Viremia control following antiretroviral treatment and therapeutic immunization during primary SIV251 infection of macaques.

Prolonged antiretroviral therapy (ART) is not likely to eradicate human immunodeficiency virus type I (HIV-I) infection. Here we explore the effect of therapeutic immunization in the context of ART during primary infection using the simian immunodeficiency virus (SIV251) macaque model. Vaccination of rhesus macaques with the highly attenuated poxvirus-based NYVAC-SIV vaccine expressing structural genes elicited vigorous virus-specific CD4 + and CD8+ T cell responses in macaques that responded effectively to ART. Following discontinuation of a six-month ART regimen, viral rebound occurred in most animals, but was transient in six of eight vaccinated animals. Viral rebound was also transient in four of seven mock-vaccinated control animals. These data establish the importance of antiretroviral treatment during primary infection and demonstrate that virus-specific immune responses in the infected host can be expanded by therapeutic immunization.

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.

Recombinant HIV-1 vaccine candidates based on replication-defective flavivirus vector.

Multiple approaches utilizing viral and DNA vectors have shown promise in the development of an effective vaccine against HIV. In this study, an alternative replication-defective flavivirus vector, RepliVax (RV), was evaluated for the delivery of HIV-1 immunogens. Recombinant RV-HIV viruses were engineered to stably express clade C virus Gag and Env (gp120TM) proteins and propagated in Vero helper cells. RV-based vectors enabled efficient expression and correct maturation of Gag and gp120TM proteins, were apathogenic in a sensitive suckling mouse neurovirulence test, and were similar in immunogenicity to recombinant poxvirus NYVAC-HIV vectors in homologous or heterologous prime-boost combinations in mice. In a pilot NHP study, immunogenicity of RV-HIV viruses used as a prime or boost for DNA or NYVAC candidates was compared to a DNA prime/NYVAC boost benchmark scheme when administered together with adjuvanted gp120 protein. Similar neutralizing antibody titers, binding IgG titers measured against a broad panel of Env and Gag antigens, and ADCC responses were observed in the groups throughout the course of the study, and T cell responses were elicited. The entire data demonstrate that RV vectors have the potential as novel HIV-1 vaccine components for use in combination with other promising candidates to develop new effective vaccination strategies.

Viral vector delivery in solid-state vehicles: gene expression in a murine prostate cancer model.

BACKGROUND: Although there are increasingly more clinical trials involving gene therapy, efficient gene transfer remains a major hurdle to success. To enhance the efficiency of delivery of viral vectors in gene therapy protocols, we evaluated the effect of various matrices to act as a vehicle for recombinant virus during intratumoral injection. METHODS: The ability of several vehicles (catgut spacer, polyglycolic acid, chromic catgut, and gelatin sponge matrix) to deliver the canarypox virus ALVAC to the cells of the murine prostate cancer cell line RM-1 was studied in vitro and in vivo. ALVAC recombinants encoding the murine cytokines interleukin 2 (IL-2), interleukin 12 (IL-12), and tumor necrosis factor-alpha (TNF-alpha) were used to assess enhancement of antitumor activity after intratumoral inoculation. Confirmatory experiments were conducted by use of another mouse prostate cancer cell line, RM-11, and a mouse bladder cancer cell line, MB-49. All statistical tests were two-sided. RESULTS: The gelatin sponge matrix proved to be the most effective solid-state vehicle for delivering viral vectors to cells in culture. In addition, this matrix statistically significantly enhanced expression of ALVAC-delivered reporter genes in tumor models when compared with fluid-phase delivery of virus (P =.037 for the RM-1 model and P =.03 for the MB-49 model). Statistically significant growth inhibition of established tumors was observed when a combination of the three recombinant ALVAC viruses expressing IL-2, IL-12, and TNF-alpha was delivered with the matrix in comparison with 1) fluid-phase intratumoral injection of the ALVAC recombinants, 2) no treatment, or 3) treatment with parental ALVAC (all P<.05). CONCLUSIONS: Viral vector delivery in a solid-state vehicle resulted in improved recombinant gene expression in vivo and translated to greater inhibition of tumor growth in an immunotherapy protocol for heterotopic tumor nodules. The efficient delivery of reporter genes described herein may prove useful in many solid tumor gene therapy protocols.

