CN1416471A - Recombinant rhabdoviruses as Live-Viral vaccines for immunodoeficiency 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 virus vaccines against immunodeficiency viruses

Government Rights in the Invention

Portions of this invention were made with government support under grant AI44340 awarded by the National Institute of Health. The Government has certain rights in this invention. Cross-references to related applications

This application claims priority based in part on US nonprovisional 09/494,262 filed January 28, 2000 under 35 U.S.C § 120.

field of invention

The invention relates to the fields of molecular biology and virology, and also relates to a method for treating HIV-1 infection, more specifically, the induction of anti-HIV-1 humoral and cellular immunity.

Background of the invention

Over the past few years, great success has been achieved in the treatment of HIV-1 infection. (Holtzer et al., Annals of Pharmacotherapy 33:198-209, 1999; Bonfanti et al., Biomedicine & Pharmacotherapy, 53:93-105, 1999). However, the development of a protective immunodeficiency virus vaccine, such as the HIV-1 virus vaccine, remains a major goal in terminating immunodeficiency virus infection. Most successful vaccines against viral diseases consist of killed or attenuated viruses (Hilleman, M.R., Nature Medicine, 4:507-14, 1998). This approach does not appear to be suitable for immunodeficiency viruses, especially HIV-1, because inactivated HIV-1 virus induces only a weak neutralizing antibody response and no cytotoxic T lymphocyte (CTL) response.

In developed countries, new antiretroviral strategies against human HIV-1 have led to a dramatic reduction in mortality among infected populations. But the development of a successful vaccine to prevent infection remains a major goal in ending HIV-1 infection. On average, every 10 seconds, a person is infected with HIV-1, and in heavily infected countries in Africa, such as Zambia and Uganda, almost 40% of young adults are HIV-1 seropositive. (1)

Currently, various HIV vaccine strategies are being investigated, including recombinant proteins (Goebel, F.D. et al., European Mutinational IMMUNO AIDS Vaccine Study Group Aids, 5:643-50, 1999; Quinnan, G.V., Jr., et al., AIDS 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., AIDS, 13:2003-12, 1999), naked DNA (Bagarazzi, M.L., et al., 1999, Journal of Infections 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; Putkonen, 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 non-replicating (replicon) live viral vectors (Berglund, P., et al., AIDS Research & Human Retroviruses, 13 : 1487-95, 1997; Mossman, S.P. et al., J.Virol., 70:1953-60, 1996; Natuk, R.J. et al., Proc.Natl.Acad.Sci.USA, 89:7777-81, 1992; Ourmanov, I., et al., J. Virol., 74:2740-2751, 2000; Schnell, M.J., et al., Proc. ), and prime-boost combination (see (5) for review). A number of these vaccine strategies have been tested in the simian immunodeficiency virus (SIV) macaque model system, but to date effective protective immunity has not been achieved although some disease amelioration has been observed. (Barouch, D.H. et al., Science, 290:486-492, 2000; David, N.L. et al., J.Virol., 74:371-8, 2000; Ourmanov, I., et al., J.Virol., 74 : 2740-2751, 2000). So far, the only effective way to protect macaques from SIV infection is to use live, attenuated SIV. Desrosiers and colleagues demonstrated that a genetically modified, nef-deleted SIV strain that does not cause disease in rhesus monkeys induced high titers of anti-SIV 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.). Animals subsequently challenged with an infectious dose of the pathogenic SIV strain were protected against infection. (Daniel, M.D. et al., Science, 258:1938-1941, 1992). A major drawback of the attenuated lentiviral vaccine approach is that even nef-deficient SIV can produce an AIDS-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 .). Another effect recently discovered regarding the use of attenuated lentiviruses is that in some cases priming virus recombines with live, attenuated SIV and even produces lethal strains. (Gundlach, B.R. et al., J. Virol., 74:3537-3542, 2000.). However, the results suggest that live viral vectors may be an excellent vaccine candidate for an HIV-1 vaccine.

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

The immune response required against HIV-1 infection is currently unknown, but a protective immune response to HIV-1 likely requires two major pathways of the immune system. A recent report on a vaccine approach with recombinant HIV-1 coat protein demonstrated that an exclusive humoral response was insufficient against HIV-1 infection, but passive transfer of three monoclonal antibodies directed against HIV-1 coat protein was essential for subsequent Rhesus monkeys challenged with pathogenic HIV-1/SIV chimeric viruses were protected. (Mascola, J.R. et al., Nature Medicine, 6:207-10, 2000.). Other studies have shown that cell-mediated responses play an important role in controlling HIV-1 infection. (Brander, C. and B.D. Walker, Current Opinion in Immunology, 11:451-9 1999; Goulder, P.J. et al., Anti-HIV cellular immunity: recent advances towards vaccine design Aids, 13:S121-36, 1999.). Exposed but not infected individuals often have HIV-1-specific CTLs but no detectable anti-HIV-1 antibodies. (Pinto, 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 a recombinant non-segmented negative-strand RNA virus expressing an immunodeficiency virus gene to act as an immunodeficiency virus vaccine (eg, HIV-1 vaccine) is disclosed. In particular, the ability of rhabdovirus-based recombinant viruses to induce an anti-HIV immune response was elucidated. After a single booster immunization in mice with a recombinant HIV-1 protein booster (gp120), the HIV-1 coat protein was stably and functionally expressed and induced a strong humoral response directed against the HIV-1 coat protein. Moreover, high neutralizing titers against HIV-1 could be detected in mouse sera. (Schnell, MJ, et al., Proc. Natl. Acad. Sci. USA, 97:3544-3549, 2000.). Little information is available on the induction of CTL responses to rhabdovirus-based vectors expressing foreign proteins. The present invention fulfills this long sought need and further relates to recombinant RV vaccines expressing HIV-1 coat protein to induce HIV-1 specific CTLLs. In particular, single inoculation of the HIV-1 virus vaccine of the present invention can induce a stable and long-lasting memory CTL response specific to HIV-1 protein. This recombinant virus is apathogenic to a wide range of animal species when administered orally or intramuscularly. In a specific embodiment, when the coding region of HIV-1 gp160 (strains NL4-3 and 89.6) is cloned into the RV glycoprotein (G) and polymerase (L ) proteins, thereby producing recombinant RVs expressing HIV-1 gp160 and expressing other RV proteins. Definition "boosted vaccine vector" is "boosted virus""boostedvirus" is "boosted vaccine vector"

Summary of the invention

The present invention relates to the use of recombinant non-fragmented negative-strand RNA viruses expressing immunodeficiency virus genes as a live virus vaccine (eg, HIV-1 vaccine) and methods for making and using such a live virus vaccine. In more detail, the present invention relates to recombinant rhabdoviruses expressing human immunodeficiency virus gene products, and also to an immunological composition that, when administered to a host, induces an immune response against immunodeficiency virus infection . When administered orally or intramuscularly, this recombinant live virus vaccine is apathogenic to a wide range of animal species and induces protective immune responses, such as neutralizing antibody responses against immunodeficiency virus and long-lasting cellular responses ( such as cytotoxic T lymphocytes).

In summary, the present invention is a recombinant non-fragmented negative-strand RNA viral vector comprising: (a) a modified negative-strand RNA viral genome modified to have one or more novel restriction sites, or without Modified to have one or more genes otherwise present in the genome; (b) a new transcription unit inserted into the genome of a modified negative-strand RNA virus to express a heterologous nucleic acid sequence; and (c) a A heterologous viral nucleic acid sequence, which is inserted into a new transcription unit, wherein the recombinant non-segmented negative-strand RNA viral vector is replication competent, and the heterologous viral nucleic acid sequence encodes an antigenic polypeptide.

In particular, in a specific embodiment of the present invention, the recombinant non-segmented negative-strand RNA virus used as a live virus vaccine is a recombinant rhabdovirus vector. The vector includes: (a) a modified rhabdovirus genome; (b) a new transcription unit inserted into the rhabdovirus genome to express a heterologous nucleic acid sequence; (c) a heterologous viral nucleic acid sequence inserted into into a novel transcription unit in which the recombinant rhabdoviral vector is replication competent and the heterologous viral nucleic acid sequence encodes an antigenic polypeptide. The modified rhabdovirus genome is, for example, a modified rabies virus genome or a modified vesicular stomatitis virus genome. Modifications in the Rhabdovirus genome include the creation of new restriction sites and/or deletion of one or more genes, such as the native G (glycoprotein) gene of Rhabdovirus, the Ψ gene of Rabies virus, etc. In some cases, the modified Rhabdovirus genome had a further modification in which the native glycoprotein was replaced by a glycoprotein derived from another virus. A glycoprotein derived from another virus is the vesicular stomatitis virus glycoprotein. In some other cases, the modified rabies virus genome had a third modification to contiguous with a structural gene that was different from the structural gene of the rhabdovirus genome after the second modification.

The term heterologous viral nucleic acid as used herein refers to a viral nucleic acid encoding an antigenic polypeptide that induces an immune response. For example, a full-length HIV coat protein, HIV gp160, HIV gag, HIV gp120, and full-length SIV coat protein are portions of antigenic polypeptides that are expressed in the recombinant viral vectors of the invention. The term heterologous viral nucleic acid as used herein does not include one or more of the native Rhabdovirus gene sequences in a recombinant Rhabdovirus, such as the VSV G gene in a recombinant RV.

