WO2001055330A2 - Recombinant rhabdoviruses as live-viral vaccines for immunodeficiency viruses - Google Patents

Abstract

This invention provides recombinant, replication-competent Rhabdovirus vaccine strain-based expression vectors for expressing heterologous viral antigenic polypeptides such as immunodeficiency virus envelope proteins or subparts thereof. An additional transcription stop/start unit within the Rhabdovirus genome is inserted to express the heterologous antigenic polypeptides. The HIV-1 gp160 protein is stably and functionally expressed, as indicated by fusion of human T cell-lines after infection with the recombinant RVs. Inoculation of mice with the recombinant Rabies viruses expressing HIV-1 gp160 induces a strong humoral response directed against the HIV-1 envelope protein after a single boost with an isolated recombinant HIV-1 gp120 protein. Moreover, high neutralization titers, up to 1:800, against HIV-1 are detected in the mouse sera. These recombinant viral vectors expressing viral antigenic polypeptides provide useful and effective pharmaceutical compositions for the generation of viral-specific immune responses.

Description

RECOMBINANT RHABDOVIRUSES AS LIVE-VIRAL VACCINES FOR IMMUNODEFICIENCY VIRUSES

GOVERNMENT RIGHTS TO THE INVENTION

This invention was made in part with government support under grant AI44340 awarded by the National Institute of Health. The government has certain rights to the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority, in part, under 35 U.S.C. §120 based upon U.S. Non- Provisional Application No. 09/494,262, filed January 28, 2000.

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

Great success has been made in the therapy of HIV-1 infection during the last several years. (Holtzer, et al., Annals of Phannacotherapy 33:198-209, 1999; Bonfanti, et al., Biomedicine & Phannacotherapy, 53:93-105, 1999). However, the development of a protective immunodeficiency virus vaccine (e.g., HIV-1 vaccine) still remains a major goal in halting immunodeficiency virus pandemics. Most successful vaccines against viral diseases have been composed of killed or attenuated viruses. (Hilleman, M. R., Nature Medicine, 4:507-14, 1998). This approach does not seem to be suitable for immunodeficiency viruses, particularly HTV-l because killed HTV-l virus induces only a poor neutralizing antibody response and no cytotoxic T lymphocyte (CTL) response.

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 Uganda, nearly 40% of young adults are HTV-1-seropositive. (1). Currently, a variety of 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. Virol, 73:4640-50, 1999), peptides (Bekyakov, I.M., et al., Journal of Clinical Investigation, 102:2072-81, 1998; Berzofsky, J.A., et al., Immunological Reviews, 170:151-72. 1999; Pinto, L.A., et al., AZDS, 13:2003-12, 1999), naked DNA (Bagarazzi, M. L., et al., 1999, Journal of Infectious Diseases, 180:1351- 5, 1999; Barouch, D. H., et al., Science, 290:486-492, 2000; Cafaro, A., et al., Nature Medicine, 5:643-50, 1999; Lu, S., et al., AIDS Research & Human Retroviruses, 14:151-5, 1998; Put onen, P., et al., Virology, 250:293-301, 1998; Robinson, H. L., Aids, 11:S109-19, 1997; Weiner, D. B., and R. C. Kennedy, Scientific American, 281:50-7, 1999.), replication- competent and incompetent (replicon) live viral vectors (Berglund, P., et al., AIDS Research & Human Retroviruses, 13:1487-95, 1997; Mossman, S. P., et al., /. Virol, 70:1953-60, 1996; Natuk, R. J., et al., Proc. Natl. Acad. Sci. USA, 89:7777-81, 1992; Ourmanov, I., et al., /. Virol, 74:2740-2751, 2000; Schnell, M. J., et al., Proc. Natl Acad. Sci. USA, 97:3544- 3549, 2000.), and prime-boost combinations, [for review see (5)]. A large number of these vaccine strategies have been tested in the simian immunodeficiency virus (SIV) macaque model system, but to date no potent protective immunity has been obtained, although some amelioration of disease course has been seen. (Barouch, D. H., et al., Science, 290:486-492, 2000; Davis, N. L., et al., J. Virol, 74:371-8, 2000; Ourmanov, I., et al., /. Virol, 74:2740- 2751, 2000.). So far, the only effective method to protect macaques from STV infection is the use of live, attenuated STV. Desrosiers and colleagues showed that a genetically modified, «e/-deleted STV strain that does not cause disease in rhesus monkeys induced high anti-STV titers of antibodies and cytotoxic T lymphocyte (CTL) activity. (Daniel, M. D., et al., Science, 258, 1938-1941, 1992; Kestler, H. W., et al., Cell, 65:651-662, 1991.). Subsequent challenge of the immunized animals with infectious doses of a pathogenic SIV strain yielded protection from infection. (Daniel, M. D., et al., Science, 258:1938-1941, 1992). A major drawback in the use of attenuated lentiviral vaccine approaches is the finding that even nef-deleted SIV can give rise to an ATDS-like disease in both neonatal and adult macaques. (Baba, T. W., et al., Science, 267:1820-5, 1995; Baba, T. W., et al., Nature Medicine, 5:194-203, 1999; Desrosiers, R. C, AIDS Research & Human Retroviruses, 10 331-2, 1994.) Additional concerns regarding the use of attenuated lentiviruses aπse from the recent finding that recombination of live, attenuated STV with challenge virus in some cases results m an even more virulent strain. (Gundlach, B. R., et al., /. Virol , 74:3537-3542, 2000 ) However, the results indicated that live- viral vectors may be excellent vaccine candidates for an HTV-l vaccine.