Effect of canarypox virus (ALVAC)-mediated cytokine expression on murine prostate tumor growth.

BACKGROUND: Canarypox virus, ALVAC, does not replicate in infected mammalian cells and has potential as a vector for gene therapy in the treatment of cancer. PURPOSE: Recombinant viruses carrying DNA sequences encoding interleukin 2 (ALVAC-IL-2), interferon gamma (ALVAC-IFN gamma), tumor necrosis factor-alpha (ALVAC-TNF-alpha), or the co-stimulatory molecule B7-1 (ALVAC-B7-1) were investigated as agents for the treatment of a newly defined mouse prostate tumor model. METHODS: RM-1 mouse prostate cancer cells, which are syngeneic (i.e., same genetic background) to C57BL/6 mice, were used. The expression of foreign gene products in vitro in infected RM-1 cells was measured by immunoprecipitation, bioassay, or flow cytometry. The effects of foreign gene product expression on RM-1 tumor cell growth in C57BL/6 mice were measured after subcutaneous injection (in the back) of 5 x 10(5) uninfected or infected cells; measurements included determinations of time to a measurable tumor size, tumor size as a function of time, and survival. The induction of protective immunity by uninfected and infected RM-1 cells was tested by injection of lethally irradiated (70 Gy) cells and subsequent challenge with uninfected cells. The generation of cytotoxic T cells was monitored by use of a 51Cr release assay. Severe combined immunodeficient (SCID) mice were used to determine whether T or B lymphocytes were involved in ALVAC vector-mediated antitumor responses. Data were analyzed by use of Pearson's modification of the chi-squared test and Kaplan-Meier survival methods. Reported P values are two-sided. RESULTS: The level of foreign gene product expression in ALVAC-infected RM-1 cells was dependent on the multiplicity of virus infection used; a multiplicity of five viruses per infected cell was chosen for subsequent experiments. RM-1 tumor growth in C57BL/6 mice was not affected by tumor cell expression of IL-2 alone, IFN gamma alone, or B7-1 alone; however, expression of TNF-alpha alone significantly delayed tumor growth at early time points (compared with parental ALVAC-infected tumors, P = .0001 at day 21 and P = .037 at day 28). Tumor cell expression of both TNF-alpha and IL-2 completely inhibited tumor growth in 60%-100% of treated mice. No protection against subsequent tumor challenge was detected in mice previously exposed to RM-1 cells expressing both TNF-alpha and IL-2. Cytotoxic T-lymphocyte activity toward RM-1 cells was not observed in C57BL/6 mice that rejected tumors. Tumor cell expression of TNF-alpha and IL-2 also resulted in tumor growth inhibition in SCID mice. CONCLUSIONS: RM-1 mouse prostate cancer cells are readily infected by ALVAC vectors, and foreign gene products are efficiently expressed. Inhibition of RM-1 tumor growth by tumor cell expression of TNF-alpha and IL-2 appears to involve nonspecific antitumor activity.

Inhibition of murine prostate tumor growth and activation of immunoregulatory cells with recombinant canarypox viruses.