In the case where the G gene of the modified Rhabdovirus genome is deleted, the sequence of the cytoplasmic domain of the Rhabdovirus G gene is fused to other sequences before cloning into the modified Rhabdovirus genome. One such example is the chimeric VSV/RV glycoprotein, where the fusion protein has the ectodomain of VSV, the transmembrane domain and the cytoplasmic domain of RV. Another such example is the chimeric HIV-1/RV glycoprotein, where the fusion protein has the ectodomain of HIV-1 gp160, the transmembrane domain and the cytoplasmic domain of RV. Thus, in some cases, heterologous viral nucleic acid is fused to the sequence of the cytoplasmic domain of the G gene of the rhabdovirus genome to generate a chimeric protein, such that the resulting chimeric protein is in the span of the heterologous protein. There is a fusion between the membrane domain and the cytoplasmic domain of the glycoprotein. In some cases, the glycoprotein gene of the recombinant Rhabdovirus was deleted, and the heterologous viral nucleic acid was fused to the sequence of the cytoplasmic domain of the G gene of the modified Rhabdovirus genome, resulting in a chimeric protein, which The protein functionally replaces the recombinant rhabdovirus glycoprotein gene.

In another specific embodiment of the present invention, a recombinant Rhabdovirus expressing a functional HIV coat protein is provided. Recombinant rhabdoviruses are replication competent. The rhabdovirus may be a recombinant rabies virus or a recombinant vesicular stomatitis virus.

Recombinant Rhabdoviruses express HIV coat proteins from any HIV-1 isolate.

In another specific embodiment of the present invention, there is provided a recombinant rhabdovirus deficient in the Ψ gene, which has a heterogeneous nucleic acid segment encoding an immunodeficiency virus coat protein or a subunit thereof. In this case, the recombinant Ψ gene-deficient rhabdovirus is rabies virus, and the immunodeficiency virus coat protein, or a subunit thereof, is of human immunodeficiency virus or simian immunodeficiency virus origin. A subunit or a fragment of an immunodeficiency coat protein includes a fragment comprising only a portion of the contiguous nucleic acid of the coat protein. These subunits or fragments include, for example, HIV gp120, HIV gp41, HIV gp40, the coat protein expressed by HIV _NL4-3 and HIV _89.6 , and subunits of other immunodeficiency viruses.

Yet in another specific embodiment of the present invention, a method of inducing an immune response in a mammal is provided. This method includes the following steps: (a) delivering to mammalian tissue a recombinant rhabdoviral vector expressing a functional immunodeficiency virus coat protein, or a subunit thereof, that efficiently induces the expression of a coat protein; (b) express coat protein or subunit thereof in vivo; (c) by delivering an effective dose of isolated immunodeficiency virus coat protein or subunit thereof in an adjuvant, or by delivering an effective dose and (d) induce a neutralizing antibody response and/or a long-lasting cellular immune response to protect the mammal against the immunodeficiency virus.

Recombinant rhabdoviruses have a rabies virus genome. In the method, a rabies virus genome deficient in the Ψ gene was used. In some cases, the rabies virus glycoprotein gene of the rabies virus genome was also defective or the rabies virus genome had a gene for a protein derived from another rhabdovirus glycoprotein in place of the rabies virus glycoprotein. Boosting of animals can be accomplished by replacing the isolated immunodeficiency virus coat protein with an effective dose of the booster vaccine vector.

In another specific embodiment of the present invention, there is provided an immunological composition comprising any one of the above-mentioned recombinant rhabdoviruses together with an adjuvant.

Yet in another embodiment of the present invention, there is provided a method of inducing an immune response in a mammal comprising the steps of: (a) administering to a mammalian tissue a non-fragmented negative-strand RNA virus expressing a A functional immunodeficiency virus coat protein, or a subunit thereof, is effective in inducing an immune response to the coat protein; (b) expressing the coat protein or a subunit thereof in vivo; (c) by administering the coat protein in an adjuvant An effective dose of an isolated immunodeficiency virus coat protein or a subunit thereof or by administering an effective dose of a booster vaccine vector to boost the animal; and (d) induce a neutralizing antibody response and/or long-lasting persistent cellular immunity response to protect mammals against immunodeficiency virus.

Methods using non-segmented negative-strand RNA viruses include rabies virus or vesicular stomatitis virus.

It is a further object of the present invention to provide a method of treating a mammal infected with an immunodeficiency virus. A non-segmented negative-strand RNA virus expressing a functional immunodeficiency virus coat protein, or a subunit thereof, is used in mammals. This RNA virus will express a functional immunodeficiency virus coat protein, or a subunit thereof. An effective dose of an isolated immunodeficiency virus coat protein or a subunit thereof, or an effective dose of a booster vaccine vector, is delivered to a mammal via an adjuvant, thereby inducing a neutralization of a functional immunodeficiency virus coat protein or a subunit thereof and antibody responses and/or long-lasting cellular immune responses. In a specific embodiment, the immunodeficiency virus is any HIV-1 virus. In another embodiment, the non-segmented negative-strand RNA virus is a Rhabdovirus. In a further embodiment, there is an induction of mucosal immunity to a functional immunodeficiency virus coat 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 coat proteins, or subunits thereof, from different immunodeficiency virus strains.

Another object of the present invention is to provide a method of protecting mammals against immunodeficiency virus infection. A non-segmented negative-strand RNA virus expressing a functional immunodeficiency virus coat protein, or a subunit thereof, is used to treat mammals. This RNA virus will express a functional immunodeficiency virus coat protein, or a subunit thereof. A neutralizing antibody response and/or a long-lasting cellular immune response to a functional immunodeficiency virus coat protein or a subunit thereof is thus induced. In a specific embodiment, the immunodeficiency virus is any HIV-1 virus. In another embodiment, the non-segmented negative-strand RNA virus is a Rhabdovirus. In a further embodiment, there is an induction of mucosal immunity to a functional immunodeficiency virus coat protein or subunit thereof. In another embodiment, the long-lasting CTL response is a cross-reactive CTL response, wherein the cross-reactive CTL is directed against coat proteins, or subunits thereof, from different immunodeficiency virus strains.

Brief description of the drawings

Figure 1. Schematic illustration of the construction method of the recombinant RV genome.

Figure 2. is a graph showing the one-step growth curve of BSR cells infected with recombinant RVs (SBN, SBN-89.6 and SBN-NL4-3).

Figure 3. Western blot analysis of recombinant rabies viruses (RVs) expressing HIV-1 gp160.

Figure 4. A composite photograph showing Sup-TI cells after infection (at 1 MOI) of these cells with SBN, SBN-89.6 and SBN-NL4-3.

Figure 5. is a graph showing the ELISA activity of mouse anti-HIV-1 gp120 sera.

Figure 6 is a Western blot analysis of mouse serum antibody responses to HIV-1 antigens.

Figure 7. is a schematic representation of the construction method of exogenous viral glycoproteins and RV-based expression vectors.

Figure 8. is a schematic representation of the construction method of RV glycoproteins of full-length and deleted RVs expressing HIV-1 gp160.

Figure 9. HIV-1 gp160 CTLs derived from immunized mice induce long-term persistent specific HIV-1 gp160 CTLs. Three 6-8-week-old female BALB/c mice (Harlan-Sprague) were inoculated peritoneally with 2×10 ⁷ colony-forming units expressing HIV-1 _NL4-3 coat protein recombinant RV in each group. Between 105 and 135 days after single inoculation, spleens were aseptically excised and single cell suspensions were prepared (see below). Stimulator cells were prepared (see below) and added back to the effector cell population at a 3:1 ratio. The cytolytic activity of cultured CTLs was determined by determination of the percentage ^of51Cr released (below).

Figure 10. Cross-inactivated target cell CTLs from HIV-1 gp160-immunized mice expressing heterologous HIV-1 coat protein. Six female BALB/c mice aged 6-8 weeks in each group were treated with 2× ¹⁰⁷ recombinant RV derived from colony-forming units expressing HIV-1 coat protein derived from NL4-3(A) or 89.6(B) strains Peritoneal inoculation. Three to four weeks after the single inoculation, the spleen was aseptically excised and splenocytes were stimulated in vitro with vaccinia virus expressing the heterologous HIV-1 coat protein (see below). Target cells were prepared by infecting vaccinia virus expressing HIV-1 coat protein derived from NL4-3 (vCB41), 89.6 (vBD3), JR-FL (VCB28) or Ba-L (vCB43) strains. Chromium release testing was done (see below). Results are shown in two different independent experiments.

Figure 11. Cytolytic activity is mediated by CD8 ⁺ T cells. Three 6-8-week-old female BALB/c mice in each group were inoculated intraperitoneally with 2×10 ⁷ recombinant RV expressing HIV-1 coat protein colony-forming units derived from NL4-3 strain. Eighteen weeks after the single inoculation, spleens were aseptically excised and splenocytes were stimulated in vitro with vaccinia virus expressing the HIV-1 _NL4-3 coat protein (see below). After 7 days of in vitro stimulation, CD8 ⁺ T cells were depleted from cell cultures (CD8 ⁻ ) and enriched with Dynabeads Mouse CD8 (Lyt2) as specified by the manufacturer. Chromium release assays were done in depleted (CD8 ⁻ ) or enriched (CD8 ⁺ ) cultures of CD8 T cells, or untreated (CD8 ⁺ /CD8 ⁻ ) cultures (see below). Target cells were prepared by infection with vaccinia virus expressing HIV-1 coat protein derived from the NL4-3 (vCB41) strain (see below). Background levels were at or below 6% of specific lysis.

