Exposure to live saprophytic Leptospira before challenge with a pathogenic serovar prevents severe leptospirosis and promotes kidney homeostasis

Reviewer #1 (Public Review):

As a reviewer for this manuscript, I recognize its significant contribution to understanding the immune response to saprophytic Leptospira exposure and its implications for leptospirosis prevention strategies. The study is well-conceived, addressing an innovative hypothesis with potentially high impact. However, to fully realize its contribution to the field, the manuscript would benefit greatly from a more detailed elucidation of immune mechanisms at play, including specific cytokine profiles, antigen specificity of the antibody responses, and long-term immunity. Additionally, expanding on the methodological details, such as immunophenotyping panels, qPCR normalization methods, and the rationale behind animal model choice, would enhance the manuscript's clarity and reproducibility. Implementing functional assays to characterize effector T-cell responses and possibly investigating the microbiota's role could offer novel insights into the protective immunity mechanisms. These revisions would not only bolster the current findings but also provide a more comprehensive understanding of the potential for saprophytic Leptospira exposure in leptospirosis vaccine development. Given these considerations, I believe that after substantial revisions, this manuscript could represent a valuable addition to the literature and potentially inform future research and vaccine strategy development in the field of infectious diseases.

Reviewer #2 (Public Review):

Summary:

The authors try to achieve a method of protection against pathogenic strains using saprophytic species. It is undeniable that the saprophytic species, despite not causing the disease, activates an immune response. However, based on these results, using the saprophytic species does not significantly impact the animal's infection by a virulent species.

Strengths:

Exposure to the saprophytic strain before the virulent strain reduces animal weight loss, reduces tissue kidney damage, and increases cellular response in mice.

Weaknesses:

Even after the challenge with the saprophyte strain, kidney colonization and the release of bacteria through urine continue. Moreover, the authors need to determine the impact on survival if the experiment ends on the 15th.

Reviewer #3 (Public Review):

Summary:

Kundu et al. investigated the effects of pre-exposure to a non-pathogenic Leptospira strain in the prevention of severe disease following subsequent infection by a pathogenic strain. They utilized a single or double exposure method to the non-pathogen prior to challenge with a pathogenic strain. They found that prior exposure to a non-pathogen prevented many of the disease manifestations of the pathogen. Bacteria, however, were able to disseminate, colonize the kidneys, and be shed in the urine. This is an important foundational work to describe a novel method of vaccination against leptospirosis. Numerous studies have attempted to use recombinant proteins to vaccinate against leptospirosis, with limited success. The authors provide a new approach that takes advantage of the homology between a non-pathogen and a pathogen to provide heterologous protection. This will provide a new direction in which we can approach creating vaccines against this re-emerging disease.

Strengths:

The major strength of this paper is that it is one of the first studies utilizing a live non-pathogenic strain of Leptospira to immunize against severe disease associated with leptospirosis. They utilize two independent experiments (a single and double vaccination) to define this strategy. This represents a very interesting and novel approach to vaccine development. This is of clear importance to the field.

The authors use a variety of experiments to show the protection imparted by pre-exposure to the non-pathogen. They look at disease manifestations such as death and weight loss. They define the ability of Leptospira to disseminate and colonize the kidney. They show the effects infection has on kidney architecture and a marker of fibrosis. They also begin to define the immune response in both of these exposure methods. This provides evidence of the numerous advantages this vaccination strategy may have. Thus, this study provides an important foundation for future studies utilizing this method to protect against leptospirosis.

Weaknesses:

Although they provide some evidence of the utility of pretreatment with a non-pathogen, there are some areas in which the paper needs to be clarified and expanded.

The authors draw their conclusions based on the data presented. However, they state the graphs only represent one of two independent experiments. Each experiment utilized 3-4 mice per group. In order to be confident in the conclusions, a power analysis needs to be done to show that there is sufficient power with 3-4 mice per group. In addition, it would be important to show both experiments in one graph which would inherently increase the power by doubling the group size, while also providing evidence that this is a reproducible phenotype between experiments. Overall, this weakens the strength of the conclusions drawn and would require additional statistical analysis or additional replicates to provide confidence in these conclusions.