BACKGROUND: Immunization with modified tumor cells carrying recombinant immunomodulatory genes is being explored as cancer immunotherapy. In this study, we examine whether canarypox ALVAC viruses carrying immunostimulatory cytokine genes (granulocyte-macrophage colony-stimulating factor, interleukin 2, interleukin 12, and tumor necrosis factor-alpha) can induce antitumor immunity (to rechallenge) in the RM-1 model of a highly aggressive, weakly immunogenic murine prostate cancer. METHODS: For antitumor activity studies, RM-1 murine prostate cancer cells were infected with the parental ALVAC virus or one or two recombinant ALVAC-cytokine viruses and then injected into male C57BL/6 mice. For rechallenge studies, other mice were first given an injection subcutaneously with irradiated (nonproliferating) recombinant ALVAC-infected RM-1 cells and then (10 days later) with untreated RM-1 cells. For the determination of which immune cells were required for antitumor activity, mice were immunodepleted of CD4, CD8, or natural killer (NK) NK1.1 cells with the corresponding monoclonal antibodies and were then given an injection of ALVAC-cytokine-infected RM-1 cells. For all experiments, tumor outgrowth and animal survival were monitored. RESULTS: After subcutaneous injection into mice, RM-1 cells infected with one (except ALVAC-interleukin 2) or two ALVAC-cytokine recombinants had statistically significantly greater antitumor activity than RM-1 cells infected with parental ALVAC (P<.001 for all; two-sided test). The antitumor activity of RM-1 cells infected with any two ALVAC-cytokine recombinants was greater than, but not statistically significantly different from, that of RM-1 cells infected with any one ALVAC-cytokine recombinant. NK1.1 cells were necessary for antitumor activity, but tumor-specific CD4(+) regulatory T cells were also induced that inhibited CD8(+) RM-1-specific cytotoxic T cells, resulting in the lack of immunity to a rechallenge by RM-1 cells. DISCUSSION: Canarypox viruses can transfer immunostimulatory cytokine genes into RM-1 prostate cancer cells. When such cells were injected into mice, the cytokines induced an antitumor response against this highly aggressive, weakly immunogenic tumor. This response, however, did not protect the mouse against a rechallenge with RM-1 cells because suppressor CD4(+) T cells were induced that inhibited tumor-specific CD8(+) cytotoxic T cells.

MAGE-1-specific precursor cytotoxic T-lymphocytes present among tumor-infiltrating lymphocytes from a patient with breast cancer: characterization and antigen-specific activation.

A potential target for development of tumor-specific immunotherapeutic strategies is the MAGE-1 gene. We have utilized a recently developed recombinant canarypox (ALVAC) virus vector containing the MAGE-1 gene (vCP235) to activate CTLs from a breast cancer patient bearing a MAGE-1+ tumor. Tumor-infiltrating lymphocytes (TILs) obtained from the tumor of a patient were stimulated in vitro with irradiated autologous peripheral blood mononuclear cells acutely infected with the vCP235 construct. These TILs preferentially expanded approximately 6-fold over a 16-day culture period and specifically recognized an allogeneic transformed B-cell line acutely infected with a vaccinia-MAGE-1 recombinant targeting vector (vP1188) in the context of HLA-A2 and/or B7. TCR V beta analysis of in vitro expanded T cells by a quantitative multiprobe RNase protection assay revealed preferential expansion of TCR V beta 6.3 and V beta 6.4. In addition, homologous T-cell receptor beta CDR3 joining sequences were found in the in vitro stimulated cultures. These results suggest that tumor antigen-specific, MHC-restricted CTLs may be derived from precursor CTLs present in TILs obtained from patients with MAGE-1+ tumors by in vitro stimulation with recombinant avipox MAGE-1 virus-infected autologous cells. Collectively, these findings provide a rationale for tumor-associated antigen-based immunization as a means of activating precursor CTLs residing in patients with tumors expressing defined tumor-associated antigens such as MAGE-1.

Poxvirus-based vectors as vaccine candidates.