Detailed Description of the Invention

Rhabdoviruses, such as rabies virus and vesicular stomatitis virus, are members of the Rhabdovirus genus family. Rabies virus has a negative-sense RNA genome of approximately 12 kb. This genome is organized in a standard manner, similar to the vesicular virus (VSV) genome. These rhabdoviruses encode five structural proteins. The five open reading frames encoding 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) initiates transcription at the 3' end of the capsidated genome, producing a short leader RNA, which is followed by the sequence synthesis of five viral RNAs. Nucleoprotein (N), phosphoprotein (P), viral polymerase (L), and genomic RNA form a helical ribonucleic acid protein complex (RNP). The ribonucleic acid protein complex (RNP) is surrounded by a host cell-derived coat membrane containing the matrix protein (M) on the inside of the cell membrane and a transmembrane glycoprotein (G) that mediates viral binding to specific receptors on the cell membrane.

The inventors have previously reported that the production of non-segmented negative-strand RNA viruses is entirely derived from cDNA. (Schnell et al., EMBO, 13:4195-4203, 1994). This method involves the intracellular expression of anti-genomic RNA in cells, and also expresses the viral proteins required for an active RNP complex formation, i.e., nucleoprotein (N), phosphoprotein (P), viral polymerase ( L). This approach avoided the antisense problems encountered when expressing non-encapsidated negative-sense genomic RNAs and positive-sense mRNAs, and the same approach was later successfully used for the recovery of another rhabdovirus, VSV. (Lawso et al., PNAS, USA, 92:4477-81, 1995).

In the present invention, large numbers of recombinant rhabdoviral vectors were produced and used to express functional genes, including the full-length HIV-1 coat protein. From the recombinant Rhabdovirus vector of the present invention, all the dominant epitopes for neutralizing antibodies, cytotoxic T lymphocytes (CTL) and antibody-dependent cellular cytotoxicity are expressed at once. The following sections illustrate the construction of different recombinant rhabdoviruses expressing HIV or SIV or other viral genes.

Recombinant rhabdovirus expression vector

Construction of several different rhabdovirus-based and replication-competent recombinant expression vectors expressing heterologous genes or gene sequences. In one aspect of the invention, an expression vector is constructed with its own glycoprotein. This recombinant expression vector genome can be expressed as: 3'-N-P-M-G-X-L-5', wherein X is a foreign gene (for example: HIV-1 160gp, HIV-1 gag, or any other HIV-1 gene, any SIV, IIIV- 2, hepatitis C gene, or any other viral antigen) (see Figure 1). X can be cloned at different gene loci to modulate expression levels.

In another aspect of the invention, an expression vector containing a glycoprotein derived from another virus or another virus serotype is constructed (see Figure 7 for an example of an RV vector with VSV glycoprotein). This vector is used to boost the virus to induce a stronger immune response. The genome of this recombinant expression vector is represented as follows: 3'-N-P-M-G (derived from another virus or viral serum)-X-L-5' (for example, 3'-NP-M-G derived from Indiana VSV serotype)-X-L-5 '), where X is a specific foreign gene (eg, HIV-1 gp160, HIV-1 gag, or any other HIV-1 gene, any SIV or HIV-2 gene or any other viral antigen). X can be cloned at different gene loci to modulate expression levels. The present invention relates to the construction of recombinant RVs (rabies virus) expressing HIV-1gp160, wherein the RV glycoprotein (G) uses a chimeric vesicular stomatitis virus (VSV) G/RV-cytoplasmic domain (Indiana or New Jersey serotype) glycoprotein instead. It should be noted that this method is not limited to VSV glycoproteins. Because Rhabdovirus has only a single surface protein on its virion, the chimeric RV/VSV virus is not neutralized by the humoral response against RV G, thus allowing a second productive infection. The utility of recombinant chimeric RV/VSV is that it can be used to display the properly folded HIV-1 coat protein on the surface of infected cells. In addition, the repeated expression of the RV nucleoprotein 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 ), may help to enhance the immune response against HIV-1 coat protein. For rabies virus (RV), the cytoplasmic domain of the RV glycoprotein is fused to a foreign glycoprotein.

It should be noted that all genes in the recombinant genome can be rearranged to attenuate the virus or to enhance the transcription of foreign genes. For example, a recombinant RV with rearranged genome, VSV glycoprotein, and HIV-1 gp160(X) can be constructed as: 3'-X-N-P-G(VSV serotype NJ)-M-L-5'.

In another aspect of the present invention, a recombinant expression vector (or RVs, or VSVs) is constructed, which contains an exogenous glycoprotein that replaces its own glycoprotein, and enters a specific host cell, for example, in order to induce a A stronger immune response mimics the tropism of other viruses (eg, HIV-1, hepatitis C) (Figure 8). Such constructs can be represented as: 3'-N-P M-HIV-1-gp160-L. Alternatively, these constructs can have their own glycoproteins (e.g., 3'-N-P-M-HIV-1-gp160 -G-L.). Furthermore, it should be noted that all genes within the recombinant genome can be rearranged to attenuate the virus or enhance transcription of foreign genes. Transgenic mice expressing human CD4 and CXCR4 were generated to analyze the induction of immune responses in G-associated RVs expressing HIV-1 gp160/RVG in vivo.

In another aspect of the present invention, a recombinant expression vector (either RV or VSV) with multiple antigens and multiple transcription termination/initiation signals was 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 HIV-1 gp160 and Y can be HIV-1 gag. An alternative construct could be 3'-N-Z-P-M-G-X-Y-L-5', where, for example, X could be HIV-1 gp160, Y could be HIV-1 gag, and Z could be HIV-1 tat.

HIV-1 virus vaccine

In a preferred embodiment, an immunodeficiency virus vaccine based on a recombinant rabies virus vector is described. Rabies virus (RV) is a negative-strand RNA virus of the Rhabdovirus family that has a relatively simple, regulatable genetic organization encoding five structural gene proteins (see above and Conzelmann, et al., Virology, 175: 485-99, 1990.). The present invention relates to a vector based on an RV vaccine strain which is apathogenic to a wide range of animal species when administered orally or intramuscularly. There are several reasons for the advantages this vector exhibits over other viral vectors. First, its adjustable genome organization makes its genetic modification easier than most of the more complex DNA and positive-strand RNA viruses. Second, rhabdoviruses have a cytoplasmic replication cycle and there is no evidence of recombination and/or integration into the host cell genome. (Rose, et al., Rhabdovirus genomes and their products, Plenum Publishing Corp., New York, 1997). Compared with most other viral vectors, the population has only a negligible seropositivity for RV, and immunization with an RV-based vector against HIV-1 will not interfere with immunity to the vector itself. In addition, RV can grow to high titers of ¹⁰⁹ colony-forming units (FFU) in various cell lines without killing cells, which may cause longer HIV-1 genes compared with a cytopathic vector. Express.

Production of recombinant vectors

Different recombinant rabies virus vectors were constructed below. A new infectious rabies virus (RV) vector was constructed that had a Ψ gene deletion (a ~ 400 base long non-coding sequence fused to a G RNA) and contained a short transcription termination/initiation signal (expressing exogenous gene) and two new transcription units at single loci (BsiWI and NheI) to introduce exogenous genes. This vector also contains a SmaI site upstream of the RV glycoprotein for deletion of the RV glycoprotein gene (G). This vector was named RV-SBN. RVs expressing the ectofunctional and transmembrane domains of HIV-1 gp160 fused to the RV G cytoplasmic domain (HIV-1 gp160-RVG) were constructed. The chimeric gp160/RVG protein is expressed by RV and incorporated into RV virions. Recombinant viruses displaying a foreign coat protein on the surface of the recombinant virus will induce a strong immune response to that antigen.

Another RV vector was also generated which is identical to RV-SBN but has an additional single PacI site downstream of the RV G protein. This vector is used to functionally replace RV G with VSV G or other viral glycoproteins. This vector was named RV-SPBN and was used as a booster vaccine vector or a booster virus.

As shown in Fig. 7, a rabies virus-based expression vector containing foreign viral glycoprotein was constructed, and the recombinant virus was recovered. For this construct, a SmaI restriction site was introduced downstream of the M/G transcriptional termination/initiation sequence and upstream of the synthetic transcriptional termination/initiation sequence for expression of foreign genes derived from RV vectors. PacI site. These two sites (SamI/Pac) can be used to replace the RV glycoprotein with glycoproteins from other viruses. In Figure 7, a chimeric VSV/RV glycoprotein (VSV ectodomain and transmembrane domain, RV cytoplasmic domain) is shown binding HIV-1 as an example. However, it should be noted that this method can be applied to each glycoprotein and exogenous antigen in different rhabdoviruses as shown in the same figure (glycoprotein is X, exogenous protein is Y).

In another experiment, recombinant RVs expressing chimeric gp160/RV G but not RV G (G-deleted RVs) were generated. These G-deleted RVs have a different tropism than wild-type RV (infecting host cells), and can only specifically infect CD4 and HIV-1 co-receptors in humans expressing HIV-1 receptors (e.g., CXCR4 or CCR5) in cells.

