Identification of the canarypox virus thymidine kinase gene and insertion of foreign genes. (1/54)

We mapped the canarypox virus (CaPV) thymidine kinase (TK) gene within a 5.8-kbp XbaI fragment of the genome by Southern blotting using the fowlpox virus (FPV) TK gene as a probe. Nucleotide sequence analysis of the fragment revealed seven open reading frames (ORFs) showing gene organization similar to that of FPV. The TK gene contained in this region had an ORF of 179 amino acids encoding a polypeptide with a putative molecular mass of 20.0 kDa. An A/T-rich region and a transcription termination signal, TTTTTAT, were found upstream and at the end of the ORF, which is consistent with poxvirus early gene regulation. The consensus sequence of the late promoter TAAAT also overlapped with the initiation codon of the ORF. The amino acid sequence similarity between the TK genes of CaPV and FPV, avipoxviruses, was 64.2%, which was lower than the similarities between vaccinia and variola orthopoxviruses (97.2%) and between Shope fibroma and myxoma leporipoxviruses (82.6%). However, the monophyly of avian clades of CaPV and FPV was supported by phylogenetic analysis. We then inserted the genes encoding lacZ, luciferase (luci), and envelope of human T-lymphotropic virus type 1 (HTLV-1 env) into the TK gene of CaPV to evaluate its suitability as an expression vector. The recombinant viruses obtained were unstable, although the foreign genes were expressed efficiently in the mammalian cells infected with the viruses.  (+info)

Antigen gene transfer to cultured human dendritic cells using recombinant avipoxvirus vectors. (2/54)

Advances in understanding the role of dendritic cells (DCs) as the major antigen (Ag)-presenting cell type of the immune system combined with the recent development of methods for the ex vivo expansion of human DCs have opened the possibility for the transfer of tumor Ags to DCs with a view toward tumor immunotherapy. In this study, we examined the feasibility of Ag transfer to cultured human DCs using the host range-restricted avipoxvirus, fowlpoxvirus (FWPV). FWPV was found to infect and express a lacZ marker gene in a number of mammalian cell lines of fibroblastic, epithelial, and hemopoietic lineage origins. LacZ recombinant FWPV (rFWPV) was found subsequently to infect human DCs that had been cultured ex vivo from peripheral blood monocytes. Using rFWPV containing lacZ under the control of a vaccinia virus (VV) early/late promoter (p7.5K) and a 10 plaque-forming units per cell multiplicity of infection, >80% of cells expressed the lacZ marker gene. Quantitative analysis showed that the level of expression continued to rise for 5 days postinfection, at which point the experiments were terminated. Replication-competent recombinant VV (rVV) was also shown to be capable of transferring the marker gene to primary DC cultures. However, neither rFWPV nor rVV were able to express transgenes under the control of late viral promoters, indicating that both rFWPV and rVV infections are arrested at an early stage in human DCs. Infection of CD83 + DCs by rFWPV was confirmed by double-staining cytochemistry. We conclude that host range-restricted FWPV can be used efficiently to transfer Ag genes to human DCs ex vivo and may have a role in the development of tumor immunotherapy protocols.  (+info)

A canarypox vaccine expressing multiple human immunodeficiency virus type 1 genes given alone or with rgp120 elicits broad and durable CD8+ cytotoxic T lymphocyte responses in seronegative volunteers. (3/54)

Induction of CD8+ cytotoxic T cells is considered one of the important correlates for the protective efficacy of candidate human immunodeficiency virus type 1 (HIV-1) vaccines. To induce CD8+ cytotoxic T lymphocytes (CTLs) along with neutralizing antibody and CD4+ T cell help, a live canarypox virus construct expressing gp120, transmembrane gp41, the gag and protease genes, and sequences containing CTL epitopes in nef and pol was given simultaneously with, or followed by, rgp120 SF2. CD8+ CTLs were detected in 61% of volunteers at some time during the trial. Three to 6 months after the last immunization, the gene-specific responses were gag, 26/81; env, 17/77; nef, 12/77; and pol, 3/16. Simultaneous immunization with the canarypox vector and the subunit, beginning with the initial immunization, resulted in earlier antibody responses. In summary, a strategy of immunization with a canarypox vector expressing multiple genes of HIV-1 given with gp120 results in durable CD8+ CTL responses to a broad range of epitopes.  (+info)

A canarypox vector expressing cytomegalovirus (CMV) glycoprotein B primes for antibody responses to a live attenuated CMV vaccine (Towne). (4/54)

To develop a vaccine against cytomegalovirus (CMV), a canarypox virus (ALVAC) expressing CMV glycoprotein (gB) was evaluated alone or in combination with a live, attenuated CMV vaccine (Towne). Three doses of 106.5 TCID50 of ALVAC-CMV(gB) induced very low neutralizing or ELISA antibodies in most seronegative adults. However, to determine whether ALVAC-CMV(gB) could prime for antibody responses, 20 seronegative adults randomly received either 106.8 TCID50 of ALVAC-CMV(gB) or 106.8 TCID50 of ALVAC-RG, expressing the rabies glycoprotein, administered at 0 and 1 month, with all subjects receiving a dose of 103.5 pfu of the Towne vaccine at 90 days. For subjects primed with ALVAC-CMV(gB), neutralizing titers and ELISA antibodies to CMV(gB) developed sooner, were much higher, and persisted longer than for subjects primed with ALVAC-RG. All vaccines were well tolerated. These results demonstrate that ALVAC-CMV(gB) primes the immune system and suggest a combined-vaccine strategy to induce potentially protective levels of neutralizing antibodies.  (+info)

