus. As shown in Fig. 7, a recombinant rabies virus based expression vector with foreign viral glycoproteins is constructed and the recombinant virus is recovered. For this construct a Smal restriction enzyme site is introduced downstream of the M/G transcription Stop/Start sequence and a Pad site upstream of the synthetic transcription Stop/Start sequence, which is used to express foreign genes from the RV vector.
• a chimeric VSV/RV glycoprotein (VSV ectodomain and transmembrane domain, RV cytoplasmic domain), in combination with HTV-l is shown as an example.
• this method can be applied to every glycoprotein and foreign antigen in different Rhabdoviruses, as shown in the same figure (glycoprotein X, foreign protein Y).
• recombinant RVs expressing chimeric gpl60/RV G without expressing RV G are generated.
• G-deleted RVs have a different tropism as compared to wild-type RV (which infects most cells) and specifically infect only cells expressing HTV-l receptor human CD4 and one of the HTV-l coreceptors (eg, CXCR4 or CCR5).
• Both the full-length and RV-glycoprotein deleted recombinant rabies RVs are constructed and recovered (Fig. 8).
• the Smal and BsiWI restriction enzyme sites are used to delete RV glycoprotein and fuse the M/G transcription Stop/Start sequence to the HTV-l/RV chimeric glycoprotein (HTV-l gpl60 ectodomain and transmembrane domain, RV cytoplasmic domain).
• the recovered RV-vector is, analogous to the HTV-l virus, specific for cells expressing human CD4 and the appropriate HIV-1 co-receptor. It should be noted that this method can be applied to every glycoprotein which supports infection of certain cell types by rhabdoviruses. It can also be used to express additional foreign antigens (HIV-1 Gag, HTV protease, SIV proteins , Hepatitis A, B or C proteins, and other viral and non-viral proteins).
• a recombinant replication-competent rabies virus expression vector having all of the above combinations can be constructed.
• a recombinant rabies virus vector having other glycoproteins (especially to construct boost viruses) without or with their own G, having genome rearrangements, and expressing multiple viral antigens from the same or different viruses e.g. HTV-l gpl60 and Hepatitis B.
• Products, methods and compositions There are provided by the invention, products, compositions and methods for assessing treating viral diseases, particularly HTV (AIDS) and administering a recombinant Rhadovirus of the invention to an organism to raise an immunological response against invading viruses, especially against immunodeficiency virus infections.
• HTV HIV
• Rhadovirus of the invention to an organism to raise an immunological response against invading viruses, especially against immunodeficiency virus infections.
• Another aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal, which involves inoculating the individual with a recombinant virus of the invention followed by the appropriate recombinant protein boost, adequate to produce antibody and/ or T cell immune response to protect the individual from infection, particularly immunodeficiency infection and most particularly HTV-l and 2 infections. Also provided are methods whereby such immunological response slows the HTV replication.
• Yet another aspect of the invention relates to a method of inducing immunological responses in an individual which comprises delivering to such individual a nucleic acid vector, sequence or ribozyme to direct the expression of HTV envelope polypeptides, or a fragment or a variant thereof, for expressing the HTV envelope polypeptide, or a fragment or a variant thereof, in vivo in order to induce an immunological response, such as, to produce antibody and/ or T cell immune response.
• Antibody and/or T cell responses include, for example, cytokine-producing T cells or cytotoxic T cells, to protect the individual, preferably a human, from the viral disease, whether that disease is already established within the individual or not.
• nucleic acid vector may comprise DNA, RNA, a ribozyme, a modified nucleic acid, a DNA/RNA hybrid, a DNA-protein complex or an RNA-protein complex.
• compositions that induce an immunological response A further aspect of the invention relates to an immunological composition that when introduced into an individual, preferably a human, capable of having induced within it an immunological response.
• the immunological response that is induced is to a polynucleotide and/or polypeptide encoded therefrom, wherein the composition comprises a recombinant Rhabdoviruses of the invention which encodes and expresses an antigen of an exogeneous viral protein, such as HTV envelope protein or polypeptide.
• the exogeneous polypeptides include antigenic or immunologic polypeptides.
• the immunological response is used therapeutically or prophylactically and takes the form of antibody immunity and/or cellular immunity, such as cellular immunity arising from CTL or CD4+ T cells.
