Respiratory Immunization With a Whole Cell Inactivated Vaccine Induces Functional Mucosal Immunoglobulins Against Tuberculosis in Mice and Non-human Primates

Abstract

Vaccination through the natural route of infection represents an attractive immunization strategy in vaccinology. In the case of tuberculosis, vaccine delivery by the respiratory route has regained interest in recent years, showing efficacy in different animal models. In this context, respiratory vaccination triggers lung immunological mechanisms which are omitted when vaccines are administered by parenteral route. However, contribution of mucosal antibodies to vaccine- induced protection has been poorly studied. In the present study, we evaluated in mice and non-human primates (NHP) a novel whole cell inactivated vaccine (MTBVAC HK), by mucosal administration. MTBVAC HK given by intranasal route to BCG-primed mice substantially improved the protective efficacy conferred by subcutaneous BCG only. Interestingly, this improved protection was absent in mice lacking polymeric Ig receptor (pIgR), suggesting a crucial role of mucosal secretory immunoglobulins in protective immunity. Our study in NHP confirmed the ability of MTBVAC HK to trigger mucosal immunoglobulins. Importantly, in vitro assays demonstrated the functionality of these immunoglobulins to induce M. tuberculosis opsonization in the presence of human macrophages. Altogether, our results suggest that mucosal immunoglobulins can be induced by vaccination to improve protection against tuberculosis and therefore, they represent a promising target for next generation tuberculosis vaccines.

Keywords: animal models; mucosal immunoglobulins; opsonization; pulmonary vaccination; tuberculosis; whole-cell vaccine.

FIGURE 1

Improved protection induced by intranasal MTBVAC HK as boost of subcutaneous BCG. (A–C) Groups of C57BL/6 adult mice where vaccinated with BCG and MTBVAC HK 4 weeks apart (107 MTBVAC HK dose when not specified). After one month, mice were intranasally challenged with H37Rv and lung bacterial load analyzed one month later. (D,E) Newborn mice were vaccinated with BCG and 8 weeks later boosted intranasally with 107 MTBVAC HK. Mice were challenged with a low-dose (D) or a high-dose (E) H37Rv inoculation and lung CFUs or survival analyzed, respectively. (F,G) Antigen-specific IFNg and IL17A production one month after MTBVAC HK vaccination, following PPD stimulation of cells from lungs (F) and spleen (G). (A–D, F,G) Data are shown as mean ± SEM and are representative of at least two independent experiments. (n = 6 mice/group). *p < 0.05; **p < 0.001; ***p < 0.001; ****p < 0.0001 by one-way ANOVA and Bonferroni post-test. (E) Data from one experiment (n = 10 mice/group) are represented in a Kaplan-Meier survival curve and statistical significance calculated by a LogRank test. **p < 0.01.

FIGURE 2

Intranasal MTBVAC HK induces mucosal immunoglobulins in respiratory airways. (A–C) Groups of C57BL/6 adult mice where vaccinated with BCG and MTBVAC HK 4 weeks apart. One month later PPD- specific IgA, IgM, IgG in BAL samples. (D–F) One month after MTBVAC HK boosting, wild-type, IgA-/- and pIgR-/- mice were intranasally challenged with H37Rv and lung bacterial load analyzed one month later. All data are mean ± SEM (A–C) Data are representative of two independent experiments (n = 6 mice/group). (D–F) Data in the graphs represent a pool of two independent experiments (n = 12 mice/group). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA and Bonferroni post-test.

FIGURE 3

Intrabronchial MTBVAC HK boosting induces local and systemic tuberculosis-specific cellular responses in NHP. (A) A study design schematic shows the time lines on a weekly basis relative to primary BCG vaccination and infectious challenge with M. tuberculosis, including booster immunization and biosampling events. (B,C) PPD-specific IFNg responses in peripheral blood were measured by ELISPOT immediately prior to and 1 week after MTBVAC HK boosting, at week 16 and 17, respectively. Individual data points are consistently colored according to the Supplementary Table 1; fat horizontal lines indicate group medians. Also, flow cytometry after intracellular cytokine staining was used to analyze IFNg responses. Dot plots of CD28 versus IFNg specific fluorescence signals of CD4 + T lymphocytes from a representative BAL sample illustrate local IFNg production (D) in the absence and (E) in the presence of PPD recall stimulation in vitro. Percentages per individual of PPD-induced IFNg-positive (F) CD4+ and (G) CD8+T lymphocytes from BAL are depicted.