For the foregoing reasons, there is a great need for the development of a protective immunodeficiency virus vaccine that is non-pathogenic for a wide range of animal species when administered orally or intramuscularly, as well as being able to induce the required neutralizing antibody and CTL responses.

The immune response(s) required to protect against HIV-1 infection is currently unknown, but a protective immune response against HIV-1 might require both major arms of the immune systems. Recent reports on vaccine approaches using recombinant HIV-1 envelope protein suggests that an exclusively humoral response is not sufficient to protect against an HIV-1 infection, but the passive transfer of three monoclonal antibodies directed against HIV-1 envelope protein resulted m protection of macaques against subsequent challenge with pathogenic HIV-1/SIV chimeric virus. (Mascola, J. R., et al., Nature Medicine, 6:207-10, 2000.). Other studies indicate that a cell-mediated response plays an important role in controlling an HIV-1 infection. (Brander, C. and B. D. Walker, Current Opinion in Immunology, 11:451-9 1999; Goulder, P. J., et al., Anti-HTV cellular immunity: recent advances towards vaccine design Aids, 13:S121-36, 1999.). Exposed but uninfected individuals often have HIV-1 -specific CTLs but no detectable antibodies against HIV-1 (Pmto, L. A., et al., Journal of Clinical Investigation, 96 867-76, 1995; Rowland- Jones, S. L., et al , Journal of Clinical Investigation, 102:1758-65, 1998.).

In the present invention, the ability of recombinant non-segmented negative-stranded RNA viruses expressing an immunodeficiency virus gene(s) as an immunodeficiency virus vaccine (e.g., HTV-l vaccine) is disclosed. Specifically, the ability of a Rhabdovirus-based recombinant viruses to induce an immune response against HIV is demonstrated. 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. Moreover, high neutralization titers against HIV-1 are detected m the mouse sera. (Schnell, M. I , et al., Proc. Natl. Acad Sci. USA, 97:3544-3549, 2000.). Little information is available regarding the induction of CTL responses against foreign proteins expressed by rhabdovirus-based vectors. 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. In a specific embodiment when 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.

DEFINITIONS

"boost vaccine vector" is "boost virus"

"boost virus" is "boost vaccine vector"

SUMMARY OF THE INVENTION

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) and methods of making and using the same. More in particular 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. These recombinant 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. In general aspects, 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.

Specifically, in one embodiment of the invention, 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, ψ gene of rabies virus, etc. In some instances, 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. In some other instances, 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.

The term heterologous viral nucleic acid as used herein refers to the viral nucleic acid that encodes the antigenic polypeptide that induces immune response. For example, a full- length HTV envelope protein, HIV gpl60, HTV gag, HTV gρl20, and full-length STV envelope protein are some of the antigenic polypeptides that are expressed in the recombinant viral vectors of the present invention. The term 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.

In the case of a modified Rhabdovirus genome where G gene is deleted, 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. Thus, in some cases, 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. In some cases, 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. In another embodiment of the invention 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.

In still another embodiment of the invention, a recombinant Ψ gene deficient Rhabdovirus having a heterologous nucleic acid segment encoding an immunodeficiency virus envelope protein or a subunit thereof is provided. In such cases, the recombinant Ψ 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. These 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.

In yet another embodiment of the invention, a method of inducing an immunological response in a mammal is provided. This method 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. In the method where the rabies virus genome is used, it is deficient in Ψ gene. In some cases, 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.

In another embodiment of the invention an immunogenic composition having any of the above mentioned recombinant Rabdo viruses along with an adjuvant is provided.

In yet another embodiment of the invention a method of inducing an immunological response in a mammal is provided 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.