A direct comparison between single and double exposure to the non-pathogen is not able to be determined. The ages of mice infected were different between the single (8 weeks) and double (10 weeks) exposure methods, thus the phenotypes associated with LIC infection are different at these two ages. The authors state that this is expected, but do not provide a reasoning for this drastic difference in phenotypes. It is therefore difficult to compare the two exposure methods, and thus determine if one approach provides advantages over the other. An experiment directly comparing the two exposure methods while infecting mice at the same age would be of great relevance to and strengthen this work.

Author response:

Public Reviews:

Reviewer #1 (Public Review):

As a reviewer for this manuscript, I recognize its significant contribution to understanding the immune response to saprophytic Leptospira exposure and its implications for leptospirosis prevention strategies. The study is well-conceived, addressing an innovative hypothesis with potentially high impact. However, to fully realize its contribution to the field, the manuscript would benefit greatly from a more detailed elucidation of immune mechanisms at play, including specific cytokine profiles, antigen specificity of the antibody responses, and long-term immunity. Additionally, expanding on the methodological details, such as immunophenotyping panels, qPCR normalization methods, and the rationale behind animal model choice, would enhance the manuscript's clarity and reproducibility. Implementing functional assays to characterize effector T-cell responses and possibly investigating the microbiota's role could offer novel insights into the protective immunity mechanisms. These revisions would not only bolster the current findings but also provide a more comprehensive understanding of the potential for saprophytic Leptospira exposure in leptospirosis vaccine development. Given these considerations, I believe that after substantial revisions, this manuscript could represent a valuable addition to the literature and potentially inform future research and vaccine strategy development in the field of infectious diseases.

We have been interested in understanding how both pathogenic and non-pathogenic Leptospira species affect each other on a mammalian reservoir host. With the current study we continue to elucidate the immune mechanisms engaged by pathogenic Leptospira interrogans versus non-pathogenic L. biflexa, as a follow up to our previous work (Shetty et al, 2021 PMID: 34249775, and Kundu et al 2022 PMID 35392072). We found that both species engaged partially overlapping myeloid immune cells and inflammatory signatures of infection. For example, some chemokines were increased, and macrophage and dendritic cells were engaged at 24h post inoculation with both species of Leptospira (PMID: 34249775). Thus, we questioned whether this robust innate immune response raised to eliminate an immunogenic but rather non-pathogenic bacterium, could also help restrain L. interrogans pathogenesis. In this study we show that L. biflexa pre-exposure to L. interrogans challenge mediates improved kidney homeostasis, mitigates leptospirosis severity and leads to increased shedding of L. interrogans in urine. This suggests an interspecies symbiotic commensalistic process that facilitates survival of the pathogenic species. These findings have high impact on the lives of millions of people in areas endemic for leptospirosis that are naturally exposed to non-pathogenic Leptospira species.

We will expand on the methodological details and will update the introduction and discussion to include answers to questions raised by the three reviewers to further clarify the importance and impact of our study.

Reviewer #2 (Public Review):

Summary:

The authors try to achieve a method of protection against pathogenic strains using saprophytic species. It is undeniable that the saprophytic species, despite not causing the disease, activates an immune response. However, based on these results, using the saprophytic species does not significantly impact the animal's infection by a virulent species.

We separate concepts of exposure to a non-virulent bacterium that establishes a brief infection with engagement of an immune response (L. biflexa), from infection established by a virulent species of Leptospira that leads to pathogenesis (L. interrogans). While trying to understand how both pathogenic and non-pathogenic Leptospira species affect each other on a mammalian reservoir host, we previously found that L. biflexa induces immune responses that should affect immunity of populations naturally exposed to this spirochete. Thus, we designed this study to answer that question.

Strengths:

Exposure to the saprophytic strain before the virulent strain reduces animal weight loss, reduces tissue kidney damage, and increases cellular response in mice.

Weaknesses:

Even after the challenge with the saprophyte strain, kidney colonization and the release of bacteria through urine continue. Moreover, the authors need to determine the impact on survival if the experiment ends on the 15th.