• compositions comprising a Rhabdovirus vector of the present invention for administration to a cell or to a multicellular organism.
• the Rhabdovirus vectors of the invention may be employed in combination with a non- sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to an individual.
• a pharmaceutical carrier suitable for administration to an individual Such compositions comprise, for instance, a media additive or a therapeutically effective amount of a recombinant virus of the invention and a pharmaceutically acceptable carrier or excipient.
• Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof.
• the formulation should suit the mode of administration.
• the invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
• the recombinant vectors of the invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
• compositions may be administered in any effective, convenient manner including, for instance, administration by intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes among others.
• the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.
• the pharmaceutical compositions of the invention are preferably administered by injection to achieve a systematic effect against relevant viral pathogens.
• the daily dosage level of the active composition of the invention will be from 10 2 FFU to 10 8 FFU of virus in the composition or 10 ⁇ g/kg tolO mg/kg of body weight of recombinant protein.
• the physician in any event will determine the actual dosage and duration of treatment which will be most suitable for an individual and can vary with the age, weight and response of the particular individual.
• the above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
• a vaccine composition is conveniently in injectable form. Conventional adjuvants may be employed to enhance the immune response.
• a suitable unit dose for vaccination is preferably administered daily and with or without an interval of at least lweek. With the indicated dose range, no adverse toxicological effects are observed with the compounds of the invention which would preclude their administration to suitable individuals.
• Recombinant RV vectors expressing an HIV-1 envelope protein In a preferred emodiment recombinant RVs expressing HTV-l envelope protein is explained. To generate RV recombinant viruses expressing HTV-l gpl60, a new vector is constructed based on the previously described infectious RV cDNA clone pSAD-L16. (Schnell, et al., EMBO Journal, 13:4195-4203, 1994).
• the ⁇ gene is deleted from the RV genome and a new transcription unit, containing a RV Stop/Start signal and two single sites (BsiWI and Nhel), is introduced into the RV genome (see also Generation of recombinant vectors, supra).
• the resulting plasmid is designated pSBN (Fig. 1).
• the SBN RV-vector is recovered by the reported methods and displayed the same growth characteristics and similar viral titers as SAD-L16, indicating that neither the deletion of the ⁇ gene nor the new transcription unit affected the RV vector (deleted).
• HIV-1 envelope genes (NL4-3 and 89.6) to be expressed from SBN are generated by PCR and cloned between the BsiWI and Nhel sites, resulting in the plasmids pSBN-NL4-3 and pSBN-89.6 (Fig.l). All constructs are checked via DNA sequencing. It should be noted that foreign genes up to at least 4kb are stable within the RV genome and a full length HIV-1 envelope protein is expressed from the recombinant RVs.
• Recombinant RVs expressing either HTV-1 NL4 . 3 or HTV-1 89 . 6 envelope proteins are recovered by transfection of cells stably expressing the T7-RNA-polymerase with plasmids encoding the RV N, P, and L proteins along with a plasmid coding for the respective RV full- length anti-genomic RNA. Three days after transfection, supematants of transfected cells are transferred to fresh cells and three days later analyzed by indirect immunofluorescence microscopy for expression of HTV-l gpl60.
• the recombinant RVs expressing HTV-l gag are also constructed and recovered with the same procedure used for the recombinant RVs expressing HIV-1 envelope protein.
• recombinant RVs expressing HTV-l envelope protein are examined. A three-fold lower titer for SBN-NL4-3 and a 10-fold titer reduction for SBN- 89.6 is noticed, as compared to wild-type SBN.
• a one-step growth curve of the recombinant RVs is performed. BSR cells are infected with a MOI of ten to allow synchronous infection of all cells. After replacing the virus inoculum with fresh medium, viral titers are determined at the indicated time-points (Fig. 2).
• HTV-l gpl60 by recombinant RVs is also examined.
• cell lysates from recombinant RV infected cells are analyzed by Western immunoblotting with an antibody directed against RV (Fig. 3, ⁇ -rabies) or HIV-1 gpl20 (Fig. 3, ⁇ -gpl20).