It is a further object of the invention to present a method of treating a mammal infected with an immunodeficiency 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. In one embodiment the immunodeficiency virus is any HTV-l virus. In another embodiment the non-segmented negative-stranded RNA virus is a Rhabdovirus. In a further embodiment there is an induction of mucosal immunity to the functional immunodeficiency virus envelope protein, or subunit thereof. In another embodiment 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. In one embodiment the immunodeficiency virus is any HIV-1 virus. In another embodiment the non-segmented negative-stranded RNA virus is a Rhabdovirus. In a further embodiment there is an induction of mucosal immunity to the functional immunodeficiency virus envelope protein, or subunit thereof. In another embodiment 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.

BRIEF DESCRIPTION OF THE FIGURES

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)

Figure 3. 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.

Figure 6. Western blot analysis of mice serum antibody response to HTV-l antigens.

Figure 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.

Figure 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 2xl0⁷ 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.

Figure 11. 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 2xl0⁷ 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). Seven days post in vitro stimulation, 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). 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). After infection, the viral polymerase-complex (P and L) begins transcription at the 3' end of the encapsidated genome to generate a short leader RNA followed by sequential synthesis of five viral RNAs. 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.

The generation of 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). This method avoids problems of antisense that are encountered when expressing the non-encapsidated negative-strand genomic RNAs, and positive strand mRNAs, and the same method was later also successful in the recovery of another Rhabdovirus, VSV. (Lawson et al, PNAS, USA, 92:4477-81, 1995). In the present invention a number of recombinant 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. 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

Several different recombinant Rhabdovirus-based and replication-competent expression vectors that express heterologous genes or gene sequences are constructed. In one aspect of the invention an expression vector with its own glycoprotein is constructed. The genome of this recombinant expression vector can be represented as: 3 -N-P-M-G-X-L-5' where X=foreign gene (e.g. HTV-l gρl60, HTV-l gag, or any other HTV-l gene; any STV , HTV-2, Hepatitis C gene, or any other viral antigen) (see Fig. 1). X can be cloned at different genome sites to regulate expression levels. In another aspect of the invention 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. The genome of this recombinant expression vector is represented as: 3 -N- P-M-G (from another virus or viral serotype)-X-L-5' (for example, 3 -N-P-M-G from VSV serotype Indiana)-X-L-5') where X=foreign gene specific (e.g. HIV-1 gpl60, HIV-1 gag, or any other HIV-1 gene; any STV or HTV-2 gene or any other viral antigen). 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). Of note, this method is not restricted to VSV glycoprotein. Because 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. The use of a recombinant chimeric RV/VSV can be used to display the properly folded HIV-1 envelope protein on the surface of the infected cell. In addition, 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. In case of Rabies Virus (RV) 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. For example, a recombinant RV with rearranged genome, VSV glycoprotein, and HTV-l gpl60 (X) can be constructed to have: 3'-X-N-P-G(VSV serotype NJ)-M-L-5\ In still another aspect of the invention 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. Alternatively these constructs can have, in addition, their own glycoproteins (e.g., 3 -N-P-M-HTV-l-gpl60-G-L). Again, 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. 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.

In still another aspect of the invention 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. For example, 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 gρl60, Y can be HIV-1 gag and Z can be HTV-l tat.

An HIV-1 virus vaccine

In a preferred embodiment, an immunodeficiency virus vaccine based on recombinant rabies virus vectors is described. Rabies virus (RV) 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. Second, 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). 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. In addition, 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.

Generation of recombinant vectors The following different recombinant rabies virus vectors are constructed. A new infectious Rabies Virus (RV) vector with a deletion of the ψ-gene (a ~ 400 bases long non- coding sequence fused to the G RNA) and new transcription unit containing a short transcription Stop/Start signal (to express foreign genes) and two single sites (BsiWI and Nhel) to introduce foreign genes is 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 (HIV-lgpl60-RVG) 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.

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. These two sites (Smal/Pac) can be used to replace the RV glycoprotein with that from other viruses. In Fig. 7 a chimeric VSV/RV glycoprotein (VSV ectodomain and transmembrane domain, RV cytoplasmic domain), in combination with HTV-l is shown as an example. However, it should be noted that 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). In another experiment, recombinant RVs expressing chimeric gpl60/RV G without expressing RV G (G-deleted RVs) are generated. These 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).

In still another aspect of the invention a recombinant replication-competent rabies virus expression vector having all of the above combinations can be constructed. For example, 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.

Methods for induction of an immune response

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. One example of administering the gene is by accelerating it into the desired cells as a coating on particles or otherwise. Such 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. Specifically, 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.

