Heterologous DNA prime-protein boost immunization with RecA and FliD offers cross-clade protection against leptospiral infection

The emergence of >300 serovars of Leptospira confounded the use of generalized bacterin, the whole cell lysate, as vaccines to control leptospirosis. Because of substantial genetic and geographic heterogeneity among circulating serovars, one vaccine strain per serovar cannot be efficacious against all the serovars. We have performed heterologous DNA prime-protein boost vaccination challenge studies in hamsters using in vivo expressed, leptospiral recombinase A (RecA) and flagellar hook associated protein (FliD). We prepared the monovalent recombinant protein, plasmid DNA, and DNA prime protein boost adjuvant vaccines. The whole cell bacterin served as a control. Our data show that (i) RecA and FliD have multiple immunogenic B and T-cell epitopes with highly conserved domains among most prevalent pathogenic Leptospira spp., (ii) humoral and cell mediated immune responses were induced remarkably, (iii) provides significant protection against homologous (Autumnalis strain N2) and cross-clade heterologous (Canicola strain PAI-1) challenge infection for the heterologous prime-protein boost (∼91–100%) and, the DNA vaccine (∼75–83%). Recombinant protein vaccine shows only partial protection (∼58–66%), (iv) RecA prime-protein boost vaccine shows sterilizing immunity, with heterologous protection. This RecA/FliD prime-protein boost strategy holds potential for vaccination against animal leptospirosis and for a better control of zoonotic transmission.


Leptospiral bacterins and recombinant proteins. Leptospiral bacterins, whole cell lysate (WCL) were
prepared from Leptospira interrogans serovar Autumnalis strain N2 as previously described 25 . RecA or FliD recombinant proteins were overexpressed and purified using E. coli expression system as described elsewhere 15 . The purified recombinant proteins were dialyzed and the protein concentration determined using a bicinchoninic acid assay (BCA) kit (Sigma, St.Louis, MO). The vaccine formulation was prepared as a proportion of 100 μg of protein in Alhydrogel (Sigma, St. Louis, MO) for first booster on day 0. On day 21, a second booster of 100 μg protein was administered by subcutaneous injections.
Construction of recA and fliD DNA vaccine. For DNA immunization, genomic DNA was extracted from L. interrogans serovar Autumnalis strain N2 using the QIAamp DNA extraction kit (Qiagen, Valencia, USA). The primers were designed for full-length leptospiral recA and fliD according to L. interrogans serovar Autumnalis strain N2 gene sequence (GenBank accession no. KF240786) using Primer 2 (Scientific & Education Software, 1991). The primer used for recA and fliD are specified in Table S1. The PCR amplification was performed in 50 μL containing 10X Taq buffer, 1.5 mM MgCl 2 , 200 μM dNTPs, 1.25 U of Dream Taq DNA polymerase (Thermo Scientific, Rockford, IL) and pair of primers (each 10 ρM) and approximately 50 ηg of template DNA. The temperature profile for amplification was initial denaturation at 95 °C for 5 min, denaturation at 95 °C for 1 min, annealing at 60 °C for 30 sec and extension at 72 °C for 1 min, for 30 cycles, followed by a final extension at 72 °C for 5 mins. Amplified recA and fliD were cloned into multiple cloning site of mammalian expression vector pEGFPN3 (Addgene, Cambridge, MA, USA). The identity and orientation of the cloned inserts were confirmed by DNA sequencing (Macrogen, South Korea). E. coli DH5α competent cells were transformed with the recA-pEGFPN3or fliD-pEGFPN3-construct, and confirmed using REA analysis and DNA sequencing. The constructed plasmids (recA-pEGFPN3 or fliD-pEGFPN3) were purified in large quantity by EndoFree Plasmid Maxi Kit (Qiagen, Valencia, USA). The quality and quantity of the purified plasmids were checked by agarose gel electrophoresis and plasmid DNA quantification using a Biophotometer (Eppendorf, Hamburg, Germany).
