Pseudovirion particle production by live poxvirus human immunodeficiency virus vaccine vector enhances humoral and cellular immune responses.
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Live-vector-based human immunodeficiency virus (HIV) vaccines are an integral part of a number of HIV vaccine regimens currently under evaluation. Live vectors that carry an intact gag gene are capable of eliciting HIV pseudovirion particle formation from infected host cells. The impact of pseudovirion particle formation on the immune response generated by live HIV vaccine vectors has not been established. In this study, a canarypox HIV vaccine candidate vector expressing HIV gag and env genes, vCP205, was modified by the introduction of a glycine-to-alanine coding change in the N-terminal myristylation site of gag to create Myr- vCP205. This substitution effectively eliminated particle formation without altering the level of protein production. vCP205 and Myr- vCP205 were then directly compared for the ability to induce HIV-specific immune responses in mice. The particle-competent vector vCP205 elicited higher levels of CD8+ T-cell responses, as indicated by gamma interferon enzyme-linked immunospot (ELISPOT) assay and intracellular cytokine staining. Humoral responses to Gag and Env were also markedly higher from animals immunized with the particle-competent vector. Furthermore, HIV-specific CD4+ T-cell responses were greater among animals immunized with the particle-competent vector. Using a human dendritic cell model of antigen presentation in vitro, vCP205 generated greater ELISPOT responses than Myr- vCP205. These results demonstrate that pseudovirion particle production by live-vector HIV vaccines enhances HIV-specific cellular and humoral immune responses. (+info)
The human immunodeficiency virus preventive vaccine research at the French National Agency for acquired immunodeficiency syndrome research.
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The human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) epidemic is of unprecedented gravity and is spreading rapidly, notably in the most disadvantaged regions of the world. The search for a preventive vaccine is thus an absolute priority. For over 10 years the French National Agency for AIDS research (ANRS) has been committed to an original program combining basic science and clinical research. The HIV preventive vaccine research program run by the ANRS covers upstream research for the definition of immunogens, animal models, and clinical research to evaluate candidate vaccines. Most researchers in 2004 believe that it should be possible to obtain partial vaccine protection through the induction of a strong and multiepitopic cellular response. Since 1992, the ANRS has set up 15 phases I and II clinical trials in order to evaluate the safety and the capacity of the candidate vaccines for inducing cellular immune responses. The tested candidate vaccines were increasingly complex recombinant canarypox viruses (Alvac) containing sequences coding for certain viral proteins, utilized alone or combined with other immunogens (whole or truncated envelope proteins). ANRS has also been developing an original strategy based on the utilization of lipopeptides. These comprise synthetic fragments of viral proteins associated with lipids that facilitate the induction of a cellular immune response. These approaches promptly allowed the assessment of a prime-boost strategy combining a viral vector and lipopeptides. (+info)
Phase I study of the intratumoral administration of recombinant canarypox viruses expressing B7.1 and interleukin 12 in patients with metastatic melanoma.
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The objective of this study was to evaluate the safety and activity of the intratumoral administration of the immune costimulatory molecule, B7.1, encoded by a vector derived from the canarypox virus, ALVAC (ALVAC-B7.1), alone and with the intratumoral injection of ALVAC encoding the immune-stimulatory cytokine, interleukin 12 (ALVAC-IL-12). Fourteen patients with metastatic melanoma who had s.c. nodules received intratumoral injections on days 1, 4, 8, and 11. Nine patients were given escalating doses of up to 25 x 10(8) plaque-forming units of ALVAC-B7.1. Five patients were given 25 x 10(8) plaque-forming units of ALVAC-B7.1 combined with ALVAC-IL-12 50% tissue culture infective dose of 2 x 10(6). Toxicity was mild to moderate and consisted of inflammatory reactions at the injection site and fever, chills, myalgia, and fatigue. Higher levels of B7.1 mRNA were observed in ALVAC-B7.1-injected tumors compared with saline-injected control tumors. Higher levels of intratumoral vascular endothelial growth factor and IL-10, cytokines with immune suppressive activities, were also observed in ALVAC-B7.1- and ALVAC-IL-12-injected tumors compared with saline-injected controls. Serum levels of vascular endothelial growth factor increased at day 18 and returned to baseline at day 43. All patients developed antibody to ALVAC. Intratumoral IL-12 and IFN-gamma mRNA decreased. Changes in serum IL-12 and IFN-gamma levels were not observed. Tumor regressions were not observed. The intratumoral injections of ALVAC-B7.1 and ALVAC-IL-12 were well tolerated at these dose levels and at this schedule and resulted in measurable biological response. This response included the production of factors that may suppress the antitumor immunologic activity of these vectors. (+info)
Tumoral and immunologic response after vaccination of melanoma patients with an ALVAC virus encoding MAGE antigens recognized by T cells.