Another novel and unexpected aspect of our findings in the single exposure experiment was that L. biflexa pre-exposure mediated a homeostatic environment in the kidney (lower ColA1, healthier renal physiology) that restrained pathogenesis of L. interrogans after challenge, which resulted in better health outcomes and increased shedding of L. interrogans in urine; in contrast, if the kidney is compromised (high ColA1) by L. interrogans (without L. biflexa pre-exposure) there was lower shedding L. interrogans in urine. Interestingly, this suggests an interspecies symbiotic commensalistic process that facilitates survival of the pathogenic species. Thus, these data suggest that higher shedding of L. interrogans in urine may not be a hallmark of increased disease, but rather it could be the opposite.

We will include these concepts in the updated discussion.

We don’t think that extending this experiment to d21 or d28 would add relevant data to our findings. We provide survival curves for both experiments up to d15 post infection.

Reviewer #3 (Public Review):

Summary:

Kundu et al. investigated the effects of pre-exposure to a non-pathogenic Leptospira strain in the prevention of severe disease following subsequent infection by a pathogenic strain. They utilized a single or double exposure method to the non-pathogen prior to challenge with a pathogenic strain. They found that prior exposure to a non-pathogen prevented many of the disease manifestations of the pathogen. Bacteria, however, were able to disseminate, colonize the kidneys, and be shed in the urine. This is an important foundational work to describe a novel method of vaccination against leptospirosis. Numerous studies have attempted to use recombinant proteins to vaccinate against leptospirosis, with limited success. The authors provide a new approach that takes advantage of the homology between a non-pathogen and a pathogen to provide heterologous protection. This will provide a new direction in which we can approach creating vaccines against this re-emerging disease.

Strengths:

The major strength of this paper is that it is one of the first studies utilizing a live non-pathogenic strain of Leptospira to immunize against severe disease associated with leptospirosis. They utilize two independent experiments (a single and double vaccination) to define this strategy. This represents a very interesting and novel approach to vaccine development. This is of clear importance to the field.

The authors use a variety of experiments to show the protection imparted by pre-exposure to the non-pathogen. They look at disease manifestations such as death and weight loss. They define the ability of Leptospira to disseminate and colonize the kidney. They show the effects infection has on kidney architecture and a marker of fibrosis. They also begin to define the immune response in both of these exposure methods. This provides evidence of the numerous advantages this vaccination strategy may have. Thus, this study provides an important foundation for future studies utilizing this method to protect against leptospirosis.

Weaknesses:

Although they provide some evidence of the utility of pretreatment with a non-pathogen, there are some areas in which the paper needs to be clarified and expanded.

The authors draw their conclusions based on the data presented. However, they state the graphs only represent one of two independent experiments. Each experiment utilized 3-4 mice per group. In order to be confident in the conclusions, a power analysis needs to be done to show that there is sufficient power with 3-4 mice per group. In addition, it would be important to show both experiments in one graph which would inherently increase the power by doubling the group size, while also providing evidence that this is a reproducible phenotype between experiments. Overall, this weakens the strength of the conclusions drawn and would require additional statistical analysis or additional replicates to provide confidence in these conclusions.

We will take these suggestions into consideration and will address as many of these issues as possible in the revised manuscript.

A direct comparison between single and double exposure to the non-pathogen is not able to be determined. The ages of mice infected were different between the single (8 weeks) and double (10 weeks) exposure methods, thus the phenotypes associated with LIC infection are different at these two ages. The authors state that this is expected, but do not provide a reasoning for this drastic difference in phenotypes. It is therefore difficult to compare the two exposure methods, and thus determine if one approach provides advantages over the other. An experiment directly comparing the two exposure methods while infecting mice at the same age would be of great relevance to and strengthen this work.

Both experiments need to be analyzed as separate but complementary as they provide different hind sights into L. interrogans pathogenesis and potential solutions to the problem. Optimal measurements of disease progression (weight loss, survival curves) require infection of mice at 8 weeks. Based on this, a new L. biflexa double exposure experiment would have to start when mice are 4 weeks old which is just after weaning, and before the mouse immune system is fully developed.