Cytotoxic T-lymphocyte induction in asymptomatic HIV-1-infected patients immunized with Retrovector-transduced autologous fibroblasts expressing HIV-1IIIB Env/Rev proteins.

OBJECTIVE: To demonstrate the safety and enhancement of HIV-1-specific immune responses in HIV-infected asymptomatic patients following treatment with retroviral vector (Retrovector)-transduced autologous fibroblasts (VTAF) expressing HIV-1IIIB Env/Rev proteins. DESIGN: A non-placebo-controlled, single arm Phase I study. PARTICIPANTS: Four HIV-1-seropositive asymptomatic volunteers were selected based on age (18-50 years), CD4/CD3 lymphocyte counts (> 600 x 10(6)/l or > 40%), and positive delayed-type hypersensitivity test to at least one recall antigen. INTERVENTIONS: Patients were treated at 2-week intervals with a total of three intramuscular injections of irradiated autologous fibroblasts transduced with a molecularly engineered, non-replicating amphotropic murine retrovector encoding the HIV-1IIIB Env/Rev proteins. MAIN OUTCOME MEASURES: The clinical status of patients was assessed by history, physical examination, serum chemistry and hematology, CD4/CD3 lymphocyte counts, HIV viral burden, and monitored throughout the study to detect potentially treatment-induced toxic or unwanted side-effects. In addition, HIV-1-specific cytotoxic T-lymphocyte (CTL) activity was measured to determine the biological activity of VTAF. RESULTS: No acute local or systemic adverse events occurred following three injections with VTAF. Furthermore, a statistically significant increase of CD8+ CTL activity against HIV-1IIIB Env/Rev-expressing targets was observed in peripheral blood mononuclear cells from two out of four patients. CONCLUSIONS: This is the first report of the administration of a gene transfer treatment to HIV-1-infected patients and provides initial support for the safety and activity of retrovector-transduced fibroblasts administered to asymptomatic patients. This treatment resulted in the detection of increased HIV-1IIIB Env/Rev-specific CTL activity in two HIV-seropositive patients and could provide a better understanding of the role of CTL activity in HIV disease progression.

Phase I trial of retroviral vector-mediated interferon (IFN)-gamma gene transfer into autologous tumor cells in patients with metastatic melanoma.

The purpose of this study was to determine the safety of treating melanoma patients with retroviral vector-mediated interferon (IFN)-gamma gene-transduced autologous tumor cells. We designed a phase I study, in which irradiated, autologous, transduced melanoma cells expressing the IFN-gamma gene were injected subcutaneously every 2 weeks with escalating cell doses for six injections. Tumor tissue was harvested from 58 patients with metastatic melanoma. Twelve patients had sufficient expansion of autologous tumor (0.56-160 x 10(7) cells) and adequate IFN-gamma expression after gene transduction (2-79,000 U/10(6) cells/24 hours) for injections. Five patients received injections. No toxicity was attributed to the IFN-gamma retroviral vector in the patients injected. One of the injected patients remains disease-free after 13 injections, following the surgical removal of brain, adrenal, and lung metastases. We found that injections of autologous tumor cells transduced by IFN-gamma gene were well tolerated. However, the ability to develop primary autologous melanoma cell lines was limited, and only a minority of patients were injected.

Cloning of the amino terminal nucleotides of the antigen I/II of Streptococcus sobrinus and the immune responses to the corresponding synthetic peptides.

A portion of the antigen I/II (spaA, B, P1) gene of Streptococcus sobrinus 6715, containing the coding sequence for the amino terminal 684 amino acids of the protein, was cloned in bacteriophage lambda GT10. Selection was by immunological detection using a polyclonal antiserum to the antigen I/II from Strep. mutans. From the amino acid sequence, peptides were synthesized, 15 amino acids in length, that covered the entire sequence. In total, 260 synthetic peptides were synthesized and evaluated for their immunogenicity in Balb/C mice. Thirty-nine peptides were immunogenic, without carrier, and the antisera generated were tested for their ability to bind cells of Strep. mutans and Strep. sobrinus in a solid-phase assay. Antisera corresponding to peptides from five regions on the I/II molecule bound cells of both bacterial species. These peptides were then evaluated for their ability to stimulate in vitro murine lymphocyte proliferation, after in vivo immunization with Strep. sobrinus cells. Two of the peptides were capable of stimulating proliferation, as determined by incorporation of [3H]-thymidine into murine lymph node cells. The sequences of these 5 peptides were then compared to sequences found in the antigen I/II from Strep. mutans (Kelly et al., 1989). As expected, there was considerable homology between the cross-reactive peptides synthesized and the analogous region from Strep. mutans. This homology was not usually contiguous and suggests that the antibodies bind a face of antigen I/II that is in an alpha-helical conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence and structural analysis of surface protein antigen I/II (SpaA) of Streptococcus sobrinus.

Streptococcal antigen I/II or the surface protein antigen A (SpaA) of Streptococcus sobrinus is an adhesin which mediates binding of the organism to tooth surfaces. The complete sequence of the gene which encodes SpaA has been determined. The gene consists of 4,584 bp and encodes a protein of 1,528 amino acid residues. The deduced amino acid sequence shows extensive homology with those of the cell surface adhesins from Streptococcus mutans serotypes c and f and from Streptococcus sanguis. Structural analysis of the N-terminal region (residues 50 to 550), which is rich in alanine and includes four tandem repeats of an 82-residue sequence, suggests that it adopts an alpha-helical coiled-coil conformation. Cell surface hydrophobicity may be associated with this region. The C-terminal region is more conserved and includes two tandem repeats of a 39-residue proline-rich sequence. A further proline-rich sequence in this region is predicted to span the cell wall. Although a hydrophobic sequence is present in the C-terminal region, it appears to be too short to span the cell membrane. Anchoring of SpaA in the cell membrane may therefore require some form of posttranslational modification or association with another membrane protein.