Recombinant rabies RVs full-length and RV glycoprotein-deleted were constructed and recovered (Fig. 8). SamI and BsiWI restriction endonuclease sites were used to delete the RV glycoprotein and to fuse M/G transcription termination/initiation sequences into the HIV-1/RV chimeric glycoprotein (HIV-1 gp160 ectodomain and transmembrane domain, RV cytoplasmic domain). The recovered RV vector complexes similarly to HIV-1 virus and is specific for cells expressing human CD4 and the appropriate HIV-1 complex receptor. It should be noted that this method can be applied to every glycoprotein supporting certain cell types infected by rhabdoviruses. It has also been used to express additional foreign antigens (HIV-1 Gag, HIV protease, SIV protein, hepatitis A, B or C protein, and other viral and non-viral proteins).

In another aspect of the present invention, a recombinant replication-competent rabies virus expression vector containing all of the above combinations was constructed. For example, having other glycoproteins (particularly to construct booster vaccines) without or with its own G, rearrangements of the genome, and expression of multiple Recombinant rabies virus vector for viral antigens.

Products, methods and compositions

The present invention provides products, compositions and methods of evaluation for the treatment of viral diseases, especially HIV (AIDS) and administration of recombinant rhabdoviruses of the invention to a subject to increase its immune response against infiltrating viruses, especially against immunodeficiency virus method of infection.

Methods of Inducing an Immune Response

Another aspect of the invention relates to a method of inducing an immune response in an individual, especially a mammal, comprising inoculating the individual with a recombinant virus of the invention and subsequently boosting the individual with a suitable recombinant protein to produce sufficient antibodies and/or The T cell immune response protects the individual against infection, especially against immunodeficiency infection, more preferably against HIV-1 and 2 infection. Also provided are methods of slowing the immune response of HIV replication.

Another aspect of the present invention also relates to a method of inducing an immune response in an individual, the method comprising delivering to the individual a nucleic acid vector, sequence or ribozyme to direct the expression of HIV coat polypeptides, or fragments and variants thereof, so that Expressing HIV coat polypeptides, or fragments and variants thereof in vivo, achieves the purpose of inducing immune responses, such as producing antibodies and/or T cell immune responses. Antibody and/or T cell responses include, for example, the production of cytokine T cells or cytotoxic T cells to protect an individual, especially a human, against a viral disease regardless of the presence or absence of disease in the individual. One example of applying a gene is to facilitate its entry into target cells by coating or other means on the particle. Such nucleic acid vectors may include DNA, RNA, ribozymes, modified nucleic acids, DNA/RNA hybrids, DNA protein complexes or RNA protein complexes.

Compositions that induce an immune response

A further aspect of the invention relates to an immunological composition having the ability to induce an immune response when introduced into an individual, preferably a human. The immune response induced is to a polynucleotide and/or polypeptide encoded thereby, wherein the composition comprises a recombinant rhabdovirus of the invention encoding and expressing an antigen of an exogenous viral protein, such as the HIV coat protein or polypeptide. In particular, foreign polypeptides include antigenic and immunogenic polypeptides. The immune response is used therapeutically or prophylactically, and employs antibody immunity and/or cellular immunity, eg in the form of cellular immunity elicited by CTL or CD4+ T cells.

In a further aspect of the invention there is provided a composition comprising a rhabdoviral vector of the invention useful in unicellular or multicellular organisms.

pharmaceutical composition

The rhabdoviral vectors of the present invention can be used in combination with non-sterile or sterile vectors, or vectors used with cells, tissues or organisms, such as pharmaceutical vectors suitable for individual use. Such compositions include, for example, a vehicle additive or a therapeutically effective amount of the recombinant virus of the invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The prescription should suit the mode of administration. The invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers containing one or more components of the above-described compositions of the invention.

The recombinant vectors of the present invention can be used alone or in combination with other compounds, such as therapeutic compounds.

Medication method

Pharmaceutical compositions may be administered by any effective and convenient method including, for example, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal methods, among others. For therapy or prophylaxis, the active factors are administered to the individual in the form of injectable compositions, for example in the form of sterile aqueous dispersions, preferably isotonic dispersions. The pharmaceutical composition of the present invention is preferably administered by injection to achieve a systemic effect against relevant viral pathogens.

For administration to mammals, especially humans, the active compositions of the invention are expected to have daily dosage levels of ¹⁰² FFU to ¹⁰⁸ FFU of virus in the composition, or 10 μg/kg to 10 mg/kg of recombinant protein by body weight. In any case, the physician will determine the actual dosage and duration of treatment which will be most suitable for the individual and may vary with the age, weight and response of the particular individual. The above doses are an example of average conditions. There may, of course, be individual instances where higher or lower dosage ranges are desirable, such instances being within the scope of this invention.

The vaccine compositions are conveniently used in injectable form. Traditional adjuvants can be used to enhance the immune response. Suitable unit doses for immunization are preferably administered daily, and may or may not be at intervals of at least 1 week. With the indicated dosage ranges, no toxicological side effects were observed with the compositions of the invention that would preclude administration to suitable individuals.

Recombinant RV vector expressing HIV-1 coat protein

In a preferred embodiment, recombinant RVs expressing HIV-1 coat protein are described. To generate HIV-1 gp160-expressing RV recombinant virus, a new vector was constructed based on pSAD-L16 of the previously infected RV cDNA clone (Schnell, et al., EMBO Journal, 13:4195-4203, 1994). Using a site-directed mutagenesis and PCR strategy, the Ψ gene was deleted from the RV genome and a new transcriptional unit including an RV stop/start signal and two unique sites (BsiWI and NheI) was introduced into the RV genome (see Recombination Vector generation, see above). The resulting plasmid was named pSBN (Figure 1). SBN RV vectors were recovered by the reported method and showed the same growth characteristics and similar viral titers as SAD-L16, indicating that neither the deletion of the Ψ gene nor the new transcriptional unit affected the RV vector (deleted) . The HIV-1 coat genes (NL4-3 and 89.6) expressed from SBN by PCR were cloned between the BsiWI and NheI sites, resulting in the pSBN-NL4-3 and pSBN-89.6 plasmids (Figure 1). All constructs were checked by DNA sequencing. It should be noted that the foreign gene is stable up to at least 4 kb in the RV genome, and that recombinant RVs express a full-length HIV-1 coat protein.

Recombinant RVs expressing HIV-1 _NL4-3 , or HIV-1 _89.6 coat protein were recovered by transfection of cells stably expressing T7-RNA-polymerase with plasmids encoding RV N, P and L proteins, There are also plasmids encoding the RNAs of the respective RV full-length anti-genomes. Three days after transfection, the suspension of transfected cells was transferred to fresh cells, and 3 days later, the expression of HIV-1 gp160 was analyzed by indirect immunofluorescence microscopy. A positive signal for gp160 in cells infected with recombinant SBN-NL4-3 and SBN-89.6 confirmed successful recovery of recombinant RVs expressing HIV coat protein. Recombinant RVs expressing HIV-1 gag were also constructed and recovered by the same process as recombinant RVs expressing HIV-1 coat protein.

Growth characteristics of recombinant RVs

The growth properties of recombinant RVs expressing the HIV-1 coat protein were examined. Note a 3-fold titer reduction for SBN-NL4-3 and a 10-fold titer reduction for SBN-89.6 compared to wild-type SBN. To examine differences in viral replication in detail, one-step growth curves of recombinant RVs were performed. BSR cells were infected at an MOI of 10 to allow simultaneous infection of all cells. Virus titers were determined at the indicated time points after replacing the virus inoculum with fresh medium (Figure 2). The two recombinant RVs expressing HIV-1 gp160 replicated at only a slightly reduced rate compared to wild-type RV, with a final titer reduction of 2.3 (SBN-NL4-3) or 8-fold. The greater than 20% genome size of recombinant RVs could not explain the slower growth of these viruses. Recombinant RV expressing the 1.9 kb gene (firefly luciferase) was grown to titers of wild-type RV. (Mebatsion, et al., Proceedings of the National Academy of Sciences of the United States of America, 93:7310-4, 1996).

Expression of exogenous glycoproteins by recombinant RVs

Expression of HIV-1 gp160 by recombinant RVs was also examined. To ensure expression of HIV-1 gp160 by recombinant virus, cell lysates from cells infected with recombinant RV were treated with antibodies directed against RV (Figure 3, α-RV rabies) or anti-HIV-1 gp120 (Figure 3, α-gp120) Western immunoblotting was analyzed. Two bands of HIV-1 gp160 and gp120 of the expected size were detected in lysates of cells infected with SBN-89.6 or SBN-NL4-3 (lanes 3 and 4), but not in mock-infected or SBN-infected cells. Not observed in cell lysates (lanes 1 and 2). Western blot probed with an αRV antibody confirmed that all viruses (lanes 2, 3 and 4) infected target cells.

Coat proteins expressed in recombinant RVs are functional

To determine whether the HIV-1 coat protein expressed in RV was functionally expressed, recombinant RVs were analyzed in a fusion assay in a human T cell line (Sup-T1). This experiment confirmed that wild-type RV was able to infect and replicate in 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). Compared with wild-type RV, large multinucleated spheroplast formation was detected in Sup-T1 cells 24 hours after infection with SNB-89.6 or SBN-NL4-3 (Fig. 4). These results indicate that the expressed HIV-1 coat protein is correctly folded, transported to the cell surface, and recognized by the HIV-1 receptor and co-receptors CD4 and CXCR4.