The advent of recombinant DNA techniques and advances in immunology have provided a means for dissecting the immunobiology of disease-causing agents. Identification and expression of individual genes from the pathogens in heterologous systems, such as VV, have yielded valuable information regarding structural properties of the gene products and their role in eliciting protective immunity. Targets of both humoral and/or cellular immunity for many disease-causing agents have been identified or confirmed using a VV expression system (Section IV). Additionally, specific VV recombinants have induced a protective immune response in experimental animals. The ability of VV recombinants to induce pertinent immune responses necessary for protection, the potential to develop polyvalent vaccines, and the successful history of VV as an immunizing agent provide the impetus for engineering VV as a live recombinant vaccine candidate. Critical to the refinement of poxviruses as recombinant immunizing agents is a more in-depth knowledge of the molecular biology of these viruses. Although significant advances have been made in this area within the past 10 years, a greater understanding of the mechanisms governing gene expression and viral virulence factors should enable the development of more safe and effective vaccine candidates. Progression of VV vector technology to other members of the poxvirus family has been successful. Development of other poxviruses as vectors may, therefore, provide a means of generating host-restricted vaccines. Fowlpox recombinant viruses, for instance, may yield candidate vaccines in the poultry industry. Interestingly, it was also demonstrated that these host-restricted recombinant viruses can be used as immunizing vehicles in other species. The ability of a nonreplicating viral vector to elicit a protective immune response is especially intriguing in light of the observation by Morgan et al. that a VV/EBV gp340/220 recombinant, derived from an avirulent VV strain, was unable to protect cottontop tamarins from a live EBV challenge.

Poxvirus-based vaccine candidates for cancer, AIDS, and other infectious diseases.

Over the past 12 years, the poxvirus vector technology has provided scientists with valuable reagents to achieve high-level expression of proteins, to address questions of structure-function relationship of specific polypeptides, to investigate the immunobiology of specific pathogens, and to develop recombinant vaccine candidates. It is this last role that has drawn enthusiasm from the medical community because of the potential this technology has to provide novel approaches for addressing urgent needs in human and veterinary medicine. From one perspective, the safety issues surrounding the use of vaccinia-based vaccine candidates have been addressed with the development of the NYVAC and ALVAC vectors. Evaluation of these novel poxvirus vectors are in progress to determine their potential impact on cancer and infectious disease.

Efficacy evaluation of prime-boost protocol: canarypoxvirus-based feline immunodeficiency virus (FIV) vaccine and inactivated FIV-infected cell vaccine against heterologous FIV challenge in cats.

OBJECTIVE: To evaluate the immunogenicity and prophylactic efficacy of immunization schemes employing a recombinant canarypoxvirus ('ALVAC')-based feline immunodeficiency virus (FIV) vaccine alone or in combination with an inactivated FIV-infected cell vaccine against homologous and heterologous FIV challenges in cats. METHODS: Specific pathogen-free cats were given a total of three immunizations with subtype A vaccines and challenged 4 weeks after the final immunization with 50 median animal infectious doses (ID50) of FIV-Petaluma, a subtype A isolate. Following the initial challenge, protected cats received a second challenge with 75 ID50 of FIV-Bangston, a subtype B isolate. FIV-specific humoral and cell-mediated responses were measured to determine the immune correlates of protection. RESULTS: Two of three cats immunized with the ALVAC FIV recombinants alone were protected from homologous FIV challenge in the presence of FIV-specific cytotoxic T-lymphocyte (CTL) responses but in the absence of FIV-specific humoral responses. All three cats immunized with the ALVAC-FIV recombinant and boosted with FIV-infected cell vaccine were also protected from homologous FIV challenge in the presence of both FIV-specific CTL and humoral responses. Partial to full protection was observed in ALVAC-FIV/FIV-infected cell vaccine-immunized cats against a heterologous FIV challenge given 8 months after the initial challenge. Two out of three cats had transient infection and the remaining cat had no sign of FIV infection at a dose at which all three control cats were readily infected. CONCLUSIONS: Immunization schemes employing ALVAC-based FIV vaccines in combination with inactivated FIV-infected cell vaccine generate protective immune responses that can cross-react with FIV isolates that are genetically distinct from the vaccine strains.

