High-dose Mycobacterium tuberculosis aerosol challenge cannot overcome BCG-induced protection in Chinese origin cynomolgus macaques; implications of natural resistance for vaccine evaluation

Abstract

This study describes the use of cynomolgus macaques of Chinese origin (CCM) to evaluate the efficacy and immunogenicity of the BCG vaccine against high dose aerosol Mycobacterium tuberculosis challenge. Progressive disease developed in three of the unvaccinated animals within 10 weeks of challenge, whereas all six vaccinated animals controlled disease for 26 weeks. Three unvaccinated animals limited disease progression, highlighting the intrinsic ability of this macaque species to control disease in comparison to macaques of other species and genotypes. Low levels of IFNγ were induced by BCG vaccination in CCM suggesting that IFNγ alone does not provide a sufficiently sensitive biomarker of vaccination in this model. An early response after challenge, together with the natural bias towards terminal effector memory T-cell populations and the contribution of monocytes appears to enhance the ability of CCM to naturally control infection. The high dose aerosol challenge model of CCM has value for examination of the host immune system to characterise control of infection which would influence future vaccine design. Although it may not be the preferred platform for the assessment of prophylactic vaccine candidates, the model could be well suited for testing post-exposure vaccination strategies and drug evaluation studies.

Figure 1

Measures of tuberculosis-induced pulmonary and clinical disease burden. (A) Kaplan Meier plot of survival of BCG vaccinated and unvaccinated NHPs after challenge with M. tb, (% weight loss from peak post-challenge weight on the day of euthanasia, (C) Red cell haemoglobin concentration on the day of euthanasia, (D) Erythrocyte sedimentation rate (ESR) on the day of euthanasia, (E) the total pathology score determined using a qualitative scoring system, (F) the score attributed to the pulmonary component as part of the total pathology; (G) number of lesions in the lung enumerated by serial sectioning and manual counting, (H) the lung to lesion volume ratio determined using MR stereology, (I) score attributed to the chest radiogram on the day of euthanasia. Graphs B-I medians shown. Black symbols = BCG vaccinated, open symbols = unvaccinated. Triangle symbols indicate animals in which disease reached levels that met endpoint criteria. p values are shown and *denotes significant difference in outcome between groups using the non-parametric Mann–Whitney U test, p ≤ 0.05. (J) representative diagram of the location and number of granulomas present in the lungs, hilar lymph nodes, spleen, liver and kidneys and *show where M. tb bacteria were isolated and cultured from.

Figure 2

IFNγ detected using ELISPOT and ELISA in CCM with and without BCG vaccination and challenged with M. tb at week 21. (A) IFNγ ELISPOT PPD-specific SFU per million cells, (B) IFNγ ELISPOT ESAT-6-specific SFU per million cells, (C) IFNγ detected using whole blood ELISA, PPD-specific. CCM were either vaccinated with BCG (n = 6) or unvaccinated controls (n = 6 to week 25, n = 5 to week 27, n = 4 to week 29 then n = 3 to week 47). Lines indicate median and dots represent individual animals. Solid line = BCG vaccinated, hashed line = unvaccinated macaques.

Figure 3

PPD-specific polyfunctional T-cell analysis of CCM with or without BCG vaccination and challenged with MTB at week 21. (A) CD4⁺ T-cells: vaccinated with BCG (n = 6), (B) CD4⁺ T-cells unvaccinated (n = 6 to week 25, n = 5 to week 27, n = 4 to week 29 then n = 3 to week 47). (C) CD8⁺ T-cells, vaccinated with BCG (n = 6). (D) CD8⁺ T-cells unvaccinated (n = 6 to week 25, n = 5 to week 27, n = 4 to week 29 then n = 3 to week 47). (E) Summed cytokine responses in CD4⁺ T-cells, (F) summed cytokine responses in CD8⁺ T-cells. Median baseline response determined from assays applied on three separate occasions prior to vaccination. Vaccination phase in blue, challenge phase in red. Interquartile range shown and black line indicates median response. Black dots show individual responses. Frequency of parent combinations of IFNγ, IL-2 and TNFα. Threshold of 0.05 (75% confidence interval). Asterisks denote significant differences between the vaccinated group and controls animals determined by Mann–Whitney test (*p ≤ 0.05).

Figure 4

IFNγ detected in serum samples, Monocyte:lymphocyte ratio, monocyte subset analysis, analysis and levels of antigen-specific IgG and MGIA assay results. (A) IFNγ detected in serum using ELISA, (B) M:L ratio across the time course, (C) correlation of CD14⁺ monocytes and lung pathology score, (D) correlation of CD16⁺ monocytes and lung pathology, (E) PPD-specific IgG detected by ELISA in naïve and vaccinated CCM at 8 weeks post-vaccination measured by optical density (OD). (F) BCG-specific IgG detected by ELISA in naïve and vaccinated CCM at 8 weeks post-vaccination measured by optical density (OD). (G) MGIA assay at 8 weeks post-vaccination. Wilcoxon matched-pairs test (p = 0.05). White = unvaccinated, black = BCG vaccinated. Mann–Whitney t-test carried out at each time point,