Coat protein (89.6) from a dual-tropic HIV-1 strain will induce cell fusion if co-expressed with CD4 and CCR5, whereas NL4-3 gp160 will only induce fusion in cells expressing CD4 and HIV-1 co-receptor CXCR4 . Infection of 3T3 murine cells expressing human CD4 did not result in cell fusion regardless of the recombinant RV used, whereas multinucleated protoplasms were detected in 3T3 cells expressing CD4 and CXCR4 following infection with SBN-NL4-3 or SBN-89.6 Groups formed. As expected, in 3T3 cells expressing CD4 and CCR5, only expression of the HIV-1 _89.6 coat protein caused fusion of these cells.

Induction of humoral immune responses in mice

Antibody responses against gp120 in mice infected with RV expressing HIV-1 gp160 were also analyzed. A possible requirement for a successful HIV-1 vaccine is the ability to induce a strong humoral response against the HIV-1 protein gp160. In order to determine whether the recombinant gp160 protein expressed by recombinant RV can induce anti-HIV-1 immune response, 5 BALB/C mice per group were treated with 10 ⁶ FFU of SBN, SBN-89.6 or 10 ⁵ FFU of SBN-NL4 on both hind paws -3 subcutaneous inoculation. Mice were bled 11, 24, and 90 days after initial infection with RV, and sera were analyzed by ELISA.

No response to HIV-1 coat protein was detected in the sera of immunized animals, but ELISA using RV glycoprotein instead of HIV-1 gp120 as antigen confirmed RV infection, and as early as 11 days after infection, high levels of Antibodies against RV. Several studies with viral vectors expressing HIV-1 gp160 have shown that a booster dose of infection or booster immunization with recombinant protein is necessary to induce detectable serum antibodies against the HIV-1 coat protein. High antibody titers detected in ELISA of RV indicated that additional infection with recombinant RV was not promising, therefore 3/5 mice per group were boosted with 10 μg of recombinant gp120 and gp41 in a Freund's complete adjuvant . Twelve days after the subunit boost, mice were bled and the immune response analyzed by HIV-1 gp120 ELISA. The results demonstrated that booster immunization of subunits of HIV-1 coat protein induced a strong immune response against HIV-1 gp120 only in mice previously infected with SBN-89.6 or SBN-NL4-3 ( FIG. 5 ). Wild-type RV (SBN)-infected mice responded only at the lowest serum dilution (1:160) after booster immunization. ELISA specific for HIV-1 gp41 was negative for all mouse sera even after booster immunization with recombinant HIV-1 gp120/gp41. These data were confirmed by Western blot analysis (Figure 6). Only sera from mice infected with SBN-89.6 or SBN-NL4-3 and subsequently boosted with the recombinant protein responded to gp120, while all other sera could not detect any HIV-1 protein. Even with gp41 subunit immunization, none of the sera had a gp41-specific band.

Induction of neutralizing antibodies

Experiments were performed to see if booster immunization with recombinant protein after primary viral infection could induce neutralizing antibodies against HIV-1. In this experiment, HIV-1 neutralizing antibody (NA) titers in MT-2 cells were determined by staining with the HIV-1 _NL4-3 essential dye. After booster injections with SBN-NL4-3 and one coat subunit of recombinant gp120 (IIIB strain), murine serum was able to neutralize tissue culture laboratory-altered (TCLA) HIV-1 _NL4 in a 1:800 dilution of serum _-3 strain, while immunization with HIV-1 NL4-3 failed to induce detectable neutralizing antibodies. These results were confirmed in two independent experiments. Sera from mice infected with wild-type RV (SBN) boosted with recombinant gp120 showed only very low 1:50 NA titers (Table 1). These results indicate that booster injections with recombinant gp120 induced high titers of NA following a primary immunization with recombinant RV expressing HIV-1 gp160.

Table 1 After infection with different RVs, the neutralizing antibody titers of the mouse sera boosted by recombinant HIV-1gp120/gp41 Booster immunization with recombinant HIV-1 gp120/gp41 HIV-1 _NL4-3 and antibody titers Experiment I Experiment II SBN-NL4-3 <1:50 1:50 SBN-NL4-3* 1:800 1:800 SBN* <1:50 <1:50

The results shown here demonstrate the generation of recombinant RV expressing the full length HIV-1 coat protein. The foreign gene was stably expressed by replication-competent RV, and a strong humoral response against the HIV-1 coat protein was induced in mice following infection with recombinant RV followed by a single booster immunization with the HIV-1 gp120 protein. Mice infected with recombinant RV expressing HIV-1 gp160 elicited a strong priming of the immune system, as indicated by a strong humoral response following a single boost of HIV-1 gp120 protein or gp41. Thus, a stronger response could be detected by boosting mice infected with additional recombinant RV containing different viral glycoproteins or with recombinant VSV expressing HIV-1 gp160.

Induction of long-lasting HIV-1 gp160-specific CTL

Recombinant RV expressing HIV-1 coat protein and primary HIV-1 isolates of a laboratory-engineered HIV-1 strain (NL4-3) show that RV-based vectors are excellent for priming B cells (see above). (Schnell, M.J., et al., Proc. Natl. Acad. Sci. USA, 97:3544-3549, 2000). The invention further relates to memory CTL responses against HIV-1 coat protein expressed by attenuated RV-based vectors. As noted, there is increasing evidence that induction of strong, long-lasting CTL responses is an important feature for a successful HIV-1 vaccine.

To analyze the efficacy of RV-based vectors in inducing cytotoxic responses against HIV-1, 2×10 ⁷ colony-forming units of recombinant RV expressing HIV-1 _NL4-3 coat protein (SBN-NL4-3) were used ( FFU) to immunize 6 mice (see above and below). Three mice were sacrificed 105 or 135 days after infection and spleens were removed. 1/3 of the spleen cell culture was infected with 1 MOI (multiplicity of infection) recombinant vaccinia virus expressing HIV-1 _NL4-3 gp160 for 16 hours, inactivated with psoralen and UV treatment, and added back as presenting cells in culture. Stimulated effector cells were assayed 7 days after activation for their ability to kill P815 target cells infected with vaccinia wild-type virus, recombinant vaccinia virus expressing HIV-1 _NL4-3 gp160 or HIV-1 Gag. As observed in Figure 9, only a strong cytotoxic response was observed against P815 target cells infected with recombinant vaccinia virus expressing HIV-1 coat protein. Only a low percentage of lysis was observed for P815 target cells infected with the other two vaccinia viruses. Notably, these responses were achieved after a single vaccination with recombinant RV expressing the HIV-1 coat protein, suggesting that RV-based vectors induce long-lasting persistent CTLs after a single vaccination.

CTLs derived from cross-inactivated target cells of HIV-1 gp160-immunized mice expressing heterologous HIV-1 coat protein

Despite significant differences in the HIV-1 coat amino acid sequence, cross-protection between divergent viruses will likely be required for a protective vaccine. To analyze the efficacy of candidate vaccines in inducing cross-reactive CTLs against gp160 of different HIV-1 strains, spleen cells from mice immunized with recombinant RV expressing HIV-1 gp160 were screened against allogeneic and heterologous HIV-1 coat proteins. P815 target cells. For this method, two groups of six mice were immunized peritoneally with 2×10 ⁷ recombinant RV expressing laboratory-engineered CXCR4-topic (NL4-3), or dua1-tropic (CXCR4 and CCR5) isolates (89.6 ) of HIV-1gp160.

Three to five weeks after immunization, three mice in each group were sacrificed, spleens were removed, and the pooled spleen cells were stimulated with recombinant vaccinia virus expressing the same HIV-1 coat protein (NL4-3 or 89.6). 7 days after stimulation, the ability of effector cells to lyse P815 target cells was analyzed. The P815 target cells were derived from laboratory-modified CXCR4-topic HIV-1 strain (NL4-3), dual-tropic strain (89.6), and two major CCR5-topic HIV-1 strains (Ba-L and JR-FL) were infected with recombinant vaccinia viruses expressing the HIV-1 coat protein. The results obtained from two different, independent experiments are shown in Figure 10A and Figure 10B, wherein Figure 10A shows the results of mice immunized with RV expressing HIV-1 _NL4-3 coat protein, and Figure 10B shows is the result of mice immunized with RV expressing the HIV-1 _89.6 coat protein. As expected, strong, specific lysis of alloantigen-expressing P815 cells was observed in both groups. More significantly, these effector cells were able to cross-kill P815 target cells expressing heterologous HIV-1 coat proteins. Viable spleen cells derived from SBN-NL4-3 immunized mice achieved specific lysis of P815 target cells expressing gp 160JR-FL, or 89.6 strains made P815 target cells in the range of 40%, with effector cells: target The cell (E:T) ratio of 50:1 was specifically lysed and also cross-killed target cells expressing HIV-1 _Ba-L gp160. Cross-inactivation was observed in effector cells derived from SBN-89.6-primed mice. Lysis of P815 target cells was to the same extent as that observed in viable spleen cells from mice immunized with SBN-NL4-3, but only about 20% of P815 cells expressing HIV-1 _NL4-3 were lysed. These data suggest that RV-based vector-induced anti-HIV-1 gp160 CTLs can be directed against distinct epitopes in the HIV-1 coat protein.