• Two bands of the expected size for HIV-1 gpl60 and gpl20 are detected in lysates from cells infected with SBN-89.6 or SBN- NL4-3 (lanes 3 and 4), but are not observed in cell lysates of mock-infected or SBN infected cells (lanes 1 and 2).
• the Western blot probed with an ocRV antibody confirmed that all viruses (lanes 2, 3, and 4) infected the target cells.
• Envelope proteins expressed in recombinant RVs are functional to determine whether the expressed HIV-1 envelope protein is functionally expressed from RV, the recombinant RVs are analyzed in a fusion assay in a human T cell-line (Sup- Tl). This experiment confirmed that wild-type RV is able to infect and replicate in human T cell-lines. Because wild-type RV infects cells by receptor-mediated endocytosis, the RV glycoprotein (G) can only cause fusion of infected cells at a low pH. (Whitt, et al., Virology, 185:681-8, 1991).
• Envelope protein from the dual-tropic HIV-1 strain (89.6) will induce cell fusion if coexpressed with CD4 and CCR5, whereas NL4-3 gpl60 will only induce fusion on cells expressing CD4 and the HTV-l coreceptor CXCR4.
• Infection of 3T3 murine cells expressing human CD4 does not result in cell fusion regardless of the recombinant RV used, whereas syncytium-formation is detected in 3T3 cells expressing CD4 and CXCR4 after infection with SBN-NL4-3 or SBN-89.6.
• SBN-NL4-3 or SBN-89.6 As expected, only expression of HTV-1 89 .6 envelope protein in 3T3 cells, expressing CD4 and CCR5, caused fusion of these cells.
• mice infected with RV expressing HTV-l gpl60 are also analyzed.
• One likely requirement for a successful HTV-l vaccine is the ability to induce a strong humoral response against the HTV-l protein gpl60.
• groups of five BALB/c mice are inoculated subcutaneously in both rear footpads with 10 6 FFU of SBN, SBN-89.6, or 10 5 FFU SBN-NL4-3. Mice are bled 11, 24, and 90 days after the initial infection with RV and the sera are analyzed by ELISA.
• mice Twelve days after the subunit boost, the mice are bled and the immune response is analyzed by an HIV-1 g l20 ELISA.
• the results demonstrate that an HTV-envelope subunit boost elicits a strong immune response against HIV-1 gpl20 only in mice previously infected with SBN-89.6 or SBN-NL4-3 (Fig. 5). Wild-type RV (SBN) infected mice reacted only in the lowest serum dilution (1:160) after the boost.
• An ELISA specific for HIV-1 gp41 is negative for all mouse sera, even after the boost with recombinant HIV-1 gpl20/gp41. These data are confirmed by Western blot analysis (Fig. 6).
• HTV-l neutralizing antibody (NA) titers are determined in MT-2 cells by a vital dye staining assay using HTV-lNL-;--.
• the mouse serum is able to neutralize a tissue culture laboratory adapted (TCLA) HTV-I N - 3 strain at a 1:800 serum dilution after immunization with SBN- NL4-3 and an envelope subunit booster injection of recombinant gpl20 (HIB strain), whereas immunization with SBN-NL4-3 did not induce detectable neutralizing antibody.
• mice with recombinant RV expressing HTV-l gpl60 results in a strong priming of the immune system, as indicated by vigorous humoral responses after a single boost with HTV-l gpl20 protein or gp41.
• boosting with another recombinant RV using a different viral glycoprotein for infection of the mice, or recombinant VSV expressing HTV-l gpl60 can be tested for an even stronger response.
• Recombinant RV expressing HTV-l envelope protein from a laboratory-adapted HTV- 1 strain (NL4-3) and a primary HTV-l isolate (89.6) show that RV-based vectors are excellent for B cell priming (supra).
• the present invention further relates to the memory CTL response against HTV-l envelope protein expressed by the attenuated RV-based vectors. As noted, increasing evidence suggests that the induction of a vigorous, long-lasting CTL response is an important feature for a successful HTV-l vaccine.
• mice were immunized with 2 x 10 7 foci forming units (FFU) of the recombinant RV expressing HIV-I N W-. envelope protein (SBN-NL4-3) (supra and infra). Three mice are sacrificed 105 or 135 days after infection and the spleens are removed. One third of the splenocyte cultures are infected with a multiplicity of infection (moi) of 1 with a recombinant vaccinia virus expressing HTV-1NL4- 3 gpl60 for 16 hours, deactivated using Psoralen and UV treatment, and added back to the culture as presenter cells.
