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.

Combination of alphavirus replicon particle-based vaccination with immunomodulatory antibodies: therapeutic activity in the B16 melanoma mouse model and immune correlates.

Induction of potent immune responses to self-antigens remains a major challenge in tumor immunology. We have shown that a vaccine based on alphavirus replicon particles (VRP) activates strong cellular and humoral immunity to tyrosinase-related protein-2 (TRP2) melanoma antigen, providing prophylactic and therapeutic effects in stringent mouse models. Here, we report that the immunogenicity and efficacy of this vaccine is increased in combination with either antagonist anti-CTL antigen-4 (CTLA-4) or agonist anti-glucocorticoid-induced TNF family-related gene (GITR) immunomodulatory monoclonal antibodies (mAb). In the challenging therapeutic setting, VRP-TRP2 plus anti-GITR or anti-CTLA-4 mAb induced complete tumor regression in 90% and 50% of mice, respectively. These mAbs had similar adjuvant effects in priming an adaptive immune response against the vaccine-encoded antigen, augmenting, respectively, approximately 4- and 2-fold the TRP2-specific CD8(+) T-cell response and circulating Abs, compared with the vaccine alone. Furthermore, while both mAbs increased the frequency of tumor-infiltrating CD8(+) T cells, anti-CTLA-4 mAb also increased the quantity of intratumor CD4(+)Foxp3(-) T cells expressing the negative costimulatory molecule programmed death-1 (PD-1). Concurrent GITR expression on these cells suggests that they might be controlled by anti-GITR mAbs, thus potentially explaining their differential accumulation under the two treatment conditions. These findings indicate that combining immunomodulatory mAbs with alphavirus-based anticancer vaccines can provide therapeutic antitumor immune responses in a stringent mouse model, suggesting potential utility in clinical trials. They also indicate that tumor-infiltrating CD4(+)Foxp3(-)PD-1(+) T cells may affect the outcome of immunomodulatory treatments.

Alphavirus replicon particles expressing TRP-2 provide potent therapeutic effect on melanoma through activation of humoral and cellular immunity.

BACKGROUND: Malignant melanoma is the deadliest form of skin cancer and is refractory to conventional chemotherapy and radiotherapy. Therefore alternative approaches to treat this disease, such as immunotherapy, are needed. Melanoma vaccine design has mainly focused on targeting CD8+ T cells. Activation of effector CD8+ T cells has been achieved in patients, but provided limited clinical benefit, due to immune-escape mechanisms established by advanced tumors. We have previously shown that alphavirus-based virus-like replicon particles (VRP) simultaneously activate strong cellular and humoral immunity against the weakly immunogenic melanoma differentiation antigen (MDA) tyrosinase. Here we further investigate the antitumor effect and the immune mechanisms of VRP encoding different MDAs. METHODOLOGY/PRINCIPAL FINDINGS: VRP encoding different MDAs were screened for their ability to prevent the growth of the B16 mouse transplantable melanoma. The immunologic mechanisms of efficacy were investigated for the most effective vaccine identified, focusing on CD8+ T cells and humoral responses. To this end, ex vivo immune assays and transgenic mice lacking specific immune effector functions were used. The studies identified a potent therapeutic VRP vaccine, encoding tyrosinase related protein 2 (TRP-2), which provided a durable anti-tumor effect. The efficacy of VRP-TRP2 relies on a novel immune mechanism of action requiring the activation of both IgG and CD8+ T cell effector responses, and depends on signaling through activating Fcγ receptors. CONCLUSIONS/SIGNIFICANCE: This study identifies a VRP-based vaccine able to elicit humoral immunity against TRP-2, which plays a role in melanoma immunotherapy and synergizes with tumor-specific CD8+ T cell responses. These findings will aid in the rational design of future immunotherapy clinical trials.

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.

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.

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.

Alphavirus replicon particles as candidate HIV vaccines.

Replicon particles based on Venezuelan equine encephalitis virus (VEE) contain a self-replicating RNA encoding the VEE replicase proteins and expressing a gene of interest in place of the viral structural protein genes. Structural proteins for packaging of replicon RNA into VEE replicon particles (VRPs) are expressed from separate helper RNAs. Aspects of the biology of VEE that are exploited in VRP vaccines include 1) expression of very high levels of immunogen, 2) expression of immunizing proteins in cells in the draining lymph node, and 3) the ability to induce mucosal immunity from a parental inoculation. Results of experiments with VRPs expressing green fluorescent protein or influenza virus hemagglutinin (HA) demonstrated that specific mutations in the VRP envelope glycoproteins affect both targeting in the draining lymph node and efficiency of the immune response in mice. VRPs expressing either the matrix-capsid portion of Gag, the full-length envelope gp160, or the secreted gp140 of cloned SIVsm H-4i were mixed in a cocktail and used to immunize macaques at 0, 1, and 4 months. Neutralizing antibodies against SIVsm H-4 were induced in 6 of 6 vaccinates and CTL in 4 of 6. An intrarectal challenge with the highly pathogenic SIVsm E660 was given at 5 months. A vaccine effect was seen in reduced peak virus loads, reduced virus loads both at set point and at 41 weeks postchallenge, and preserved or increased CD4 counts compared to controls. A candidate VRP HIV vaccine expressing Clade C Gag contains a sequence that is very close to the South African Clade C consensus and was selected from a recent seroconverter in the Durban cohort to represent currently circulating genotypes in South Africa. A GMP lot of this vaccine has been manufactured and tested for a phase I trial in the first months of 2002.