Assessing vaccine-mediated protection in an ultra-low dose Mycobacterium tuberculosis murine model

  1. C.R. Plumlee
  2. H.W. Barrett
  3. D.E. Shao
  4. K.A. Lien
  5. L.M. Cross
  6. S.B. Cohen
  7. P.T Edlefsen
  8. K.B. Urdahl

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

Despite widespread immunization with Bacille-Calmette-Guerin (BCG), the only currently licensed tuberculosis (TB) vaccine, TB remains a leading cause of mortality globally. There are many TB vaccine candidates in the developmental pipeline, but the lack of a robust animal model to assess vaccine efficacy has hindered our ability to prioritize candidates for human clinical trials. Here we use a murine ultra-low dose (ULD) Mycobacterium tuberculosis (Mtb) challenge model to assess protection conferred by BCG vaccination. We show that BCG confers a reduction in lung bacterial burdens that is more durable than that observed after conventional dose challenge, curbs Mtb dissemination to the contralateral lung, and, in a small percentage of mice, prevents detectable infection. These findings are consistent with the ability of human BCG vaccination to mediate protection, particularly against disseminated disease, in specific human populations and clinical settings. Overall, our findings demonstrate that the ultra-low dose Mtb infection model can measure distinct parameters of immune protection that cannot be assessed in conventional dose murine infection models and could provide an improved platform for TB vaccine testing.

Article activity feed

  1. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    This manuscript characterizes the ULD mouse model as a new platform for pre-clinical TB vaccine testing. Using the current tuberculosis (TB) vaccine, BCG, the manuscripts shows that three distinct parameters of protective immunity can be assessed in this model: 1) reduction of bacterial burden (which is shown to be more durable in this model than in the conventional model); 2) prevention of dissemination to the contralateral lung; and 3) prevention of detectable infection. The last parameter of protection is notable because vaccines have not been previously shown to be capable of preventing Mycobacterium tuberculosis (Mtb) infection in the mouse model, and in fact, it has been widely believed that mice lack the immune effector mechanisms necessary to prevent detectable infection. We show here that this is not true. When mice are challenged with a physiologic infectious dose of Mtb, vaccine-induced immunity can indeed prevent detectable infection. Thus, we believe this physiologic dose challenge model, provides potential for an improved platform for preclinical vaccine testing, as it allows for measurement of protective parameters that could not previously be assessed and may provide a window to assess meaningful differences between vaccine candidates. We were happy that both reviewers recognized the significance of this work, noting that the study “offers a new avenue for evaluation of TB vaccines, especially vaccines to prevent establishment of long-term infection” and “is clinically useful to test new TB vaccine candidates by improving the conventional TB vaccine model.”

    We thank the reviewers for their time and excellent comments. We have addressed the Reviewer’s comments as outlined below. Most could be fully addressed by minor modifications to the manuscript’s texts or figures. Reviewer 2 requested additional studies to further assess the model’s durability at timepoints later than day 125 post-infection. In response to this comment, we have modified the manuscript to soften our conclusions about the model’s durability. However, we do not believe that performing additional experiments (which would take up to a year to perform) to further examine BCG’s durability in this model is necessary to support the manuscript’s conclusions. Changes made to the manuscript in response to the reviewers’ comments are underlined.

    This report provides evidence that the ULD infection model of M. tuberculosis provides the capacity to detect 3 types of protection by BCG: 1) durable reductions in Mtb burden, 2) inhibition of Mtb dissemination, 3) prevention of detectable bacteria. In general, this is well-reasoned, well-written, transparently presented and easy to follow. A few points.

    Major issues:

    In Figure 2A, 2B, 3A - the authors have a highly skewed distribution with a bimodal distribution between detected bacterial counts and zero bacterial counts. This type of distribution does not lend itself to a t-test. What is the mean of two exclusive categories? For these graphs, the authors should consider plotting the median and interquartile range, then applying a non-parametric test. I suspect the p-values will be as significant, if not more, and there will be some comparisons where the median of the BCG group will be 0 CFU.