• moi multiplicity of infection
• Stimulated effector cells are analyzed 7 days after activation for their ability to kill P815 target cells infected with vaccinia wild-type virus, a recombinant vaccinia virus expressing HIV-1 N L4 -3 gpl60 or HIV-1 Gag.
• a strong cytotoxic response is detected only against P815 target cells infected with the recombinant vaccinia virus expressing HTV-l envelope protein. Only a low percentage of lysis is observed for P815 cells infected with the other two vaccinia viruses.
• these responses are achieved after a single inoculation with recombinant RV expressing HTV-l envelope protein, which indicates that RV-based vectors are able to induce long-lasting CTLs after a single vaccination.
• HTV-l envelope amino acid sequences There is a significant difference in HTV-l envelope amino acid sequences but cross- protection between divergent viruses will be a likely requirement for a protective HTV-l vaccine.
• splenocytes from mice immunized with a recombinant RV expressing HIV-1 gpl60 are screened against P815 target cells expressing homologous and heterologous HIV-1 envelope proteins.
• mice are immunized intraperitoneally (i.p) either with 2 x 10 recombinant RV expressing HTV-l gpl60 from a laboratory-adapted, CXCR4-tropic (NL4-3) or a dual-tropic (CXCR4 and CCR5) isolate (89.6).
• mice from each group are sacrificed, the spleens are removed, and the pooled splenocytes are stimulated with a recombinant vaccinia virus expressing the homologous HtV-1 envelope protein (NL4-3 or 89.6).
• effector cells are analyzed for their ability to lyse P815 cells infected with recombinant vaccinia viruses expressing HIV-1 envelope protein from the laboratory-adapted, CXCR4-tropic HTV-l strain (NL4-3), the dual-tropic strain (89.6), and two primary, CCR5-tropic HTV-l strains (Ba-L and JR-FL).
• Activated splenocytes from SBN-NL4-3 immunized mice achieved a specific lysis of P815 cells expressing gpl60 JR-FL or 89.6 in the 40% range at an effector:target (E:T) ratio of 50:1 and are also able to cross-kill target cells expressing HTV-l ⁇ a - L gpl60. Cross-killing is also observed with effector cells from SBN-89.6 primed mice.
• P815 target cells are lysed in the same range as observed for activated splenocytes from mice immunized with SBN-NL4-3, but lysed only about 20% P815 cells expressing HTV-l N - 3 - These data indicate that CTLs against HTV-l gpl60 induced by RV-based vectors may be directed against different epitopes within the HIV-1 envelope protein.
• HIV-1 -specific CTL activity is mediated by CD8 + T-cells
• mice The phenotype of the T-cell subpopulation mediating cytolytic activity is assessed by selective depletion.
• Three mice are immunized with 2 x 10 FFU of recombinant RV expressing HTV-IN I - S envelope protein, eighteen weeks later the spleens are removed.
• Splenocytes are re-stimulated with a recombinant vaccinia virus expressing the homologous
• Immuno-magnetic bead cell separation is completed to both deplete and positively isolate CD8 + T- cells from the activated splenocyte culture.
• Chromium release assays are completed using cultures depleted of CD8 + T-cells (CD8 " ), cultures of isolated CD8 cells (CD8 + ) or unprocessed cultures (CD8 + /CD8 " ).
• P815 target cells are infected with vaccinia virus expressing HTV-I N 14- 3 gpl60 or HTV-l gag.
• the CD8 + T-cell depleted cultures show no activity while the CD8 + T-cell enriched and unprocessed cultures show high specific lysis at E:T ratios of 25:1 and 12.5:1, respectively.
• the CD8 + T-cell enriched population is also enriched in lytic units, as the CTL activity is still on a plateau at 12.5:1, in contrast to the unselected population.