Identification of five MAGE-A1 epitopes recognized by cytolytic T lymphocytes obtained by in vitro stimulation with dendritic cells transduced with MAGE-A1. (5/54)

MAGE genes are expressed by many human tumors of different histological types but not by normal cells, except for male germline cells. The Ags encoded by MAGE genes and recognized by T cells are therefore strictly tumor-specific. Clinical trials involving therapeutic vaccination of cancer patients with MAGE antigenic peptides or proteins are in progress. To increase the range of patients eligible for therapy with peptides, it is important to identify additional MAGE epitopes recognized by CTL. Candidate peptides known to bind to a given HLA have been used to stimulate T lymphocytes in vitro. In some instances, CTL clones directed against these synthetic peptides have been obtained, but these clones often failed to recognize tumor cells expressing the relevant gene. Therefore, we designed a method to identify CTL epitopes that selects naturally processed peptides. Monocyte-derived dendritic cells infected with a recombinant canarypoxvirus (ALVAC) containing the entire MAGE-A1 gene were used to stimulate CD8+ T lymphocytes from the blood of individuals without cancer. Responder cell microcultures that specifically lysed autologous cells expressing MAGE-A1 were cloned using autologous stimulator cells either transduced with a retrovirus coding for MAGE-A1 or infected with recombinant Yersinia-MAGE-A1 bacteria. The CTL clones were tested for their ability to lyse autologous cells loaded with each of a set of overlapping MAGE-A1 peptides. This strategy led to the identification of five new MAGE-A1 epitopes recognized by CTL clones on HLA-A3, -A28, -B53, -Cw2, and -Cw3 molecules. All of these CTL clones recognized target cells expressing gene MAGE-A1.  (+info)

Phase I study in cancer patients of a replication-defective avipox recombinant vaccine that expresses human carcinoembryonic antigen. (6/54)

PURPOSE: A phase I clinical trial in patients with advanced carcinoma was conducted, using a replication-defective avipox vaccine containing the gene for the human carcinoembryonic antigen (CEA). The canarypox vector, designated ALVAC, has the ability to infect human cells but cannot replicate. PATIENTS AND METHODS: The recombinant vaccine, designated ALVAC-CEA, was administered intramuscularly three times at 28-day intervals. Each cohort of six patients received three doses of either 2.5 x 10(5), 2.5 x 10(6), or 2.5 x 10(7) plaque-forming units of vaccine. RESULTS: The vaccine was well tolerated at all dose levels and no significant toxicity was attributed to the treatment. No objective antitumor response was observed during the trial in patients with measurable disease. Studies were conducted to assess whether ALVAC-CEA had the ability to induce cytolytic T-lymphocyte (CTL) responses in patients with advanced cancer. Peripheral blood mononuclear cells (PBMCs) from patients with the MHC class I A2 allele were obtained before vaccine administration and 1 month after the third vaccination. Peripheral blood mononuclear cells were incubated with the CEA immunodominant CTL epitope carcinoembryonic antigen peptide-1 and interleukin 2 and quantitated using CTL precursor frequency analysis. In seven of nine patients evaluated, statistically significant increases in CTL precursors specific for CEA were observed in PBMCs after vaccination, compared with before vaccination. CONCLUSION: These studies constitute the first phase I trial of an avipox recombinant in cancer patients. The recombinant vaccine ALVAC-CEA seems to be safe and has been demonstrated to elicit CEA-specific CTL responses. These studies thus form the basis for the further clinical exploration of the ALVAC-CEA recombinant vaccine in phase I/II studies in protocols designed to enhance the generation of human T-cell responses to CEA.  (+info)

Efficient human immunodeficiency virus ( HIV)-1 Gag-Env pseudovirion formation elicited from mammalian cells by a canarypox HIV vaccine candidate. (7/54)

Canarypox viruses undergo abortive replication in mammalian cells. Despite this restriction on replication in mammalian cells, significant immune responses have been shown in animals and in humans receiving recombinant canarypox vaccine vectors expressing heterologous immunogens. A recombinant canarypox vaccine candidate (vCP205), which expresses human immunodeficiency virus (HIV)-1 Gag, Env, and protease proteins, is presently under investigation in phase I and phase II human trials in the United States and elsewhere. In this study, the ability of vCP205 to elicit HIV Gag-Env pseudovirion formation in avian and mammalian cells was investigated. Gag-Env pseudovirions were produced from both avian and mammalian cell lines infected by this vaccine vector. A subset of mammalian cells was identified in which pseudovirion production and release was very efficient, surpassing the production from infected avian cells. The production of Gag-Env pseudovirions by canarypox HIV vaccine vectors may have important implications for future HIV vaccine design.  (+info)

Viral vector delivery in solid-state vehicles: gene expression in a murine prostate cancer model. (8/54)

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.  (+info)

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