HIV-1 recombinant poxvirus vaccine induces cross-protection against HIV-2 challenge in rhesus macaques.

Rhesus macaques were immunized with attenuated vaccinia or canarypox human immunodeficiency virus type 1 (HIV-1) recombinants and boosted with HIV-1 protein subunits formulated in alum. Following challenge with HIV-2SBL6669, three out of eight immunized macaques resisted infection for six months and another exhibited significantly delayed infection, whereas all three naive controls became infected. Immunizations elicited both humoral and cellular immune responses; however, no clear correlates of protection were discerned. Although more extensive studies are now called for, this first demonstration of cross-protection between HIV-1 and -2 suggests that viral variability may not be an insurmountable problem in the design of a global AIDS vaccine.

Loss of CD4 membrane expression and CD4 mRNA during acute human immunodeficiency virus replication.

Using mAbs and genomic probe to the CD4 molecule, the HIV receptor, we demonstrated that HIV replication induces the disappearance of its functional receptor from the cell surface by two distinct mechanisms. First, after being expressed onto the cell surface, HIV envelope gp110 will complex CD4, efficiently masking the CD4 epitope used by the virus to bind its receptor. This phenomenon occurs on the surface of each infected cell and is not due to the release of soluble gp110; infection with recombinant HIV/vaccinia viruses expressing a mutated HIV env gene designed to prevent gp110 release from the cell surface induces a similar gp/CD4 complexes formation. Second, virus replication induces a dramatic and rapid loss of CD4 mRNA transcripts, preventing new CD4 molecules from being synthesized. These two mechanisms of receptor modulation could have been developed to avoid reinfection of cells replicating the virus as well as to produce more infectious particles. These results suggest that the classical virus interference documented for other retroviruses might not only be due to receptor/envelope interaction, but might also depend on receptor gene expression.

Vaccine protection of chimpanzees against challenge with HIV-1-infected peripheral blood mononuclear cells.

Because human immunodeficiency virus (HIV) can be transmitted as cell-free virus or as infected cells (cell-associated virus), vaccines must protect against infection by both viral forms. Vaccine-mediated protection of nonhuman primates against low doses of cell-free HIV-1, HIV-2, or simian immunodeficiency virus (SIV) has been demonstrated. It is now shown that multiple immunizations of chimpanzees with HIV-1 antigens protected against infection with cell-associated virus. Protection can persist for extended periods (one animal had not been exposed to viral antigens for 1 year before challenge). These results show that it is possible to elicit long-lasting protective immunity against cell-associated HIV-1.

Hepatitis C virus structural proteins do not prevent human dendritic cell maturation.

AIM: Infection with hepatitis C virus (HCV) results in chronic hepatitis in more than 70% of cases. Alterations in the maturation of dendritic cells (DC) might play a role in the immune system's inability to eliminate the virus, although viral factors that could be involved have not been identified. This study in vitro investigated whether HCV structural proteins affect maturation of monocyte-derived DC. METHODS: HCV proteins (core, E1, E2) were expressed by transduction with recombinant adenoviruses of immature DC. The ability of these transduced DC to respond to a maturation stimulus was evaluated by measuring cell surface markers, allogenic lymphocyte stimulation and interleukin (IL)-12 production. RESULTS: Expression of HCV structural proteins did not modify DC maturation in the presence of lipopolysaccharide, as determined by their phenotype and stimulatory functioning. IL-12 secretion was not affected by HCV protein expression in mature DC. CONCLUSION: Our results suggest that HCV structural proteins do not affect maturation of monocyte-derived DC by lipopolysaccharide. These findings are important for further studies to clarify the pathogenesis of chronic HCV infection and towards the rational design of cellular vaccine approaches for immunotherapy against hepatitis C.

Carboxyl-terminal and central regions of human immunodeficiency virus-1 NEF recognized by cytotoxic T lymphocytes from lymphoid organs. An in vitro limiting dilution analysis.

