title: The use of directed evolution to create a stable and immunogenic recombinant BCG expressing a modified HIV-1 Gag antigen.
abstract: Numerous features make Mycobacterium bovis BCG an attractive vaccine vector for HIV. It has a good safety profile, it elicits long-lasting cellular immune responses and in addition manufacturing costs are affordable. Despite these advantages it is often difficult to express viral antigens in BCG, which results in genetic instability and low immunogenicity. The aim of this study was to generate stable recombinant BCG (rBCG) that express high levels of HIV antigens, by modification of the HIV genes. A directed evolution process was applied to recombinant mycobacteria that expressed HIV-1 Gag fused to the green fluorescent protein (GFP). Higher growth rates and increased GFP expression were selected for. Through this process a modified Gag antigen was selected. Recombinant BCG that expressed the modified Gag (BCG[pWB106] and BCG[pWB206]) were more stable, produced higher levels of antigen and grew faster than those that expressed the unmodified Gag (BCG[pWB105]). The recombinant BCG that expressed the modified HIV-1 Gag induced 2 to 3 fold higher levels of Gag-specific CD4 T cells than those expressing the unmodified Gag (BCG[pWB105]). Mice primed with 10(7) CFU BCG[pWB206] and then boosted with MVA-Gag developed Gag-specific CD8 T cells with a frequency of 1343±17 SFU/10(6) splenocytes, 16 fold greater than the response induced with MVA-Gag alone. Levels of Gag-specific CD4 T cells were approximately 5 fold higher in mice primed with BCG[pWB206] and boosted with MVA-Gag than in those receiving the MVA-Gag boost alone. In addition mice vaccinated with BCG[pWB206] were protected from a surrogate vaccinia virus challenge.
authors: Division of Virology, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
authors: Rosamund
authors: R
authors: Chapman
authors: Division of Virology, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
authors: William R
authors: WR
authors: Bourn
authors: Division of Virology, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa; Medical Research Council, Cape Town, South Africa; Department of Medicine Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
authors: Enid
authors: E
authors: Shephard
authors: Division of Virology, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
authors: Helen
authors: H
authors: Stutz
authors: Division of Virology, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
authors: Nicola
authors: N
authors: Douglass
authors: Division of Virology, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
authors: Thandi
authors: T
authors: Mgwebi
authors: Department of Molecular and Cell Biology, Faculty Of Science, University of Cape Town, Cape Town, South Africa.
authors: Ann
authors: A
authors: Meyers
authors: Division of Virology, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
authors: Nyasha
authors: N
authors: Chin'ombe
authors: Division of Virology, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa; National Health Laboratory Services, Cape Town, South Africa.
authors: Anna-Lise
authors: AL
authors: Williamson
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D019020:Directed Molecular Evolution; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D009163:Mycobacterium bovis / Q000235:genetics* / Q000276:immunology; D014614:Vaccines, Synthetic / Q000235:genetics* / Q000276:immunology; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics* / Q000276:immunology
chemical_list: D014614:Vaccines, Synthetic; D054301:gag Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins