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

Previous studies demonstrated that Leptospira biflexa, a saprophytic species, triggers innate immune responses in the host during early infection. This raised the question of whether these responses could suppress a subsequent challenge with pathogenic Leptospira. We inoculated C3H/HeJ mice with a single or a double dose of L. biflexa before challenge with a pathogenic serovar, L. interrogans serovar Copenhageni FioCruz (LIC). Pre-challenge exposure to L. biflexa did not prevent LIC dissemination and colonization of the kidney. However, it rescued weight loss and mouse survival thereby mitigating disease severity. Unexpectedly, there was correlation between rescue of overall health (weight gain, higher survival, lower kidney fibrosis marker ColA1) and higher shedding of LIC in urine. This stood in contrast to the L. biflexa unexposed LIC challenged control. Immune responses were dominated by increased frequency of effector T helper (CD4+) cells in spleen, as well as significant increases in serologic IgG2a. Our findings suggest that exposure to live saprophytic Leptospira primes the host to develop Th1 biased immune responses that prevent severe disease induced by a subsequent challenge with a pathogenic species. Thus, mice exposed to live saprophytic Leptospira before facing a pathogenic serovar may withstand infection with far better outcomes. Furthermore, a status of homeostasis may have been reached after kidney colonization that helps LIC complete its enzootic cycle.


Significance
Previous evidence of host innate immunity induced by live saprophytic Leptospira in mice led us to posit that these responses might mitigate leptospirosis severity upon a subsequent challenge with a pathogenic serovar.In this study, we validated our hypothesis.This is important for development of novel strategies to control leptospirosis and for understanding the epidemiologic risk factors of this and other infectious diseases transmitted by direct contact between pathogen and host.Unexpectedly, these studies also show that there is a correlation between kidney health after L. interrogans infection (less fibrosis marker ColA1) and higher shedding of this spirochete in urine.This suggests that a status of homeostasis may be reached after kidney colonization by L. interrogans that helps the spirochete fulfill its enzootic cycle. .

Introduction
Leptospirosis, a neglected re-emerging enzootic spirochetal disease, affects millions of people worldwide causing an overall mortality rate of 65000 per year (1).In addition, it causes serious health problems in animals of agricultural interest which leads to substantial economic losses mostly in tropical and subtropical countries.Assessing the true severity of leptospirosis can be incredibly challenging, especially when early diagnosis is difficult due to nonspecific symptoms overlapping with other illnesses (2).Recent outbreaks of both human and canine leptospirosis in New York and California in 2020 to 2022 (3,4) underscores the need for development of effective strategies to control this disease.Although serovar-specific vaccines are available for animals and at least one is available for humans, no broadly effective vaccine is available for either (5).The absence of an effective cross-protective vaccine candidate increases the risk of disease re-emergence on a global scale.Efforts to use leptospiral surface antigens in various vaccine formulations have shown limited success in conferring protection against leptospiral dissemination and shedding, as well as severe disease.Leptospira' immune evasion strategies contribute to the complexities of finding good vaccine candidates.
The genus Leptospira is broadly categorized into 2 major clades P and S (Pathogens and Saprophytes) and further categorized into 4 subclades, P1, P2, S1 and S2 based on their virulence properties, growth conditions and genetic make-up (6,7) .CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made The copyright holder for this preprint (which this version posted July 1, 2024.; https://doi.org/10.1101/2024.03.01.582981 doi: bioRxiv preprint phenotypically not well-characterized) (8).Leptospira survives in moist conditions and are freeliving organisms naturally found in soil and water bodies (9,10).The spread of infection occurs through contaminated water contact with breached skin or mucosal surfaces (11).Saprophytic strains of Leptospira, such as L. biflexa (S1), are unable to establish disease due to the lack of certain virulence factors (7) and have been found in natural environments around the world alongside pathogenic serovars (6,11,12).Moreover, L. biflexa exhibits certain niche-specific adaptations that allow them to persist in both environmental and host setting (13,14).
Our previous studies (15,16) demonstrated that L. biflexa triggers a robust innate immune response in mice during the acute phase of infection.This raised the question of whether saprophytic Leptospira induced immune responses could confer any degree of resistance or immune memory that could suppress a subsequent challenge with a pathogenic serovar of Leptospira.Answering that question was the main goal of the current study.) after counting Leptospira under a dark-field microscope (Zeiss USA, Hawthorne, NY) using a Petroff-Hausser chamber.

