Expression of Vaccinia E3L and K3L Genes by a Novel Recombinant Canarypox HIV Vaccine Vector Enhances HIV-1 Pseudovirion Production and Inhibits Apoptosis in Human Cells

Abstract

Poxviruses that are attenuated for growth in human cells provide a safe means of HIV antigen expression and are capable of eliciting HIV-specific immune responses, including CD8+ cytotoxic T-lymphocyte (CTL) responses. HIV-1 antigen expression in human cells by attenuated poxvirus vectors may be limited by interferon-mediated host defense mechanisms. To enhance HIV antigen expression in human cells, the vaccinia virus E3L and K3L genes were inserted into a canarypox vector that expresses HIV-1 Gag, Env, and a Nef/Pol polyepitope string. E3L and K3L markedly reduced the activation of the double-stranded RNA-dependent protein kinase, PKR, and led to a significant reduction in apoptosis in HeLa cells. Production and release of HIV-1 antigen in the form of pseudovirions was enhanced in both duration and magnitude by this vector modification. The addition of immunomodulatory genes to attenuated poxviruses represents a novel strategy for enhancing antigen production by live vector HIV vaccine candidates.

Pseudovirion Particle Production by Live Poxvirus Human Immunodeficiency Virus Vaccine Vector Enhances Humoral and Cellular Immune Responses

“…2A and B and D and E). As noted previously (12), the particles were predominantly immature in appearance. Particles were readily found budding from murine cells (Fig.…”

“…The cells were incubated for an additional 8 h, and then Gag proteins were immunoprecipitated with HIV patient sera and analyzed by SDS-PAGE and autoradiography. In a separate experiment, the p24 contents of cell lysates and supernatants were measured at 36 h postinfection with vCP205 or Myr Ϫ vCP205 by p24 antigen capture enzyme-linked immunosorbent assay (ELISA) as previously described (12). For electron microscopy, BSC-40 and C2C12 cells were infected with vCP205 or Myr Ϫ vCP205 at an MOI of 10.…”

“…Cells were harvested at 24 h postinfection, washed in phosphate-buffered saline (PBS), and fixed in 2.5% glutaraldehyde in PBS. Processing and analysis by transmission electron microscopy were performed as previously described (12). Imaging was performed using a Philips CM12 electron microscope.…”

“…Thus, live vectors that include a complete gag gene and elicit sufficient production of Gag protein in the cytoplasm of infected cells will generate pseudovirions. Pseudovirion production has been demonstrated to occur in tissue culture cells infected by vaccinia virus vectors (22), modified vaccinia Ankara (MVA) vectors (34), canarypox (ALVAC) vectors (11,12), and adenoviral vectors (28) expressing Gag proteins. Expression of Gag together with the envelope protein (Env) by live vaccine vectors leads to the release of pseudovirions bearing Env on their surfaces (12,19,21,28).…”

Direct Comparison of Antigen Production and Induction of Apoptosis by Canarypox Virus- and Modified Vaccinia Virus Ankara-Human Immunodeficiency Virus Vaccine Vectors

“…After six rounds of plaque purification and selection using staining with X-Gluc (5-bromo-4-chloro-3-indolyl-␤-D-glucuronic acid), a master stock of MVA205 was generated. One experiment in this paper describes the use of vCP1452, a vector that includes the vaccinia virus E3L and K3L genes, whose construction was described in detail previously (20). MVA-GFP was generously provided by Mark Feinberg (11).…”