Establishment of an aerosol challenge model of tuberculosis in rhesus macaques and an evaluation of endpoints for vaccine testing

Abstract

The establishment of an aerosol challenge model in nonhuman primates (NHPs) for the testing of vaccines against Mycobacterium tuberculosis would assist the global effort to optimize novel vaccination strategies. The endpoints used in preclinical challenge studies to identify measures of disease burden need to be accurate and sensitive enough to distinguish subtle differences and benefits afforded by different tuberculosis (TB) vaccine regimens when group sizes are inevitably small. This study sought to assess clinical and nonclinical endpoints as potentially sensitive measures of disease burden in a challenge study with rhesus macaques by using a new protocol of aerosol administration of M. tuberculosis. Immunological and clinical readouts were assessed for utility in vaccine evaluation studies. This is the first example of TB vaccine evaluation with rhesus macaques where long-term survival was one of the primary endpoints. However, we found that in NHP vaccine efficacy studies with maximum group sizes of six animals, survival did not provide a valuable endpoint. Two approaches used in human clinical trials for the evaluation of the gamma interferon (IFN-gamma) response to vaccination (enzyme-linked immunospot [ELISpot] assay and enzyme-linked immunosorbent assay [ELISA]) were included in this study. The IFN-gamma profiles induced following vaccination were found not to correlate with protection, nor did the level of purified protein derivative (PPD)-specific proliferation. The only readout to reliably distinguish vaccinated and unvaccinated NHPs was the determination of lung lesion burden using magnetic resonance (MR) imaging combined with stereology at the end of the study. Therefore, the currently proposed key markers were not shown to correlate with protection, and only imaging offered a potentially reliable correlate.

FIG. 1.

Experimental study design. PM, postmortem.

FIG. 2.

Immune response to vaccination. (A) Frequency of PPD-specific IFN-γ-secreting cells measured by ELISpot assay. (B) Frequency of Ag85A-specific IFN-γ-secreting cells measured by ELISpot assay. (C) Quantity of IFN-γ secreted by PBMC during 6 days of culture of PBMC with PPD measured by ELISA. (D) Frequency of CD3⁺ T cells that secreted IFN-γ following stimulation with PPD. Vaccination with BCG at week 0 is indicated by the dotted line, and vaccination with MVA85A indicated by the dashed line at week 12.

FIG. 3.

Polyfunctional T-cell analysis after vaccination. (A to C) Responding CD4⁺ T-cell populations following stimulation with Ag85A peptide pools. (D to H) Responding CD4⁺ T-cell populations following stimulation with PPD. (I and J) Responding CD8⁺ T cells following stimulation with PPD.

FIG. 4.

Kaplan-Meier plot of survival of vaccinated and unvaccinated NHPs after challenge with M. tuberculosis.

FIG. 5.

Measures of tuberculosis-induced pulmonary and clinical disease burden. (A) Ratio of lung volume to lesion volume determined by MR stereology. (B) Number of lesions in the lung enumerated by serial sectioning and manual counting. (C) Total pathology score determined by using a qualitative scoring system. (D) Scores attributed to the pulmonary component as part of the total pathology score. (E) Score attributed to the chest radiogram on the day of euthanasia. (F) Percent weight loss from peak postchallenge weight on the day of euthanasia. (G) ESR on the day of euthanasia. (H) Bacterial load in the lung.

FIG. 6.

Immune responses after aerosol challenge with M. tuberculosis. (A to C) Frequency of mycobacterium-specific IFN-γ-secreting cells after challenge measured by ELISpot assay. (A) ESAT6; (B) Ag85A; (C) PPD. (D) Profile of IFN-γ secretion by PPD-stimulated whole blood measured by ELISA.