Hamsters were vaccinated with DNA in two doses (100 μg/dose) by intramuscular (IM) injection. For prime boost vaccination strategy, first dose of 100 µg DNA, followed by the second protein booster of 100 µg was administered. Forty-two days after the first immunization, hamsters were challenged intraperitoneally with 5x median lethal dose (LD50) of L. interrogans serovar Autumnalis strain N2 (1.37 × 10 7 leptospires) and L. interrogans serovar Canicola strain PAI-1 (1.6 × 10 3 leptospires) as homologous and heterologous infection as described in our earlier studies 26 . Two independent animal cohort experiments were conducted for the evaluation of the vaccination strategy. Animals were monitored daily and those which survived were euthanized on day 70 after challenge. Blood samples were collected from the retro orbital venous plexus on 14 th , 21 st (before booster) and 42 nd days (before challenge), and the sera were stored at −80 °C. The in vivo expression of DNA vaccine constructs of recA and fliD were performed by Western blot analysis 27 with specific rabbit hyper immune sera 15 . Animal experiments described in this study were carried out in strict accordance with the recommendations approved by the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), Bharathidasan University Ethics Committee in Animal Experimentation (BDU/IAEC/2011/29) and Bharathidasan University Institutional Biosafety Committee (BT/BS/17/29/2000 PID).
Humoral immune response. ELISA was performed to monitor the humoral immune response. Briefly, polystyrene 96 well microtitre plates (Nunc; Thermo Scientific, USA) were coated overnight at 4 °C with 0.2 µg/well of rRecA/rFliD as antigen using carbonate coating buffer (pH 9.6). The plates were washed three times with PBS containing 0.05% [v/v] Tween 20 (PBST) and blocked with 4% non-fat milk in PBST. Hamster sera were added at a dilution of 1:200 for 1 h at 37 °C, followed by three washes with PBST. RNA isolation, cDNA synthesis, and qRT-PCR. Total RNA was isolated from the spleen cells using the TRIzol reagent (Invitrogen, Carlsbad, CA) and cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). Quantitative reverse transcriptase PCR (qRT-PCR) was performed using a CFX96 Touch TM Real-Time PCR detection system (Bio-Rad, Hercules, CA, USA). The qRT-PCR using SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA, USA) and primers 28,29 was carried out in a 25 μL reaction volume (50 ng cDNA, 12.5 μL Master Mix, 0.5 μM of each primer). Primer details are given in Table S2. The cycling conditions consisted of 95 °C for 10 min (denaturation), followed by target DNA amplification for 45 cycles (95 °C for 5 s, 60 °C or 61 °C for 30 s, and a variable extension time at 72 °C). The melting curves were analyzed immediately after amplification at a linear temperature transition rate of 0.1 °C/s from 55 to 95 °C, with continuous fluorescence acquisition. The relative CT (ΔΔCT) method was used to quantify cytokine gene expression 28 . Briefly, the fold change of each target mRNA was normalized to βactin housekeeping gene CT (ΔCT), and compared to a calibrator sample, the same normalized gene in the pre-immune sera sample (ΔΔCT). The final value represents the mean of triplicate relative fold between immunized and non-immunized hamster.
Sterilizing immunity by Culture and Real-Time PCR. The animals that survived up to 28 days post challenge were euthanized. Sterilizing immunity was determined by culture isolation of leptospires from kidney tissues as described previously 30 . For quantification of leptospiral DNA, DNA was extracted from freshly isolated kidney tissues from immunized hamsters using DNeasy blood and tissue kit (Qiagen, Valencia, CA). 16 S rRNA gene based real-time PCR was performed on a CFX96 Touch TM Real-Time PCR system (Bio-Rad, Hercules, CA, USA) 29 .
Statistical analysis. The Fisher exact test and log-rank test were used to determine significant differences in mortality and survival rates, respectively, among the experimental groups. Antibody levels were analyzed with two-way ANOVA to compare differences between the groups. The Student's t-test was used to determine significant differences among the mRNA relative expression analyses. A P ≤ 0.05 was considered significant in all analyses. The data were computed with either GraphPad Prism version 7.0 or SigmaPlot version 11.0. High degree identify templates were used to generate the initial 3D structure of RecA and FliD protein using Modeller 9.19.
