Randomized, double-blind, Phase 1 trial of an alphavirus replicon vaccine for cytomegalovirus in CMV seronegative adult volunteers.

Development of a cytomegalovirus (CMV) vaccine is a priority. We evaluated a two component alphavirus replicon particle vaccine expressing CMV gB or a pp65/IE1 fusion protein, previously shown to induce robust antibody and cellular immune responses in mice, in a randomized, double-blind Phase 1 clinical trial in CMV seronegative subjects. Forty subjects received a lower dose (LD) or higher dose (HD) of vaccine or placebo by intramuscular or subcutaneous injection at Weeks 0, 8 and 24. The vaccine was well tolerated, with mild to moderate local reactogenicity, minimal systemic reactogenicity, and no clinically important changes in laboratory parameters. All vaccine recipients developed ex vivo, direct IFN-gamma ELISPOT responses to CMV antigens (maximal mean spot-forming cells per 10(6) PBMC in LD and HD groups of 348 and 504 for pp65, 83 and 113 for IE1, and 138 and 114 for gB), and neutralizing antibodies (maximal geometric mean titer 110 with LD and 218 with HD). Polyfunctional CD4(+) and CD8(+) T cell responses were detected by polychromatic flow cytometry. This alphavirus replicon particle vaccine was safe and induced neutralizing antibody and multifunctional T cell responses against three CMV antigens that are important targets for protective immunity.

Development and preclinical evaluation of an alphavirus replicon particle vaccine for cytomegalovirus.

We used a replication-incompetent, single-cycle, alphavirus replicon vector system to produce virus-like replicon particles (VRP) expressing the extracellular domain of human cytomegalovirus (CMV) glycoprotein B or a pp65/IE1 fusion protein. Efficient production methods were scaled to produce pilot lots and clinical lots of each alphavirus replicon vaccine component. The vaccine induced high-titered antibody responses in mice and rabbits, as measured by ELISA and CMV neutralization assays, and robust T-cell responses in mice, as measured by IFN-gamma ELISPOT assay. A toxicity study in rabbits showed no adverse effects in any toxicology parameter. These studies support clinical testing of this novel CMV alphavirus replicon vaccine in humans.

A novel alphavirus vaccine encoding prostate-specific membrane antigen elicits potent cellular and humoral immune responses.

PURPOSE: Prostate-specific membrane antigen (PSMA) is an attractive target for active immunotherapy. Alphavirus vaccines have shown promise in eliciting immunity to tumor antigens. This study investigated the immunogenicity of alphavirus vaccine replicon particles (VRP) that encode PSMA (PSMA-VRP). EXPERIMENTAL DESIGN: Cells were infected with PSMA-VRP and evaluated for PSMA expression and folate hydrolase activity. Mice were immunized s.c. with PSMA-VRP or purified PSMA protein. Sera, splenocytes, and purified T cells were evaluated for the magnitude, durability, and epitope specificity of the anti-PSMA response. Antibodies were measured by flow cytometry, and cellular responses were measured by IFN-gamma enzyme-linked immunospot and chromium release assays. Cellular responses in BALB/c and C57BL/6 mice were mapped using overlapping 15-mer PSMA peptides. A Good Laboratory Practice-compliant toxicology study was conducted in rabbits. RESULTS: PSMA-VRP directed high-level expression of active PSMA. Robust T-cell and B-cell responses were elicited by a single injection of 2 x 10(5) infectious units, and responses were boosted following repeat immunizations. Anti-PSMA responses were detected following three immunizations with 10(2) infectious units and increased with increasing dose. PSMA-VRP was more immunogenic than adjuvanted PSMA protein. Responses to PSMA-VRP were characterized by Th-1 cytokines, potent CTL activity, and IgG2a/IgG2b antibodies. T-cell responses in BALB/c and C57BL/6 mice were directed toward different PSMA peptides. Immunogenic doses of PSMA-VRP were well tolerated in mice and rabbits. CONCLUSIONS: PSMA-VRP elicited potent cellular and humoral immunity in mice, and specific anti-PSMA responses were boosted on repeat dosing. PSMA-VRP represents a promising approach for immunotherapy of prostate cancer.

Cellular and humoral immune responses to alphavirus replicon vaccines expressing cytomegalovirus pp65, IE1, and gB proteins.

Development of vaccines against cytomegalovirus (CMV) is an important public health priority. We used a propagation-defective, single-cycle RNA replicon vector system derived from an attenuated strain of an alphavirus, Venezuelan equine encephalitis virus, to produce virus-like replicon particles (VRP) expressing various combinations of pp65, IE1, or gB proteins of human CMV. Protein expression in VRP-infected cells was highest with single-promoter replicons expressing pp65, IE1, a pp65/IE1 fusion protein, or the extracellular domain of gB and with double-promoter replicons expressing pp65 and IE1. Protein expression was lower with double- and triple-promoter replicons expressing gB, especially the full-length form of gB. BALB/c mice immunized with VRP expressing gB developed high titers of neutralizing antibody to CMV, and mice immunized with VRP expressing pp65, IE1, or a pp65/IE1 fusion protein developed robust antigen-specific T-cell responses as measured by gamma interferon enzyme-linked immunospot assay. Three overlapping immunodominant pp65 peptides contained a nine-amino-acid sequence (LGPISGHVL) that matches the consensus binding motif for a major histocompatibility complex H2-D(d) T-cell epitope. These data provide the basis for further development and clinical evaluation of an alphavirus replicon vaccine for CMV expressing the pp65, IE1, and gB proteins.