Cytotoxic T lymphocytes (CTL) specific for human immunodeficiency virus (HIV) proteins have been analyzed in lymphoid organs from seropositive patients. Indeed, an active HIV replication coexists with a major CD8+ lymphocytic infiltration in these organs. We have shown in a previous report that HIV-seropositive patients lungs were infiltrated by HIV specific CD8+ lymphocytes. In the present report, we show that HIV-specific CTL responses can also be detected in lymph nodes and spleens, and were mainly directed against the ENV, GAG, and NEF HIV-1 proteins. The primary NEF-specific CTL responses were further characterized by epitope mapping. Determination of epitope-specific CTL frequencies were performed by limiting dilution analysis. Our results indicated that, in addition to the central region of NEF (AA66-148), a new immunodominant region is recognized by CTL. This region corresponds to the carboxyl-terminal domain of NEF (amino acids 182-206). AA182-206 is recognized in association with at least two common human histocompatibility leukocyte antigen (HLA) molecules (HLA-A1 and B8), with clonal frequencies of one CTL per 10(-5) to 10(-6) splenic lymphocytes. Our data indicate that lymphoid organs may represent a major reservoir for in vivo activated HIV-specific CTL. Furthermore, the carboxyl-terminal domain of NEF was found to be conserved among several HIV strains. Therefore, our finding is of interest for further HIV vaccines development.

Malaria vaccine development: progress and challenges.

A safe and effective malaria vaccine would contribute greatly to the control and prevention of the disease. Although a review of global activity in malaria vaccine development does reflect significant activity, progress has remained slow. This article discusses the current vaccine candidates, with emphasis on those in the clinic, and explains the numerous challenges to making and evaluating malaria vaccines, which have resulted in only a few approaches being adopted and repeatedly evaluated. Against a parasite with more than 5200 genes, the lack of definitive knowledge regarding the nature of protective immunity and absence of reliable surrogates of protection are among the key challenges to a rational evaluation and prioritization of candidate vaccines. Pursuing the current R&D strategies may not result in the availability of a vaccine with characteristics suitable to impact significantly on disease morbidity in developing countries. Therefore, it is critical that the main challenges to malaria vaccine development be unambiguously identified and collectively addressed.

The E2 trans-activating protein of bovine papillomavirus type 1 (BPV1) is serine-phosphorylated in vivo.

The E2 open reading frame of bovine papillomavirus 1 (BPV1) encodes both positive and negative transcriptional regulatory factors. The full-length E2 gene polypeptide is a strong transcriptional transactivator that acts on enhancers within the papillomavirus long control region (LCR), and two shorter E2 proteins function as transcription repressors. A vaccinia recombinant virus harboring the full length E2 coding sequence of BPV1 directs the synthesis of a 48 kD phosphoprotein with specific DNA binding activity. We show that in BPV1-transformed cells the full-length transactivator is a phosphoprotein, whereas truncated E2 proteins were not detectably phosphorylated.

Single peptide and anti-idiotype based immunizations can broaden the antibody response against the variable V3 domain of HIV-1 in mice.

The third variable (V3) domain of the human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein gp120 is a major target of neutralizing antibodies in infected persons and in experimental immunized animals. Given the high degree of sequence variability of V3, the humoral response toward this region is very type-specific. In the present study, we evaluated the potential of a single peptide and an anti-idiotypic antibody to broaden the anti-V3 antibody specificity in BALB/c mice. We show that a synthetic peptide derived from the V3 determinant of HIV-1 MN isolate (V3MN), when used as an immunogen, was able to induce an antibody response to multiple (up to six) HIV-1 strains. The extent of this cross-reactivity, which tended to enlarge as the injections increased, appeared to be inversely correlated with the binding affinity to V3MN peptide. These data thus present evidence that, despite its great sequence heterogeneity, the V3 loop encompasses conserved amino-acid positions and/or stretches which may be less immunogenic than their variable counterparts. We additionally demonstrate that a rabbit anti-idiotype (Ab2), recognizing a binding site related idiotype on a V3-specific mouse monoclonal antibody (Ab1), could mount a broadened humoral response (Ab3) in mice. Unlike nominal antibody Ab1 which strictly reacted with the European HIV-1 LAI isolate, elicited Ab3 recognized the two divergent HIV-1 strains SF2 and 1286, originating respectively from North America and Central Africa, in addition to LAI. The reasons accounting for this Ab2-induced enlargement of the V3 antibody response are discussed. Our findings suggest that single peptide and anti-idiotype based immunizations may provide viable approaches to overcome, at least in part, HIV epitope variability.

Confronting the hypervariability of an immunodominant epitope eliciting virus neutralizing antibodies from the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1)--II. Synthetic peptides linked to HIV-1 carrier proteins gag and nef.