Because, we saw the proteins to be conserved among all pathogenic leptospires we then asked whether these proteins have immunogenic potential to induce strong B and T cell response. We performed mapping of B and T-cell epitopes of RecA/FliD by BcPred and NetMHCcons and NetMHCII analysis. BcPred analysis showed 4 and 6 immunogenic B-cell specific epitopes for RecA and FliD respectively ( Fig. 1A and B) with VaxiJen score ranging between 0.85 to 0.99 ( Table S3). The predicted epitopes were conserved among the prevalent pathogenic leptospiral species as seen from the heat map analysis (Fig. 1C and D). The alleles selected for binding affinity belong to both MHC I and MHC II alleles. The lists of alleles with strong binder epitopes (IC50 < 50 nM) were considered as potential binders ( Table S4). The total no of strong binders for RecA and FliD was found to be high among the MHC alleles used in the present study, which account for 97% of HLA allelic variants in most ethnicities.
Among the different alleles A0203, A0206, A1101, DRB0101, DRB0701, IAd, IAs, TAP had the least IC50 value (IC50 value < 50) and considered to be potential binders (Fig. 1E-H). Because in silico analysis evidenced the immunogenic potential of RecA and FliD proteins, we then asked whether genetic vaccines preparation from these proteins will offer protective immunization against leptospiral infection. We next prepared DNA vaccines by cloning the CDs of RecA and FliD in to pEGFPN3 vector. The cloned recA-pEGFPN3 and fliD-pEGFPN3 vaccine constructs were confirmed by REA analysis (Fig. S4) followed by sequencing. For subunit vaccine, the rRecA and rFliD proteins were over expressed in E. coli as described previously 15 . Bacterin vaccine (WCL) was prepared from L. interrogans serovar Autumnalis strain N2 and the sterility check showed absence of leptospiral growth in bacterin inoculated EMJH media. All vaccine candidates were given intraperitoneally and the in vivo expression of cloned recA-pEGFPN3 and fliD-pEGFPN3 were monitored by western blot analysis of hamster hind leg tissue lysates probed with the corresponding hyper immune sera. Western blot analysis showed expression of RecA/FliD in hamsters immunized with the corresponding DNA vaccines (Fig. S5).
Humoral immune response. The specific humoral immune response in immunized hamsters was evaluated using an IgG-ELISA with serum samples collected on days 0, 14, 21, and 42 postimmunization (p.i.). To ensure the specific reactivity of the serum samples against recombinant RecA/FliD, we performed an IgG ELISA using unrelated leptospiral proteins rLigA and rLipL32 with N-terminal 6x-His-tag. The sera was highly specific for the recombinant RecA/FliD ruling out the possibility of immunization not against the potentially immunogenic 6x His-tag (Fig. S6).
Significant levels of circulating anti-RecA/FliD antibodies were detected (P < 0.001) (Fig. 2). At 21 and 42 days p.i., there was a significant induction of humoral immune response in hamsters immunized with the RecA/FliD subunit preparation compared to that in the negative control group (PBS-Alhydrogel/pEGFPN3-Alhydrogel) (P < 0.001). The IgG response in hamsters immunized using the prime-boost strategy differed significantly from that in the negative-control group on day 42 p.i. (P < 0.001). In the group immunized with the DNA vaccine, there were detectable levels of IgG, but this was found to lower even after 42 days p.i (P < 0.05) (Fig. 2).
Cytokine expression profile. The induction of both Th1 and Th2 type cytokines were evaluated from total mRNA isolated from spleen samples of vaccinated hamsters. mRNA abundance of Th1 and Th2 cytokines were measured by qRT-PCR analysis (Fig. 3). The groups that received prime boost/DNA vaccines showed significant differences (P < 0.001) in cytokine levels when compared to control groups. In contrast, vaccination with WCL/ recombinant proteins elicited cytokine response which was comparably less significant (P < 0.05).
