Research ArticleIMMUNOLOGY

A systematic approach to simultaneously evaluate safety, immunogenicity, and efficacy of novel tuberculosis vaccination strategies

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Science Advances  04 Mar 2020:
Vol. 6, no. 10, eaaz1767
DOI: 10.1126/sciadv.aaz1767
  • Fig. 1 Schematic representation of vaccination/infection model.

    Six- to 8-week-old female naïve C57BL/6 mice were vaccinated via the SC or IT route or left unvaccinated. Twenty days later, animals received a booster vaccination via the same or alternate route or did not receive a booster. During the vaccination period, mice were weighed weekly and assigned daily a clinical health score. Sixty days after prime vaccination, some mice from each group were euthanized, and clearance of bacilli from the lungs and spleen, cellular immune responses in the lung interstitium and airways, lung histology, and serum cytokine/chemokine profiles were studied (vaccine safety and immunogenicity). At day 90 after prime vaccination, remaining mice were aerosol infected with Mtb H37Rv. At day 118, Mtb colony-forming units (CFU) from lung tissue was enumerated, and lung histopathology was analyzed (vaccine efficacy). Three recombinant strains of BCG were investigated: BCG::pYUB (standard BCG reconstituted with an empty control plasmid conferring resistance to hygromycin, serving as the control strain), BCG::RD1, and BCG::RD1 ESAT-6 Δ92–95. Together with unvaccinated mice, this resulted in a total of 25 experimental groups.

  • Fig. 2 Vaccine safety.

    BCG CFU recovered from (A) lung tissue and (B) splenic tissue. (C) Representative images of iBALT-like structures (green arrowhead), generalized inflammation (yellow arrowhead), and normal lung tissue (red arrowhead) at ×40 magnification. (D) Representative H&E staining of lung sections from each experimental group at ×40 magnification. (E) Morphometric quantitation of histological changes presented as a percentage of the total lung area. (F) Weekly weight changes and health scores. Weight changes (top row) are presented as mean values from two pooled independent experiments (n = 6 to 13 mice per group). Health score results (bottom row) are presented as mean values from one representative experiment (n = 7 mice per group). (A to C) Presented as means ± SEM from two pooled independent experiments (n = 10 mice per group). The statistical significance of differences between each experimental group relative to unvaccinated controls is shown. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. The P values were determined using a one-way ANOVA followed by a Dunnett’s multiple comparison test.

  • Fig. 3 Systemic cytokine/chemokine profile and serum IgA levels before Mtb infection.

    (A) Serum cytokines/chemokines 60 days following prime vaccination presented in a heat map. (B) Serum levels of IL-1β, IL-6, and TNF-α used as measures of systemic inflammation for vaccine safety studies. (C) Serum levels of key TH1 cytokines (IFN-γ, IL-2, and IL-12p70). (B and C) Presented as means ± SEM from two pooled independent experiments (n = 10 mice per group). (D) Serum levels of IgA used as a measure of humoral immunity for immunogenicity studies (n = 3 mice per group). The statistical significance of differences between each experimental group relative to unvaccinated controls is shown. *P < 0.05 and **P < 0.01. The P values were determined using a one-way ANOVA followed by a Dunnett’s multiple comparison test.

  • Fig. 4 Airway immune cell profiles following vaccination.

    Sixty days following prime vaccination, animals were euthanized, and BALF was harvested for FACS analysis. (A) Representative FACS plots and enumeration of CD44hi T lymphocyte subsets TCM, TEM, and TRM. Indicated significance is of total memory T lymphocyte numbers. (B) Representative FACS plots and quantification of CD4+CD8 and CD8+CD4 and CD4CD8 (DN) TRM. Indicated significance is of total TRM numbers. (C) Total numbers of CD4+, CD8+, and DN TRM. (D) Representative FACS plots showing the gating strategy to enumerate tetramer+ cells and total numbers of tetramer+ TRM. (E) Proportion of tetramer+ and tetramer CD4+ TRM are also shown. (A to D) Results are presented as means ± SEM from two pooled independent experiments (n = 5 to 10 mice per group). The statistical significance of differences between each experimental group relative to unvaccinated controls is shown. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. The P values were determined using a one-way ANOVA followed by a Dunnett’s multiple comparison test.

  • Fig. 5 Vaccine-induced protection and pulmonary pathology following Mtb infection.

    (A) Twenty-eight days following Mtb infection, animals were sacrificed, and the lungs were assessed for numbers of viable Mtb. (B) H&E-stained lung sections were analyzed for histopathology. (C) Representative H&E staining of lung sections from each group at ×40 magnification. (A and B) Presented as means ± SEM from two pooled independent experiments. (A) n = 7 to 12 mice per group and (B) n = 6 to 8 mice per group. The statistical significance of differences between each experimental group relative to unvaccinated controls is shown. Scale bar, 600 μm. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. The P values were determined using a one-way ANOVA followed by a Dunnett’s multiple comparison test.

  • Fig. 6 Vaccine empirical integrated model.

    Simultaneous comparison of safety, immunogenicity, and efficacy through integration of measurements into single measures of their summed effects (Eqs. 1 to 3 and Materials and Methods). (A to C) Vaccine strategies ordered from right to left based on increased safety, immunogenicity, or efficacy. (D) Vaccines plotted on overall performance of immunogenicity versus efficacy. The size of circles (D) is scaled to safety effect, where the safest strategy is represented by the largest circle. The overall ranking of each vaccine strategy is indicated by the number next to the circle and as a bar graph.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/10/eaaz1767/DC1

    Fig. S1. Lung parenchymal immune cell profiles following vaccination.

    Fig. S2. FACS gating strategy to identify tetramer+ CD4+ TRM.

    Fig. S3. Vaccine empirical integrated model.

    Table S1. Health assessment scoring criteria.

    Table S2. Raw data for VEIM.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Lung parenchymal immune cell profiles following vaccination.
    • Fig. S2. FACS gating strategy to identify tetramer+ CD4+ TRM.
    • Fig. S3. Vaccine empirical integrated model.
    • Table S1. Health assessment scoring criteria.
    • Table S2. Raw data for VEIM.

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