Combining of subtype specific peptides from the hypervariable loop of the envelope glycoprotein gp120 of divergent HIV-1 isolates may help in designing a broadly protective immunogen against HIV-1 infection. To enhance the immunogenicity of such a polyvalent antigen, in the absence of oil-containing adjuvants, it is necessary to link the peptides to a protein carrier. It is preferable to use as carriers those proteins from HIV-1 itself which may contribute to eliciting protective immunity. The structural and non-structural proteins, gag P18 and nef, respectively, which can be prepared in high yields by recombinant DNA techniques in Escherichia coli, were selected for this purpose. The corresponding peptide-protein conjugates, each containing 21 distinct peptides, were prepared using the cross-linking reagents N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) or m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS). Conjugates prepared by the second method elicited approximately 10-100 times higher levels of antibodies recognizing the homologous peptides and the HIV-1 envelope glycoproteins. The sulfo-MBS conjugation procedure preserved the antigenicity of both gag P18 and nef and the respective conjugates elicited an immune response to these proteins. Despite the low immunization dose of single peptides (10 micrograms) present in the mixture of peptides collectively linked to the carriers, antibody responses to most of the individual peptides were high (dilution endpoints 1: greater than 16,000, 1: greater than 80,000 for the nef and gag P18 conjugates, respectively). Conjugates consisting of a multitude of HIV-1 envelope-derived peptides in combination with gag P18 and nef carriers are expected to elicit broadly protective immunity against distinct HIV-1 subtypes.

Qualitative and quantitative analysis of human cytotoxic T-lymphocyte responses to HIV-1 proteins.

OBJECTIVE: To study the degree of immunogenicity of each HIV-1 protein. DESIGN: In most viral systems, antiviral cytotoxic T-lymphocytes (CTL) from a given donor preferentially recognize only one or a small number of viral proteins. METHODS: Anti-HIV CTL were generated by in vitro stimulation of peripheral blood mononuclear cells from seropositive donors and tested against multiple HIV-1 proteins or groups of proteins encoded by seven genes (env, gag, pol, nef, rev, tat and vif). Using autologous target cells infected with recombinant vaccinia viruses expressing one of the HIV-1LAI proteins, we compared the cytolytic activities obtained from bulk culture with those found in limiting dilution analysis (LDA). RESULTS: Our results were noteworthy for the following reasons. (1) Each responding donor reacted simultaneously to multiple proteins; this is very unusual in other viral systems. Anti-Gag CTL were detected in most, and anti-Pol in approximately three-quarters, of the patients, together with very high amounts of the corresponding CTL precursors in LDA. CTL against Env and Nef were found in two-thirds of the patients, while Vif- and Rev-specific CTL were less frequent. Finally, Tat was seldom recognized by CTL, but its antigenicity was revealed in LDA. (2) All responding cells revealed in bulk cultures as well as in LDA were CD8+ T-cells, and their in vitro differentiation did not require the help of CD4+ T-cells. (3) Proteins from the HIV-1LAI isolate were recognized with high frequency by CTL from seropositive donors, most certainly being infected by other isolates, which suggests that relatively conserved epitopes are predominant targets of CTL. CONCLUSION: Taken together, these results are encouraging for vaccine purposes, since anti-HIV-1 CTL stimulation is thought to be a requirement for such a vaccine.

An HIV-1 and HIV-2 cross-reactive cytotoxic T-cell epitope.

The HLA-B27-restricted HIV gag cytotoxic T-lymphocyte (CTL) epitope, 265-279, is highly conserved amongst HIV-1 isolates, only one, HIV-1ELI, having a single amino acid substitution. Over the same region HIV-2 differs by five amino acids. As a broadly cross-protective AIDS vaccine should protect against infection from all isolates of HIV-1 and HIV-2, we tested CTL specific for the HIV-1 265-279 epitope with peptide analogues from HIV-1ELI, HIV-2 and two simian immunodeficiency virus (SIV) isolates, and with recombinant vaccinia viruses expressing the respective gag genes, to determine whether there was any cross-reactivity for this CTL epitope. CTL from the HIV-1-infected donor could recognize the HIV-1ELI peptide, the HIV-2 peptide and recombinant vaccinia virus-infected target and one of the two SIV peptide-treated targets. Epitopes that exhibit such cross-reactivity may be valuable in vaccine design.

Targeted macrophage cytotoxicity using a nonreplicative live vector expressing a tumor-specific single-chain variable region fragment.