Infection of mice and study design
We carried out two experiments set apart by a single or double exposure to a saprophytic serovar of Leptospira (L.biflexa) before challenge with a pathogenic serovar (L.interrogans).
Groups of mice were inoculated with 10 8 Leptospira intraperitoneally (IP) both for exposure to L. biflexa and for challenge with L. interrogans.Each experiment was reproduced once.In the single exposure study (Fig 1A), Group 1 (n=3) was the naive control which received PBS (PBS

ELISA
Leptospiral extract for Leptospira biflexa and Leptospira interrogans were prepared as described previously (19).Briefly, Leptospira was cultured in EMJH media and once confluency was observed, cells were centrifuged to obtain a pellet.This pellet was then incubated with BugBuster® solution (1mL) at RT in a shaker incubator (100 rpm) for 20 min and homogenized by vortexing.Stocks were stored at -20°C.This whole-cell extract of Leptospira was then diluted in 1X sodium carbonate coating buffer.Nunc MaxiSorp flat-bottom 96 well plates (eBioscience, San Diego, CA) were coated with extracts prepared from 10 7 -10 8 bacteria per well and incubated at 4℃ overnight.Cells were washed using 1X PBST the following day and blocked for 1 h using 1% BSA solution, followed by another wash with 1X PBST.Serum samples (1:100) were added to the antigen coated wells and incubated at 37°C for 1 h, washed twice with 1X PBST, followed by HRP conjugate secondary anti-mouse-IgG1, IgG2a and IgG3 (1:10000) which was incubated for 30 mins.After washing the plate 3 times with 1X PBST the color was developed using TMB Sureblue followed by Stop solution before the absorbance was measured at OD 450 nm using an ELISA plate reader (Molecular Devices Spetramax).

Flow cytometry
Spleens were chopped into small pieces and macerated to prepare single cell suspensions on the same day of euthanasia to avoid loss of cell viability.RBC lysis was performed using ACK lysis buffer (Gibco).AO/PI dual staining was used to count live/dead cells on a Luna counter (Logos Biosystems, South Korea).10 6 cells were seeded in a 96 well microtiter plate after washing with 1X PBS twice.Blocking was performed with anti-mouse CD16/32 antibody (1:100), followed by 20 minutes incubation on ice.Fluorochrome-conjugated antibodies (Table S1) were used to stain specific cell surface markers after 30 mins incubation in the dark at 4°C.Freshly prepared flow staining buffer was used for washing stained cells.Cells were fixed using 4% Paraformaldehyde for 10-15 mins at room temperature.Beads were stained using specific fluorochrome conjugated antibodies and used for compensation, while FMO prepared with spleen cells simultaneously were used for gating controls.Cells were resuspended in flow staining buffer and the BioRad ZE5 cell analyzer was used for data acquisition.Data analysis was done using Flow Jo software.Gating strategy used for immunophenotyping of spleen cells is provided in supplementary Fig S1.

Statistical analysis
One-way ANOVA with Tukey's multiple comparison test and unpaired t-test with Welch's correction were used to analyze differences between experimental groups.GraphPad prism software was used to plot graphs; a value of p<0.05 is considered significant.

Results
Exposure to saprophytic Leptospira before infection with a pathogenic serovar prevents disease and increases survival of C3H-HeJ mice.We inoculated adult C3H-HeJ male mice with a single dose of L. biflexa two weeks before challenge with L. interrogans (LB  3D).
In addition, we were able to collect kidneys from experimental mice subjected to the double exposure of L. biflexa because they all survived subsequent challenge with L.
interrogans.As such, we did a comparative gross morphological analysis between the 4 groups (Fig S3).We found that kidney from LB Serologic IgG2a responses to L. interrogans were significantly higher in mice pre-exposed to L.
biflexa before challenge with L. interrogans.