In a further aspect of the invention there are provided compositions comprising a Rhabdovirus vector of the present invention for administration to a cell or to a multicellular organism.

Phartnaceutical compositions

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. 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.

Methods of administration The pharmaceutical compositions may be administered in any effective, convenient manner including, for instance, administration by intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes among others. Li therapy or as a prophylactic, 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.

For administration to mammals, and particularly humans, it is expected that the daily dosage level of the active composition of the invention will be from 10² FFU to 10⁸ FFU of virus in the composition or 10 μg/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). Using site directed mutagenesis and a PCR strategy, the Ψ 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 Ψ gene nor the new transcription unit affected the RV vector (deleted). The 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-1₈₉.₆ 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. A positive signal for gpl60 in cells infected with recombinant SBN-NL4-3 and SBN-89.6 confirmed the successful recovery of recombinant RVs expressing HTV-l envelope protein. 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.

Growth characteristics of recombinant RVs

Growth characteristics of 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. To examine the differences in virus replication in detail, 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). Both recombinant RVs expressing HIV-1 gpl60 replicated at only a slightly reduced rate compared to wild-type RV, with the final titers being 2.3- (SBN-NL4-3) or 8- fold (SBN-89.6) reduced. The 20% longer genome size of the recombinant RVs cannot explain the slower growth of these viruses. A recombinant RV expressing a 1.9 kb gene (firefly lucif erase) grew to wild-type RV titers. (Mebatsion, et al., Proceedings of the National Academy of Sciences of the United States of America, 93:7310-4, 1996).

Expression of foreign glycoprotein by recombinant RVs Expression of HTV-l gpl60 by recombinant RVs is also examined. To ensure the expression of HTV-l gpl60 by the recombinant viruses, cell lysates from recombinant RV infected cells are analyzed by Western immunoblotting with an antibody directed against RV (Fig. 3, α-rabies) or HIV-1 gpl20 (Fig. 3, α-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). In contrast to wild-type RV, large syncytium-formation is detected in Sup- Tl cells 24 hours after infection with SBN-89.6 or SBN-NL4-3 (Fig. 4). These results indicate that the expressed HTV-l envelope proteins are properly folded, transported to the cell surface, and are recognized by the HTV-l receptor and coreceptor, CD4 and CXCR4.

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. As expected, only expression of HTV-1₈₉.6 envelope protein in 3T3 cells, expressing CD4 and CCR5, caused fusion of these cells.

Induction of a humoral immune response in mice Anti-gpl20 antibody response in mice infected with RV expressing HTV-l gpl60 is 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. To determine whether the recombinant g l60 proteins expressed by recombinant RV are able to induce an anti-HTV-1 immune response, groups of five BALB/c mice are inoculated subcutaneously in both rear footpads with 10⁶ FFU of SBN, SBN-89.6, or 10⁵ 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.

No response to the HIV-1 envelope is detected in the sera of immunized animals, but an ELISA using RV glycoprotein, instead of HIV-1 gpl20, as an antigen confirmed the RV infection and detected high level of antibodies against RV as early as 11 days after infection. Several studies on viral vectors expressing HTV-l gpl60 indicated that a booster infection or a boost with a recombinant protein is necessary to induce detectable serum antibody response against HIV-1 envelope protein. The high antibody titer detected in the RV ELISA indicated that an additional infection with the recombinant RV would not be promising, therefore 3 out of 5 mice from every group were boosted with lOμg of recombinant gpl20 and gp41 in complete Freund adjuvant. 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). Only sera from mice infected with SBN-89.6 or SBN-NL4-3 and subsequently boosted with recombinant protein are able to react with g l20, whereas all other sera failed to detect any HTV-l protein. None of the sera had gp41-specific bands, even with a gp41 subunit immunization. Induction of neutralizing antibodies

An experiment is also carried out to see whether primary virus infection followed by recombinant protein boost induces neutralizing antibodies against HTV-l. In this experiment, 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. These results are confirmed in two independent experiments. The sera from wild-type RV (SBN) infected mice which received a recombinant gpl20 boost displayed only a very low NA titer of 1:50 (Table 1). These results indicate that a boost injection with recombinant gρl20 following the priming with recombinant RV expressing HIV-1 gpl60 elicits high titers of NA.

Table 1. Neutralizing antibody totres of sera from mice infected with different RVs followed by boost injection of recombinant HIV-1 gpl20/gp41.

