Purification of Ag-specific T lymphocytes after direct peripheral blood mononuclear cell stimulation followed by CD25 selection. I. Application to CD4(+) or CD8(+) cytomegalovirus phosphoprotein pp65 epitope determination. (1/35)

The two main constraints that currently limit a broader usage of T cell therapy against viruses are the delay required to obtain specific T cells and the safety of the selection procedure. In the present work we developed a generally applicable strategy that eliminates the need for APC for timing reasons, and the need for infectious viral strains for safety concerns. As a model, we used the selection of T lymphocytes specific for the immunodominant CMV phosphoprotein pp65. PBMC from healthy seropositive donors were first depleted of IL-2R alpha-chain CD25(+) cells and were then stimulated for 24-96 h with previously defined peptide Ags or with autologous PBMC infected with a canarypox viral vector encoding the total pp65 protein (ALVAC-pp65). Subsequent immunomagnetic purification of newly CD25-expressing cells allowed efficient recovery of T lymphocytes specific for the initial stimuli, i.e., for the already known immunodominant epitope corresponding to the peptides used as a model or for newly defined epitopes corresponding to peptides encoded by the transfected pp65 protein. Importantly, we demonstrated that direct PBMC stimulation allowed recovery not only of CD8(+) memory T lymphocytes, but also of the CD4(+) memory T cells, which are known to be crucial to ensure persistence of adoptively transferred immune memory. Finally, our analysis of pp65-specific T cells led to the identification of several new helper and cytotoxic epitopes. This work thus demonstrates the feasibility of isolating memory T lymphocytes specific for a clinically relevant protein without the need to prepare APC, to use infectious viral strains, or to identify immunodominant epitopes.  (+info)

Recombinant canarypox vaccine-elicited CTL specific for dominant and subdominant simian immunodeficiency virus epitopes in rhesus monkeys. (2/35)

Since virus-specific CTL play a central role in containing HIV replication, a candidate AIDS vaccine should generate virus-specific CTL responses. In this study, the ability of a recombinant canarypox virus expressing SIV Gag-Pol-Env (ALVAC/SIV gag-pol-env) was assessed for its ability to elicit both dominant and subdominant epitope-specific CTL responses in rhesus monkeys. Following a series of five immunizations, memory CTL responses specific for a dominant Gag epitope could be demonstrated in the peripheral blood of vaccinated monkeys. Memory CTL responses to a subdominant Pol epitope were undetectable in these animals. Following challenge with SIVmac251, the experimentally vaccinated animals developed high frequency CTL responses specific for the dominant Gag epitope that emerged in temporal association with the early containment of viral replication. Interestingly, the experimentally vaccinated, but not the control vaccinated animals, developed CTL responses to the subdominant Pol epitope that were detectable only after containment of early viremia. Thus, recombinant canarypox vaccination elicited low frequency, but durable memory CTL populations. The temporal association of the emergence of the dominant epitope-specific response with early viral containment following challenge suggests that this immune response played a role in the accelerated clearing of early viremia in these animals. The later emerging CTL response specific for the subdominant epitope may contribute to the control of viral replication in the setting of chronic infection.  (+info)

Expression of vaccinia E3L and K3L genes by a novel recombinant canarypox HIV vaccine vector enhances HIV-1 pseudovirion production and inhibits apoptosis in human cells. (3/35)

Poxviruses that are attenuated for growth in human cells provide a safe means of HIV antigen expression and are capable of eliciting HIV-specific immune responses, including CD8+ cytotoxic T-lymphocyte (CTL) responses. HIV-1 antigen expression in human cells by attenuated poxvirus vectors may be limited by interferon-mediated host defense mechanisms. To enhance HIV antigen expression in human cells, the vaccinia virus E3L and K3L genes were inserted into a canarypox vector that expresses HIV-1 Gag, Env, and a Nef/Pol polyepitope string. E3L and K3L markedly reduced the activation of the double-stranded RNA-dependent protein kinase, PKR, and led to a significant reduction in apoptosis in HeLa cells. Production and release of HIV-1 antigen in the form of pseudovirions was enhanced in both duration and magnitude by this vector modification. The addition of immunomodulatory genes to attenuated poxviruses represents a novel strategy for enhancing antigen production by live vector HIV vaccine candidates.  (+info)