    We completely agree that a t-test would not be appropriate for data with bimodal distribution, such as if the mice with detected bacterial counts and those with zero bacterial counts were both included in this analysis. We apologize that we did not sufficiently explain that only mice with detectable bacterial counts were included in our analysis of BCG’s ability to reduce bacterial burdens; those with zero bacterial counts were excluded. We recognize that doing this may underestimate the ability of a vaccine to reduce bacterial burdens as a handful of mice that might have had detectable bacterial burdens in the absence of vaccination are not included in the analysis. However, including mice all mice with undetectable bacterial burdens would be confounded by the fact that some mice in the ULD model are never infected at all, at least measurably, even in the unvaccinated group. We decided that the best way to disentangle these issues would be to analyze reduction of bacterial burdens and proportion of mice with zero detectable CFUs separately. Thus, for the former, only those with detectable CFUs are considered, and separately, we compare the proportion of mice with mice with undetectable bacterial burdens in the vaccinated mice compared to the unvaccinated controls (Figure 5). For these reasons, we believe the t-test is an appropriate test for this analysis. However, in response to this comment we have made changes to the text, figure legend, and Methods, to clearly state how and why the analysis was done this way. We thank the reviewer for these comments, as we believe the original manuscript was not sufficiently clear in this respect, and it is very important to convey how the analysis is being performed.

    In 3B, instead of presenting a model of the data, can the authors present the raw data across all experiments as datapoints with violin plots, or some other form of data visualization? They could present the fixed effect model as a supplementary figure. The fixed effect model works better for plotting proportions in 4A.

    We thank the reviewer for this comment. In Figure 3B of the revised manuscript, the raw data across all experiments is now shown as datapoints with violin plots.

    Minor points:

    Abstract, line 35. The authors state that BCG does A, B, C in a small percentage of mice. It is not clear whether this means all of A, ,B and C happen in a small percentage. Or rather whether the small percentage refers to C alone. Perhaps this can be rewritten for clarity.

    We thank the reviewer for this comment. The small percentage was meant to refer to C alone, but we agree this was not clear as it was written. In the revised manuscript, this sentence in the abstract is written as follows, “We show that BCG confers a reduction in lung bacterial burdens that is more durable than that observed after conventional dose challenge, curbs Mtb dissemination to the contralateral lung, and, in a small percentage of mice, prevents detectable infection.”

    Line 123. 12/20 vs 10/20. Hard to say it appeared to prevent based on these numbers. Perhaps may prevent?

    In the revised manuscript we have changed “appeared to prevent” to “may prevent”, as suggested.

    Line 128. The authors use the word colonized here but don't use this term elsewhere. What is the difference between colonized and infected, and why is colonized used only here?

    In the revised manuscript we have changed “colonized” to “infected”, as suggested.

    The power calculations could be a supplementary table if space is tight.

    We are amenable to moving the power calculations to a supplementary table if this is the preference of the editor.

    Reviewer #2

    Manuscript "Assessing vaccine-mediated protection in an ULD mycobacterium tuberculosis murine model" is an interesting, well-documented and comprehensive study to develop new TB murine model used to assess new TB vaccine candidates. Although TB vaccine are urgently needed, many TB vaccine candidates remain in the development pipeline mainly because the conventional vaccine evaluation strategy is hindered by the lack of reliable animal models that mimic human TB pathogenic cycle. This manuscript used the ULD mouse infection model to resembles human Mtb infection to test the ability of the ULD model as a TB vaccine testing strategy by assessing the BCG vaccination in I. durability in lung bacterial burden, II. the capacity to protect Mtb dissemination to other organs, and III. levels to protect Mtb infection. Overall, this study is quite extensive and potential interest as the result can be readily used for clinical settings.

    Major This study started based upon one of the biggest problems of conventional TB infection model, in which the protection efficacy can be misinterpreted as CFU burden dissipates at later time points due to relatively high burden of initial infection load. To propose that vaccine efficacy test outcomes could be better in ULD murine model compared to that of conventional TB infection model as initial infection burden in ULD model is pathogenetically similar to human infection case. This reviewer is concerned about the authors' interpretation of the results as authors monitored all experimental outcomes at maximum day 125 when lung CFU was ~ 50 fold lower than conventional TB model. Because authors didn't monitor longer period of time, it is not clear if the ULD murine model is optimal to prevent lung CFU dissipation or dissemination to other lung lobes or organs. Authors need to provide additional evidence if the ULD model results are still positive to support authors' hypothesis or the BCG vaccine efficacy in the ULD model was attributed simply to yet lower bacterial burden.