FIGURE 4

TB disease measures and IFNg responses after infectious challenge. (A–C) Total arbitrary score of pathological involvement at endpoint and for lung and extra-thoracic disseminated disease, respectively. (D) Post-mortem enumeration of bacterial burden in the lung. (E–H) PPD-specific and (I–K) ESAT6-CFP10 fusion protein (E6C10) specific IFNg responses by specific ELISPOT analysis of PBMC, at indicated timepoints relative to primary vaccination or infectious challenge. Individual data points are consistently colored according to the Supplementary Table 1; fat horizontal lines indicate group medians.

FIGURE 5

Intrabronchial MTBVAC HK induces mucosal immunoglobulins in respiratory airways from non-human primates. (A–C) PPD-specific IgG, IgM, and IgA were measured in sera samples from the different individuals throughout the vaccination phase (until week 20). (D–G) PPD- specific IgA, IgM, IgG, as well as J chain were analyzed in BAL samples harvested at week 13 (before MTBVAC HK) and week 20 (after MTBAVC HK) from three individuals from the MTBVAC HK group. Data in the graphs show values for each individual (H) Direct binding of IgA, IgM, and IgG to Mtb surface. H37Rv bacteria were incubated with BAL samples and immunoglobulin binding measured by flow cytometry using specific secondary antibodies. Representative overlay histograms are shown. Black line: unvaccinated; Red line: BCG vaccinated; Blue line: BCG/MTBVAC HK vaccinated. Data in the graphs show the mean fluorescence intensity (MFI) obtained with each BAL compared to the measured when bacteria are incubated with PBS. (A–G) Data show individual from one experiment. (F) Data are shown as mean ± SEM and are representative of two independent experiments. (A–C, H) *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA and Bonferroni post-test. (A–C) Comparisons of the different timepoints with week 20 are shown. (D–G) *p < 0.05; **p < 0.01 by paired t-student test.

FIGURE 6

Mucosal immunoglobulins induced by MTBVAC HK opsonize Mtb in vitro. GFP-expressing H37Rv coated with BAL samples were added to THP-1 human monocytes. (A) Percentage of infected cells was determined by flow cytometry 4 h post-infection. Representative dot-plots from one individual are shown in the left panel. Data in the graph is represented as the fold-change of the percentage obtained with the BAL from BCG-only or BCG/MTBVAC HK compared with the BAL from unvaccinated NHP. (B) Representative images of infected cells stained with lysotracker and Hoechst reagents. Data in the graph show colocalization values for each individual from MTBVAC HK group comparing colocalization obtained with BAL from week 13 and 20. (C) C3b binding to H37Rv surface was analyzed by flow cytometry using an antibody against C3b. A representative overlay histogram is shown. Black line: unvaccinated; Red line: BCG vaccinated; Blue line: BCG/MTBVAC HK vaccinated. Data in the graph show the MFI fold-change obtained following incubation with BAL from week 13 and 20 compared to the measured with the value when bacteria are incubated with PBS. (D) THP1 cells were infected with H37Rv previously incubated with BAL from week 20 in the presence or absence of a neutralizing antibody antiC3b. (E) Fold-change comparison of infected- THP1 cells at 48 h versus 0 h for each experimental condition. (A,E) Data in the graphs are mean ± SEM from a pool of three independent experiments. **p < 0.01; ****p < 0.0001 by one-way ANOVA and Bonferroni post-test. (B) Data show mean values of at least six images for each experimental condition from one experiment. (C) Data show individual from one experiment. (B,C) *p < 0.05 by paired t-student test. (D) Data in the graphs are mean ± SEM from a pool of two independent experiments. *p < 0.05 by two-way ANOVA and Sidak’s multiple comparison test.