Induction of p53-specific immune responses in colorectal cancer patients receiving a recombinant ALVAC-p53 candidate vaccine. (4/35)

PURPOSE: The tumor-associated auto-antigen p53 is commonly overexpressed in various types of human cancer, including colorectal cancer. Experiments in preclinical models have shown that it can serve as a target for T-cell-mediated tumor-eradication. The feasibility of a p53-specific therapeutic vaccination was investigated in cancer patients. EXPERIMENTAL DESIGN: A Phase I/II dose-escalation study was performed that evaluated the effect of a recombinant canarypoxvirus (ALVAC) vaccine encoding wild-type human p53 in 15 patients with advanced colorectal cancer. Each group of five patients received three i.v. doses of one-tenth of a dose, one-third of a dose, or 1 dose of the vaccine [1 dose = 1 x 10(7.5) cell culture infectious dosis (CCID)50]. RESULTS: Potent T-cell and IgG antibody responses against the vector component of the ALVAC vaccine were induced in the majority of the patients. Enzyme-linked immunosorbent-spot assay (ELISPOT) analysis of vaccine-induced immunity revealed the presence of IFN-gamma-secreting T cells against both ALVAC and p53, whereas no significant interleukin-4 responses were detected. Vaccine-mediated enhancement of p53-specific T-cell immunity was found in two patients in the highest-vaccine-dose group. CONCLUSIONS: This study demonstrated the feasibility, even in patients with advanced cancer, to elicit immune responses against the ubiquitously expressed tumor-associated auto-antigen p53. Our results form the basis for additional studies that will explore the antitumor capacity of p53 containing multivalent vaccines in cancer patients with limited tumor burden.  (+info)

Preparation of clinical-grade recombinant canarypox-human immunodeficiency virus vaccine-loaded human dendritic cells. (5/35)

Preclinical data are reported that support a human immunodeficiency virus (HIV) vaccine strategy using recombinant canarypox-HIV vectors (ALVAC-HIV) to load human dendritic cells (DCs) with HIV antigens. Clinical-grade DCs were infected with good manufacturing practice-grade ALVAC-HIV vaccine constructs. ALVAC infection, HIV gene expression, and DC viability and function were monitored by use of immunohistochemistry, flow cytometry, blastogenesis assays, antigen-specific interferon (IFN)-gamma enzyme-linked immunospot assay, and enzyme-linked immunosorbent assay protein detection. The vaccines infected both immature and mature DCs, and intracellular HIV-1 Gag protein was detected within hours. ALVAC-HIV induced DC maturation that was mediated by tumor necrosis factor-alpha and induced DC apoptosis that was directly related to the length of vaccine exposure. Of importance, the infected DCs remained functional in T cell stimulation assays and induced HIV antigen-specific CD8(+) T cell production of IFN-gamma from cells of HIV-1-infected individuals. These data support an ongoing HIV vaccine trial comparing conventional vaccine delivery routes with ex vivo vaccine-loaded autologous DCs for immunogenicity in HIV-1-uninfected volunteers.  (+info)

DNA immunization with hepatitis C virus (HCV) polycistronic genes or immunization by HCV DNA priming-recombinant canarypox virus boosting induces immune responses and protection from recombinant HCV-vaccinia virus infection in HLA-A2.1-transgenic mice. (6/35)