CD8 ⁺ T cell-mediated HIV-1 specific CTL activity

The phenotype of T cell subsets that mediate cytolytic activity is defined by selective deletion. Three mice were immunized with 2×10 ⁷ FFU of recombinant RV expressing HIV-1 _NL4-3 coat protein, and their spleens were removed 18 weeks later. Spleen cells were restimulated for 7 days with recombinant vaccinia virus expressing the same HIV-1 coat protein. Immunomagnetic bead cell isolation of deletion-type and isolated positive CD8 ⁺ T-cells from active spleen cell cultures was performed. Complex release assays were performed with depleted CD8 ⁺ T-cell (CD8 ⁻ ) cultures, isolated CD8 cell (CD8 ⁺ ) cultures or untreated cultures (CD8 ⁺ /CD8 ⁻ ).

P815 target cells were infected with vaccinia virus expressing HIV-1 _NL4-3 gp160 or HIV-1 gag. As shown in Figure 11, cultures depleted of CD8 ⁺ T-cells showed no activity, whereas at E:T ratios of 25:1 and 12.5:1, enriched CD8 ⁺ T-cells and untreated cultures Each showed high specific dissolution. In fact, the enriched CD8 ⁺ T-cell population was also enriched in lysing monoclonals, as CTL activity was still 12.5:1 at a plateau compared to the unselected population. These data suggest that cytolytic activity is mediated by a subset of CD8 ⁺ T cells. Moreover, these results imply that in addition to antibodies, recombinant RV vectors also generate long-term active anti-CD8 ⁺ T cell responses.

discuss

The present invention relates to RV-based vectors for the expression of HIV-1 coat proteins. These vectors were able to induce a humoral immune response against HIV-1 gp160 following a single immunization followed by a booster injection with recombinant HIV-1 gp120. (Schnell, M.J., et al., Proc. Natl. Acad. Sci. USA, 97:3544-3549, 2000). Accumulating evidence indicates that CTL responses play an important role in the antiviral immune response against HIV-1. (Brander, C. and B.D. Walker, Current Opinion in Immunology, 11:451-9, 1999.). Therefore, the development of an effective preventive HIV-1 vaccine requires the screening of HIV-1 antigens capable of inducing long-lasting and broadly active CTL responses. The invention further relates to RV-based vectors that induce such responses.

Mice single-vaccinated with recombinant RV expressing the HIV-1 coat protein elicited a strong CTL response against the HIV-1 coat protein compared to the observed humoral responses. In addition, these responses were stable for at least 135 days after immunization. One explanation for this strong response is that RV was grown in various cell lines that did not kill the cells, which resulted in longer expression of HIV-1 genes compared to a cytopathic viral vector. In addition, the expression of RV nucleoprotein 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;), may help to enhance the overall immune response against HIV-1 coat protein after a single immunization.

The recombinant RVs of the present invention can induce cross-reactive CTLs against various HIV-1 coat proteins. Previous studies have shown that a single amino acid exchange can abrogate CTL cross-inactivation, whereas other experiments have shown that single and even double amino acid substitutions generally do not abrogate cross-inactivation. (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, it remains questionable whether recombinant RVs induce CTLs directed against different epitopes. However, several studies have shown that individuals derived from HIV-1 infection, even from different HIV-1 clades, exhibit cross-reactivity; suggesting that extensive cross-reactivity is an important requirement for HIV-1 vaccines. (Cao, H., et al., J. Virol., 71:8615-23, 1997; Rowland-Jones, S.L., et al, Journal of Clinical Investigation, 102:1758-65, 1998.). Only one study has shown that canarypox-based HIV-1 vaccines induce cross-clade CTL activity in an uninfected volunteer. (Ferrari, G., et al., Proc.Natl.Acad.Sci.USA, 94:1396-401, 1997.) The inventors of the present invention are currently analyzing whether the CTLs induced by recombinant RV to HIV-1gp160 HIV-1 coat proteins of clades other than B are also cross-reactive.

In summary, the present invention demonstrates the ability of murine sera to neutralize strains of HIV-1. The present invention thus demonstrates that recombinant RVs are excellent vectors for B cell priming. The present invention also demonstrates that single vaccination with recombinant RV expressing HIV-1 coat proteins induces strong, long-lasting CTL-specific responses against HIV-1 proteins, such as the coat proteins of different HIV-1 strains. These results further emphasize the utility of RV as an HIV-1 vaccine.

Compared with most other viral vectors, there is only a negligible seropositivity for RV in the human population, and immunization with RV-based vectors against HIV-1 does not interfere with immunity against the vector itself . Oral immunization with RV vaccine strains against RV was successful and non-pathogenic in chimpanzees (Report of the forth WHO Consultation on oral immunization of dogs against rabies.unpublished document WHO/Rab.Res/93.42.1993. ), so RV-based vectors will also be promising in inducing mucosal immunity against HIV-1. Thus, the present invention fulfills a long-standing but unfulfilled need for a method of treating HIV-1 infection. With the recombinant RVs of the present invention, all major antigenic determinants for neutralizing antibodies, cytotoxic lymphocytes and antibodies dependent on cell cytotoxicity are simultaneously expressed, thereby inducing anti-HIV-1 humoral and cell-mediated immunity.

Example

The following examples further illustrate the invention but do not in any way limit the scope of the invention. The following examples are carried out using standard techniques which are well known and routine to those skilled in the art and otherwise are described in detail. The examples are illustrative, but not limiting, of the invention. All animal methods of treatment and prophylaxis described herein are preferably applicable to mammals, most preferably humans.

Embodiment 1: Construction of plasmid

Figure 1 is a schematic illustration of the method for constructing a recombinant RV genome. Shown at the top is the wild-type RV genome (SAD L16) with 5 open reading frames. Using PCR methods and site-directed mutagenesis, the entire Ψ gene was deleted and a new transcriptional unit containing two single-site tiny RVs was introduced between the G and L genes (SBN). The cDNA sequence encoding HIV-1 _89.6 or HIV-1 _NL4-3 gpl60 was inserted with BsiWI and NheI sites, resulting in plasmid pSBN-89.6 or pSBN-NL4-3 (bottom).

Two single sites were subjected to site-directed mutagenesis (GeneEditor ^TM Promega Inc.) was introduced upstream of the RV cDNA pSAD L16 of the previously described G (SmaI) and Ψ genes (NheI), resulting in plasmid pSN. The pSN was the target for the introduction of a new transcription termination/initiation sequence, along with the introduction of a single BsiWI site using the polymerase chain reaction (PCR) method. First, PCR was performed from pSN using Vent polymerase (New England Biolabs Inc.) and forward primer RP1 5'TTTTGCTAGCTTATAAAGTGCTGGGTCATCTAAGC-3' (SEQ ID NO: 3) or RP10 5'-CACTACAAGTCAGTCGAGACTTGGAATGAGATC-3' (SEQ ID NO: 4) Two fragments are amplified. The reverse primers were RP18 5'-TCTCGAGTGTTCTCTCTCCAACAA-3' (SEQ ID NO: 5) and RP17 5'- AAGCTAGCAAAACGTACGGGAGGGGTGTTAGTTTTTTTCATGGACTTGGATCGTTGAAAGGACG -3' (SEQ ID NO: 6). RP17 contains an RV transcription termination/initiation sequence (underlined) and a BsiWI and NheI site (in italics). The PCR product was digested with NheI, ligated, and the 3.5 kb band was eluted from the agarose gel. After gel elution, this band was digested with ClaI/MluI and ligated to pSN previously digested with ClaI/MluI. The plasmid was named pSBN.

The gene of HIV-1 gp160 encoding the coat protein of HIV-1 strain 89.6 and NL4-3 strain was amplified by PCR with Vent polymerase, forward primer 5'-GGGCTGCAGCTCGAGCGTACGAAAATGAGAGTGAAGGAGATCAGG-3' (SEQ ID NO: 7) containing PstI /XhoI/BsiWI site (italics), and the reverse primer 5'-CCTCTAGATTATAGCAAAGCCCTTTCCAAG-3' (SEQ ID NO: 8) contains an XbaI site (italics). The PCR product was digested with PstI and XbaI, and cloned into pBluescript II SK+ (Stratagene). After the sequence was formed, the HIV-1 gp160 gene was digested with BsiWI and XbaI, and connected to pSBN that had been digested with BsiWI and NheII, and the resulting plasmids were named pSBN-89.6 and pSBN-NL4-3.

Example 2: Recovery of RV infection derived from cDNA

For rescue experiments with recombinant RVs, a previously described vaccinia virus lacking the RV recovery system was used (see Finke, et al., Journal of Virology, 73:3818-25, 1999). Briefly, using a Ca ₂ PO ₄ transfection kit (Stratagene), following the manufacturer's instructions, 5 μg of full-length RV cDNA and plasmids (2.5 μg , 1.25 μg and 1.25 μg) were transfected into BSR-T7 cells stably expressing T7 RNA polymerase (a generous gift of Drs.S.Finke and K.-K.Conzelmann). Three days after transfection, the tissue culture suspension was transferred to fresh BSR cells, and 3 days later infected RV was detected by immunostaining against the N protein of RV (Centocor).