• the present invention relates to RV-based vectors expressing HTV-l envelope proteins. These vectors are able to induce a humoral response against HTV-l gpl60 after a single immunization followed by a boost injection with recombinant HIV-1 gpl20. (Schnell, M. J., et al, Proc. Natl. Acad. Sci. USA, 97:3544-3549, 2000.). Expanding evidence suggests that CTL responses play a major role in the anti- viral immune response against HTV-l. (Brander, C. and B. D. Walker, Current Opinion in Immunology, 11:451-9, 1999.). The development of an effective prophylactic HTV-l vaccine therefore requires the selection of HTV-l antigen(s) capable of inducing long-lasting and broadly reactive CTL responses. The present invention further relates to RV-based vectors to induce such responses.
• RV nucleoprotein which was previously shown to be an exogenous superantigen (Lafon, M., Research in Immunology, 144:209-13, 1993; Lafon, M., et al., Nature, 358:507-10, 1992), might help to enhance a general immune response against the HTV-l envelope after a single immunization.
• the recombinant RVs of the present invention are able to induce cross-reactive CTLs against a variety of different HTV-l envelope proteins.
• Previous studies showed that single amino acid exchanges can abrogate CTL cross-reactivity, whereas other examinations indicated that single or even double amino acid substitutions frequently did not abrogate cross- lling. (Cao, H., et al., J. Virol, 71:8615-23, 1997; Johnson, R. P., et al., Journal of Experimental Medicine, 175:961-71, 1992; Johnson, R. P., et al., Journal of Immunology, 147:1512-21, 1991.).
• the inventors of the present invention are currently analyzing if CTLs against HTV-l g ⁇ l60 induced by recombinant RV are also cross-reactive against HTV-l envelope protein from clades other than B. In summery, the present invention demonstrates the ability of the murine sera to neutralize HIV-1 strain. Thus the present invention shows that recombinant RVs are excellent vectors for B cell priming.
• the present invention also shows that a single vaccination with recombinant RV expressing HIV-1 envelope protein elicits a strong, long- lasting CTL response specific against HIV-1 proteins, such as the envelope protein of different HIV-1 strains. These results further emphasize the use of RV as an HTV-l vaccine.
• the present invention fulfills a long felt, yet unfulfilled need, for a method of treating HIV-1 infections.
• RVs of the present invention Using the recombinant RVs of the present invention, all of the dominant epitopes for neutralizing antibodies, cytotoxic lymphocytes, and antibody dependent cell cytotoxicity are expressed at one time, thereby eliciting both humoral and cell-mediated immunity against HTV-l. Examples
• Example 1 Plasmid construction. Shown in Fig. 1 is a schematic representation of a method for the construction of recombinant RV genomes. At the top, the wild-type RV genome with its five open reading frames is shown (SAD L16). Using a PCR strategy and site directed mutagenesis the entire ⁇ gene is removed and a new minimal RV transcription unit containing two single sites is introduced between the G and L genes (SBN). The cDNA sequence encoding HTV-1 89 . 6 or HTV-1 NL 4- 3 gpl60 is inserted using the BsiWI and Nhel sites resulting in the plasmids, pSBN- 89.6 or pSBN-NL4-3 (bottom).
• pSN is the target used to introduce a new transcription Stop/Start sequence, as well as a single BsiWI site using a polymerase chain reaction (PCR) strategy.
• PCR polymerase chain reaction
• TTTTGCTAGCTTATAAAGTGCTGGGTCATCTAAGC-3' SEQ ID NO: 3
• RP10 5'- CACTACAAGTCAGTCGAGACTTGGAATGAGATC-3' SEQ ID NO: 4
• the reverse primers were RP18 5'-TCTCGAGTGTTCTCTCTCCAACAA-3' (SEQ ID NO: 5) and RP17 5'- AAGC ⁇ AGCAAAACG ⁇ ACGGGAGGGGTGTTAGTTTTTTTCATGGACTTGGATCGTT
• RP17 contains a RV transcription Stop/Start sequence (underlined) and a BsiWI and Nhel site (shown in italics). PCR products are digested with Nhel, ligated, and the 3.5 kb band eluted from an agarose gel. After gel elution the band is digested with Clal/Mlul and ligated to the previously Clal/Mlul digested pSN. The plasmid is designated pSBN.