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PURPOSE: To evaluate the toxicity, antitumoral effectiveness, and immunogenicity of repeated vaccinations with ALVAC miniMAGE-1/3, a recombinant canarypox virus containing a minigene encoding antigenic peptides MAGE-3(168-176) and MAGE-1(161-169), which are presented by HLA-A1 and B35 on tumor cells and can be recognized by cytolytic T lymphocytes (CTLs). MATERIALS AND METHODS: The vaccination schedule comprised four sequential injections of the recombinant virus, followed by three booster vaccinations with the MAGE-3(168-176) and MAGE-1(161-169) peptides. The vaccines were administered, both intradermally and subcutaneously, at 3-week intervals. RESULTS: Forty patients with advanced cancer were treated, including 37 melanoma patients. The vaccines were generally well tolerated with moderate adverse events, consisting mainly of transient inflammatory reactions at the virus injection sites. Among the 30 melanoma patients assessable for tumor response, a partial response was observed in one patient, and disease stabilization in two others. The remaining patients had progressive disease. Among the patients with stable or progressive disease, five showed evidence of tumor regression. A CTL response against the MAGE-3 vaccine antigen was detected in three of four patients with tumor regression, and in only one of 11 patients without regression. CONCLUSION: Repeated vaccination with ALVAC miniMAGE-1/3 is associated with tumor regression and with a detectable CTL response in a minority of melanoma patients. There is a significant correlation between tumor regression and CTL response. The contribution of vaccine-induced CTL in the tumor regression process is discussed in view of the immunologic events that could be analyzed in detail in one patient. (+info)
Enhanced viral and tumor immunity with intranodal injection of canary pox viruses expressing the melanoma antigen, gp100.
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BACKGROUND: The route of administration and extent of helper T-cell activation are factors that are likely to be important for the development of effective cancer vaccines. In order to optimize CD8(+) cytotoxic T-lymphocyte (CTL) responses, the immunologic effects of direct lymph node (LN) injections of canary pox virus (ALVAC) vectors (expressing the melanoma antigen, gp100) and immunogenic gp100 peptides, along with concomitant injections of the helper adjuvant, tetanus toxoid, were studied in high-risk HLA-A*0201(+) patients. METHODS: Forty-two patients were vaccinated using six different protocols. Twenty-three patients were 'primed' with ALVAC(2)-gp100m and 'boosted' with gp100 peptides, either subcutaneously or into an LN. Intranodal (IN) peptides, alone, were administered to six patients. Thirteen patients were given tetanus toxoid initially, and with each gp100 vaccination. Toxicity was recorded and immunologic responses were determined in 35 patients by enzyme-linked immunospot (ELISPOT) and gp100-tetramer binding assays and anti-ALVAC(2) enzyme-linked immunosorbent assays (ELISAs). RESULTS: All vaccine protocols were tolerated well. Using stringent criteria for immunologic response, 8 of 18 patients responded to the viral vaccines, in striking contrast to peptides only (0 of 6 patients) or with help in trans from tetanus-reactive T-cells (1 of 11 patients). Changes in gp100-reactive CTL frequencies and ALVAC antibodies were greatest when viruses were injected directly into LNs. CONCLUSIONS: IN injections of ALVAC(2)-gp100m viruses are feasible, safe, and may be a superior method of vaccination in humans. CTL responses to this vaccine were not enhanced by tetanus toxoid. (+info)
Two families of rep-like genes that probably originated by interspecies recombination are represented in viral, plasmid, bacterial, and parasitic protozoan genomes.
