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 107 CFU BCG[pWB206] and then boosted with MVA-Gag developed Gag-specific CD8 T cells with a frequency of 1343±17 SFU/106 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.

HIV-1 Subtype C Mosaic Gag Expressed by BCG and MVA Elicits Persistent Effector T Cell Responses in a Prime-Boost Regimen in Mice

“…In this study, we demonstrated that BCG-Gag M persists in the tissues of vaccinated mice as determined by the presence of BCG-Gag M colonies in the peripheral lymphoid organs (spleen and lymph nodes) of these mice 11.5 weeks post vaccination ( Fig 2 ). Our group and others have also shown that rBCG persists in vivo up to 20 weeks post vaccination [ 28 , 59 – 61 ]. Future studies of BCG-Gag M should include experiments to confirm the long term persistence of HIV-specific T cell responses.…”

Engineering Tobacco Mosaic Virus and Its Virus‐Like‐Particles for Synthesis of Biotemplated Nanomaterials

“…The iteration of these processes can generate protein mutants with optimized or even new properties such as producing VLPs with enhanced structural stability. [ 87 ] Although directed evolution has not been widely used for VLP engineering, the recent development of new directed evolution methods, including assisted machine learning, [ 88 ] which identifies more promising mutations to be constructed and screened, and in vivo continuous directed evolution, [ 89 ] which streamlines the genetic engineering process, will accelerate the engineering of VLPs.…”

Engineering Tobacco Mosaic Virus, Barley Stripe Mosaic Virus, and their Virus-Like-Particles for Synthesis of Biotemplated Nanomaterials

“…The selected variants can then be mutated to introduce additional genetic diversity and subject to another round of selection. The iteration of these processes can generate protein mutants with optimized or even new properties such as producing VLPs with enhanced structural stability [84]. Although directed evolution has not been widely used for VLP engineering, the recent development of new directed evolution methods, including assisted machine learning [85], which identifies more promising mutations to be constructed and screened, andin vivo continuous directed evolution [86], which streamlines the genetic engineering process, will accelerate the engineering of VLPs.…”