Recombinant Mycobacterium bovis bacillus Calmette-Guerin elicits human immunodeficiency virus type 1 envelope-specific T lymphocytes at mucosal sites

Abstract

A successful vaccine vector for human immunodeficiency virus type 1 (HIV-1) should induce anti-HIV-1 T-cell immune responses at mucosal sites. We have constructed recombinant Mycobacterium bovis bacillus Calmette-Guérin (rBCG) expressing an HIV-1 group M consensus envelope (Env) either as a surface, intracellular, or secreted protein as an immunogen. rBCG containing HIV-1 env plasmids engineered for secretion induced optimal Env-specific T-cell gamma interferon enzyme-linked immunospot responses in murine spleen, female reproductive tract, and lungs. While rBCG-induced T-cell responses to HIV-1 envelope in spleen were lower than those induced by adenovirus prime/recombinant vaccinia virus (rAd-rVV) boost, rBCG induced comparable responses to rAd-rVV immunization in the female reproductive tract and lungs. T-cell responses induced by rBCG were primarily CD4(+), although rBCG alone did not induce anti-HIV-1 antibody. However, rBCG could prime for a protein boost by HIV-1 envelope protein. Thus, rBCG can serve as a vector for induction of anti-HIV-1 consensus Env cellular responses at mucosal sites.

FIG. 1.

Expression of HIV-1 group M consensus gp120 and gp140 envelope proteins in BCG strains. (A) Expression of intact CON6 gp120 in rBCG-P transformed by the surface expression plasmid pJH152-gp120 (lane 2), the intracellular expression plasmid pJH153-gp120 (lane 3), and the secreted expression plasmid pJH154-gp120 (lane 4) in rBCG-P. The expression of CON6 gp140CF was also determined in rBCG-P transformed by the surface expression plasmid pJH152-gp140CF (lane 5). (B) The secreted expression of CON6 gp120 in pJH154-gp120 (lane 8), the surface expression of gp140CF in pJH152-gp140CF (lane 9), and the intracellular expression of gp140 in pJH153-gp140CF (lane 10) were demonstrated in rBCG-D. Both the intact and partially cleaved gp140 products were shown using gp120 MAb T8 (anti-C1 gp120 region), gp41-specific MAb 7B2, anti-gp140 MAb 3B3, and anti-gp140 MAb 16H3. Lane M is molecular weight standards in thousands (Bio-Rad, CA). Negative controls were untransformed BCG-D (lane 6) and rBCG strains transformed by empty plasmid pJH222 (lanes 1 and 7).

FIG. 2.

Antigen-specific T-cell responses induced by different modes of insert expression by rBCG. HIV-1 envelope-specific (A) and BCG vector-specific (B) T-cell responses were assessed by using IFN-γ ELISPOT assays. Mice were immunized i.p. with various doses of rBCG strains or untransformed BCG strains such as BCG-P or BCG-D. The mean (± standard error of the mean) SFC per 10⁶ cells derived from five mice per group are shown on the y axis. Mouse groups immunized with the indicated doses and designs of rBCG or control constructs are shown on the x axis. The mouse group indicated as CON6 was immunized with CON6 protein only. The mouse group indicated as rAd-rVV was primed with rAd-CON6 and boosted with rVV-CON6 as a positive control.

FIG. 3.

HIV-1 envelope-specific and vector-specific T-cell responses in spleen, lungs, and FRT of mice immunized with rBCG-P/120secPas. HIV-1-specific (A to C) and BCG vector-specific (D to F) T-cell responses in spleen (A and D), lungs (B and E), and FRT (C and F) were assessed by using IFN-γ ELISPOT assays. “CON6” indicates mice immunized with only CON6 protein. “rAd-rVV” indicates mice that were primed with rAd5 expressing CON6 Env and boosted with rVV expressing CON6 Env, used as positive controls. The mean (± standard error of the mean) SFC per 10⁶ cells are shown on the y axis. The indicated immunization groups and doses are shown on the x axis. Assays were performed and analyzed on pooled lungs and FRT samples as described in Materials and Methods. Statistical significance was assessed by comparing rBCG-P/120secPas with control groups such as BCG-P or CON6 (*, P < 0.0001; #, P < 0.001).

FIG. 4.

M. tuberculosis WCL-specific IFN-γ production is dependent on CD4⁺ splenocytes. In order to access the T-cell population responsible for IFN-γ production following immunization with 120secPas and BCG-P, via the i.p. route, we removed the respective CD4 or CD8 population using magnetically labeled antibodies from the prepared splenocytes. CD4⁺ T-cell responses were predominant when mice were immunized with rBCG. The mean (± standard error of the mean) SFC per 10⁶ cells derived from 15 mice per group are shown on the y axis.

FIG. 5.

HIV envelope-specific isotype induction by rBCG-P/120secPas. IgG subclasses including IgG1 (empty columns), IgG2a (gray columns), IgG2b (hatched columns), and IgG3 (solid columns) of HIV-1 envelope-specific serum antibodies induced by immunization with various immunization regimens (shown on the x axis) were determined by ELISA. The geometric mean log endpoint antibody titers (± standard errors of the means; n = 15) were plotted on the y axis.

FIG. 6.

Induction of antibody response with rBCG-P expressing HIV-1 gp120 in BALB/c mice with combinations of immunogen formulations. To evaluate the immunogenicity in mice of the rBCG-P expressing CON6 Env proteins, mice were immunized once with either killed or live rBCG-P/120secPas with or without IFA. The geometric mean endpoint titers (± standard errors of the means) for each group of mice (n = 5 per group) are shown. Abbreviations: 120secPas, rBCG-P expressing HIV-1 CON6 gp120 as the secreted protein; CON6, recombinant CON6 gp140CF protein.