Cross-protection in NYVAC-HIV-1-immunized/HIV-2-challenged but not in NYVAC-HIV-2-immunized/SHIV-challenged rhesus macaques.

OBJECTIVES: Immunization with attenuated poxvirus-HIV-1 recombinants followed by protein boosting had protected four of eight rhesus macaques from HIV-2SBL6669 challenge. The present study was designed to confirm this result and to conduct the reciprocal cross-protection experiment. METHODS: Twenty-four macaques were primed with NYVAC (a genetically attenuated Copenhagen vaccinia strain) recombinants with HIV-1 and HIV-2 env and gag-pol or NYVAC vector alone and boosted with homologous, oligomeric gp160 proteins or adjuvant only. Binding and neutralizing antibodies, cytotoxic T-lymphocytes (CTL) and CD8 T cell antiviral activity (CD8AA) were evaluated. One half of each immunization and control group were intravenously challenged with SHIV(HXB2) the other half was challenged with HIV-2SBL6669,. Protective outcome was assessed by monitoring virus isolation, proviral DNA and plasma viral RNA. RESULTS: Both immunization groups developed homologous binding antibodies; however, homologous neutralizing antibodies were only observed in NYVAC-HIV-2-immunized macaques. While no cross-reactive neutralizing antibodies were detected, both immunization groups displayed cross-reactive CTL. Significant CD8AA was observed for only one NYVAC-HIV-2-immunized macaque. Virological assessments verified that both NYVAC-HIV-1 and NYVAC-HIV-2 immunization significantly reduced viral burdens and partially protected against HIV-2 challenge, although cross-protection was not at the level that had been previously reported. Humoral antibody and/or CTL and CD8AA were associated with protection against homologous HIV-2 challenge, while cellular immune responses seemed more important for cross-protection. No significant protection was observed in the SHIV-challenged macaques, although NYVAC-HIV-1 immunization resulted in significantly lower viral burdens compared with controls. CONCLUSIONS: Further delineation of cross-reactive mechanisms may aid in the development of a broadly protective vaccine.

Induction of immune responses to HIV-1 by canarypox virus (ALVAC) HIV-1 and gp120 SF-2 recombinant vaccines in uninfected volunteers. NIAID AIDS Vaccine Evaluation Group.

OBJECTIVE: To determine the ability of live attenuated canarypox virus expressing HIV antigens to induce CD8+ cytotoxic T-cell responses and to prime for neutralizing antibody responses to boosting with purified recombinant gp120 subunit vaccine. DESIGN: A prospective, double-blind, randomized, immunogenicity and safety study was conducted in healthy adults at low risk for acquiring HIV infection and who were seronegative for HIV. METHODS: CD8+ cytotoxic T-cells directed against Env or Gag expressing target cells were measured after live recombinant canarypox-HIV-1 vaccine priming (vaccine given at days 0, 7, 14 and 21). Neutralizing antibodies were measured after subunit boosting (vaccine given at days 28 and 84). RESULTS: CD8+ CTL were induced in 64% of volunteers by the live recombinant canarypox-HIV-1 vaccine. All volunteers who received two doses of subunit vaccine after live recombinant canarypox priming developed neutralizing antibodies directed against laboratory strains of HIV-1 and seven out of eight volunteers tested developed neutralizing antibodies to the primary isolate, BZ167, but to none of eight other primary isolates. Unprimed controls had low or absent neutralizing antibodies after two doses of subunit vaccine. CONCLUSIONS: The live canarypox vector was safe, stimulated cytotoxic T-cells and primed for a vigorous neutralizing antibody response upon boosting with subunit gp120 vaccine. This vaccine combination should be evaluated further for inducing protection against HIV infection.

Immunogenicity of whole-cell tumor preparations infected with the ALVAC viral vector.

