Generation of mucosal anti-human immunodeficiency virus type 1 T-cell responses by recombinant Mycobacterium smegmatis

Abstract

A successful vaccine vector for human immunodeficiency virus type 1 (HIV-1) should induce anti-HIV-1 immune responses at mucosal sites. We have generated recombinant Mycobacterium smegmatis vectors that express the HIV-1 group M consensus envelope protein (Env) as a surface, intracellular, or secreted protein and have tested them in animals for induction of both anti-HIV-1 T-cell and antibody responses. Recombinant M. smegmatis engineered for expression of secreted protein induced optimal T-cell gamma interferon enzyme-linked immunospot assay responses to HIV-1 envelope in the spleen, female reproductive tract, and lungs. Unlike with the induction of T-cell responses, priming and boosting with recombinant M. smegmatis did not induce anti-HIV-1 envelope antibody responses, due primarily to insufficient protein expression of the insert. However, immunization with recombinant M. smegmatis expressing HIV-1 Env was able to prime for an HIV-1 Env protein boost for the induction of anti-HIV-1 antibody responses.

FIG. 1.

Expression of HIV-1 CON6 gp120 and gp140CF envelope proteins in M. smegmatis. (A) Western blot analysis of CON6 Env protein expression in various M. smegmatis designs, including molecular weight (Mr.) standards (lane 1), untransformed M. smegmatis (lane 2), and recombinant M. smegmatis transformed with either empty plasmid (lane 3), the surface expression plasmid pJH152-CON6 gp120 (lane 4), the intracellular expression plasmid pJH153-CON6 gp120 (lane 5), the secreted expression plasmid pJH154-CON6 gp120 (lane 6), or the surface expression plasmid pJH152-CON6 gp140 (lane 7) or pJH222-CON6 gp140 (lane 8). Both the intact (89 kDa for gp140, 79 kDa and 74 kDa for gp120) and partially cleaved (49 kDa and 34 kDa) CON6 Env protein products, as indicated by arrows, were found with gp120 MAb T8 (anti-C1 gp120 region), gp41-specific MAb 7B2, and V3 loop-specific MAb 7B9. (B) Schematic representation of the full-length (top) and cleaved (bottom) gp140, indicating the binding locations of MAbs T8, 7B2, and 7B9. Full-length gp140 could be detected by T8, 7B2, or 7B9, while the cleaved 34-kDa Env protein band could be detected only by MAb T8. A 50-kDa protein band could be detected by both MAbs 7B2 and 7B9. (C) Western blot analysis of supernatants from untransformed M. smegmatis (lane 1) and recombinant M. smegmatis transformed with the empty plasmid (lane 2) or pJH154-CON6gp140CF (lane 3) for detecting secreted CON6 Env protein expression. A truncated gp140 protein (lane 3) could be detected by 7B9, as indicated by the arrow. A high-molecular-weight protein band, as indicated by an arrowhead, was nonspecific, as it was also detected in recombinant M. smegmatis transformed with pJH154-CON6gp140CF (lane 3) and in the negative control lanes 1 and 2.

FIG. 2.

HIV-1 envelope-specific and vector-specific T-cell responses in the spleen, lungs, and FRT of mice immunized with recombinant M. smegmatis. HIV-1-specific (A to C) and vector-specific (D to F) T-cell responses in the spleen (A and D), lungs (B and E), and FRT (C and F) were assessed by IFN-γ ELISPOT assays. Mice that were primed with rAd-CON6 and boosted with rVV-CON6 were used as positive controls. Unimmunized mice were used as negative controls. Empty bars represent immunization groups that received prime/boost immunizations. Solid bars represent immunization groups that received priming immunization only. Mean (± standard error of the mean) numbers of 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 with pooled lung and FRT samples as described in Materials and Methods.

FIG. 3.

Antigen-specific T-cell responses induced by different modes of insert expression by recombinant M. smegmatis. HIV-1 envelope-specific (A and B) and vector-specific (C and D) T-cell responses were assessed by IFN-γ ELISPOT assays. Mice were immunized i.p. (A and C) or i.d. (B and D) with various designs of recombinant M. smegmatis or untransformed recombinant M. smegmatis, as indicated. Mean (± standard error of the mean) numbers of 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 recombinant M. smegmatis 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. 4.

Isotype of anti-HIV envelope antibodies induced by pJH154-gp120 and -gp140CF. IgG subclasses, including IgG1 (empty bars), IgG2a (gray bars), IgG2b (hatched bars), and IgG3 (solid bars), 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 = 5) are plotted on the y axis.

FIG. 5.

Induction of antibody responses with recombinant M. smegmatis is limited by the amount of HIV-1 Env expressed in recombinant M. smegmatis pJH154-gp120. To evaluate the immunogenicity of recombinant M. smegmatis expressing CON6 env in mice, mice were immunized once with either killed or live recombinant M. smegmatis expressing CON6 gp120 with or without IFA adjuvant. The geometric mean log endpoint antibody titers (± standard errors of the means; n = 5) are shown on the y axis. Abbreviations: rMsmeg, recombinant M. smegmatis expressing HIV-1 CON6 Env; CON6, recombinant CON6 gp140CF protein.