Discussion
Understanding host immune responses to Leptospira infection is crucial for advancing our ability to develop new control measures for leptospirosis (19,(21)(22)(23)(24)(25).Given their widespread presence in nature, the likelihood of humans or animals getting exposed to non-pathogenic serovars of Leptospira is likely high (10,11).Our previous studies showed innate immunity engagement during saprophytic L. biflexa infection in mice (15,16).In addition, L. biflexa extracts can be used to detect Leptospira-specific antibody in up to 67% of serum from patients with clinically confirmed leptospirosis (19) which points to a high degree of immunodominant cross-reactive epitopes between L. biflexa and pathogenic Leptospira.Further, the current hypothesis regarding evolution of Leptospira species is that symbiosis of Leptospira with eukaryotes emerged from free-living ancestral species (26); in other words, pathogenic Leptospira may have evolved from an environmental ancestor (6).Thus, we hypothesized that these highly cross-reactive immunodominant epitopes may also induce cross-protective immune responses.The objective of the current study was to assess whether exposure to a live saprophytic serovar of Leptospira provides any heterologous cross-species protection against a subsequent challenge with a pathogenic serovar in a mammalian host (mouse).
In the initial analysis of pathogenesis (Fig  While other researchers have explored vaccination strategies using live attenuated or mutant strains of pathogenic serovars, our approach was to utilize a live saprophytic bacterial strain which is unique in the field (21,23,31,32).We previously showed that oral delivery of a probiotic strain, Lactobacillus plantarum, reduces the severity of leptospirosis by recruiting myeloid cells (21) which suggests that a general phenomenon of trained immunity may be involved.
Current vaccines based in inactivated pathogenic species provide equivalent protection to the one achieved in this study (5,33) with the caveat of being serovar specific (32-34).Although our current study conclusively shows protection from severe leptospirosis after heterologous challenge, it remains to be shown if protection extends to multiple pathogenic serovars of Leptospira.Using a live saprophytic strain of Leptospira as control strategy could pave the way for development of novel broadly effective vaccines against leptospirosis.Such a vaccine could have a substantial economic impact if applied to animals of agricultural interest.
Another interesting aspect of our current study is that it shows that exposure to a live saprophytic strain of Leptospira provides protection against a pathogenic serovar.Thus, in the real-life scenario where individuals or animals may naturally encounter a saprophytic Leptospira Male C3H/HeJ mice (n=6-8/group) were purchased from The Jackson Laboratory (Bar Harbor, ME) and were maintained in a pathogen-free environment in the Laboratory Animal Care Unit of the University of Tennessee Health Science Center (UTHSC).All experiments were performed in compliance with the UTHSC Institutional Animal Care and Use Committee (IACUC), Protocol no.19-0062.