The immunogenicity of recombinant canarypox (ALVAC) viral vectors within murine whole-cell tumor vaccines was evaluated using the T cell thymic lymphoma STF10 and the B16 melanoma. Tumor cells were modified with the recombinant ALVAC vectors and injected into syngeneic mice. Control mice receiving cells alone all developed tumors, while mice injected with tumor variants bearing parental and recombinant vectors either completely rejected their tumors, or exhibited a significant delay in tumor formation. Rechallenge of mice receiving STF10-variant vaccines yielded a protective effect against parental tumor cells only when a modified regimen incorporating two vaccinations was utilized. Notably, the parental ALVAC virus was equivalent to all other recombinant ALVAC viruses in conferring antitumor immunity when using a prime-and-boost protocol. Tumorigenicity experiments in nude mice revealed that the effector mechanism mediating rejection of tumor cells bearing ALVAC vectors is multifactorial, in that the immunogenicity of STF10/ALVAC vaccines is reduced, but not completely abolished in these mice. Finally, in vitro experiments revealed that cytotoxic T cells specific for parental STF10 cells could be generated as a result of in vivo immunization with STF10/ALVAC vaccines.

Canarypox virus-mediated interleukin 12 gene transfer into murine mammary adenocarcinoma induces tumor suppression and long-term antitumoral immunity.

The antitumoral activity of recombinant canarypox virus vectors (ALVAC) expressing murine interleukin 12 (IL-12) was evaluated in the syngeneic, nonimmunogenic murine mammary adenocarcinoma model (TS/A). Seven-day preestablished subcutaneous tumors (5- to 6-mm mean diameters) were injected on days 7, 10, 14, 17, 21, and 24 with the vector ALVAC-IL12 at 2.5 x 10(5) TCID50 (50% tissue culture infective dose). Total tumor regression occurred in 40 to 50% of the treated mice. Furthermore, 100% of the cured mice were protected against a contralateral subsequent challenge with the TS/A parental cells on day 28. The ALVAC-IL12 treatment is not effective in nude mice, suggesting the critical role of T cells. CD4 and CD8 T cells infiltrated the tumors treated with ALVAC-IL12 in the BALB/c model. Furthermore, in vivo depletion of CD4+ T cells totally abrogated the induction of the long-term antitumoral immune response by ALVAC-IL12. Interestingly, some tumor growth inhibition was also observed with ALVAC-betaGal treatment and a vaccinal effect was found in 33% of the treated animals, suggesting an adjuvant effect of the vector itself. Other ALVAC vectors expressing murine cytokines (IL-2, GM-CSF, IFN-gamma) were evaluated in the same model. Major antitumoral activity was observed with ALVAC-GM-CSF. However, a combination of ALVAC-GM-CSF and ALVAC-IL12 had no synergistic effect. These results suggest that in vivo gene transfer with canarypox virus expressing IL-12 may provide an effective and safe strategy for the treatment of human cancers.

Role of the vaccinia virus E3L and K3L gene products in rescue of VSV and EMCV from the effects of IFN-alpha.

Vaccinia virus (VV) has been shown to be relatively resistant to the antiviral effects of interferon-alpha (IFN-alpha) and to rescue replication of IFN-sensitive viruses, such as encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV), from the antiviral effects of IFN. The E3L and K3L gene products have been implicated in the IFN resistance of VV. We have investigated the role that these VV-encoded functions play in the rescue of VSV and EMCV from the effects of IFN. Transient expression of the E3L open reading frame (ORF) was sufficient to rescue VSV but not EMCV from the IFN-induced antiviral state. Rescue of VSV by mutants of E3L correlated with the ability of the mutated E3L gene products to bind dsRNA. Conversely, transient expression of the K3L ORF was sufficient to partially rescue EMCV but not VSV from the effects of IFN. Results with VV deleted of either the K3L or E3L ORFs were consistent with results obtained by transient expression of these genes. These results demonstrate that the VV E3L gene products are likely responsible for the VV-mediated rescue of VSV from the effects of IFN and the K3L gene product is likely at least partly responsible for rescue of EMCV.

