Protective Efficacy of Inhaled BCG Vaccination Against Ultra-Low Dose Aerosol M. tuberculosis Challenge in Rhesus Macaques

Abstract

Ten million cases of tuberculosis (TB) were reported in 2018 with a further 1.5 million deaths attributed to the disease. Improved vaccination strategies are urgently required to tackle the ongoing global TB epidemic. In the absence of a validated correlate of protection, highly characterised pre-clinical models are required to assess the protective efficacy of new vaccination strategies. In this study, we demonstrate the application of a rhesus macaque ultra-low dose (ULD) aerosol M. tuberculosis challenge model for the evaluation of TB vaccination strategies by directly comparing the immunogenicity and efficacy of intradermal (ID) and aerosol BCG vaccination delivered using a portable vibrating mesh nebulizer (VMN). Aerosol- and ID-delivered Bacille Calmette-Guérin (BCG) induced comparable frequencies of IFN-γ spot forming units (SFU) measured in peripheral blood mononuclear cells (PBMCs) by ELISpot, although the induction of IFN-γ SFU was significantly delayed following aerosol immunisation. This delayed response was also apparent in an array of secreted pro-inflammatory and chemokine markers, as well as in the frequency of antigen-specific cytokine producing CD4 and CD8 T-cells measured by multi-parameter flow cytometry. Interrogation of antigen-specific memory T-cell phenotypes revealed that vaccination-induced CD4 and CD8 T-cell populations primarily occupied the central memory (TCM) and transitional effector memory (TransEM) phenotype, and that the frequency of CD8 TCM and TransEM populations was significantly higher in aerosol BCG-vaccinated animals in the week prior to M. tuberculosis infection. The total and lung pathology measured following M. tuberculosis challenge was significantly lower in vaccinated animals relative to the unvaccinated control group and pathology measured in extra-pulmonary tissues was significantly reduced in aerosol BCG-vaccinated animals, relative to the ID-immunised group. Similarly, significantly fewer viable M. tuberculosis CFU were recovered from the extra-pulmonary tissues of aerosol BCG-vaccinated macaques relative to unvaccinated animals. In this study, a rhesus macaque ULD M. tuberculosis aerosol challenge model was applied as a refined and sensitive system for the evaluation of TB vaccine efficacy and to confirm that aerosol BCG vaccination delivered by portable VMN can confer a significant level of protection that is equivalent, and by some measures superior, to intradermal BCG vaccination.

High dose aerosol challenge with Mycobacterium tuberculosis fails to overcome BCG vaccination-induced protection in cynomolgus macaques of Chinese origin: implications of natural resistance for TB vaccine evaluation.

“…Six macaques (Group A) were immunised intradermally (ID) in the upper left arm with 100 μl BCG vaccine, Danish strain 1331 (SSI, Copenhagen, Denmark). The method is as described elsewhere 13,29 but in brief; BCG vaccine was prepared for ID administration according to manufacturer's instructions for preparation of vaccine for administration to human adults, by addition of 1 ml Sautons diluent to a vial of vaccine, to give a suspension of BCG at an estimated concentration of 2 x 10 6 to 8 x 10 6 CFU/ml. All vaccinations were administered within one hour of vaccine reconstitution.…”

“…Prior to blood sample collection, vaccination, aerosol challenge and euthanasia, animals were weighed, examined for gross abnormalities and body temperature measured. As in previous studies 29 , red blood cell haemoglobin (RBC[Hb] levels were measured using a HaemaCue haemoglobinometer (Haemacue Ltd, Dron eld, UK) to identify the presence of anaemia, and erythrocyte sedimentation rates (ESR) were measured using the Sediplast system (Guest Medical, Edenbridge, UK) to detect and monitor in ammation induced by infection with M. tb. The time of necropsy, if prior to the end of the planned study period, was determined by experienced primatology staff and based on a combination of the following adverse indicators: depressed or withdrawn behaviour, abnormal respiration (dyspnoea), loss of 20% of peak post-challenge weight, ESR levels elevated above normal (>20 mm), haemoglobin level below normal limits (< 100g/dL), increased temperature (>41 o C) and severely abnormal chest X-ray.…”

“…There appears to be a much larger population of terminally differentiated memory (TEM2) cells in CCM in comparison to rhesus macaques, where memory T-cell populations were more biased towards transitional effector memory cells in naïve animals 18,29 . These terminally differentiated memory cells are considered to be a short lived population capable of exerting a rapid but more limited range of functional parameters 17 .…”

“…None of the animals had been used previously for experimental procedures and each socially compatible group was randomly assigned to a study treatment. All animal procedures and study design were approved by the Public Health England, Porton Down Establishment Animal Welfare and Ethical Review Body, and authorized under an appropriate UK Home O ce project license and as in previous studies 13,28,29 . Study was carried out in compliance with the ARRIVE guidelines.…”

“…The enhanced natural ability to control M. tb-induced disease may also play a role in the strong protective e cacy afforded by BCG vaccination to cynomolgus macaques that is in contrast to the lower level of protection conferred by BCG to rhesus macaques 31,29 . Therefore, the natural resistant to TB of CCM populations is likely to confound e cacy measures when this species is used for the evaluation of prophylactic vaccines; however, this same feature is a useful attribute for the study of latent or chronic disease states.…”