1 LIC 1 ) 1 LIC 1 ) 1 ) 1 ) 1 LIC 1 ) 2 LIC 2 ) 2 LIC 2 ) 2 ) 2 LIC 2 mice 2 ) 2 ) 2 )
at 8 weeks (Fig 1A) and measured a significant rescue of weight loss over a period of 15 days as compared to mice infected at 8 weeks that did not receive L. biflexa (PBS (p<0.0001);unchallenged control mice that received L. biflexa (LB 1 ) or PBS (PBS gained weight throughout the corresponding 15 days (Fig 1B).Survival curves were generated after the mice reached the following endpoint criteria: 20% weight loss or 15 days post challenge with L. interrogans or 15 days post inoculation with L. biflexa/PBS for the controls (Fig 1C).All mice infected with L. reached the 20% weight loss endpoint criteria between d9 and d12 post infection.In contrast, 3/4 (75%) of the mice that received one dose of L. biflexa before challenge with L. interrogans (LB survived and gained significant body weight (Fig 1B)which was similar to the naïve control that received only PBS.Analysis of bacterial dissemination was done by qPCR of the Leptospira 16S gene in genomic DNA purified from blood, kidney tissue and urine.Of note, although 16S rRNA primers can amplify L. biflexa 6h post infection(20), we processed the tissue samples 30 days or 45 days post L. biflexa exposure.We also found that a single exposure to L. biflexa before challenge did not prevent dissemination of pathogenic L. interrogans in blood (Fig S2A) or shedding in urine (Fig 1D), or kidney colonization (Fig 1E).Culture of kidney in EMJH media showed presence of ~ 2500 motile, morphologically intact L. interrogans under dark field microscopy which was confirmed by 16S qPCR (Fig 1F) both on d3 and d5 post culture of kidney collected from LB 1 positive cultures by dark field microscopy or 16S qPCR.In the double exposure study, mice were inoculated with 2 bi-weekly doses of L. biflexa two weeks before challenge with L. interrogans (LB at 10 weeks in comparison with the respective controls (Fig 2A).As expected, mice infected at 10 weeks with LIC that did not receive L. biflexa (PBS lost ~ 11 % of weight on d11 post challenge and did not recover (Fig 2B).In contrast, mice that received a double dose of L. biflexa two weeks before challenge at 10 weeks with L. interrogans (LB 2 LIC lost a maximum of 5% weight on d10 and recovered fully by d15 post infection; unchallenged control mice that received L. biflexa (LB 2 ) or PBS (PBS 2 ) gained weight throughout the 15 days (Fig 2B).Survival curves generated after the mice reached endpoint criteria (Fig 2C) show that all experimental groups survived LIC infection.Analysis of bacterial dissemination showed that a double exposure to L. biflexa before challenge did not prevent dissemination of pathogenic L. interrogans in blood (Fig S2B), or shedding in urine (Fig 2D), or kidney colonization (Fig 2E).Culture of kidney in EMJH media showed presence of 5,000-10,000 motile, morphologically intact L. interrogans on d3 and d5 post culture of kidney collected from PBS in contrast to 1,000 to 2,500 live L. interrogans observed in culture from kidney collected from LB produce positive cultures by dark field microscopy or 16S qPCR.L. biflexa exposure before challenge with L. interrogans mitigates renal histopathological changes.As expected, H&E staining of histological slices of all kidneys from mice challenged with L. interrogans in both single and double exposure experiments (inflammation with increased immune cell infiltration (Fig 3Aand 3C).In contrast, H&E staining of kidney slices from the groups of mice exposed to L. biflexa before L. showed reduced immune cell infiltration.We also measured expression of a marker (ColA1) for kidney fibrosis.In both experiments, kidneys from mice challenged with L. interrogans (had significantly higher expression of ColA1 as compared to the controls; in contrast, kidneys from mice challenged with L. interrogans after exposure to L. biflexa (were not different than the controls(Figs 3B and contrast to kidneys from mice challenged with L. interrogans that were not previously exposed to L. biflexa (

