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

A vaccine to protect individuals from human immunodeficiency virus (HIV) infection or disease is urgently needed to combat the ongoing epidemic of AIDS. A variety of approaches to generate HIV-specific cellular and humoral responses are presently under evaluation in preclinical development and in human trials. Among the most promising approaches are those employing live-virus-derived vector systems that express HIV proteins efficiently within host cells. Viral vectors presently undergoing testing in human HIV vaccine trials include poxviruses, adenoviruses, and alphaviruses, while a variety of additional vector systems are in earlier stages of development (6, 8, 31, 43). Vaccination regimens employing live viral vectors expressing HIV genes have provided protection from disease following simian-human immunodeficiency virus (SHIV) 89.6P challenge in macaques (1, 39, 41). The factors that influence the immunogenicity of live-virus-based HIV vaccines remain incompletely defined. One factor that may contribute to the immunogenicity of these vaccine vectors is their ability to generate HIV virus-like particles, or pseudovirions. The HIV Gag protein drives the process of particle assembly and is sufficient to generate the production of virus-like particles, or pseudovirions, in the absence of additional viral gene products (16, 49). 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). Production of Gag or Gag-Env pseudovirions by live viral vectors in vivo may be beneficial to the immune response. Live vectors are likely to predominantly infect cells that are not professional antigen-presenting cells (APCs), such as skeletal muscle cells. Because these cells do not express class II molecules or costimulatory molecules on their surfaces, they are unlikely to function well as antigen-presenting cells. Uptake of released or particulate antigen by professional APCs, such as macrophages and dendritic cells, may then be required for the induction of potent adaptive immune responses to vector-encoded gene products. This uptake can be accomplished through the phagocytosis of particulate antigens released from vector-infected cells, followed by processing and presentation of vector-encoded antigen. We propose that pseudovirion formation by live vectors increases the availability of antigen to be taken up by professional APCs directly as particulate antigen and in this way enhances the production of a broad immune response. In support of this hypothesis, HIV particles have been shown to be taken up by dendritic cells in vitro, allowing HIV antigens to enter the class I presentation pathway through a process known as antigen cross-priming or the alternative class I presentation pathway (15, 25, 26). If this mechanism is relevant for HIV vaccine strategies, then we predict that a particle-competent vector would be superior in CD8+ T-cell responses to one that lacks this capacity. In this study, we compared the immunogenicity of a poxvirus-based vector that elicits particle formation to that of a vector in which a single nucleotide substitution altered the myristylation receptor glycine residue of Gag and eliminated pseudovirion formation. We found that HIV-specific CD8+ T-cell responses were superior in mice receiving the particle-competent vector. The titers of antibodies to Gag and Env were also substantially higher in mice administered the particle-competent vector. Furthermore, the particle-competent vector markedly enhanced HIV-specific CD4+ T-cell responses. These results provide the first direct evidence that pseudovirion particle formation by live-vector HIV vaccines enhances HIV-specific humoral and cellular immune responses.