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Review 1: "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 1: "A "suicide" BCG Strain provides Enhanced Immunogenicity and Robust Protection against Mycobacterium Tuberculosis in Macaques"
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key-enterThis Pub is a Review of
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.



This preprint introduces BCG-TetOFF-DL, a new BCG strain regulated by tetracyclines, displaying enhanced protection against Mtb in macaques. It triggers robust CD4-positive T helper cell responses, indicating potential as an advanced TB vaccine candidate targeting the host's immune response.

The manuscript discusses constructing and evaluating two BCG strains, BCG-TetON-DL and BCG-TetOFF-DL, that could be efficiently killed by adding or removing tetracyclines. Both strains show enhanced immunogenicity and protection against Mycobacterium tuberculosis (Mtb) in vitro and the mouse model. The study provides detailed information on the construction of these strains, the evaluation of BCG-TetOFF-DL in macaques, and the potential implications for developing tuberculosis vaccines. The study had a logical flow of experiments upscaled from in vitro to mouse model and eventually to non-human primates. The document provides a comprehensive description of the construction and evaluation of the BCG strains, including the intravenous route used for vaccination, challenge, and assessment of immune responses. BCG-TetOFF-DL showed improved CD4 T cell response, better protection (6/8 vs. 2/8 macaques were sterile), and rapid elimination after the doxycycline administration was stopped. Suggests potential as an advanced TB vaccine candidate. The study's findings have significant implications for developing safer and more effective tuberculosis vaccines, a critical area of research given the global burden of tuberculosis.

The conclusions drawn in the manuscript are supported well by the data presented. The experiments conducted included appropriate controls, replication, and adequate sample sizes (limitation to which has been stated as a future direction). The statistical analysis showed rigor as well. The supplementary data was mentioned but not provided in the manuscript. All other figures for data were well-labelled and illustrated in the manuscript. Providing the supplementary data is required. The submission is unambiguous, with minor suggestions that may be rectified. In line 70, the acronym "WT tetO" is not described. Lines 175-179 mention stimulation with M. tuberculosis H37Rv whole cell-lysate, but this needs to be discussed more if mentioned, and there was no data provided to support it either. The statement in line 256 about the doxy regimen not affecting WT BCG needs to be supported by some reference. The BAL data for 20 weeks post IV-vaccination was not provided anywhere.

Overall, the manuscript provides valuable insights into the development of BCG strains with enhanced immunogenicity, with minor considerations in the preprint highlighted above.

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