The results presented herein demonstrate that a recombinant RV expressing a full- length HTV-l envelope protein is generated. The foreign gene is stably expressed by replication competent RV and induces a strong humoral response in mice against HTV-l envelope protein after infection with recombinant RV and a single subsequent boost of HTV-l gpl20 protein. Infection of 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. Thus, 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.

Induction of long-lasting HIV-1 gplόO-specific CTL.

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). (Schnell, M. J., et al., Proc. Natl. Acad. Sci. USA, 97:3544-3549, 2000.). 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.

To analyze the potency of RV-based vectors to induce a cytotoxic response against HIV-1, six mice were immunized with 2 x 10⁷ foci forming units (FFU) of the recombinant RV expressing HIV-I_NW-. 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. 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. As can be observed in Figure 9, 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. Of note, 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.

CTLs from HIV-1 gplόO immunized mice cross-kill target cells expressing a heteroloeous HTV-l envelope protein

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. To analyze the potency of the vaccine candidate to induce cross-reactive CTLs against gpl60 from different HTV-l strains, 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. For this approach, two groups of six 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).

Three and five weeks after the immunization, three 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). Seven days after the stimulation, 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). The results from two different, independent experiments are shown in Figure 10A for mice immunized with a RV expressing HIV-1 _NL4-₃ Env and in Figure 10B for mice immunized with RV expressing HTV- 1₈₉.₆ Env. As expected, a strong, specific lysis of P815 cells expressing the homologous antigen is observed for both groups. More striking, these effector cells are able to cross-kill P815 target cells expressing heterologous HTV-l envelope proteins. 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-lβ_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

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

HTV-l envelope protein for 7 days. 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_N14-₃ gpl60 or HTV-l gag. As illustrated in Figure 11, 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. Indeed, 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. These data indicate that the cytolytic activity is mediated by the CD8⁺ T-cell sub-population. Furthermore, these results imply that in addition to antibodies, recombinant RV vectors also generate long-lived anti-HTV-1 CD8⁺ T-cell responses.

Discussion

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.

In contrast to the observed humoral response, a single inoculation of mice with a recombinant RV expressing HTV-l envelope protein results in a vigorous CTL response against HIV-1 Env. In addition, these responses are stable for at least 135 days after immunization. One explanation for these strong responses is that RV grows in various cell- lines without killing the cells, which results in longer expression of HIV-1 genes compared to a cytopathogenic viral vector. In addition, the expression of the 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.). Therefore, the question remains if CTLs induced by recombinant RVs are directed against different epitopes. However, several studies indicating that CTLs from HIV-1 infected individuals show cross-reactivity even with different clades of HIV-1, indicating a broad cross-reactivity, is an important requirement for an HIV-1 vaccine. ( Cao, H., et al., /. Virol, 71:8615-23, 1997; Rowland- Jones, S. L., et al, Journal of Clinical Investigation, 102:1758-65, 1998.). There is currently only one study showing cross-clade CTLs reactivities induced with a canarypox-based HIV-1 vaccine in uninfected volunteers. (Ferrari, G., et al., Proc. Natl. Acad. Sci. USA, 94:1396-401, 1997.). The inventors of the present invention are currently analyzing if CTLs against HTV-l gρl60 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.

In contrast to most other viral vectors, only a negligible sero-positivity exists in the human population to RV and immunization with a RV-based vector against HIV-1 will not interfere with immunity against the vector itself. Because oral immunization against RV with a RV vaccine strain is successful and apathogenic in chimpanzees (Report of the forth WHO Consultion on oral immunization of dogs against rabies, unpublished document WHO/Rab.Res./93.42, 1993.), a RV-based vector will also be promising in inducing mucosal immunity against HIV-1. Therefore, the present invention fulfills a long felt, yet unfulfilled need, for a method of treating HIV-1 infections. 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

The following examples further illustrate the present invention, but of course are not in any way limiting its scope. The examples below are carried out using standard techniques, that are well known and routine to those of skill in the art, except where otherwise described in detail. The examples are illustrative, but do not limit the invention. All animal methods of treatment or prevention described herein are preferably applied to mammals, most preferably to humans.

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 Ψ 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-1₈₉.₆ or HTV-1_NL4-₃ gpl60 is inserted using the BsiWI and Nhel sites resulting in the plasmids, pSBN- 89.6 or pSBN-NL4-3 (bottom).