Antigen-specific recognition and subsequent destruction of tumor cells is the goal of vaccine-based immunotherapy of cancer. Often, however, tumor antigen-specific cytotoxic T lymphocytes (CTLs) are either not available or in a state of anergy. In addition, MHCI expression on tumor cells is often downregulated. Either or both of these situations can allow tumor growth to proceed unchecked by CTL control. We have shown previously that tumor antigen-specific monoclonal antibodies can be expressed in vaccinia virus and that activated macrophages infected with this virus acquire the ability to kill tumor cells expressing that antigen. Here we show that a membrane-anchored form of the scFv portion of the MUC1 tumor antigen-specific monoclonal antibody, SM3, can be expressed on activated macrophages with the highly attenuated poxvirus, modified vaccinia Ankara (MVA), as a gene transfer vector. Cells infected with the MVA-scFv construct were shown to express the membrane-bound scFv by Western blot and FACS analysis. That cells expressing the membrane-anchored scFv specifically bind antigen was shown by FACS and by BIAcore analysis. GM-CSF-activated macrophages were infected with the construct and shown to recognize specifically MUC1-expressing tumor cells as measured by IL-12 release. Furthermore, activated macrophages expressing the membrane-bound scFv specifically lyse target cells expressing the MUC1 antigen but not cells that do not express MUC1.

New versatile cloning and sequencing vectors based on bacteriophage M13.

A new pair of cloning and sequencing vectors based on bacteriophage M13mp7 has been developed. These vectors (M13tg130 and M13tg131) contain, in addition to the EcoRI, BamHI, HindIII, SmaI, SalI and PstI sites present in other vectors [cf., M13mp8 and M13mp9, Messing and Vieira, Gene 19 (1982) 269-276], unique restriction recognition sequences for the enzymes EcoRV, KpnI, SphI, SstI and XbaI. A restriction site for the enzyme BglII has been incorporated into the polylinker region of one of the vector pair to permit rapid discrimination between the two vectors.

Characterization of polyoma virus early proteins expressed from vaccinia virus recombinants.

We previously reported that live recombinant vaccinia viruses (VV) encoding either the large T (LT) or middle T (MT) antigens of polyoma virus (PyV) were able to induce rejection of tumors caused by PyV-transformed cells [Lathe et al., Nature 326 (1987) 878-880]. Here we present evidence that PyV early proteins expressed by the recombinants retain the biochemical characteristics of their authentic counterparts despite the cytopathic effect of VV infection. VV-encoded LT is a nuclear phosphoprotein, with specific DNA binding, ATPase and nucleotide-binding activities. VV-expressed MT associates with cellular kinases, particularly with pp60c-src, by which it is phosphorylated in vitro. Expression levels of LT and MT reached 10(6) molecules per infected cell. The use of VV as a vector is encouraged by the high expression level obtained and because VV infection does not seem to prevent appropriate post-translational processing of proteins encoded by VV recombinants.

Expression of heterologous genes in Mycobacterium bovis BCG: induction of a cellular response against HIV-1 Nef protein.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) has been used as a live bacterial vaccine to immunize more than two billion people against tuberculosis. In an attempt to use this vaccinal strain as a vehicle for protective antigens, the human immunodeficiency virus type 1 gene encoding the Nef protein was cloned in a mycobacteria-Escherichia coli shuttle plasmid and transferred into BCG. The nef gene was expressed under the control of an expression cassette carrying the promoter of the groES/groEL1 operon from Streptomyces albus and a synthetic ribosome-binding site. Lymph node cells from mice immunized with BCG-nef proliferated vigorously in response to purified Nef protein. This first report of a proliferative response suggests that recombinant BCG strains may be used to immunize against pathogens for which T-cell-mediated responses are important for protection.

Sulphation of hirudin in BHK cells.

Hirudin, a thrombin inhibitor of the leech, was expressed in BHK cells; the alpha 1-antitrypsin signal peptide was used to direct secretion into the culture medium. The recombinant hirudin so produced inhibited thrombin and was shown by labelling experiments with [35S]sulphate to have been posttranslationally modified.

DNA binding factors that bind to the negative regulatory element of the human immunodeficiency virus-1: regulation by nef.