Canarypox virus expressing wild type p53 for gene therapy in murine tumors mutated in p53.

The antitumor activity of a recombinant canarypox virus expressing wild type murine p53 (ALVAC-p53) was investigated in two murine syngeneic tumors harboring an endogenous p53 mutation (CMS4 and TS/A). Direct intratumor injections of ALVAC-p53 in CMS4 pre-established subcutaneous tumors induced total tumor regression in 66% of mice. Furthermore, 100% of the cured mice was protected against a contralateral subsequent challenge with the parental tumor cells. The intravenous treatment of experimental lung metastasis by ALVAC-p53 also induced significant tumor growth inhibition in both models. The antitumor effect of ALVAC-p53 was only observed in immunocompetent animals and was associated with the generation of a specific antitumor immune response. ALVAC-p53 induced the expression of a functional p53 wild type protein as demonstrated by up-regulation of p21waf1 and induction of apoptosis. A vaccine strategy using intravenous or subcutaneous ALVAC-p53/NYVAC-p53 prime boost protocol failed to induce CTL against p53 wild type used as target tumor antigen, and failed to protect mice against challenge with the mutated tumor cells. The mechanism of the curative and protective effects observed after direct intratumor injections results from the induction of a specific antitumor response directed against other antigens than p53. Our results suggest that the local induction of tumor apoptosis, combined with the adjuvant effect of ALVAC vector, enhances the immunogenicity of the intratumor environment and allows induction of specific antitumor immune response.

Recombinant viruses as a tool for therapeutic vaccination against human cancers.

Viral vectors can be used to express a variety of genes in vivo, that encode tumor associated antigens, cytokines, or accessory molecules. For vaccination purposes, the ideal viral vector should be safe and enable efficient presentation of expressed antigens to the immune system. It should also exhibit low intrinsic immunogenicity to allow for its re-administration in order to boost relevant specific immune responses. Furthermore, the vector system must meet criteria that enable its industrialization. The characteristics of the most promising viral vectors, including retroviruses, poxviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, and alphaviruses, will be reviewed in this communication. Such recombinant viruses have been successfully used in animal models as therapeutic cancer vaccines. Based on these encouraging results, a series of clinical studies, reviewed herein, have been undertaken. Human clinical trials, have as of today, allowed investigators to establish that recombinant viruses can be safely used in cancer patients, and that such recombinants can break immune tolerance against tumor-associated antigens. These promising results are now leading to improved immunization protocols associating recombinant viruses with alternate antigen-presentation platforms (prime-boost regimens), in order to elicit broad tumor-specific immune responses (humoral and cellular) against multiple target antigens.

Human immunodeficiency virus type 1 envelope-specific cytotoxic T lymphocytes response dynamics after prime-boost vaccine regimens with human immunodeficiency virus type 1 canarypox and pseudovirions.

Virus-specific cytotoxic T lymphocytes (CTLs) may represent significant immune mechanisms in the control of human immunodeficiency virus (HIV) infection and, therefore, CTL induction may be a fundamental goal in the development of an efficacious acquired immunodeficiency syndrome (AIDS) vaccine. In the current study, prime-boost protocols were used to investigate the potential of noninfectious human immunodeficiency virus type 1 (HIV-1) pseudovirions (HIV PSV) in enhancing HIV-specific CTL responses in Balb/c mice primed with the recombinant canarypox vector, vCP205, encoding HIV-1 gp120 (MN strain) in addition to Gag/Protease (HIB strain). The prime-boost immunization regimens were administered intramuscularly and involved injections of vCP205 followed by boosts with HIV PSV. Previous vaccination strategies solely involving vCP205 had induced good cellular immune responses in uninfected human volunteers, despite some limitations. The use of genetically engineered HIV PSV was a logical step in the evaluation of whole noninfectious virus or inactivated virus vaccine strategies, particularly as a potential boosting agent for vCP205-primed recipients. Based on this current study, HIV PSV appeared to have the capability to effectively induce and boost cell-mediated HIV-1-specific responses. In order to observe the immune effects of HIV PSV in a prime-boost immunization strategy, both HIV vaccine immunogens required careful titration in vivo. This suggests that careful consideration should be given to the optimization of immunization protocols destined for human use.

