Vaccine platform for prevention of tuberculosis and mother-to-child transmission of human immunodeficiency virus type 1 through breastfeeding

Abstract

Most children in Africa receive their vaccine against tuberculosis at birth. Those infants born to human immunodeficiency virus type 1 (HIV-1)-positive mothers are at high risk of acquiring HIV-1 infection through breastfeeding in the first weeks of their lives. Thus, the development of a vaccine which would protect newborns against both of these major global killers is a logical yet highly scientifically, ethically, and practically challenging aim. Here, a recombinant lysine auxotroph of Mycobacterium bovis bacillus Calmette-Guérin (BCG), a BCG strain that is safer than those currently used and expresses an African HIV-1 clade-derived immunogen, was generated and shown to be stable and to induce durable, high-quality HIV-1-specific CD4(+)- and CD8(+)-T-cell responses. Furthermore, when the recombinant BCG vaccine was used in a priming-boosting regimen with heterologous components, the HIV-1-specific responses provided protection against surrogate virus challenge, and the recombinant BCG vaccine alone protected against aerosol challenge with M. tuberculosis. Thus, inserting an HIV-1-derived immunogen into the scheduled BCG vaccine delivered at or soon after birth may prime HIV-1-specific responses, which can be boosted by natural exposure to HIV-1 in the breast milk and/or by a heterologous vaccine such as recombinant modified vaccinia virus Ankara delivering the same immunogen, and decrease mother-to-child transmission of HIV-1 during breastfeeding.

FIG. 1.

Construction of BCG.HIVA. (A) A synthetic GC-rich HIVA gene was fused to the region encoding the 19-kDa lipoprotein signal sequence and inserted into the episomal pJH222 or integrative pJH223 E. coli-mycobacterium shuttle plasmid. These plasmids both contained kanamycin resistance (aph) and complementing lysA genes and an E. coli origin of replication (oriE). In addition, pJH222 contained the mycobacterial origin of replication (oriM) and pJH223 carried the attachment site (attP) and the integrase gene (int) of mycobacteriophage L5. The BALB/c mouse T-cell and MAb Pk epitopes used in this work are depicted. Pα-Ag, M. tuberculosis α-antigen promoter; P_HSP60, heat shock protein 60 gene promoter. (B) Western blot of lysates of rBCG containing pJH222.HIVA (lanes 1 and 2) and pJH223.HIVA (lanes 3 and 4) and of BCG.p (lane 5) are shown. HIVA was detected using the anti-Pk MAb followed by horseradish peroxidase-protein A and enhanced chemiluminescence. wt, wild type. (C) Stability of rBCG harboring pJH222.HIVA. Mice were injected i.p. with 10⁷ CFU of BCG.HIVA, and rBCG was recovered from homogenized spleens 15 weeks later and plated (without kanamycin). Ten randomly picked mycobacterial colonies were tested for kanamycin resistance, and two of these colonies were tested for the presence of the HIVA gene by HIVA gene-specific PCR. Lanes 1 and 8, molecular markers; lane 2, BCG.p; lane 3, no template; lane 4, plasmid pJH222.HIVA; lanes 5 and 6, kanamycin-resistant colonies; and lane 7, BCG.HIVA vaccine stock. nd, not done.

FIG. 2.

Induction of multifunctional HIV-1-specific CD8⁺ T cells by the BCG.HIVA priming-MVA.HIVA boosting regimen. (A) Mice were either left unimmunized or immunized with 10⁶ CFU of p.BCG or BCG.HIVA and subsequently given a booster dose of 10⁶ PFU of MVA.HIVA as indicated. (B) Analysis of bifunctional vaccine-elicited CD8⁺ T cells. The top panels provide examples of dot blots as generated for group 6 and epitope H, and the bottom panels summarize the data obtained for each vaccination group by using the H (top) and P (bottom) epitopes. For the IFN-γ/CD107a/b and TNF-α/CD107a/b analyses, the frequencies of nondegranulating (empty bars) and degranulating (full bars) cells producing cytokine are shown. For the IFN-γ/TNF-α analysis, group average frequencies corresponding to dot blot quadrants I, II, and III are plotted. Data are presented as means ± standard deviations (SD; n, 4 to 5 mice). (C) Results of a 12-h procedure of in vivo killing of syngeneic peptide-pulsed cells from naïve and vaccinated animals. The left panel is an example dot blot showing data for splenocytes reisolated from naïve mice. Right panels show H- and P-specific killing as means ± SD (n = 5).

FIG. 3.