Example 3: One-step growth curve

As shown in Figure 2, it is a one-step growth curve diagram representing recombinant RV BSR cells infected with recombinant RVs (SBN, SBN-89.6, and SBN-NL4-3). At the indicated time points, titers of dual virus were determined.

BSR cells (one BHK-21 clone) were plated in 60 mm dishes and infected 16 hours later with SBN, SBN-89.6, or SBN-NL4-3 at a total multiplicity of infection (MOI) of 5 in a volume of 2 ml. After incubation for 1 hour at 37°C, the inoculum was removed and the cells were washed 4 times with phosphate buffered saline (PBS) to remove any unadsorbed virus. An additional 3 ml of complete medium was returned, and 100 μl of tissue culture suspension was removed at 4, 16, 24 and 48 hours post-infection. Viral aliquots were titrated in duplicate on BSR cells. Shown in Figure 3 is the Western blot analysis of recombinant RVs expressing HIV-1 gp160. Infect Sup-T1 cells with 2 MOI of SBN, SBN-89.6, or SBN-NL4-3, and dissolve for more than 24 hours. Proteins were separated by SDS-PAGE and analyzed by Western blotting. In lysed cells infected with SBN-89.6 or SBN-NL4-3, an antibody directed against gp120 detected two bands of HIV-1 gp160 and gp120 at the expected positions (α-gp120, lanes 3 and 4). No signal was detected in mock or SBN-infected cells ([alpha]-gp120, lanes 1 and 2). Cells successfully infected by recombinant RVs were confirmed with a polyclonal antibody directed against RV (α-rabies, lanes 2, 3, 4).

As shown in Figure 4, Sup-T1 cells were infected with 1 MOI of SBN, SBN-89.6, or SBN-NL4-3. 24 hours post-infection, syncytium formation was detected in cell cultures infected with HIV-1 gp160 expressing recombinant RV (SBN-89.6 group and SBN-NL4-3 group), indicating expression of functional HIV-1 coat protein. No cell fusion was detected in cultures infected with wild-type RV (SBN group).

Example 4: Immunization

Each group of 5 BALB/C female mice at the age of 4-6 weeks obtained from the Jackson laboratory was treated with 10 ⁶ colony forming units (FFU) of SBN, SBN-89.6 or 10 in DMEM+10% FBS (fetal bovine serum). ⁵ NL4-3 was inoculated subcutaneously on both hind paws. Three months after infection, 3 out of 5 mice in each group were boosted intraperitoneally with 10 μg of recombinant gp41 (IIIB, Intracel Inc.) and 10 μg of recombinant gp120 (IIIB, Intracel Inc.) in 100 μl of Freund's complete adjuvant.

Example 5: Enzyme-linked immunosorbent assay (ELISA)

Recombinant HIV-1 gp120 (strain IIIB, Intracel) was resuspended in coating buffer (50 mM Na ₂ CO ₃ , pH 9.6) at a concentration of 200 ng/ml, and 100 μl per well was plated in a 96-well ELISA Maxisorp plate. Incubate overnight at 4°C, wash the plate 3 times (PBS pH 7.4, 0.1% Tween-20), block with blocking buffer (PBS, pH 7.4, 5% dry milk powder) at room temperature for 30 minutes, use Serially diluted sera were incubated for 1 hour. Plates were washed three times, followed by the addition of horseradish peroxidase (HRP) (1:5000, Jackson ImmunoResearch Laboratories) conjugated to a goat anti-mouse IgG (H+L) secondary antibody. After incubation at 37° C. for 30 minutes, the plate was washed 3 times and 200 μl of OPD-substrate (o-phenylenediamine dihydrochloride, Sigma) was added to each well. _The reaction was terminated by adding 50 μl of 3M _H2SO4 per well. The determined density of visible light is 490 nm. Shown in Figure 5 is a graph depicting the ELISA activity of anti-HIV-1 gp120 mouse sera. Each of 5 mice was immunized with recombinant RVs (SBN, SBN-89.6 or SBN-NL4-3), and 3 months after the initial infection, 3 mice in each group were immunized with recombinant HIV-1 gp120 and gp41 (SBN*, SBN-89.6* or SBN-NL4-3*) booster immunization. Each data point on the curve represents the mean number of mice per group in three independent experiments. One mouse in the SBN-89.6 group did not respond to the booster injection and was not included in the curve. Error bars represent standard deviation.

Example 6: Western blotting

Human T-lymphocyte cells (Sup-T1) were infected with 2 MOI for 24 hours and lysed in 50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS, and 1× Protease Inhibitor Cocktail (Sigma) Resuspend in buffer for 5 minutes. The protein suspension is transferred to a microtube and spun at 10,000 x g for 1 min to remove cellular debris. Proteins were separated by 10% SDS-PAGE and transferred to a membrane loaded with PVDF (polyvinylidene fluoride) (Osmonics). After blocking for 1 hour (5% dry milk powder in PBS, pH 7.4), blots were mixed with sheep α-gp120 antibody (ARRRP) (1:1000) or human α-rabies serum (1:500) in blocking buffer Incubate in solution for 1 hour. Secondary antibody, goat α-human or donkey α-sheep, horseradish peroxidase (HRP) (Jackson ImmunoResearch Laboratories) conjugated antibody (1:50000) was added and blots were incubated for 1 hour. Each incubated antibody was then washed three times with WB-wash buffer (PBS pH 7.4, 0.1% Tween-20). (NEN) Chemifluorescence was performed following the manufacturer's directions.

Except that the α-human IgG conjugate in mouse serum was replaced by alkaline phosphatase conjugated goat anti-mouse IgG (H+L) at a dilution of 1:5000 (Jackson ImmunoResearch Laboratories), the rest were according to the manufacturer's instructions. Instructions, Western Blot analysis was performed with a commercially available Western Blot Kit (QualiCode HIV-1/2 Kit, Immunetics) to detect anti-HIV-1 antibodies. Shown in Figure 6 is the Western Blot (Western Blot) analysis of the reaction of the mouse serum antibody to the HIV-1 antigen. Immunized with RVs (α-SBN, α-SBN-89.6 or α-SBN-NL4-3) or with recombinant gp120 and gp41 (α-SBN*, α-SBN-89.6* or α-SBN-NL4-3* ) and booster injections (SBN, SBN-89.6, and SBN-NL4-3) from one mouse in each group were tested at a 1:100 dilution. Highly positive and weakly positive human control sera were used to detect HIV-1 protein locations. SC indicates control serum.

Example 7: Detection of virus neutralization.

The HIV-1 strain was recovered in 293T cells, the preserved virus was expanded in MT-2 cells (HIV-1 NL4-3), frozen at -75°C, and titrated on MT-2 cells. The neutralization assay was according to Montefiori et al. (Journal of Clinical Microbiology, 26, 231-5, 1998.). Briefly, approximately 5000 _TCID50 (half the tissue culture infectious dose) of HIV-1 _NL4-3 was incubated with serially diluted mouse sera for 1 hour. Add MT-2 cells and incubate for 4-5 days at 37°C, 5% CO ₂ . 100 μl of cells were transferred to a poly-L-leucine plate and stained with Neutral Red (ICN) for 75 minutes. The cells were washed with PBS, dissolved with acidic ethanol, and analyzed at 550nm with a colorimetric reagent. Protection at least 50% viral suppression was estimated.

All publications and references cited in this specification, including but not limited to patent applications, are hereby incorporated by reference as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein. here.

However, for certain specific examples, a reference is used to illustrate the invention, and variations of methods and compositions employed will be apparent to those of ordinary skill in the art, and it is indicated that, except as specifically described herein, the present invention Inventions can be practiced. Accordingly, the present invention includes all modifications included within the spirit and scope of the present invention as defined by the claims.

Example 8: Preparation of spleen cells

Spleen cells were aseptically excised to prepare a single cell suspension. Red blood cells were lysed with ACK lysis buffer (Bio Whitaker), and spleen cells were kept in RPMI-10 medium containing 10% fetal bovine serum and washed twice. Spleen cells divide into effector and stimulator cells. Stimulator cells were prepared by infecting at a multiplicity of infection (MOI) 1 for 1 hour with recombinant vaccinia virus (MOI=10) expressing the coat protein of HIV-1. Cells were washed once with PBS to remove excess virus, and incubated at 37°C for 16 hours. After incubation, vaccinia virus was inactivated with psoralen (Sigma) (see below). Stimulator cells were added back to the effector cell population at a 3:1 ratio, and 10% T-STIM (Collaborative Biomedical Products) was added as a source of interleukin-II (IL-2).

Inactivation of virus with psoralen

Spleen cells were subsequently incubated with vaccinia virus and the virus was inactivated with psoralen (Sigma). Psoralen was added to the cells to a final concentration of 5 g/ml. After incubation at 37°C for 10 minutes, the cells were treated with long-wave UV (365 nm) for 4 minutes and washed twice with PBS.

