Construction and characterization of recombinant fowlpox viruses expressing human papilloma virus E6 and E7 oncoproteins.

Human papilloma virus (HPV)-16 is the most prevalent high-risk mucosal genotype and the expression of the E6 and E7 proteins, which can bind to the p53 and p105Rb host cell-cycle regulatory proteins, is related to its tumorigenicity. Virus-like-particle (VLP)-based immunogens developed recently are successful as prophylactic HPV vaccines. However, given the high number of individuals infected already with HPV and the absence of expression of the L1 structural protein in HPV-infected or HPV-transformed cells, an efficient therapeutic vaccine targeting the non-structural E6 and E7 oncoproteins is required. In this study, two new fowlpox virus (FPV) recombinants encoding the HPV-16 E6 and E7 proteins were engineered and evaluated for their correct expression in vitro, with the final aim of developing a therapeutic vaccine against HPV-related cervical tumors. Although vaccinia viruses expressing the HPV-16 and HPV-18 E6 and E7 oncoproteins have already been studied, due to their natural host-range restriction to avian species and their ability to elicit a complete immune response, FPV recombinants may represent efficient and safer vectors also for immunocompromised hosts. The results indicate that FPV recombinants can express correctly the E6 and E7 oncoproteins, and they should represent appropriate vectors for the expression of these oncoproteins in human cells.

Molecular and biological characterization of simian-human immunodeficiency virus-like particles produced by recombinant fowlpox viruses.

Virus-like particles (VLPs) mimicking the simian-human immunodeficiency virus SHIV89.6P (VLPSHIV) were produced by co-infection of Vero cells with fowlpox SIVgag/pol (FPgag/polSIV) and fowlpox HIV-1env89.6P (FPenv89.6P) recombinant viruses. As a necessary prerequisite for a more efficient vaccine approach, ultrastructural, functional and molecular characterizations of VLP(SHIV) were performed in the SHIV-macaque model to verify the similarity of these particles to SHIV89.6P. Here we show that VLPSHIV can infect T cells by fusion without replication, as demonstrated by the absence of new viral progeny in VLPSHIV-infected C8166 cells. Biochemical characterization showed identical protein profiles of VLPSHIV and SHIV89.6P, and ultrastructural analysis of Vero cells releasing VLPSHIV also confirmed the morphological similarity of these pseudovirions to SHIV89.6P particles. Viral mRNAs were also found packaged inside the core of VLPSHIV by RT-PCR and reverse transcriptase assays.

Evaluation in rabbits of different anti-SHIV vaccine strategies based on DNA/fowlpox priming and virus-like particle boosting.

Two different prime-boost immunization protocols were tested in rabbits and their immune response was evaluated and compared with the final aim of defining a vaccine strategy that might be able to protect non-human primates from infection with the pathogenic simian/human immunodeficiency virus, SHIV(89.6P). The two regimens were based on three priming immunizations with either an expression plasmid plus a fowlpox (FP) recombinant vector or with two FP recombinant vectors, each one expressing either the SIV(mac239) gag/pol or the HIV-1env(89.6P) genes. In both protocols, priming immunizations were followed by two boosts with SHIV-mimicking virus-like particles (VLP). A complete SHIV-specific response was observed in all animals. Interestingly, the DNA vaccine was three to 10 times more efficient than the FP recombinant in inducing an anti-gag humoral response. Real-time PCR confirmed the memory effect on T-cell subsets secreting interleukin-4 and interferon-gamma, as a consequence of stimulation of both arms of the immune system. Although both protocols were almost equally effective in eliciting homologous neutralizing antibodies and highlighted the efficacy of VLP administration for boosting, protocol A seemed to be more effective in promoting a balanced T-cell memory immune response and appears more promising for vaccine purposes.