• the HTV-l gpl60 genes encoding the envelope protein of the HTV-l strains 89.6 and NL4-3, are amplified by PCR using Vent polymerase, the forward primer 5 - GGGCTGCAGCTCGAGCGTA CGAAAATGAGAGTGAAGGAGATCAGG-3' (SEQ TD NO: 7) containing PstT/XhoI/BsiWI sites (italics), and the reverse primer 5 - CC ⁇ C ⁇ AGATTATAGCAAAGCCCTTTCCAAG-3' (SEQ ID NO: 8) containing a Xbal (italics) site.
• the PCR products are digested with Pstl and Xbal and cloned to pBluescript II SK + (Stratagene). After conformation of the sequence, the HTV-l gpl60 genes are excised with BsiWI and Xbal and ligated to pSBN, which had been digested with BsiWI and Nhel. The resulting plasmids are entitled pSBN-89.6 and pSBN-NL4-3.
• Example 2 Recovery of infectious RV from cDNA.
• the previously described vaccinia virus-free RV recovery system is used (see Finke, et al, Journal of Virology, 73:3818-25, 1999).
• BSR-T7 cells which stably express T7 RNA polymerase (a generous gift of Drs. S. Finke and K.-K. Conzelmann) are transfected with 5 ⁇ g of full-length RV cDNA in addition to plasmids coding for the RV N-, P-, and L-proteins (2.5 ⁇ g, 1.25 ⁇ g, and 1.25 ⁇ g) respectively, using a Ca 2 PO transfection kit (Stratagene) as indicated by the vendor.
• tissue culture supematants are transferred onto fresh BSR cells and infectious RV is detected three days later by immunostaining against RV the N protein (Centocor).
• Example 3 One-Step Growth Curve Shown in Fig. 2 is a graph showing One-step growth curves of recombinant RV BSR cells that are infected with the recombinant RVs (SBN, SBN-89.6, and SBN-NL4-3). The viral titers are determined in duplicate at the indicated time-points.
• BSR cells (a BHK-21 clone) are plated in 60 mm dishes and 16 hours later infected
• FIG. 3 the Western blot analysis of recombinant RVs expressing HTV-l gpl60 is shown.
• Sup-Tl cells are infected with a MOI of 2 with SBN, SBN-89.6, or SBN-NL4-3 and lysed 24 h later. Proteins are separated by SDS-PAGE and analyzed by Western blotting.
• An antibody directed against gpl20 detected two bands at the expected size for HTV-l gpl60 and gpl20 in cell-lysates infected with SBN-89.6 or SBN-NL4-3 ( ⁇ -gpl20, lanes 3 and 4).
• mice Groups of five 4-6 week old female BALB/c mice obtained from Jackson Laboratories are inoculated subcutaneously in both rear footpads with 10 6 foci forming units (FFU) SBN, SBN-89.6, or 10 5 NL4-3 in DMEM + 10% FBS. Three out of five mice in each group are boost immunized intraperitonealy three months after infection with 10 ⁇ g recombinant gp41 (inB, Intracel Inc.) and 10 ⁇ g recombinant gpl20 (IIIB, Intracel Inc.) in 100 ⁇ l complete Freunds adjuvant.
• FFU foci forming units
• Example 5 Enzyme-linked Immunosorbent Assay (ELISA ).
• Recombinant HTV-l gpl20 (TUB strain, Intracel) is resuspended in coating buffer (50 mM Na 2 C0 3 , pH 9.6) at a concentration of 200 ng/ml and plated in 96 well ELISA MaxiSorp plates (Nunc) at 100 ⁇ l in each well. After overnight incubation at 4°C, plates are washed three times (PBS pH 7.4, 0.1% Tween-20), blocked with blocking buffer (PBS, pH 7.4, 5% dry milk powder) for 30 minutes at room temperature, and incubated with serial dilutions of sera for 1 hour.
• coating buffer 50 mM Na 2 C0 3 , pH 9.6
• Nunc 96 well ELISA MaxiSorp plates
• HRP horseradish peroxidase-conjugated
• H+L horseradish peroxidase-conjugated
• OPD-substrate o-phenylenediamine dihydrochloride, Sigma
• mice Five mice each are immunized with recombinant RVs (SBN, SBN-89.6, or SBN-NL4-3) and 3 months after the initial infection three mice from each group are boosted with recombinant HTV-l gpl20 and gp41 (SBN*, SBN-89.6*, or SBN-NL4-3*).
