A Mycobacterium tuberculosis-specific subunit vaccine that provides synergistic immunity upon co-administration with Bacillus Calmette-Guérin

Given the encouraging clinical results of both candidate subunit vaccines and revaccination with Bacillus Calmette-Guérin (BCG) against tuberculosis (TB), there is support for combining BCG and subunit vaccination for increased efficacy. BCG and Mycobacterium tuberculosis (Mtb) share ~98% of their genome and current subunit vaccines are almost exclusively designed as BCG boosters. The goal of this study is to design a TB subunit vaccine composed of antigens not shared with BCG and explore the advantages of this design in a BCG + subunit co-administration vaccine strategy. Eight protective antigens are selected to create an Mtb-specific subunit vaccine, named H107. Whereas traditional vaccines containing BCG-shared antigens exhibit in vivo cross-reactivity to BCG, H107 shows no cross-reactivity and does not inhibit BCG colonization. Instead, co-administering H107 with BCG leads to increased adaptive responses against both H107 and BCG. Importantly, rather than expanding BCG-primed T cells, H107 broadens the overall vaccine repertoire with new T cell clones and introduces ‘adjuvant-imprinted’ qualities including Th17 responses and less-differentiated Th1 cells. Collectively, these features of H107 are associated with a substantial increase in long-term protection.


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In our standardized murine aerosol Mtb challenge model, we powered the experiments for detecting a treatment effect (protection) of 0.5 log10 CFU reduction in lungs compared to non-treated controls with a type I error rate of 5% (!=0.05), a power of 80% and standard deviation of 0.35 log10 CFU (based on previous experiments). This results in n=8 mice per group for primary analysis. For comparative T cell magnitudes and phenotypical analysis, 4-6 mice per group were used based on our previous studies (e.g. PMIDs: 27554293, 33879592) comparing T cell responses in immunized and Mtb-infected mice for these and similar parameters. A minimum of 4 mice per condition was used determine, being increased to improved statistical robustness up to 6 mice as feasible given the number of animals to compare at a single time point under a single analysis. Experimental design that allowed for paired analysis (such as in Fig. 4) was included, as possible, to maximize statistical power within such sample sizes. For the study with human samples, no sample size calculation was performed, but based on previous studies (PMIDs 27409590, 32499216, 29602771) from our group we expect a difference in magnitude of response when comparing MTB300 reactivity in QFT+ vs. QFT-individuals. The final sample size used was based on the samples available from the clinical site.
No data was excluded from the analyses All experimental replications were successful and supported the data presented. Among the studies performed once: The TCR sequence analysis (performed by ENPICOM) was included as an exploratory approach to increase the understanding of the vaccineinduced T cell imprint. These analyses were time and resource intensive and since the data were consistent with the flow cytometry analysis, we did not find a repeat experiment necessary. The human participant study was based on the statistical significance of the results acquired and the samples available from the clinical site, and the long-term Mtb infection time point was due to the consistency with the shorter term data, the statistical significance of the results, ethical considerations, and time to publication reporting considerations.
Mice were randomly assigned to cages and treatment upon arrival to our animal facility. For each time point in the study, mice were randomly selected for primary analysis. Human participants were allocated into experimental groups based on IGRA status depicting their previous Mtb exposure (QFT+/-) as that was the intended comparison of the study.
The investigator was not involved in CFU data collection. Organ homogenization, plating, and CFU counting was performed by an experienced technician, who was not involved in study design and/or data analysis. The investigator was not specifically blinded during analysis and interpretation, however the statistical comparisons to be performed between groups (ANOVA with Tukey's posttest) was pre-determined, and therefore was not impacted. Similarly, the sample preparation and data acquisition of T cell immunological data was performed by an effectively-blinded technician who was not involved in the study design. The investigator applied uniform gating strategies, etc to all samples being compared. However, as analysis of the T cells responses would inherently reveal the vaccine status/history of the animals, blinding of the investigator was not realistically possible for the studies performed. TCR sequence analysis was performed by a third party entity (ENPICOM), who performed statistical comparisons without scientific investment in the outcome or conclusions. Individuals performing the experiments and collecting the raw data from the human participants were blinded to cohort assignment QFT+ vs QFT-. Participants from both groups were included in each experiment. Individuals analyzing the data were not blinded since the comparisons were dependent on knowing the respective cohort.