2 .
5A).We measured decreased frequencies in T cells when LIC infected mice were compared to the respective controls but not between PBS 5B).No differences were observed in NK cells between any of the groups (Fig 5C).We measured increased frequencies in helper T cells between all groups; of note, (Fig 5D).We also measured decreased frequencies in cytotoxic T cells between all groups; of note PBS Fig 5E).Furthermore, T cell subset typing (Fig 6) showed that frequency of early effector CD4+ T helper cells (Fig 6B, CD44-CD62L-) and effector T helper cells (Fig 6C, CD44+CD62L-) were significantly increased when compared between the LIC challenged groups and the respective controls (and that frequency of early effector and effector T helper cells were higher in the LB No major changes were measured in memory CD4+ T helper cells (Fig 6D, CD44+CD62L+).In the CD8+ cytotoxic T cell subsets, we measured significant decreases in frequency of naïve T cells between LIC infected groups and the respective controls (Fig 6E, CD62L+CD44-) and this was replicated in the CD8+ cytotoxic memory except that differences with the LIC infected groups were not significant (Fig 6H, CD44+CD62L+).
interrogans dissemination in blood, shedding in urine and kidney colonization of the kidney.Histological inspection of kidney slices(Fig 3)  showed that exposure to a saprophytic Leptospira before challenge supported normal structural morphology and prevented infiltration of immune cells in both single and double exposure experiments; in addition, it significantly reduced a fibrosis marker (ColA1) in the single exposure experiment.In the double exposure experiment, differences in ColA1 fibrosis marker are not significant between the two LIC infected groups because 10-week old C3H-HeJ infected with LIC are more resistant to pathology resultant from infection.Our findings are intriguing as they suggest that while prior live saprophytic exposure did not prevent infection or leptospiral dissemination, it may confer protection against kidney fibrosis.challenge, induced higher antibody titer in the serum, specifically IgG2a antibodies against L. interrogans in both single and double exposure experiments (Fig4).Increased IgG2a response in serum is associated with induction of a Th1 biased immune response.Others have recently found that saprophytic L. biflexa induced Th1 responses, higher T cell proliferation and IFN-g producing CD4+ T cells (28).Persistent IgM and strain specific IgG responses was observed during a homologous leptospiral challenge in C57BL6/J mice(22).In our study, exposure to a saprophyticLeptospira induced antibody responses that may provide heterologous protection against the pathogenic strain of Leptospira.This supports a promising broad-spectrum efficacy.Thus, live vaccines derived from a saprophytic strain of Leptospira could offer broader protection and overcome the limitation of serovar specificity often observed with killed whole-cell vaccines based on pathogenic strains.Differences in antibody titer among the L. interrogans infected group pre-exposed to saprophytic L. biflexa can be attributed to the robust trafficking and differentiation of B and helper T cells (CD4+) measured in spleen (Fig 5 and Fig 6).Presence of effector helper T (CD4+) cells in the spleen indicate a robust cellular immune response as these cells produce cytokines that play a pivotal role in activating other immune cells, including antibody-producing B cells.Moreover, our findings align with another observation which further reinforces the potential immunostimulatory properties of components (polar lipids) derived from saprophytic L. biflexa, indicating that these components could play a crucial role in inducing robust B cell responses (29).Induction of helper T cell responses along with dynamic transition from naïve to early effector and effector without T helper memory reflects an orchestrated immune response upon pathogenic challenge in the saprophytic pre-exposed group that is typical of effective responses to vaccines.Previous studies have highlighted the significance of activated CD4+ T cells during Leptospira infection in providing protective immunity to the host and mitigating the severity of subsequent adaptive immune responses, such as the activation of CD4+ T cells or the production of specific antibodies, provides insights into how innate immune signals drive the adaptive immune response against a pathogenic threat.It may also aid in identifying key signaling molecules or pathways that could be targeted for therapeutic interventions or vaccine design.

Figure 1 :
Figure 1: Weight loss, kidney colonization, shedding in urine and survival to challenge with

Figure 2 :
Figure 2: Weight loss, kidney colonization, shedding in urine and survival to challenge with

Figure 3 :
Figure 3: Kidney histopathology and quantification of renal fibrosis.Representative H & E-

Figure 4 :
Figure 4: Detection of IgG1, IgG2a and IgG3 specific to L. interrogans in serum from

Figure 5 :
Figure 5: Frequency of lymphocytes in spleen of mice subjected to a double exposure of L.

Figure 6 :
Figure 6: Frequency of T cell subsets (CD62L/CD44) in spleen of mice subjected to a double by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made In both single and double L. biflexa exposure experiments we measured anti-L.interroganstotalIgM, total IgG, and IgG subtypes IgG1, IgG2a and IgG3 in serum collected 2 weeks after challenge with L. interrogans (IgG subtypes shown in Figs 4A and4B).In both experiments, total IgM and IgG were significantly increased in PBS-LIC and LB-LIC when compared to the respective controls, but not between PBS-LIC and LB-LIC 1, Fig 2, Fig S2) we observed that prior exposure to one or two doses of saprophytic L. biflexa rescues weight loss in mice challenged ).In both experiments, mice exposed to L. biflexa before challenge with LIC, produced evidence of L.
species, they may develop immune responses that mitigate severe disease outcomes if the host later encounters a pathogenic strain of Leptospira.By exploring the immune dynamics during the co-exposure to different Leptospira serovars, this study could open avenues of research on strategies that leverage natural exposure to saprophytic species to devise safe control measures against leptospirosis.This concept is important for understanding the epidemiological risk factors of leptospirosis and it should be applicable to other infectious diseases caused by direct contact between the pathogen and mucosal membranes or abraded host skin.Importantly, we found that in mice pre-exposed to live saprophytic Leptospira there was a correlation between kidney health after LIC infection (less infiltration of immune cells in kidney and less fibrosis marker ColA1) and higher shedding of live LIC in urine.This suggest that a status of homeostasis was reached after kidney colonization that helps the spirochete Adler B. Vaccines against leptospirosis.Curr Top Microbiol Immunol.2015;387:251-72.