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Two families of genes related to, and including, rolling circle replication initiator protein (Rep) genes were defined by sequence similarity and by evidence of intergene family recombination. The Rep genes of circoviruses were the best characterized members of the "RecRep1 family." Other members of the RecRep1 family were Rep-like genes found in the genomes of the Canarypox virus, Entamoeba histolytica, and Giardia duodenalis and in a plasmid, p4M, from the Gram-positive bacterium, Bifidobacterium pseudocatenulatum. The "RecRep2 family" comprised some previously identified Rep-like genes from plasmids of phytoplasmas and similar Rep-like genes from the genomes of Lactobacillus acidophilus, Lactococcus lactis, and Phytoplasma asteris. Both RecRep1 and RecRep2 proteins have a nucleotide-binding domain significantly similar to the helicases (2C proteins) of picorna-like viruses. On the N-terminal side of the nucleotide binding domain, RecRep1 proteins have a domain significantly similar to one found in nanovirus Reps, whereas RecRep2 proteins have a domain significantly similar to one in the Reps of pLS1 plasmids. We speculate that RecRep genes have been transferred from viruses or plasmids to parasitic protozoan and bacterial genomes and that Rep proteins were themselves involved in the original recombination events that generated the ancestral RecRep genes. (+info)
Avipoxvirus phylogenetics: identification of a PCR length polymorphism that discriminates between the two major clades.
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Avipoxvirus infections have been observed in an extensive range of wild, captive and domesticated avian hosts, yet little is known about the genome diversity and host-range specificity of the causative agent(s). Genome-sequence data are largely restricted to Fowlpox virus (FWPV) and Canarypox virus (CNPV), which have been sequenced completely, showing considerable divergence between them. It is therefore proving difficult, by empirical approaches, to identify pan-genus, avipoxvirus-specific oligonucleotide probes for PCR and sequencing to support phylogenetic studies. A previous preliminary study used the fpv167 locus, which encodes orthologues of vaccinia virus core protein P4b (A3). PCR per se did not discriminate between viruses, but restriction-enzyme or sequence analysis indicated that the avipoxviruses clustered either with FWPV or with CNPV. Here, further study of the P4b locus demonstrated a third cluster, from psittacine birds. A newly identified locus, flanking fpv140 (orthologue of vaccinia virus H3L), confirms the taxonomic structure. This locus is particularly useful in that viruses from the fowlpox-like and canarypox-like clusters can be discriminated by PCR on the basis of fragment size, whilst sequence comparison allows discrimination for the first time between Pigeonpox virus and Turkeypox virus. Except within the psittacines, virus and avian host taxonomies do not show tight correlation, with viruses from the same species located in very different clades. Nor are all the existing recognized avipoxvirus species, defined primarily by avian host species (such as CNPV and Sparrowpox virus), resolved within the present structure. (+info)
Recombinant nipah virus vaccines protect pigs against challenge.
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Nipah virus (NiV), of the family Paramyxoviridae, was isolated in 1999 in Malaysia from a human fatality in an outbreak of severe human encephalitis, when human infections were linked to transmission of the virus from pigs. Consequently, a swine vaccine able to abolish virus shedding is of veterinary and human health interest. Canarypox virus-based vaccine vectors carrying the gene for NiV glycoprotein (ALVAC-G) or the fusion protein (ALVAC-F) were used to intramuscularly immunize four pigs per group, either with 10(8) PFU each or in combination. Pigs were boosted 14 days postvaccination and challenged with 2.5 x 10(5) PFU of NiV two weeks later. The combined ALVAC-F/G vaccine induced the highest levels of neutralization antibodies (2,560); despite the low neutralizing antibody levels in the F vaccinees (160), all vaccinated animals appeared to be protected against challenge. Virus was not isolated from the tissues of any of the vaccinated pigs postchallenge, and a real-time reverse transcription (RT)-PCR assay detected only small amounts of viral RNA in several samples. In challenge control pigs, virus was isolated from a number of tissues (10(4.4) PFU/g) or detected by real-time RT-PCR. Vaccination of the ALVAC-F/G vaccinees appeared to stimulate both type 1 and type 2 cytokine responses. Histopathological findings indicated that there was no enhancement of lesions in the vaccinees. No virus shedding was detected in vaccinated animals, in contrast to challenge control pigs, from which virus was isolated from the throat and nose (10(2.9) PFU/ml). Based on the data presented, the combined ALVAC-F/G vaccine appears to be a very promising vaccine candidate for swine. (+info)