Protective efficacy of vaccines. The protective efficacy of RecA/FliD in hamsters against homologous challenge (Autumnalis strain N2) or heterologous challenge (Canicola strain PAI-1) was determined in two independent experiments. The survival analysis in heterologous or homologous challenged hamsters showed ~58-100% survival among vaccinated animals over a period of 28 days ( Table 1). On the contrary, groups that received PBS or pEGFPN3 vector control group showed a median survival of <8 days (Fig. 4). Among the vaccinated groups, hamsters that received DNA vaccines recA-pEGFPN3/fliD-pEGFPN3) display a protection of ∼75-83% against N2 infection (P < 0.01) and ∼83-91% against PAI-1 infection (P < 0.01). This is significantly (P < 0.10) higher than the protection offered by RecA recombinant protein vaccination. Though the recombinant protein vaccinated groups show ∼58-66% protection, when used along with DNA vaccines in the form of prime boost vaccine increased its efficacy to ∼91-100% (P < 0.01). Next, we asked whether sterilizing immunity is offered by vaccination. To test this, presence of leptospires from kidneys of the surviving hamsters were evaluated by reisolation in EMJH media. Approximately 58.4-100% of vaccinated hamsters' kidney show sterilizing immunity with striking immunity offered by prime boost vaccination with recA-pEGFPN3 + rRecA (100%). Additionally, our re-isolation of leptospires were confirmed by rea-time PCR. Our 16S rRNA gene based real-time PCR analysis shows complete reduction of leptospiral DNA 28 days post-infection compared to 0 day (~10 7 leptospires) controls (P < 0.001).

Discussion
The currently available vaccines for leptospirosis are far behind, due to their inadequacy to protect the animals from different serovars confronted from various animal and environmental sources 31 . In concise, they indeed lack cross clade protection from one serovar to another serovar. Humans are the prime susceptible host for leptospirosis and used to consociate leptospirosis through infected animal sources. Especially the pet animals like the dogs and the domesticated cattle are reported to be the major source for human infection apart from their recreational and other environmental activities 32 . Habitually, the domesticated animals are the transient carriers and their incidence is through wild rodents. Predominantly, wild rodents are reported to be a carrier for serovar Autumnalis and the canine for Canicola. Consequently, in the current scenario in developing countries any vaccine candidate should offer protection against Canicola and Autumnalis infection and cross clade protection. Considering these actualities, we have investigated the prevalence of leptospirosis among canine population, which has evidenced an overall seroprevalence of 37.7% (51/135) with the predominant serovar to be Autumnalis 9.6%, followed by Canicola 8.14% 33 . Next, we assessed the efficiency of the present day available vaccine (MEGAVAC-6). The humoral immune response of the Megavac-6 vaccinated dogs against the leptospiral whole cell lysate, lipopolysaccharide (LPS), recombinant LipL32, rGroEL, rLK73.5 and rLigA were assessed in different time intervals. Among all the antigens the whole cell sonicated antigens followed by leptospiral LPS exhibited the highest antibody production among the vaccinated dogs after 64 days from the initial vaccination and drops down after 9 months. This may be due to the dominance of serovar specific leptospiral LPS, which may be the dominant immunogenic antigen in inactivated bivalent leptospiral vaccines 34 .
Although vast information is available about the immunogenic proteins of Leptospira 35 , especially the outer surface membrane proteins are of great interest, and several studies have demonstrated their potential uses in vaccine development 7 . Among them Lig (Leptospiral immunoglobulin-like) family proteins are well-studied with proven vaccine efficacy to be 90-100% in case of both recombinant protein and as DNA vaccination [36][37][38] . However, recent studies reported failure of complete protection to animals using Lig family proteins in addition to other surface leptospiral proteins 39 . The lack of success in developing vaccination was due to the following reasons: (1) lack of protein conservation among all pathogenic leptospiral species 40 , (2) lack of sterilizing immunity 36 , (3) promising model animals to perform leptospiral vaccination study (survival of control animals in challenge experiments were demonstrated 14 , and finally (4) lack of cross-clade protection. This prompted us to identify the intensive vaccine candidates, which might show a cross clade protection for leptospirosis. Adopting an expression gene library screen of virulent Leptospira interrogans serovar Autumnalis strain N2, we identified RecA and FliD with unique antigenic properties 15 . Apparently, these proteins were expressed only during natural infection of the host and remained undetectable in in vitro cultures. We hypothesized targeting in vivo expressed  The relative CT (ΔΔ CT) method was used to quantify cytokine gene expression: CTs were normalized against the GAPDH gene CT (Δ CT) and then compared to the same normalized gene in the PBS or pEGFPN3 immunized hamster group (calibrator). The control groups were set to 1. ns-no significance, *P < 0.05, **P < 0.01, ***P < 0.001. P values were obtained through comparison with the negative control (PBS or pEGFPN3) using a Tukey's multiple comparisons test by 2way ANOVA.  proteins as vaccine candidate may offer warranted protection against leptospiral infection. The high immunogenic nature (Fig. 1) and the conservation of these proteins among pathogenic leptospires (Fig. S1) prompted us to develop a DNA vaccine using RecA and FliD.