Highly attenuated HTLV type Ienv poxvirus vaccines induce protection against a cell-associated HTLV type I challenge in rabbits.

The entire envelope protein of the human T cell leukemia/lymphoma virus type I (HTLV-I)1711, obtained from the DNA of a West African healthy HTLV-I-infected patient, was expressed in the highly attenuated poxvirus vaccine vectors ALVAC and NYVAC. These live recombinant vaccine candidates were used to immunize New Zealand White rabbits. Immunization regimens included inoculation of the poxvirus recombinant alone as well as prime/boost protocols using gp63 HTLV-I envelope precursor protein in Alum as the subunit boost. All animals were exposed to an HTLV-I cell-associated challenge (5 x 10(4) cells) from a primary culture of the HTLV-IBOU isolate. The results indicated that two inoculations of the ALVAC-based HTLV-Ienv vaccine candidate protected animals against viral challenge 5 months following the last immunization. However, a combination protocol with ALVAC-env and two additional boosts of gp63 surprisingly failed to confer protection, suggesting that administration of the subunit preparation might be deleterious. Further, in the case of the NYVAC HTLV-Ienv recombinant, protection was afforded as early as 2 months following the first immunization. Last, all the protected animals in the NYVAC and ALVAC trials were challenged 5 months following the initial challenge exposure with 5 ml of blood from an HTLV-IBOU-infected animal, and subsequently became infected. Protection conferred by the attenuated HTLV-Ienv recombinant poxvirus vaccine in the rabbit model might be instrumental for optimizing the immunogenicity of poxvirus-based vaccine candidates against human immunodeficiency virus (HIV), particularly because of the need to enhance protection against cell-to-cell transmission.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple immunizations with attenuated poxvirus HIV type 2 recombinants and subunit boosts required for protection of rhesus macaques.

Vaccine protocols involving multiple immunizations with molecularly attenuated vaccinia virus (NYVAC) or naturally attenuated canarypox virus (ALVAC) HIV-2 recombinants and subunit boosts have conferred longlasting protection against HIV-2 infection of macaques. Similar complex protocols using HIV-1 NYVAC and ALVAC recombinants and subunit boosts have provided cross-protection against HIV-2 challenge. Here a simplified three-immunization regimen over 24 weeks was tested in 18 juvenile rhesus macaques. Twelve macaques were immunized twice with NYVAC or ALVAC recombinants carrying HIV-2 env, gag, and pol genes. Subsequently, macaques in groups of three received either an additional recombinant immunization or an HIV-2 gp160 boost. Six control macaques received three immunizations of NYVAC or ALVAC vector alone and additionally alum at the third immunization. Macaques primed with ALVAC recombinant exhibited sporadic T cell proliferative activity, and all but one failed to develop neutralizing antibodies. In contrast, macaques primed with NYVAC recombinants had no T cell proliferative activity but exhibited neutralizing antibody titers (highest in the three recombinant group) that declined by the time of challenge. None of the macaques exhibited significant cytotoxic T lymphocyte activity. Following challenge at 32 weeks with HIV-2SBL6669 all macaques became infected. Thus, the three-immunization regimen is not sufficient to confer protective immunity in the HIV-2 rhesus macaque model. However, delayed infection in macaques immunized with the NYVAC-HIV-2 recombinant may have been associated with the development of memory B cells capable of providing a neutralizing antibody response on challenge.