    We thank the reviewer for these comments. While we agree that it would be interesting to see if the protective effects of BCG immunization were durable even beyond 125 days post-infection, we don’t believe that defining the durability further is necessary to complete the study or to support our conclusions. In many ways, we believe we have been quite comprehensive and rigorous in this study, examining over 1,000 mice at timepoints ranging from 14 to 125 days post-infection. We believe we have conclusively shown that BCG’s reduction of lung bacterial burdens is more durable in the ULD model than with a conventional dose challenge (50-100 CFU); while the difference is maintained out to days 90-125 in the former, it wanes in the latter. Similarly, BCG’s ability to prevent dissemination to the contralateral lung, a parameter that cannot be assessed in the conventional dose model, is also durable to days 90-125. Finally, because we used a large number of mice, we showed for the first time that BCG can prevent detectable infection in mice challenged with physiologic Mtb dose (pIn response to the reviewer’s comments, we have softened our statements regarding showing that BCG confers durable reductions in lung bacterial burdens in the ULD model. Now, throughout the abstract and manuscript, we say that we show that BCG confers a reduction in lung bacterial burdens that is more durable than observed with conventional dose challenge.

    Minor Fig. 1 - 10 out of 20 mouse in BCG vaccinated condition didn't show bacterial burden in the lung at any time. It is not clear that this even is attributed to the failed infection or BCG vaccination mediated protection.

    We agree. In this same experiment, 7 out of 20 of the unvaccinated control mice also didn’t show bacterial burden in the lung. One of the features of the ULD model is that we use such a low dose that we intentionally leave some of the mice uninfected, even in the unvaccinated controls. We believe that it is necessary to do this to achieve many of the advantages of the model (e.g., assess dissemination to the contralateral lung and prevention of detectable infection), however, an inherent challenge of the model is that in a single experiment you cannot discern whether an individual mouse with no detectable lung bacteria had infection prevented or whether it would never have been infected in the first place. In the manuscript, we do not claim the difference observed in Figure 1 (7/10 vs. 10/20 with zero CFU) is meaningful. We state in lines 123-125 that “we also observed that 7/20 of the unimmunized mice and 10/20 of the BCG-immunized mice had no detectable infection in either lung (Figure 2A), a difference that was not statistically significant in this single experiment (p=0.53).” We go on to show (Figure 5), that if results from several experiments are pooled, the difference becomes highly significant (p

    As shown in Fig. 1 and 2A, at 42 day post infection, conventional TB infection model reached 5 X 106 CFU in an unimmunized condition and 5 X 105 CFU in a BCG vaccinated condition. In this ULD model, the CFU was 1 X 105 CFU in an unimmunized condition and 1 X 104 CFU in an BCG vaccinated model even at 63 day post infection. If we directly compare the CFU between ULD and conventional TB infection model, the difference was ~ 50 folds. Authors may need to show the bacterial CFU burden is still plateaued and stable bacterial dissemination even after a longer period of infection.

    We have shown that differences in lung bacterial burdens and bacterial dissemination are durable as long as we’ve looked, which is to days 90-125. As discussed above, we believe this is sufficient to support our conclusions and the goals of the study.

    Fig. 2 - The conventional TB model may be included as a negative control.

    We show results of BCG efficacy in the conventional TB model in Figure 1. Because conventional dose and ULD infections are different doses, they cannot be in the same infection chamber at the same time and therefore they need to be shown as separate experiments. Nevertheless, the results shown in Figure 1 are highly reproducible, as shown by us and by several other groups (as referenced).

    Fig. 5A - Why total challenged mouse number gets increased ?

    We presume the reviewer is asking why there are more mice challenged at later timepoints than at early timepoints. Our early experiments suggested that there might be relatively more vaccinated mice with undetectable infection at late timepoints than at earlier ones. As a result, we assessed more experiments at late timepoints than at earlier ones.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Manuscript "Assessing vaccine-mediated protection in an ULD mycobacterium tuberculosis murine model" is an interesting, well-documented and comprehensive study to develop new TB murine model used to assess new TB vaccine candidates.