In the present study, the humoral immune response developed with DNA vaccination was significantly lower when compared to immunization with recombinant proteins (Fig. 2). On the contrary, in challenge experiments, DNA vaccination offered more protection both against homologous and heterologous infection (Fig. 4). Remarkably, high level of protection was conferred by prime boost vaccination reaching a survival percentage between 91 and 100%. Though animals immunized with the recombinant proteins display increased IgG response, only 58-66% of animals survived against the challenge. The reciprocal relationship between the humoral immune response and survivability, predicts humoral immune response alone is not sufficient to eliminate these spirochetes from the host. Additionally, the positive correlation established in the present between the cytokine levels in vaccinated groups and survivability substantiated an indispensable role for cell-mediated immunity (CMI) to clear pathogens from the host system. Our results are in compliance with previous report where the outer membrane leptospiral protein LipL32 impart protection when used as a DNA vaccine but failed to do so in the form of recombinant protein 41,42 . Hence, an effective leptospiral vaccine must be a vital combination option that would not only prevent invasion of spirochetes but also should eliminate those that somehow escaped the immune response and have formed successful niche in various organs.
All animals in the non-vaccinated groups died before day 12 in the present study, which shows the virulence of the strain, and the potential of the designed experimental animal model. The key success of vaccine development is to achieve sterilizing immunity and to confer heterologous protection. Majority of vaccine candidates available till data fail to offer sterilizing immunity 7 against leptospiral infection. Because, leptospirosis spreads through shedding of leptospires in urine of carrier animals 43 . Therefore, use of effective vaccine candidates that provide sterilizing immunity could have more prophylactic applications for the prevention of leptospirosis. Though sterilizing immunity was offered by LigB (131-645) subunit vaccine, heterologous protection was overlooked in the study making it very preliminary 44 . Interestingly in our present study, the prime boost vaccine conferred protection in heterologous challenge with sterilizing immunity. Being an in vivo expressed protein, the use of recA or fliD as a prime boost vaccine candidate could have a promising role in reducing the disease burden of leptospirosis. Due to their effective protection against homologous and heterologous challenge infection these candidates are valued for protection of domesticated animals from Autumnalis and Canicola infection, a common threat from rodent carriers.
In an earlier study, the TcG2/TcG4 DNA prime boost vaccine provided protective immunity against Trypanosoma cruzi infection and Chagas disease 45 . Apart from eliciting a strong immune response, sterilizing immunity was achieved in RecA prime-boost immunized hamsters. This is considered as an important finding of our study, as sterilizing immunity as most vaccine candidates failed to induce sterilizing immunity against leptospirosis 6,38 . Hamsters immunized with recombinant Mycobacterium bovis BCG expressing LipL32 or LigBrep-based prime-boost vaccination strategy conferred high-level of immunization and sterilizing protection against lethal infection in the hamster model of leptospirosis 46,47 .
SCiEnTifiC REPORTS | (2018) 8:6447 | DOI:10.1038/s41598-018-24674-8 recombinant proteins as boosting dose effectively stimulate antigen-specific memory B cells to differentiate into antibody-secreting cells thus resulting in production of high-titer, antigen-specific antibodies 50 . To the best of our knowledge, this is the first report documenting a prime-boost vaccination strategy that provides significant protection against homologous (Autumnalis strain N2) and cross-clade heterologous (Canicola strain PAI-1) challenge infections.
In conclusion, our outcomes further demonstrate that recA or fliD prime boost vaccine could impart a significant level of protection as revealed by enhanced survival, increased humoral and cell mediated response and sterilizing immunity after challenge. Finally, we conclude that our in vivo antigen based DNA prime protein boost vaccine can activate a strong humoral and CMI thus seems to be an ideal vaccine candidate for leptospirosis and may be a better control of zoonotic transmission to humans.