RESUMO
The well-established safety profile of the tuberculosis vaccine strain, Mycobacterium bovis bacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ΔleuCD transformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging both in vitro and in vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCD vaccine expressing HIV gp120 that retained stable full-length expression after 10(24)-fold amplification in vitro and following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >10(68)-fold amplification in vitro and induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCD lots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches.
Assuntos
Antígenos Virais/biossíntese , Portadores de Fármacos , Produtos do Gene gag/biossíntese , Instabilidade Genômica , Proteína gp120 do Envelope de HIV/biossíntese , Mycobacterium bovis/genética , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Animais , Antígenos Virais/genética , Produtos do Gene gag/genética , Vetores Genéticos , Proteína gp120 do Envelope de HIV/genética , Camundongos Endogâmicos C57BL , Vacinas contra a SAIDS/genética , Vacinas contra a SAIDS/imunologia , Linfócitos T/imunologiaRESUMO
Immunovirological consequences of a switch to a maraviroc/raltegravir dual therapy were analyzed in 16 HIV-infected patients with persistent viral load below 50âcopies/ml. At 26-week postswitch, the CD4/CD8 ratio decreased and the CD8 T-cell activation increased. A decrease in classical monocytes was associated with a shift toward a proinflammatory monocyte profile and negatively correlated with ultrasensitive viral load. Thus, this therapeutic switch induced a proinflammatory profile probably driven by a slight loss of virus control.