Two single sites are introduced in the previously described RV cDNA pSAD LI 6 upstream of the G (Smal) and Ψ gene (Nhel) by site directed mutagenesis (GeneEditor™ Promega Inc.) using the primers RP11 5 - CCTCAAAAGACCCCGGGAAAGATGGTTCCTCAG-3' (SEQ ID NO: 1) and RP12 5'- GACTGTAAGGACYGGCTAGCCTTTCAACGATCCAAG-3' (SEQ ID NO: 2) resulting in the plasmid pSN. 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. First, two fragments are amplified by PCR from pSN using Vent polymerase (New England Biolabs Inc.) and the forward primers RP1 5 -

TTTTGCTAGCTTATAAAGTGCTGGGTCATCTAAGC-3' (SEQ ID NO: 3) or RP10 5'- CACTACAAGTCAGTCGAGACTTGGAATGAGATC-3' (SEQ ID NO: 4). The reverse primers were RP18 5'-TCTCGAGTGTTCTCTCTCCAACAA-3' (SEQ ID NO: 5) and RP17 5'- AAGCΓAGCAAAACGΓACGGGAGGGGTGTTAGTTTTTTTCATGGACTTGGATCGTT

GAAAGGACG-3' (SEQ ID NO: 6). 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 - CCΓCΓAGATTATAGCAAAGCCCTTTCCAAG-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. For rescue experiments of the recombinant RVs, the previously described vaccinia virus-free RV recovery system is used (see Finke, et al, Journal of Virology, 73:3818-25, 1999). In brief, BSR-T7 cells, which stably express T7 RNA polymerase (a generous gift of Drs. S. Finke and K.-K. Conzelmann) are transfected with 5 μg of full-length RV cDNA in addition to plasmids coding for the RV N-, P-, and L-proteins (2.5 μg, 1.25 μg, and 1.25 μg) respectively, using a Ca₂PO transfection kit (Stratagene) as indicated by the vendor. Three days after transfection, 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

(7xl0⁶ cells) with a multiplicity of infection (MOI) of 5 with SBN, SBN-89.6, or SBN-NL4-3 in a total volume of 2 ml. After incubation at 37°C for 1 hour, inocula are removed and cells are washed four times with phosphate-buffered saline (PBS) to remove any unabsorbed virus.

Three milliliters of complete medium is added back and 100 μl of tissue culture supematants are removed at 4,16, 24 and 48 hours after infection. Virus aliquots are titered in duplicate on BSR cells.

In 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 (α-gpl20, lanes 3 and 4). No signal is detected either in the mock or SBN infected cells (α-gpl20, lanes 1 and 2). Successful infection of the cells by the recombinant RVs is confirmed with a polyclonal antibody directed against RV (α-rabies, lanes 2, 3, and 4). Shown in Fig. 4. are Sup-Tl cells which are infected using a MOI of 1 with SBN,

SBN-89.6, or SBN-NL4-3. Twenty-four hours after infection, syncytia-formation is detected in cell cultures infected with recombinant RV expressing HIV-1 gpl60 (panel SBN-89.6 and SBN-NL4-3), indicating expression of functional HIV-1 envelope protein. No cell fusion is detected in cultures infected with wild-type RV (panel SBN).

Example 4: Immunization.

Groups of five 4-6 week old female BALB/c mice obtained from Jackson Laboratories are inoculated subcutaneously in both rear footpads with 10⁶ foci forming units (FFU) SBN, SBN-89.6, or 10⁵NL4-3 in DMEM + 10% FBS. Three out of five mice in each group are boost immunized intraperitonealy three months after infection with 10 μg recombinant gp41 (inB, Intracel Inc.) and 10 μg recombinant gpl20 (IIIB, Intracel Inc.) in 100 μl complete Freunds adjuvant.

Example 5: Enzyme-linked Immunosorbent Assay (ELISA ). Recombinant HTV-l gpl20 (TUB strain, Intracel) is resuspended in coating buffer (50 mM Na₂C0₃, pH 9.6) at a concentration of 200 ng/ml and plated in 96 well ELISA MaxiSorp plates (Nunc) at 100 μl 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. Plates are washed three times followed by the addition of horseradish peroxidase-conjugated (HRP) goat anti-mouse-IgG (H+L) secondary antibody (1:5000, Jackson IxnmunoResearch Laboratories). After a 30 minute incubation at 37°C, plates are washed three times and 200 μl OPD-substrate (o-phenylenediamine dihydrochloride, Sigma) is added to each well. The reaction is stopped by the addition of 50 μl of 3 M H₂S0₄ per well. Optical density is determined at 490 nm. Shown in Fig. 5 is a graph depicting ELISA reactivity of mouse sera against HTV-l gpl20. 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.

Example 6: Western Blotting.