Preparation of chromium-labeled target cells

Prepare target cells (P815) by infecting with 10 MOI of recombinant vaccinia virus expressing HIV-1 coat protein (see the legend of the specific figure for the specific protein) for 1 hour, wash the target cells to remove excess virus, and incubate at 37°C for 16 hours . To measure background, target cells were infected with vaccinia (vP1170) expressing HIV-1 Gag (vP1287) or wild type. Wash target cells once in PBS, label cells with 100 μCi ⁵¹ Cr for 1 hour, wash twice in PBS, add to effector cells at various E:T ratios (see figure), 37°C, 4 hours. The specific percentage ^of51Cr released is calculated as 100X (experimental release-spontaneous release)/(maximum release-spontaneous release). The maximum release was determined by adding 5% Triton X-100 to the lysed cell suspension. Spontaneous release was determined by incubating target cells without addition of effector cells.

Preparation of CD8+ depleted T-cells

After several days of in vitro stimulation, CD8+ T cells were depleted (CD8-) from cell cultures and enriched (CD8+) with DynabeadsMouse CD8(Lyt2) as instructed by the manufacturer.

Sequence Listing

SEQUENCE LISTING <110>Thomas Jefferson University<120>Recombinant rhabdovirus as live virus vaccine for immunodeficiency virus<130>P02US06731<140>PCT/US01/01989<141>2001-01-22<150>US 09/494,262 <151>2000-01-28<160>8<170>FastSEQ for Windows Version 4.0<210>1<211>33<212>DNA<213>Artificial sequence<220><223>Primer<400>1cctcaaaaga ccccgggaaa gatggttcct CAG 33 <210> 2 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 2GACTGTAGGGGCTAGC CTTTCAACGA TCCAAG 36 <210> 3 <212> DNA <213> Artificial artificial Sequence <220> <223> Primers <400> 3tttttgCTAGC TTATAAGTG CTGGGTCTC TAAGC 35 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence >5<211>24<212>DNA<213>artificial sequence<220><223>primer<400>5tctcgagtgt tctctctcca acaa 0 <artificial sequence>DNA 24<210>6<211>64<212> 223>引物<400>6aagctagcaa aacgtacggg aggggtgtta gtttttttca tggacttgga tcgttgaaag        60gacg                                                                     64<210>7<211>45<212>DNA<213>人工序列<220><223>引物<400>7gggctgcagc tcgagcgtac gaaaatgaga gtgaaggaga tcagg                        45<210> 8<211>30<212>DNA<213>Artificial sequence<220><223>Primer<400>8cctctagatt atagcaaagc cctttccaag 30

Claims (34)

1. A recombinant rhabdovirus vector, comprising: a) a modified rhabdovirus genome; b) a new transcription unit, which is inserted into the Rhabdovirus genome to express a heterologous nucleic acid sequence; and c) a heterologous viral nucleic acid sequence inserted into said new transcription unit, Wherein the recombinant Rhabdovirus vector is capable of replication, and the nucleic acid sequence of the heterologous virus encodes an antigenic polypeptide. 2. The recombinant rhabdovirus vector according to claim 1, wherein said modified rhabdovirus genome is a modified rabies virus genome. 3. The recombinant rhabdovirus vector according to claim 2, wherein said modified rabies virus genome has a second modification, so that a glycoprotein derived from other viruses replaces the glycoprotein of rabies virus . 4. The recombinant rhabdovirus vector according to claim 3, wherein said glycoprotein derived from other viruses is glycoprotein of vesicular stomatitis virus. 5. The recombinant rhabdovirus vector according to claim 3, wherein said modified rabies virus genome has a third modification, so that after said second modification, there is a Concatenation of structural genes in the modified rhabdovirus genome. 6. The recombinant rhabdovirus vector according to claim 1, wherein the nucleic acid sequence of the heterologous virus encodes an antigenic polypeptide selected from a group consisting of full-length HIV-1 coat protein, HIV gp160, HIV gp120 , and the full-length SIV coat protein protein set. 7. The recombinant rhabdovirus vector according to claim 6, wherein the heterologous viral nucleic acid is fused to the sequence of the cytoplasmic domain of the glycoprotein gene of the modified rhabdovirus genome, resulting in A chimeric protein, whereby said chimeric protein has a fusion between a transmembrane domain of said foreign protein and a cytoplasmic domain of said glycoprotein. 8. The recombinant rhabdovirus vector of claim 1, further comprising a deletion of a recombinant rhabdovirus gene, and wherein said heterologous viral nucleic acid is fused to said modified rhabdovirus genome In the sequence of the cytoplasmic domain of the glycoprotein, a chimeric protein was produced, which functionally replaced the recombinant rhabdovirus glycoprotein gene. 9. A recombinant Rhabdovirus expressing a functional HIV coat protein, wherein said recombinant Rhabdovirus is replication competent. 10. The recombinant rhabdovirus according to claim 9, wherein said rhabdovirus is a recombinant rabies virus or a recombinant vesicular stomatitis virus. 11. The recombinant rhabdovirus according to claim 9, wherein said HIV coat protein is derived from any HIV-1 isolate. 12. An immunogenic composition comprising any one of the recombinant Rhabdovirus vectors of claims 1-9 and an adjuvant. 13. A recombinant rabies virus deficient in the Ψ gene, comprising a heterologous nucleic acid segment encoding an immunodeficiency virus coat protein, or a subunit thereof. 14. The recombinant rabies virus defective in the [Psi] gene of claim 13, wherein said rhabdovirus is a rabies virus. 15. The recombinant rabies virus defective in Ψ gene according to claim 13, wherein said immunodeficiency virus coat protein, or its subunit is derived from human immunodeficiency virus. 16. The recombinant rabies virus deficient in the Ψ gene according to claim 13, wherein said immunodeficiency virus coat protein, or a subunit thereof, is derived from a simian immunodeficiency virus. 17. A method of inducing an immune response in a mammal comprising: (a) delivering a recombinant rhabdovirus vector expressing a functional immunodeficiency virus coat protein or a subunit thereof to said mammalian tissue, and it can effectively induce an immune response to said coat protein. (b) expressing said coat protein or subunit thereof in vivo; (c) boosting said mammal by delivering an effective dose of the isolated immunodeficiency virus coat protein or subunit thereof in an adjuvant, or by delivering an effective dose of a booster vaccine vector; and (d) inducing a neutralizing antibody response and/or a long-lasting cellular immune response to protect said mammal against immunodeficiency virus. 18. The method of claim 17, wherein said recombinant rhabdovirus comprises a rabies virus genome. 19. The method of claim 18, wherein said rabies virus genome is deficient in the Ψ gene. 20. The method of claim 18, wherein said rabies virus genome is a rabies virus glycoprotein gene deficient. 21. The method according to claim 19, wherein in the rabies virus genome, there is a glycoprotein gene derived from other rhabdoviruses instead of the rabies virus glycoprotein. 22. A method of inducing an immune response in a mammal comprising: (a) delivering a non-fragmented negative-strand RNA expressing a functional immunodeficiency virus coat protein or a subunit thereof to said mammalian tissue, and it can effectively induce an immune response to said coat protein . (b) expressing said coat protein or subunit thereof in vivo; (c) boosting said mammal by delivering an effective dose of the isolated immunodeficiency virus coat protein or subunit thereof in an adjuvant, or by delivering an effective dose of a booster vaccine vector; and (d) inducing a neutralizing antibody response and/or a long-lasting cellular immune response to protect said mammal against immunodeficiency virus. 23. The method of claim 22, wherein said non-segmented negative-strand RNA virus is rabies virus or vesicular stomatitis virus. 24. A recombinant non-fragmented negative-strand RNA viral vector, comprising: a) a negative-strand RNA viral genome of a Ψ gene-deficient modification; b) a new transcription unit, which is inserted into the modified negative-strand RNA viral genome to express a heterologous nucleic acid sequence; and c) a heterologous viral nucleic acid sequence inserted into a new transcription unit, wherein said recombinant non-fragmented negative-strand RNA viral 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-strand RNA virus expressing a functional immunodeficiency virus coat protein, or a subunit thereof; b) expressing said functional immunodeficiency virus coat protein, or a subunit thereof; c) boosting immunization of said mammal by delivering an effective dose of the isolated immunodeficiency virus coat protein or subunit thereof in an adjuvant, or by delivering an effective dose of a booster vaccine vector; and d) Inducing a neutralizing antibody response and/or a long-lasting cellular immune response to said functional immunodeficiency virus coat protein or its subunit. 26. The method of claim 25, wherein said immunodeficiency virus is any HIV-1 virus. 27. The method of claim 25, wherein said non-segmented negative-sense RNA virus is a Rhabdovirus. 28. The method of claim 25, further comprising the induction of mucosal immunity to said functional immunodeficiency virus coat protein, or a 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 coat proteins derived from different immunodeficiency virus strains , or its subunits. 30. A method of protecting a mammal against infection by an immunodeficiency virus comprising: a) administering to said mammal a non-segmented negative-strand RNA virus expressing a functional immunodeficiency virus coat protein, or a subunit thereof; b) expressing said functional immunodeficiency virus coat protein, or a subunit thereof; c) boosting said mammal by delivering an effective dose of the isolated immunodeficiency virus coat protein or subunit thereof in an adjuvant, or by delivering an effective dose of a booster vaccine vector; and d) Inducing a neutralizing antibody response and/or a long-lasting cellular immune response to said functional immunodeficiency virus coat protein or its subunit. 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-sense RNA virus is a rhabdovirus. 33. The method of claim 30, further comprising the induction of mucosal immunity to said functional immunodeficiency virus coat protein, or a 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 coat proteins derived from different immunodeficiency virus strains , or its subunits.

Images