    Although TB vaccine are urgently needed, many TB vaccine candidates remain in the development pipeline mainly because the conventional vaccine evaluation strategy is hindered by the lack of reliable animal models that mimic human TB pathogenic cycle. This manuscript used the ULD mouse infection model to resembles human Mtb infection to test the ability of the ULD model as a TB vaccine testing strategy by assessing the BCG vaccination in I. durability in lung bacterial burden, II. the capacity to protect Mtb dissemination to other organs, and III. levels to protect Mtb infection. Overall, this study is quite extensive and potential interest as the result can be readily used for clinical settings.

    Major

    This study started based upon one of the biggest problems of conventional TB infection model, in which the protection efficacy can be misinterpreted as CFU burden dissipates at later time points due to relatively high burden of initial infection load. To propose that vaccine efficacy test outcomes could be better in ULD murine model compared to that of conventional TB infection model as initial infection burden in ULD model is pathogenetically similar to human infection case. This reviewer is concerned about the authors' interpretation of the results as authors monitored all experimental outcomes at maximum day 125 when lung CFU was ~ 50 fold lower than conventional TB model. Because authors didn't monitor longer period of time, it is not clear if the ULD murine model is optimal to prevent lung CFU dissipation or dissemination to other lung lobes or organs. Authors need to provide additional evidence if the ULD model results are still positive to support authors' hypothesis or the BCG vaccine efficacy in the ULD model was attributed simply to yet lower bacterial burden.

    Minor

    Fig. 1 - 10 out of 20 mouse in BCG vaccinated condition didn't show bacterial burden in the lung at any time. It is not clear that this even is attributed to the failed infection or BCG vaccination mediated protection. As shown in Fig. 1 and 2A, at 42 day post infection, conventional TB infection model reached 5 X 106 CFU in an unimmunized condition and 5 X 105 CFU in a BCG vaccinated condition. In this ULD model, the CFU was 1 X 105 CFU in an unimmunized condition and 1 X 104 CFU in an BCG vaccinated model even at 63 day post infection. If we directly compare the CFU between ULD and conventional TB infection model, the difference was ~ 50 folds. Authors may need to show the bacterial CFU burden is still plateaued and stable bacterial dissemination even after a longer period of infection.

    Fig. 2 - The conventional TB model may be included as a negative control.

    Fig. 5A - Why total challenged mouse number gets increased ?

    Significance

    This study is clinically useful to test new TB vaccine candidates by improving the conventional TB vaccine model.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    This report provides evidence that the ULD infection model of M. tuberculosis provides the capacity to detect 3 types of protection by BCG: 1) durable reductions in Mtb burden, 2) inhibition of Mtb dissemination, 3) prevention of detectable bacteria. In general, this is well-reasoned, well-written, transparently presented and easy to follow. A few points.

    Major issues:

    In Figure 2A, 2B, 3A - the authors have a highly skewed distribution with a bimodal distribution between detected bacterial counts and zero bacterial counts. This type of distribution does not lend itself to a t-test. What is the mean of two exclusive categories? For these graphs, the authors should consider plotting the median and interquartile range, then applying a non-parametric test. I suspect the p-values will be as significant, if not more, and there will be some comparisons where the median of the BCG group will be 0 CFU.

    In 3B, instead of presenting a model of the data, can the authors present the raw data across all experiments as datapoints with violin plots, or some other form of data visualization? They could present the fixed effect model as a supplementary figure. The fixed effect model works better for plotting proportions in 4A.

    Minor points:

    Abstract, line 35. The authors state that BCG does A, B, C in a small percentage of mice. It is not clear whether this means all of A, ,B and C happen in a small percentage. Or rather whether the small percentage refers to C alone. Perhaps this can be rewritten for clarity.

    Line 123. 12/20 vs 10/20. Hard to say it appeared to prevent based on these numbers. Perhaps may prevent?

    Line 128. The authors use the word colonized here but don't use this term elsewhere. What is the difference between colonized and infected, and why is colonized used only here?

    The power calculations could be a supplementary table if space is tight.

    Significance

    This is a strong paper and the data are compelling.
    The significance is that this offers a new avenue for evaluation of TB vaccines, especially vaccines to prevent establishment of long-term infection.

  4. Version published to 10.1101/2023.03.22.533820v2 on bioRxiv
  5. Version published to 10.1101/2023.03.22.533820v1 on bioRxiv