Stable Expression of Lentiviral Antigens by Quality-Controlled Recombinant Mycobacterium bovis BCG Vectors.
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.
Human Vaccine Institute and Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.
Bryan E
BE
Hart
Human Vaccine Institute and Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.
Rose
R
Asrican
Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.
So-Yon
SY
Lim
Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.
Jaimie D
JD
Sixsmith
Howard Hughes Medical Institute and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, USA.
Regy
R
Lukose
Human Vaccine Institute and Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.
Sommer J R
SJ
Souther
Human Vaccine Institute and Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.
Swati D G
SD
Rayasam
Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina, USA.
Joseph W
JW
Saelens
Human Vaccine Institute and Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.
Ching-Ju
CJ
Chen
Human Vaccine Institute and Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.
Sarah A
SA
Seay
Howard Hughes Medical Institute and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, USA.
Linda
L
Berney-Meyer
Howard Hughes Medical Institute and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, USA.
Leslie
L
Magtanong
Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.
Kim
K
Vermeul
Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.
Priyadharshini
P
Pajanirassa
Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.
Amanda E
AE
Jimenez
Howard Hughes Medical Institute and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, USA.
Tony W
TW
Ng
Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina, USA.
David M
DM
Tobin
Howard Hughes Medical Institute and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, USA.
Steven A
SA
Porcelli
Howard Hughes Medical Institute and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, USA.
Michelle H
MH
Larsen
Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.
Joern E
JE
Schmitz
Human Vaccine Institute and Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.
Barton F
BF
Haynes
Howard Hughes Medical Institute and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, USA.
William R
WR
Jacobs
Human Vaccine Institute and Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.
Sunhee
S
Lee
Human Vaccine Institute and Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA richard.frothingham@duke.edu.
Richard
R
Frothingham
D016915:AIDS Vaccines / Q000235:genetics / Q000276:immunology; D000818:Animals; D000956:Antigens, Viral / Q000096:biosynthesis* / Q000235:genetics; D004337:Drug Carriers; D015683:Gene Products, gag / Q000096:biosynthesis* / Q000235:genetics; D005822:Genetic Vectors; D042822:Genomic Instability; D015699:HIV Envelope Protein gp120 / Q000096:biosynthesis* / Q000235:genetics; D008810:Mice, Inbred C57BL; D009163:Mycobacterium bovis / Q000235:genetics*; D018449:SAIDS Vaccines / Q000235:genetics / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology
D016915:AIDS Vaccines; D000956:Antigens, Viral; D004337:Drug Carriers; D015683:Gene Products, gag; D015699:HIV Envelope Protein gp120; D018449:SAIDS Vaccines; C115853:simian immunodeficiency virus gag p55