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Review 2: "A "suicide" BCG Strain provides Enhanced Immunogenicity and Robust Protection against Mycobacterium Tuberculosis in Macaques"

Reviewers suggested modifying the title to highlight safety and comparable protection, verifying statistical calculations, providing supplementary data, and conducting further experiments to support conclusions.

Published onJan 11, 2024
Review 2: "A "suicide" BCG Strain provides Enhanced Immunogenicity and Robust Protection against Mycobacterium Tuberculosis in Macaques"
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A “suicide” BCG strain provides enhanced immunogenicity and robust protection against Mycobacterium tuberculosis in macaques
A “suicide” BCG strain provides enhanced immunogenicity and robust protection against Mycobacterium tuberculosis in macaques

Abstract Intravenous (IV) BCG delivery provides robust protection against Mycobacterium tuberculosis (Mtb) in macaques but poses safety challenges. Here, we constructed two BCG strains (BCG-TetON-DL and BCG-TetOFF-DL) in which tetracyclines regulate two phage lysin operons. Once the lysins are expressed, these strains are cleared in immunocompetent and immunocompromised mice, yet induced similar immune responses and provided similar protection against Mtb challenge as wild type BCG. Lysin induction resulted in release of intracellular BCG antigens and enhanced cytokine production by macrophages. In macaques, cessation of doxycycline administration resulted in rapid elimination of BCG-TetOFF-DL. However, IV BCG-TetOFF-DL induced increased pulmonary CD4 T cell responses compared to WT BCG and provided robust protection against Mtb challenge, with sterilizing immunity in 6 of 8 macaques, compared to 2 of 8 macaques immunized with WT BCG. Thus, a “suicide” BCG strain provides an additional measure of safety when delivered intravenously and robust protection against Mtb infection.

RR:C19 Evidence Scale rating by reviewer:

  • Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.



The authors of this manuscript have generated a new BCG strain that dies after vaccination. This strain is safer than classic BCG, because it disappears after time, yet it retains the immunogenicity and protective properties of wild-type BCG.

The authors have used clever molecular methods to engineer a BCG strain that is killed upon cue and then releases antigens to the host. This part is robust and well-detailed. 

With this vaccine, they asked whether it performs comparable to WT BCG as a vaccine. This is important because it has been classically observed that killed MTB or killed BCG was not as good a vaccine as live BCG. 

Questions that were answered: 

  1. Is BCG Tet-Off cleared in vivo? Yes 

  2. Is BCG Tet-Off immunogenic (prior to Mtb challenge), despite being cleared? Yes, comparable to WT BCG.

  3. Does BCG Tet-off retain protective efficacy, despite being cleared? Yes, protection was not lost. Hypothesis that protection is compromised was clearly rejected. 

  4. Is BCG Tet-off more protective, in mice? No. Equal protection in lungs, seemingly less protection in spleens (although ns according to statistical testing done).

  5. Is BCG Tet-off protective, in NHP? When removing the historic controls, there was a trend that BCG Tet-off appeared to be more protective in NHP. However, in this experiment, BCG WT sterilized 2/8 which is not as impressive as Darragh 2020 where WT BCG sterilized 6/10. The finding that BCG Tet-off provided sterilizing immunity in 6/8 is therefore not clearly better than the previous published findings.

  6. Immune studies after challenge (Figure 6) are confounded by huge variance within each group, including sterile vs. non-sterile, 6 log difference in CFU. Therefore, it is not clear that these are about immunogenicity rather than the host containment of the Mtb challenge, in the two groups.

In sum, BCG Tet-off has been made, it is safe, despite it getting cleared, it is both immunogenic and protective, providing comparable immunogenicity in mice and NHP, and some potentially divergent protection data, tending to be less protective in mice but more protective in NHP. While the more protective is potentially exciting, the BCG Tet-off sterilized at about the same degree as the 2020 historical paper on iv WT BCG. 


  1. Re-verify stats:
    There are some comparisons that look to be hugely different with ns p-values (spleen in mice, lung in NHP) and other comparisons that look to have considerable overlap on raw data with hugely significant p-value (CD4 data in Figure 6). Is it possible that some of these comparisons were significant, despite the ns, based on the test done? It is best to check calculations before publishing. I also suggest that if there are two experimental groups here plus historic data as a positive control, the statistical test is really BCG WT vs. BCG tet-off, and the authors may not need to adjust for 3-way comparisons. 

  2. Modify title to highlight the key findings, which are:
    Suicide BCG strain is safer than WT BCG, yet confers immunogenicity and protection against TB comparable to WT BCG, in mouse and NHP models of TB.

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