A defined tuberculosis vaccine candidate boosts BCG and protects against multidrug-resistant Mycobacterium tuberculosis

Abstract

Despite the widespread use of the childhood vaccine against tuberculosis (TB), Mycobacterium bovis bacillus Calmette-Guérin (BCG), the disease remains a serious global health problem. A successful vaccine against TB that replaces or boosts BCG would include antigens that induce or recall the appropriate T cell responses. Four Mycobacterium tuberculosis (Mtb) antigens--including members of the virulence factor families PE/PPE and EsX or antigens associated with latency--were produced as a single recombinant fusion protein (ID93). When administered together with the adjuvant GLA-SE, a stable oil-in-water nanoemulsion, the fusion protein was immunogenic in mice, guinea pigs, and cynomolgus monkeys. In mice, this fusion protein-adjuvant combination induced polyfunctional CD4 T helper 1 cell responses characterized by antigen-specific interferon-γ, tumor necrosis factor, and interleukin-2, as well as a reduction in the number of bacteria in the lungs of animals after they were subsequently infected with virulent or multidrug-resistant Mtb strains. Furthermore, boosting BCG-vaccinated guinea pigs with fusion peptide-adjuvant resulted in reduced pathology and fewer bacilli, and prevented the death of animals challenged with virulent Mtb. Finally, the fusion protein elicited polyfunctional effector CD4 and CD8 T cell responses in BCG-vaccinated or Mtb-exposed human peripheral blood mononuclear cells. This study establishes that the protein subunit vaccine consisting of the fusion protein and adjuvant protects against TB and drug-resistant TB in animals and is a candidate for boosting the protective efficacy of the childhood BCG vaccine in humans.

Fig. 1

ID93 protein construct and characterization. (A) Schematic of the ID93 fusion protein. (B – D) SDS-PAGE and immunoblot of three lots of ID93. (B) 2 μg per lane of ID93 (lanes 1 – 3) were run in reducing and non-reducing conditions on a 4 – 20 % Tris glycine gel. (C) Immunoblot of ID93 with mouse antibody to ID93 and rabbit anti-sera to E. coli (50 ng and 1 μg of ID93, respectively). (D) Immunoblot of ID93 with mouse antibody to Rv3619, Rv1813, Rv3620, and Rv2608 (50 ng of ID93). EC, E. coli protein standards; ID93, lanes 1 – 3; lane 4, Rv3619; lane 5, Rv1813; lane 6, Rv3620; lane 7, Rv2608.

Fig. 2

Human PPD⁺ CD4 and CD8 T cells respond to ID93 antigen stimulation. PBMC from 7 healthy PPD⁺ subjects (3 with a history of BCG vaccination) with diverse HLA types were incubated for 8 h with medium, ID93 (20 μg/ml), PPD (10 μg/ml) or SEB (1 μg/ml). T cells were identified by intracellular cytokine staining based on CD3 expression, and further gated as CD4/CD45RO⁺ or CD8/CD45RO⁺ memory T cells. Percent of CD4 and CD8 T cells expressing IFN-γ, TNF, IL-2, or combinations of the three cytokines in response to antigen stimulation are shown.

Fig. 3

Immunogenicity and efficacy of ID93/GLA-SE against M. tuberculosis H37Rv and MDR TN5904 strains. (A) ID93-specific IgG1 and IgG2c antibodies were determined on day 49 in sera from animals immunized 3x 3 wks apart with GLA-SE (20 μg TLR-4 agonist) or ID93 (8 μg) + GLA-SE. Mean of reciprocal dilution + SD and IgG2c:IgG1 ratio are shown. (B) T cell in vitro cytokine recall responses to ID93 were measured 3 wks after the last immunization by intracellular cytokine staining and flow cytometry. CD4 T cells were identified based on CD3 expression, and further gated as CD44^hi. Bars show the percentage of cells expressing IFN-γ in response to ID93 stimulation (mean + SD, n = 3 mice), and pie charts show proportion of cells expressing one, two or the three cytokines IFN-γ, TNF, and IL-2. Data shown are representative of two experiments. (C and D) The number of viable bacteria in the lungs was determined 3 or 4 wks after challenge with aerosolized TN5904 (D) or H37Rv (C) strains, respectively. One way ANOVA followed by Dunnett’s Multiple Comparison Test was used for statistical analysis (vaccine groups were compared to saline control group); ** P < 0.01. (E and F) Histopathological evaluation of lung tissues post-challenge with H37Rv (E) or TN5904 (F). Granuloma (g) formation are shown in H & E-stained sections (scale 20 μm), and AFB ( → ) were evaluated (scale 5 μm). Data shown are representative of 4 mice/group in two independent experiments.

Fig. 4

Lung cellular infiltrates post-challenge. Mice were immunized 3x, 3 wks apart with saline, 20 μg GLA-SE, 8 μg ID93/GLA-SE, or once with 5×10⁴ live BCG. Four weeks after the last boost, mice were challenged with an aerosol dose of 50 – 100 H37Rv bacilli. Lung cell phenotype and in vitro cytokine recall responses to ID93 were measured by intracellular cytokine staining and flow cytometry 4 wks after challenge. (A) Number of Gr-1⁺ cells in the lungs. (B) Number of T cells (x 10³), identified based on CD3 expression, and further gated as CD4^-CD44^lo, CD4^-CD44^hi, CD4⁺CD44^lo or CD4⁺CD44^hi. (C) Number of CD44^hiCD4⁺ and CD44^hiCD8⁺ T cells expressing TNF and/or IFN-γ in response to media (“-Ag”), ID93 (10 μg/ml) or PPD (10 μg/ml) stimulation. Mean + SD (n = 2 pools of 3 mice each) are shown for a representative experiment. (D) Proportion of cells expressing IFN-γ, TNF, or TNF/IFN-γ effector cytokines in response to ID93 in vitro stimulation.

Fig. 5

ID93/GLA-SE vaccine confers long term protection against Mtb in guinea pigs in a BCG prime/protein boost regimen. Guinea pigs received either a single injection of saline (one of three groups) or of BCG (two groups). Four months later, BCG-primed animals received the ID93/GLA-SE vaccine or saline. (A) Serum ID93-specific IgG1 and IgG2 antibodies mean reciprocal dilution + SD. (B and C) Weekly weight monitoring after Mtb challenge for overt signs of disease and weight loss (B), resulting in euthanasia of the sick animals (C). Log Rank Test was used for statistical comparisons of median animal survival among the experiment groups; P < 0.01 compared to *saline or ** BCG → saline. (D and E) H&E-stained sections of lung and spleen, respectively, of each vaccinated animal on day 432 after challenge with H37Rv (scale: 0.2 mm). Guinea pig 547-834 in the BCG → saline group was euthanized on day 418 because of morbidity and excessive weight loss. (F). Granulomas (g) are shown in H&E-stained sections, and Masson’s trichrome (TC) stain was used to assess the amount of lung fibrosis in blue (scale: 20 μm). Data shown are representative of 4 animals/group.

Fig. 6

Cytokine responses to vaccine antigen in immunized macaques. Peripheral blood collected from ID93/GLA-SE-immunized animals was stimulated with ID93 or saline after which plasma was collected and assayed for cytokines by multiplexed bead-array assay. Background-subtracted antigen-specific cytokine responses are shown for blood collected pre-immunization (day 0, ○) and after third immunization (day 71, ●). * P ≤ 0.05, **P ≤ 0.01 by Wilcoxon Rank-Sum Test (n = 6).