Review 3: "A "suicide" BCG Strain provides Enhanced Immunogenicity and Robust Protection against Mycobacterium Tuberculosis in Macaques"

RR:C19 Evidence Scale rating by reviewer:

• Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

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Review:

BCG protects against extrapulmonary TB in children. The quest for clinically and biologically surrogate markers of immune protection in TB is still ongoing – and was applied to a number of new TB vaccine candidates. These may represent booster vaccines – on top of BCG – or novel TB – vaccines, some of them genetically altered BCG – strains. For practical and also regulatory reasons, TB vaccines have been delivered intradermally. This is of conceptually challenging since the entry of MTB is (in most cases) happens via the airway system. Although MTB infection manifests in the lung – it is a systemic infection: which resulted in 2 key reports some 4 years ago showing that i.v. administration of viable BCG or endo-tracheal application appears to provide superior protection against MTB. If we ignore the practical challenge of these applications for mass – vaccinations, the Achilles heel was the increased risk for BCG-itis in immune-compromised individuals. If a vaccine delivers indeed superior results as compared to the standard i.d. BCG vaccination, efficient ways for alternate delivery routes applicable for vaccination of larger populations will be found. The real challenge is the risk of unwanted side-effects, also reflected in up to 8% severe risks of patients with bladder cancer receiving intra-vesical BCG application. The current report shows that the new BCG candidates are indeed safe and effective. This is elegantly demonstrated using a murine model as well as a non-human primate (NHP) model with subsequent virulent MTB challenge. The solution to the challenge to make viable BCG safer, while maintaining its efficacy (upon i.v. or endo-tracheal application) was the construction of genetically engineered BCG strains where phage lysing operons are regulated by the antibiotic tetracycline. Using the elegant TetON system, the authors showed that i.v. application of such genetically engineered BCG strains elicited a strong CD4 and CD8 + T-cell responses in the lung - associated with immune protected against MTB challenge. From a mechanistic viewpoint, the typical pro-inflammatory Th1 cytokines were produced in T-cells harvested from pulmonary tissue – being aware that the first passage after i.v. injection are the capillaries of the lung - as well as increased frequencies of CD153positive T-cells: CD153 serves as the ligand for the TNF-receptor CD30 and has been associated with immune protection in TB. Testing of immune reactivity of immune responses directed against MTB (H37Rv) lysates showed no major differences between different vaccine strains. Since the “gemisch” of a lysate is rather undefined, a more molecularly defined matrix of synthetic antigens would have been helpful to detect different immunological immune response patterns identified by the new vaccine candidates as compared to controls. The search for “protective targets” is not over reflected reflected in the comment from Ogongo and Ernst Finding antigens for TB vaccines. The good, the bad and the useless (Ref: https://doi.org/10.1038/s41591-022-02123-4). The model described here provides previous immunological material to identify such relevant immune targets – further studies will hopefully follow. Since one of the key mechanisms of the engineered vaccines is the release of immunogenic products, vaccine-component may also induce antibodies. Although the role in immune protection of serum and tissue - associated antibodies, including mucosal IgA, is still debated, the readers may be curious to learn about the different nature of the quality and quantity of molecular target specific antibodies: there may be differences between the “standard” and the new vaccine candidates which were not picked up by the cell-based assay. Antibody profiling can easily be done and the available material may hold also biologically relevant information(s). It is also clinically interesting that small granulomas were found in 2 animals in different vaccine groups – yet no viable bacteria could be detected. A more recent report analyzing the spatial anatomy of (sarcoidosis associated) granulomas using single cell analysis ( Krausgruber et al., 2023, Immunity 56, 289–306 February 14, 2023 ª 2023 https://doi.org/10.1016/j.immuni.2023.01.014) ) showed that these structures resemble lymphoid structures driven by IL-17. It would have been mechanistically interesting whether this was also the case in the limited number of granulomas described here. In summary, this work provides a clinically relevant and elegant solution to administer a smart genetically engineered “enhanced” live BCG which provides increased efficacy associated with i.v. application while ensuring safety. This paves the way to more mechanistic studies to gauge for markers of immune protection and calls for the design of smart, very well controlled safety and feasibility phase I clinical trials.