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). After blocking for 1 hour (5% dry milk powder in PBS pH 7.4), blots are incubated with sheep -gpl20 antibody (ARRRP) (1:1000) or human α-rabies sera (1:500) in blocking buffer for 1 hour. Secondary antibodies of goat -human or donkey α-sheep horseradish peroxidase-conjugated (HRP) antibodies (1:5000) (Jackson ImmunoResearch Laboratories) are added and blots incubated for one hour. Each antibody incubation is followed by three washes with WB-wash buffer (PBS pH 7.4, 0.1% Tween-20).. Chemiluminescence (NEN) is performed, as directed by the manufacturer.

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 α-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 (α-SBN, α-SBN-89.6 or α-SBN-NL4-3), or immunized and boost injected with recombinant gpl20 and gp41 (α-SBN*, α-SBN-89.6* or α-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% C0₂ for 4-5 days. 100 μl 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.

All publications and references, including but not limited to patent applications, cited in this specification, are herein incorporated by reference in their entirety as if each individiual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth.

While this invention has been described with a reference to specific embodiments, it will be obvious to those of ordinary skill in the art that variations in these methods and compositions may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the claims.

Example 8: Preparation of splenocytes

Spleens are aseptically removed and single cells suspensions are prepared. Red blood cells are lysed with ACK lysing buffer (BioWhitaker) and the remaining splenocytes are washed twice in RPMI-10 media containing 10% fetal bovine serum. Splenocytes are divided into effector and stimulator cells. Stimulator cells are prepared by infection with recombinant vaccinia virus (moi = 10) expressing an envelope protein from HTV-l at a multiplicity of infection (moi) of 1 for two hours. Cells are washed with PBS once to remove excess virus and incubated for 16 hours at 37°C. After incubation, the vaccinia virus is inactivated using Psoralen (Sigma) (infra). Stimulator cells are added back to the effector cell population at a ratio of 3:1 and 10% T-STTM (Collaborative Biomedical Products) is added as a source of interleukin-II (TL-2). Inactivation of virus with Psoralen

Following incubation of splenocytes with the vaccinia virus, 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.

Preparation of chromium labeled target cells.

Target cells (P815) 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. To measure background, 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 μCi ⁵¹Cr 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 ⁵¹Cr 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.

Preparation ofCD8+ depleted T cells.

Seven days post in-vitro stimulation, CD8⁺ T-cells are depleted from the cell culture (CD8^~) and enriched (CD8⁺) using Dynabeads Mouse CD8 (Lyt2), as described by the manufacturer.

Claims

CLAIMSWhat is claimed is:

1. A recombinant Rhabdovirus vector comprising:

(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 said new transcription unit, wherein the recombinant Rhabdovirus vector is replication competent, and said heterologous viral nucleic acid sequence encodes an antigenic polypeptide.

2. The recombinant Rhabdovirus vector of Claim 1, wherein said modified rhadovirus genome is a modified rabies virus genome.

3. The recombinant Rhabdovirus vector of Claim 2, wherein said modified rabies vims genome has a second modification to have a glycoprotein from another class of vims in place of a rabies viras glycoprotein.

4. The recombinant Rhabdovims vector of Claim 3, wherein said glycoprotein from another class of virus is vesicular stomatitis vims glycoprotein.

5. The recombinant Rhabdovims vector of Claim 3, wherein said modified rabies virus genome has a third modification to have contiguity of structural genes different from that in said modified rhabodvirus genome after said second modification.

6. The recombinant Rhabdovims vector of Claim 1, wherein said heterologous viral nucleic acid encodes an antigenic polypeptide selected from the group consisting of a full- length HIV envelope protein, HIV gpl60, HIV gag, HTV gpl20, and full-length STV envelope protein.

7. The recombinant Rhabdovims vector of Claim 6, wherein said heterologous viral nucleic acid is fused to a sequence of a cytoplasmic domain of a glycoprotein gene of said modified Rhabdovims genome to produce a chimeric protein such that said chimeric protein has a fusion between a transmembrane domain of said heterologous protein and a cytoplasmic domain of said glycoprotein.

8. The recombinant Rhabdovims vector of Claim 1 further comprising a deletion of a recombinant Rhabdovims glycoprotein gene, and wherein said heterologous viral nucleic acid is fused to a sequence of a cytoplasmic domain of a glycoprotein gene of said modified Rhabdovirus genome to produce a chimeric protein which functionally substitutes for said recombinant Rhabdovims glycoprotein gene.

9. A recombinant Rhabdovims that expresses a functional HTV envelope protein wherein said recombinant Rhabdovirus is replication-competent.

10. The recombinant Rhabdovims of Claim 9, wherein said Rhabdovims is a recombinant rabies vims or a recombinant vesicular stomatitis virus.

