Stable Expression of Lentiviral Antigens by Quality-Controlled Recombinant Mycobacterium bovis BCG Vectors

Author:

Hart Bryan E.,Asrican Rose,Lim So-Yon,Sixsmith Jaimie D.,Lukose Regy,Souther Sommer J. R.,Rayasam Swati D. G.,Saelens Joseph W.,Chen Ching-ju,Seay Sarah A.,Berney-Meyer Linda,Magtanong Leslie,Vermeul Kim,Pajanirassa Priyadharshini,Jimenez Amanda E.,Ng Tony W.,Tobin David M.,Porcelli Steven A.,Larsen Michelle H.,Schmitz Joern E.,Haynes Barton F.,Jacobs William R.,Lee Sunhee,Frothingham Richard

Abstract

ABSTRACTThe well-established safety profile of the tuberculosis vaccine strain,Mycobacterium bovisbacille 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 ΔleuCDtransformants 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 bothin vitroandin vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCDvaccine expressing HIV gp120 that retained stable full-length expression after 1024-fold amplificationin vitroand following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >1068-fold amplificationin vitroand induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCDlots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches.

Publisher

American Society for Microbiology

Subject

Microbiology (medical),Clinical Biochemistry,Immunology,Immunology and Allergy

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