Heterologous Boost Following Mycobacterium bovis BCG Reduces the Late Persistent, Rather Than the Early Stage of Intranasal Tuberculosis Challenge Infection

Abstract

Adults are the leading population affected by tuberculosis (TB) epidemic and death. Developing an effective vaccine against adult TB is urgently needed. Mycobacterium bovis Bacillus Calmette-Guerin (BCG) prime-heterologous boost strategy has been explored extensively to protect adults against primary TB infection, but the majority of experimental regimens have not improved the protection primed by the BCG vaccine. The reason attributed to the failure remains unknown. In this study, CTT3H-based vaccines, namely DMT adjuvanted CTT3H subunit or DNA vaccine (pCTT3H-DMT), and recombinant adenovirus rAdCTT3H were constructed. Protective efficacy and immunogenicity of BCG prime-CTT3H based boosters were compared in C57BL/c mice models of primary or late persistent TB infection. Similar protective efficacy against early intranasal infection was provided by different CTT3H-based vaccines alone in vaccinated mice, and their protection was inferior to that of the BCG vaccine. In addition, CTT3H-based heterologous boosters did not enhance the protection conferred by the BCG vaccine against primary infection. However, all of these three boosters provided stronger protection against late persistent TB infection than BCG alone, regardless of vaccine types. Although BCG prime-boosters elicited Th1-biased responses to the antigen CTT3H, the number of CTT3H-sepcific IFN-γ-expressing T_EM (CD62L^loCD44^hi) and IL-2-expressing T_CM (CD62L^hiCD44^hi) cells in the spleen was not improved before exposure to Mycobacterium tuberculosis infection. In contrast, IFN-γ⁺ T_EM and IL-2⁺ T_CM cells in spleens, especially in lungs were significantly increased in BCG prime-boosters after exposure vaccination. Our results indicate that BCG prime-boost strategy might be a promising measure for the prevention against late persistent TB infection by induction of IFN-γ⁺ T_EM and IL-2⁺ T_CM cells in the lung, which can be used as alternative biomarkers for guiding the clinical practice and future development of TB vaccine for adults.

Keywords: BCG; early stage of tuberculosis; heterologous boost; late persistent tuberculosis; memory T cells.

Figure 1

Construction and identification of rAdCTT3H and pCTT3H. (A) The structural diagram of recombinant replication-deficient adenovirus serotype 5-based rAdCTT3H. (B) The structural diagram of the recombinant eukaryotic expression plasmid pCTT3H. Identification of the recombinant protein CTT3H expressed in the supernatant or lysates of rAdCTT3H infected HEK293T cells (C) or pCTT3H transfected HEK293T cells (D) by western blotting with anti-CTT3H antibody.

Figure 2

Comparison of protective efficacy among different regimens. (A) The immunization and challenge schedules of three CTT3H-based vaccines alone immunized C57BL/6 mice against primary M. tuberculosis infection (n = 6). C57BL/6 mice were immunized with CTT3H-DMT, pCTT3H-DMT, and rAdCTT3H, respectively. BCG was used as positive control. PBS, DMT, pVAX-1, or Adnull were used as negative controls. At the tenth week after immunization, mice were challenged i.n. with approximately 100 CFU virulent M. tuberculosis H37Rv strain. Four weeks post-challenge, spleen, and lung were aseptically removed from each mouse and the number of M. tuberculosis in both organs was cultured and enumerated. The results are shown as (mean ± SEM) log₁₀ CFU/lung (B) or spleen (C) of different groups. (D) The protocol of BCG prime-CTT3H-based boosters in mice against primary infection (n = 6). Bacterial load in the lung (E) and spleen (F) of the different groups were shown as (mean ± SEM) log₁₀ CFU/organ. (G) The protocol of BCG prime-CTT3H-based boosters against mice late persistent TB infection (n = 6). Bacterial load in the lung (H) and the spleen (I) of different groups was shown as (mean ± SEM) log₁₀ CFU/organ. All experiments were repeated twice with similar results.

Figure 3

Comparison of CTT3H-specific Th1 responses among three CTT3H-based vaccines immunized C57BL/6 mice (n = 6). The immunization schedule was described in Figure 2A. At the tenth week after immunization, sera from each C57BL/6 mice of different groups were collected and titers of anti-CTT3H antibodies IgG (A), IgG2a (B), and IgG1 (C) were detected by ELISA. The results are shown as (mean ± SEM) log₁₀ endpoint titers. (D) The ratio of IgG2a/IgG1 of different vaccinated groups. (E) The levels of CTT3H-specific IFN-γ secreted by splenocytes. The concentration of IFN-γ in supernatants was detected by ELISA and the results are expressed as mean ± SD (pg/mL). (F) Comparison of CTL response to TB10.4 among different vaccinated groups (mean ± SEM). ^*p < 0.05 vs. the BCG group. All experiments were repeated twice with similar results.