11. The recombinant Rhabdovims of Claim 9, wherein said HTV envelope protein is from any HIV-1 isolate.

12. An immunogenic composition comprising a recombinant Rhabdovims vector as in any one of claims 1 to 9 and an adjuvant.

13. A recombinant Ψ gene deficient rabies virus comprising a heterologous nucleic acid segment encoding an immunodeficiency virus envelope protein, or a subunit thereof .

14. The recombinant Ψ gene deficient rabies vims of Claim 13, wherein said Rhadovims is a rabies vims.

15. The recombinant Ψ gene deficient rabies vims of Claim 13, wherein said immunodeficiency virus envelope protein, or a subunit thereof, is from a human immunodeficiency virus.

16. The recombinant Ψ gene deficient rabies vims of Claim 13, wherein said immunodeficiency viras envelope protein, or a subunit thereof, is from a simian immunodeficiency viras.

17. A method of inducing an immunological response in a mammal, comprising: a) delivering to a tissue of said mammal a recombinant Rhabdovims vector that expresses a functional immunodeficiency viras envelope protein, or a subunit thereof, effective to induce an immunological response to said envelope protein; b) expressing said envelope protein, or the subunit thereof, in vivo; c) boosting said mammal by delivering an effective dose of an isolated immunodeficiency viras 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 said mammal from an immunodeficiency virus.

18. The method of Claim 17, wherein said recombinant Rhabdovims comprises a rabies virus genome.

19. The method of Claim 18, wherein said rabies virus genome is deficient in Ψ gene.

20. The method of Claim 18, wherein said rabies vims genome is deficient in a rabies vims glycoprotein gene.

21. The method of Claim 19, wherein said rabies vims genome has glycoprotein gene from another class of Rhabdovims in place of a rabies vims glycoprotein.

22. A method of inducing an immunological response in a mammal, comprising: a) delivering to a tissue of said 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 said envelope protein; b) expressing said envelope protein, or the subunit thereof, in vivo; c) boosting said mammal by delivering an effective dose of an isolated immunodeficiency viras 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 said mammal from an immunodeficiency virus.

23. The method of Claim 22, wherein said non-segmented negative-stranded RNA virus is a Rabies virus or a Vesicular Stomatitis vims.

24. A recombinant non-segmented negative-stranded RNA vims vector comprising: a) a modified negative-stranded RNA vims genome that is deficient in ψ gene; b) a new transcription unit that is inserted into said modified negative-stranded RNA virus genome to express heterologous nucleic acid sequences; and c) a heterologous viral nucleic acid sequence that is inserted into said new transcription unit, wherein said recombinant non-segmented negative-stranded RNA virus vector is replication competent, and said heterologous viral nucleic acid sequence encodes an antigenic polypeptide.

25. A method of treating a mammal infected with an immunodeficiency virus, comprising: a) administering to said mammal a non-segmented negative-stranded RNA virus that expresses a functional immunodeficiency vims envelope protein, or subunit thereof; b) expressing said functional immunodeficiency virus envelope protein, or subunit thereof; c) boosting said mammal 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 to said functional immunodeficiency viras envelope protein, or subunit thereof.

26. The method of Claim 25, wherein said immunodeficiency vims is any HTV-l vims.

27. The method of Claim 25, wherein said non-segmented negative-stranded RNA virus is a Rhabdovirus.

28. The method of Claim 25, further comprising an induction of mucosal immunity to said functional immunodeficiency vims envelope protein, or subunit thereof.

29. The method of Claim 25, wherein said long-lasting cellular response further comprises a cross-reactive CTL response wherein said cross-reactive CTLs are directed against envelope proteins, or subunits thereof, from different immunodeficiency vims strains.

30. A method of protecting a mammal from an immunodeficiency virus infection, comprising: a) administering to said mammal a non-segmented negative-stranded RNA virus that expresses a functional immunodeficiency viras envelope protein, or subunit thereof; b) expressing said functional immunodeficiency vims envelope protein, or subunit thereof; c) boosting said mammal 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 to said functional immunodeficiency vims envelope protein, or subunit thereof.

31. The method of Claim 30, wherein said immunodeficiency virus is any HIV-1 virus.

32. The method of Claim 30, wherein said non-segmented negative-stranded RNA virus is a Rhabdovirus.

33. The method of Claim 30, further comprising an induction of mucosal immunity to said functional immunodeficiency viras envelope protein, or subunit thereof.

34. The method of Claim 30, wherein said long-lasting cellular response further comprises a cross-reactive CTL response wherein said cross-reactive CTLs are directed against envelope proteins, or subunits thereof, from different immunodeficiency vims strains.