Induction of a polarized Th1 response by insertion of multiple copies of a viral T-cell epitope into adenylate cyclase of Bordetella pertussis.

The adenylate cyclase (CyaA) of Bordetella pertussis delivers the N-terminal catalytic domain into the cytosol of a large number of eukaryotic cells, in particular, professional antigen-presenting cells. This allows the delivery of CD8(+) T-cell epitopes to the major histocompatibility complex class I presentation pathway. We have previously shown that immunization of mice with CyaA carrying a single CD8(+) T-cell epitope leads to antiviral protection as well as to protective and therapeutic antitumor immunity associated with the induction of specific cytotoxic T-lymphocyte (CTL) responses. Here, we evaluated the capacity of CyaA carrying one to four copies of the CD8(+) CD4(+) T-cell epitope from the nucleoprotein of the lymphocytic choriomeningitis virus to induce T-cell responses. Both CTL and Th1-like specific responses were detected in mice immunized with recombinant CyaA with or without adjuvant. Although the insertion of the larger peptides resulted in partial loss of the invasive capacity of recombinant CyaA, insertion of several copies of the same epitope led to a strong enhancement of Th1 responses and, to a lesser degree, CTL responses. These results underscore the potency of CyaA for vaccine design with a new impact on diseases in which the Th1 response has been described to have a beneficial effect.

In vivo induction of CTL responses by recombinant adenylate cyclase of Bordetella pertussis carrying multiple copies of a viral CD8(+) T-cell epitope.

Bordetella pertussis adenylate cyclase toxin (ACT) is one of the few known protein toxins penetrating directly into the cytosol of target cells across their cytoplasmic membrane without the need for endocytosis. This capacity of ACT was recently exploited for in vivo delivery of single viral CD8(+) T-epitopes into MHC class I-presenting cells and induction of protective antiviral cytotoxic T-cell (CTL) responses. Here, we have explored the potential of the cell-invasive adenylate cyclase domain of the toxin to deliver larger antigens by evaluating the epitope-specific CTL responses induced by constructs bearing one to four copies of the CD8(+) T-epitope from the nucleoprotein of the lymphocytic choriomeningitis virus. The increase in the number of copies of the epitope was accompanied by a moderate decrease of the specific cell invasiveness of the ACT protein and did not lead to further enhancement of the level of induced epitope-specific CTL cells in mice, as compared to ACT with a single copy of the epitope. These results demonstrate the capacity of ACT to deliver larger heterologous antigens comprising several epitopes for antigenic presentation in vivo.

Lymphoproliferative response to synthetic V3 loop P18 peptide and HIV-1 envelope glycoprotein among individuals immunized with gp160 candidate vaccines.

Because T-cell responses are critical for defense against viral infections, the synthetic P18 peptide (RIQRGPGRAFVTIGK) and active component of gp160 protein has previously been shown to induce cytotoxic and helper T-lymphocyte responses. In order to further define the T-helper cells, responses which are known to play a role in enhancing the immunological response to foreign antigen, we studied the response of individuals immunized with HIV gp160 candidate vaccines. We investigated the proliferative cellular response of peripheral blood mononuclear cells (PBMC) derived from individuals immunized with gp160 antigens in three different protocols. We found a PBMC proliferative response to synthetic P18 peptide in healthy immunized individuals induced by gp160 antigen with or without vaccinia virus. There was correlation between the proliferative response to P18 peptide and other antigens such as HIV-like proteins and gp160 molecule. HLA-DR typing revealed the possible presentation of P18 peptide by several different class II molecules. Since these class II molecules occur frequently in the general population, P18 peptide appears to contain broadly reactive epitopes and thus is presented by multiple HLA class II molecules. Due to its broad reactivity P18 peptide is one of the candidates for inclusion as a subunit vaccine against HIV-1.

HIV-1 soluble antigens induced CD8+ cytotoxic T-cell responses in an immunized individual.

In an attempt to determine whether immunization of healthy HIV-1 seronegative individuals with a soluble gp160 candidate vaccine could induce an anti-HIV specific immune response, volunteers were immunized by two injections of a water-in-oil emulsion containing a mixture of gp160 antigen together with selected peptides. Following immunization, lymphocytes were collected and stimulated in vitro with autologous HIV-1-infected cells. The results showed that immunization with soluble HIV-1 envelope was able to generate CD3+ CD8+ CTLs directed to gp160 antigen. The CTL response was restricted to class I molecule HLA-A2. The CTL response was comparable to that elicited by immunization with HIV-1-envelope recombinant vaccinia virus.

Cytotoxic T lymphocytes specific for the synthetic VEINCTR peptide, a sequence found within the Fas molecule and env gp120 in the blood of HIV-1 seropositive individuals.

We have previously identified a VEINCTR peptide common to both the Fasmolecule and HIV-1 gp120. Here we report the characterization in PBMCs from HIV-1-infected individuals of a CD8+ class I restricted CTL activity directed towards this peptide. The peptide is highly conserved in various HIV-1 strains, being located at amino acid 287-293 (VEINCTR), within an epitope known as cell T epitope on the env protein of human immunodeficiency virus type-1. Cell cultures were obtained by polyclonal activation using autologous blast cells and CTL lines generated from frozen peripheral blood lymphocytes of HIV-1 seropositive donors by stimulation with the peptide and recombinant interleukin-2. The env-specific CTL turned out to kill autologous target cells infected with a recombinant vaccinia virus containing the env gene of HIV-1 or pulsed with peptide. Specificity was determined using shorter peptides. The CTL activity was directed against autologous target cells presenting the heptapeptide which is site located in the Fas molecule, known to be functionally involved in T-cell apoptosis.

Detection of CTL activity in PBMCS taken from HIV-ENV immunized individuals after in vitro viral infection.

The cellular immune response to the AIDS virus in healthy individuals immunized with HIV-1 antigens has not yet been entirely understood. Unlike HIV-1 infected patients where direct measurements of anti HIV-1 CTL activities can be readily performed with fresh peripheral blood mononuclear cells, uninfected volunteers immunized against HIV-1 antigens have fewer circulating CTL necessitating an in vitro activation in order to amplify the cytotoxic signal and make it measurable. This study presents experiments where specific CTLs are successfully obtained simply by in vitro infection of PBMCs from HIV-1 Envelope immunized individuals.

Cytotoxic T lymphocytes specific for HIV-1 gp160 antigen and synthetic P18IIIB peptide in an HLA-A11-immunized individual.

Cytotoxic T cell determinants should be an important component of an anti-human immunodeficiency virus (HIV) vaccine. The epitopes of proteins can be defined with short synthetic peptides for class I-restricted CTLs. An immunodominant CTL epitope from the HIV-1 IIIB envelope protein gp160 comprising 15 amino acids (residues 315-329: RIQRGPGRAFVTIGK) (P18IIIB) has been identified that is recognized by class I MHC molecule H-2d-restricted murine CD8+ CTLs. We have investigated the epitope specificity of anti-HIV-1 CTLs in immunized individuals and we found that the CTL response was restricted by more than one class I MHC molecule, including HLA-A2 and HLA-A3. In the present work, we also show that the response against P18IIIB peptide is restricted by the HLA-A11 molecule in an individual immunized by vaccinia virus expressing gp160 protein. This peptide could thus be recognized in association with different HLA class I allotypes. This work has implications for vaccine strategies, using the P18 peptide.