We studied immune responses to hepatitis C virus (HCV) genes delivered as DNA encoding the entire HCV protein coding genome in two polycistronic plasmids encoding HCV capsid-E1-E2-NS2-NS3 and HCV NS3-NS4-NS5 in HLA-A2.1-transgenic mice. Immune responses to HCV DNA prime and recombinant canarypox virus boost were also studied with the above constructs. At 8 weeks after a canarypox virus boost, the DNA prime/canarypox virus boosting regimen induced potent cellular immune responses to HCV structural and nonstructural proteins on target cells expressing the HLA-A2.1 allele. High frequencies of gamma interferon-secreting cells, as detected by enzyme-linked immunospot assay, were obtained in response to several endogenously expressed HCV proteins. We also observed cytotoxic-T-lymphocyte reactivity in response to endogenously expressed HCV proteins in fresh spleen cells without in vitro expansion. Upon challenge with a recombinant vaccinia virus expressing HCV proteins at 2 months postimmunization, the HCV DNA prime/canarypox virus-immunized mice showed a complete reduction in vaccinia virus titers compared to HCV DNA prime/boost- and mock-immunized controls. Immune responses were still detectable 4 months after canarypox virus boost in immunized mice. Interestingly, at 10 months postimmunization (8 months after canarypox virus boost), the protection in HCV DNA prime/boost-immunized mice against recombinant HCV-vaccinia virus challenge was higher than that observed in HCV DNA prime/canarypox virus boost-immunized mice.  (+info)

Induction of HLA-G-restricted human cytomegalovirus pp65 (UL83)-specific cytotoxic T lymphocytes in HLA-G transgenic mice. (7/35)

The non-classical major histocompatibility complex class I molecule HLA-G is expressed mainly by extravillous trophoblasts at the materno-foetal interface. HLA-G has been found to bind endogenously processed nonameric peptides but its function as a restriction element for a cytotoxic T cell response to viruses with tropism for trophoblastic cells has never been demonstrated. In this study, candidate viral peptides derived from human cytomegalovirus (HCMV) pp65 (UL83), which stabilized the HLA-G molecule on HLA-G-transfected T2 cells, were identified. The specific anti-pp65 cytotoxic T lymphocyte (CTL) response restricted by HLA-G in triple transgenic mice (HLA-G, human beta2m, human CD8alpha) was then investigated by injection of dendritic cells loaded with synthetic pp65-derived peptides or by infection with canarypox virus expressing pp65. Results showed that CTLs from HLA-G mice have the capacity to kill target cells either infected with recombinant vaccinia viruses expressing pp65 or loaded with specific pp65-derived peptides using HLA-G as an antigen-presenting molecule. It was also demonstrated that these HLA-G-restricted pp65-specific T cells are able to kill the human astrocytoma cell line U373, which was transfected with HLA-G and infected with HCMV. Moreover, using HLA-G tetramers refolded with a synthetic pp65-derived peptide, peptide-specific CD8(+) cells restricted by HLA-G have been detected in vivo. These findings provide the first evidence that HLA-G can select anti-HCMV-restricted CTLs in vivo, although the potency of this cytolytic response is limited (20-25 %). The weak HLA-G-restricted anti-HCMV response is probably due to HLA-G-mediated inhibitory signals on the development of an antiviral CTL response.  (+info)

Human immunodeficiency virus (HIV) seropositivity among uninfected HIV vaccine recipients. (8/35)

Since 1987, >10,000 individuals worldwide have received immunizations with human immunodeficiency virus (HIV) preventive vaccine constructs. Many constructs elicit antibodies detected by standard serologic tests (enzyme immunoassays, rapid tests, and Western blots) and result in vaccine recipients' serum being identified as reactive and indicative of HIV infection. To determine the frequency of vaccine-induced HIV antibody among uninfected HIV vaccine trial participants and to identify factors associated with these results, serum samples from HIV-uninfected participants from selected United States phase I/II HIV-1 vaccine trials were tested with 6 serologic screening tests. Reactive specimens were tested by use of Western blot. Overall, 490 serum specimens from 461 vaccine recipients were tested; 100 (20.4%) reacted on at least 1 serologic test, and 65 (13%) were determined to be positive by Western blot. Canarypox or vaccinia vaccine recipients' serum with or without HIV envelope glycoprotein (gp120 or gp160) boosts accounted for all positive Western blot results; no positive Western blot results were obtained from gp120 subunit recipients. The potential for vaccine recipients being misclassified as HIV infected increased with vaccine complexity.  (+info)

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