• Each data point on the graph indicates the average of mice from each group in three independent experiments.
• One mouse of the SBN-89.6 group did not react to the boost injection and is not included in the graph.
• the error bars indicate the standard deviations.
• Human T-lymphocytic cells (Sup-Tl) cells are infected with a MOI of 2 for 24 hours and resuspended in lysis buffer 50mM Tris, pH 7.4; 150 mM NaCl, 1% NP-40, 0.1% SDS, and lx protease inhibitor cocktail (Sigma) for 5 minutes. The protein suspension is transferred to a microfuge tube and spun for 1 minute at 10,000 x g to remove cell debris. Proteins are separated by 10% SDS-PAGE and transferred to a PVDF-Plus membrane (Osmonics).
• blots are incubated with sheep -gpl20 antibody (ARRRP) (1:1000) or human ⁇ -rabies sera (1:500) in blocking buffer for 1 hour.
• RRRRP sheep -gpl20 antibody
• HRP horseradish peroxidase-conjugated antibodies
• Western blot analysis to detect anti-HXV-1 antibody is performed using a commercial Western Blot kit (QualiCode HTV- 1/2 Kit, Immunetics) according to the manufacturer's instructions, except for the mouse sera in which ⁇ -human IgG conjugate is substituted with a 1:5000 dilution of an alkaline phosphatase-co jugated goat anti-mouse IgG (H+L) (Jackson ImmunoResearch Laboratories). Shown in Fig. 6 is the Western blot analysis of mice serum antibody response to HTV-l antigens.
• Sera from one mouse of each group (SBN, SBN-89.6, or SBN-NL4-3), which are immunized by the RVs ( ⁇ -SBN, ⁇ -SBN-89.6 or ⁇ -SBN-NL4-3), or immunized and boost injected with recombinant gpl20 and gp41 ( ⁇ -SBN*, ⁇ -SBN-89.6* or ⁇ -SBN-NL4-3*), are tested at 1:100 dilutions.
• a highly positive and weakly positive human control serum is used to detect the position of the HTV-l proteins.
• SC indicates the serum control.
• Example 7 Virus Neutralization Assays.
• H V-l strains are recovered on 293T cells and virus stocks are expanded on MT-2 cells (HTV-l NL4-3), frozen at -75° C and titered on MT-2 cells.
• Neutralization assays are performed according to Montefiori et al., (Journal of Clinical Microbiology, 26, 231-5, 1998). Briefly, ⁇ 5000 TCTDsc of HTV-1 NL4 - 3 are incubated with serial dilutions of mouse sera for 1 hour. MT-2 cells are added and incubated at 37°C, 5% C0 2 for 4-5 days. 100 ⁇ l of cells are transferred to a poly-L-lysine plate and stained with neutral red dye (Neutral Red, ICN) for 75 minutes. Cells are washed with PBS, lysed with acid alcohol and analyzed using a colorimeter at 550 nm. Protection is estimated to be at least 50% virus inhibition.
• the virus is inactivated using psoralen (Sigma). Psoralen is added to cells to achieve a final concentration of 5 Dg/ml. Following a ten minute incubation at 37°C the cells were treated with long-wave UV (365 nm) for 4 minutes and washed twice with PBS.
• Target cells are prepared by infection with vaccinia virus expressing the HTV- 1 protein (see specific figure legend for specific protein) for one hour at a moi of 10, washed to remove excess virus, and incubated for 16 hours at 37°C.
• target cells are infected with vaccinia virus expressing HTV-l Gag (vP1287) or wild-type vaccinia (vP1170).
• Target cells are washed once in PBS, incubated with 100 ⁇ Ci 51 Cr for one hour to label the cells, washed two times in PBS and added to effector cells at various E:T ratios (see figures) for four hours at 37°C.
• the percent specific 51 Cr release is calculated as 100 x (experimental release - spontaneous release)/(maximum release - spontaneous release). Maximum release was determined from supematants of cells that were lysed by the addition of 5% Triton X-100. Spontaneous release was determined from target cells incubated without added effector cells.
• CD8 + T-cells are depleted from the cell culture (CD8 ⁇ ) and enriched (CD8 + ) using Dynabeads Mouse CD8 (Lyt2), as described by the manufacturer.

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