Effect of the BCG.HIVA priming on the induction of HIV-1-specific CD8⁺ T cells. (A) Immunization groups. Mice were either left unimmunized or primed with increasing doses of BCG.HIVA and given a booster dose of 10⁶ PFU of MVA.HIVA on a schedule similar to that shown in Fig. 2A. (B) The top right panels provide examples of dot blots for the analysis of bifunctional vaccine-elicited CD8⁺ T cells as generated for group 6 and epitope H. The bottom panels summarize the data obtained for each vaccination group by using the H (top) and P (bottom) epitopes. For the IFN-γ/CD107a/b and TNF-α/CD107a/b analyses, the frequencies of nondegranulating (empty bars) and degranulating (full bars) cells producing cytokine are shown. For the IFN-γ/TNF-α analysis, average frequencies corresponding to dot blot quadrants I, II, and III are plotted. Data are presented as means ± SD (n, 4 to 5 mice). (C and D) Analyses of trifunctional vaccine-elicited T cells. The two left panels indicate the gating. The right panels give the frequencies of trifunctional cells corresponding to the upper right quadrants for individual mice (circles) and groups (bars; values are means for the groups) as obtained with the H (top) and P (bottom) epitopes. Frequencies are expressed as percentages of CD8⁺ IFN-γ⁺ (C) and CD8⁺ TNF-α⁺ (D) cells.

FIG. 4.

Immunogenicity of the pTHr.HIVA DNA priming-BCG.HIVA boosting regimen for CD8⁺ T cells. (A) Mice were left unimmunized or primed with 10⁶ CFU of BCG.HIVA or BCG.p or 100 μg of pTHr.HIVA DNA, groups 5 and 6 were given booster doses of 10⁶ CFU of BCG.p or BCG.HIVA, and then all groups were challenged with 4 × 10⁶ PFU of WR.HIVA. (B) The top panels provide examples of dot blots for the analysis of bifunctional CD8⁺ T cells as generated for group 6 and epitope H. The bottom panels summarize the data obtained for each vaccination group by using the H (top) and P (bottom) epitopes. For the IFN-γ/CD107a/b and TNF-α/CD107a/b analyses, the frequencies of nondegranulating (empty bars) and degranulating (full bars) cells producing cytokine are shown. For the IFN-γ/TNF-α analysis, average frequencies corresponding to dot blot quadrants I, II, and III are plotted. Data are presented as means ± SD (n, 4 to 5 mice). (C and D) Analyses of trifunctional vaccine-elicited T cells. The two left panels indicate the gating. The right panels give the frequencies of trifunctional cells corresponding to the upper right quadrants for individual mice (circles) and groups (bars; values are means for the groups) as obtained with the H (top) and P (bottom) epitopes. Frequencies are expressed as percentages of CD8⁺ IFN-γ⁺ (C) and CD8⁺ TNF-α⁺ (D) cells.

FIG. 5.

Induction of high-quality HIV-1-specific CD4⁺ T cells and complete protection against surrogate virus challenge. The mice and the treatment groups (1 through 6) were the same as those described in the legend to Fig. 4A. (A) The leftmost panels summarize the data obtained for each cytokine and vaccination group. Data are presented as means ± SD (n, 4 to 5 mice). The middle panels demonstrate the gating for IFN-γ-, TNF-α-, and IL-2-producing CD4⁺ T cells as generated by group 6 for a cocktail of three MHC class II epitopes. The rightmost panels give the upper-right-quadrant data for trifunctional HIV-1-specific CD4⁺ T cells from individual mice (circles) and groups (bars). Data are presented as means ± SD (n, 4 to 5 mice). (B) Mice were either left naïve (1) or vaccinated with BCG.p (2), BCG.HIVA (3), pTHr.HIVA DNA (4), pTHr.HIVA DNA and BCG.p (5), or pTHr.HIVA and BCG.HIVA (6) and challenged with WR.HIVA. The WR.HIVA loads in ovaries were determined 4 days later. Data for individual mice (circles) and group means (bars; n, 4 to 5 mice) are shown.

FIG. 6.

Immune responses and protection against M. tuberculosis challenge by BCG.HIVA. (A) The mice and the treatment groups were the same as those described in the legend to Fig. 3A (immunized mice were primed with increasing doses of BCG.HIVA). Left panels show examples of bifunctional analyses using PPD as an antigenic stimulus. On the right, data corresponding to dot blot quadrants I, II, and III for each immunization group are shown as means ± SD (n, 4 to 5 mice). (B) Mice were left naïve or immunized subcutaneously in their left hind legs with the presently used BCG vaccine, a parental BCG, or lysine auxotrophic BCG.HIVA and challenged with inhaled M. tuberculosis. M. tuberculosis loads in lungs (left) and spleens (right) were determined 4 weeks later. Data for individual mice (circles) and geometric means for each group (n, 7 to 9 mice) are shown.