The nef gene has been reported to be a silencer of human immunodeficiency virus (HIV) transcription that requires the presence of the negative regulatory element (NRE) located at the 5' end of the HIV long terminal repeat (LTR) to exert its negative effect. We have examined nuclear extracts from human, nontransformed T cells for factors that bind to the NRE of HIV-1 and to determine whether binding of factors to this region can be affected by the nef gene. Using gel retardation and methylation interference assays, we have observed several DNA binding factors that bind to a region between nucleotides -315 and -240 upstream of the cap site, within the NRE segment of the 5' LTR. Furthermore, the precise locations of the binding sites for two of these factors, termed here A1 and R, were determined. Factor A1 appears to belong to a family of cellular activation associated factors (called here A1-A4), but it is distinct in that it is the only DNA binding factor so far observed that appears to be downregulated by the nef gene or its product and that it has been found only in cells undergoing lymphokine-driven cell division. In contrast to the A factors, factor R appears to be associated with cellular quiescence and binds to a nearby but distinct site in the NRE. Experiments in which extracts were mixed before gel retardation suggest that the binding of factors R and A1 are mutually exclusive. Based on these observations we propose a model in which the nef gene aids in the maintenance of HIV latency by downregulating the binding of proliferation associated DNA binding factor, which we have called A1.

Redirected cellular cytotoxicity by infection of effector cells with a recombinant vaccinia virus encoding a tumor-specific monoclonal antibody.

Cytotoxicity is an important function of the immune system that results in the destruction of cellular targets by humoral and/or cellular mechanisms. We wanted to assess the possibility of targeting the lytic function of immune cells toward cancer cells, which express the gene coding for a known tumor antigen (Ag) (GA733-2/epithelial cell adhesion molecule), using a viral vector encoding a monoclonal antibody (mAb) specific for said tumor Ag (CO17-1A). To this end, we have constructed recombinant vaccinia viruses expressing the sequences corresponding to mAb CO17-1A, which recognizes a specific Ag (GA733-2) that is present on the surface of most gastrointestinal carcinomas. The recombinant vectors encoding either a secreted or membrane-anchored form of CO17-1A mAb were used to infect effector cells, which were subsequently assessed for their cytotoxic activity. The recombinant viruses were able to infect both granulocyte-macrophage colony-stimulating factor-activated human macrophages and Ag-stimulated murine cytotoxic T lymphocytes. Infected granulocyte-macrophage colony-stimulating factor-activated macrophages were found to be able to kill GA733-2-expressing tumor cells. Likewise, infected cytotoxic T lymphocytes, although conserving their original alloreactivity, gained the capability of killing GA733-2-expressing cancer cells.

Recombinant vaccinia viruses expressing immunoglobulin variable regions efficiently and selectively protect mice against tumoral B-cell growth.

The variable regions of the immunoglobulin (Ig) expressed on the surface of a malignant B cell can be considered tumor-specific antigens and, as such, could be targets for immunotherapeutic approaches. However, because until now the immunization procedures have been complex and have given only partial protection, it was necessary to find new methods of immunotherapy. Here, we present a successful method of vaccination against B-cell tumors in a murine model. We produced recombinant vaccinia viruses (rVV) expressing the heavy and the light chain of surface Ig of a patient's malignant B cells and we tested the ability of these rVV to protect immunized mice against tumor growth of transfectomas producing the same human Ig. The protection of the mice was complete and specific to the variable region of the immunizing heavy chain although specific lymphoproliferative and cytotoxic responses were not detectable in vitro. The protection was strictly dependent on the presence of CD4 T cells and asialo GM1+ cells. Furthermore, tumor protection clearly required gamma-interferon and was partially inhibited by blocking the Fas-Fas ligand interaction. We also show, in a murine syngeneic model, that rVV expressing a poorly mutated Ig protects against the growth of Ig-producing tumor.

Recombinant polyoma--vaccinia viruses: T antigen expression vectors and anti-tumor immunization agents.

Live vaccinia virus recombinants expressing viral antigens have recently been developed as effective anti-viral vaccines. We have examined the possibility of extending this approach to specific anti-tumor immunity, using tumors induced by the polyoma virus (PyV) as a model system. Three recombinant vaccinia viruses, separately encoding the three early proteins of the polyoma virus (large, middle and small tumor (T) antigens) were constructed. Each recombinant efficiently expresses the appropriate T antigen, which exhibits biochemical properties and subcellular localization of the authentic PyV protein. The potential of the recombinants to elicit immunity towards PyV-induced tumors was assessed in rats by a challenge injection of syngeneic PyV-transformed cells. After prior immunization with the large-T or the middle-T viruses, small tumors developed, which later regressed and were eliminated in more than 50% of the animals. In contrast, the small-T virus failed to elicit tumor rejection. Established tumors could also be eliminated by curative vaccinations. No circulating antibodies directed against PyV large-T or middle-T antigens were detected in animals vaccinated with the large-T or middle-T viruses, suggesting that rejection may be due to a cell-mediated immune response.