Figure 4

Th1 biased response induced by BCG prime-CTT3H-based boosters (n = 6). The immunization schedule was described in Figure 2D. ELISA was used to determine titers of anti-CTT3H IgG (A), IgG2a (B), and IgG1(C) in the serum from each C57BL/6 mouse in BCG prime-booster vaccinated groups at the eighteenth week. (D) The ratio of IgG2a/IgG1 of different vaccinated groups. (E) The levels of CTT3H-specific IFN-γ secreted by splenocytes (mean ± SD pg/mL). (F) CTL response to TB10.4 (mean ± SEM). ^*p < 0.05 vs. the BCG repeated vaccination group. All experiments were repeated twice with similar results.

Figure 5

CTT3H antigen-specific T cells in spleens induced by BCG prime-CTT3H-based boosters before exposure (n = 6). The immunization protocol was described in Figure 2D. At the eighteenth week, splenocytes from heterologous boosters with PBS, CTT3H-DMT, pCTT3H-DMT, rAdCTT3H, and BCG vaccinated mice were collected and counted. 2.5 × 10⁶ cells were seeded in each well of a 24-well plate and stimulated with CTT3H (10 μg/mL) as described in detail in the section of Materials and Methods. The absolute number of CTT3H-specific IFN-γ⁺ (or IL-2 ⁺) CD4 ⁺ (or CD8 ⁺) T cells, IFN-γ⁺ CD4 ⁺ (or CD8 ⁺) T_EM cells, and IL-2⁺ CD4 ⁺ (or CD8 ⁺) T_CM cells from spleens were detected by flow cytometry. Results are shown as mean ± SD (n = 6). (A,B) Flow cytometry analysis strategy, (C) comparing BCG prime and BCG repeated boost strategy, (D,F) comparing BCG prime, subunit vaccine prime, and BCG prime and boost with subunit vaccine including protein vaccine (D), DNA vaccine (E), and recombinant adenovirus vaccine (F).

Figure 6

CTT3H antigen-specific T cells in spleens induced by BCG prime-CTT3H-based boosters after exposure (n = 6). The immunization and infection protocols were described in Figure 2G. At the nineteenth week, the absolute number of CTT3H-specific IFN-γ⁺ (or IL-2 ⁺) CD4 ⁺ (or CD8 ⁺) T cells, IFN-γ⁺ CD4 ⁺ (or CD8 ⁺) T_EM cells, and IL-2⁺ CD4 ⁺ (or CD8 ⁺) T_CM cells from spleens were detected by flow cytometry. Results are shown as mean ± SD (n = 6). (A–C) Comparing BCG prime, BCG repeated boosting, and BCG prime and boosted with subunit vaccine strategies including DNA vaccine (A), protein vaccine (B), and recombinant adenovirus vaccine (C).

Figure 7

CTT3H antigen-specific T cells in lungs induced by BCG prime-CTT3H-based boosters after exposure (n = 6). The immunization and infection protocols were described in Figure 2G. At the nineteenth week, the absolute number of CTT3H-specific IFN-γ⁺ (or IL-2 ⁺) CD4 ⁺ (or CD8 ⁺) T cells, IFN-γ⁺ CD4 ⁺ (or CD8 ⁺) T_EM cells, and IL-2⁺ CD4 ⁺ (or CD8 ⁺) T_CM cells in pneumonocytes were detected by flow cytometry. Results are shown as mean ± SD (n = 6). (A–C) Comparing BCG prime, BCG repeated boosting, and BCG prime and boosted with subunit vaccine strategies including DNA vaccine (A), protein vaccine (B), and recombinant adenovirus vaccine (C).

Figure 8

Comparison of histopathological changes in lungs among different regimens. The immunization and challenge schedules were shown in Figures 2A,D,G. Arrowheads indicate AF positive bacteria. HE, scale bar = 400 μm; AF staining, scale bar = 50 μm. (A) The representative lung pathological changes of three CTT3H-based vaccines alone immunized C57BL/6 mice against primary M. tuberculosis infection and (B) lung pathological scores (n = 3). (C) The representative lung pathological changes of BCG prime-CTT3H-based boosters in mice against primary infection and (D) lung pathological scores (n = 3). (E) The representative lung pathological changes of BCG prime-CTT3H-based boosters against mice late persistent TB infection and (F) lung pathological scores (n = 3).