Prototype Pentavalent Bovine Leptospira Vaccines Blended with Oil Adjuvant Provides Protection and Prevents Renal Colonization in Guinea Pig Model

Leptospirosis is a widespread zoonotic disease caused by Leptospira spp that affects virtually all mammals. Bovine Leptospirosis is characterised by subclinical infection, fever, jaundice, mastitis, abortion and infertility (Thiermann, 1984; Balakrishnan, 2009). It significantly impacts the economy of livestock farmers by causing reproductive failure and production losses in bovines (Ellis 1994; Faine et al., 1999; Guitian et al., 2001). The successful control of bovine leptospirosis requires a combination of vaccination programme, biosecurity measures and chemotherapeutic regimens. In addition to protection from clinical disease, vaccination also reduces/prevents renal colonisation of Leptospires and in turn reduces excretion of leptospires in urine which is a major source of environmental contamination, infection to human and other animals.
       
The immune responses produced by leptospires are serogroup specific (Faine et al., 1999). Therefore a successful vaccine against Leptospires requires inclusion of serogroups prevalent in that geographical region (Srivastava, 2011). Although several preliminary attempts were made for developing of a successful vaccine against leptospirosis by incorporating prevalent serogroups (Seshagiri Rao and Keshavamurthy, 1985; Venugopal and Ratnam, 1992; Balakrishnan and Roy, 2014; Rani Prameela et al., 2015) there are no commercial vaccines currently available in India.
       
Bovine Leptospiral vaccines used in the United States are inactivated whole-cell vaccines containing L. interrogans serovars Hardjo (type hardjoprajitno), Canicola, Pomona and Icterohaemorrhagiae and L. kirschneri serovar Grippotyphosa (Hanson et al., 1972; Naiman et al., 2001). These Leptospira vaccines provide adequate protection against clinical disease. However, cattle vaccinated with vaccines are vulnerable to infection with serovar Hardjo despite the presence of high titres of serovar specific anti-LPS antibody (Bolin et al., 1989). It has been suggested that a strong cell-mediated immune response to serovar Hardjo is necessary for protection (Ellis et al., 2000; Naiman et al., 2001; Brown et al., 2003).
       
Selection of appropriate adjuvant is a key factor for the success of any vaccine. An ideal adjuvant should stimulate early and long lasting antibody substantial antibody titres and also stimulate cellular immune response. Montanide series of oil adjuvants have been shown to be safe in cattle and goat (Dar et al., 2013; Patil et al., 2002).
       
This study was undertaken to develop a prototype pentavalent vaccine using the prevalent serovars namely Australis, Hardjo, Hebdomadis, Hebdomadis, Javanica and Pomona. Montanide adjuvants namely ISA 61 VG, ISA71 VG, ISA 201 VG and aluminium hydroxide gel adjuvant were used in the vaccine formulations. Immunogenicity of the vaccines and their safety were assessed experimentally in Guinea pigs. The ability of these vaccines to protect Guinea pigs against virulent Leptospires challenge was also demonstrated.

Culture of leptospires
 
L.interrogans serogroup Australis serovar Australis, serogroup Sejore serovar Hardjo, Serogroup Hebdomadis serovar Hebdomadis, serogroup Javanica serovar Poi and serogroup Pomona serovar Pomona available in Zoonoses Research Laboratory which were collected from Regional Medical Research Centre, Portplair were cultured in EMJH medium. The serovars were characterised using serogroup specific polyclonal serum. The serovars were cultured in EMJH medium by gradually reducing 0.1% Bovine Serum Albumin (BSA) concentration at each level from 1% to 0.5%. The time taken to obtain 2×10⁸ organisms per ml at each concentration of BSA was measured by counting Leptospires after the third passage using Petroff- Hausser counting chamber (M/s Hausser Scientific, U.S.A). The leptospires cultured in EMJH medium containing 0.5% BSA was used for the preparation of vaccine. This research work was carried out in Zoonoses Research Laboratory, Tamil Nadu Veterinary and Animal Sciences University during 2015.
 
Preparation of pentavalent vaccines prototypes
 
All five serovars were cultured separately in 100 ml of EMJH medium in spinner culture flask. Each serovar was inactivated by adding 0.4% (v/v) formalin (final concentration) and incubated 37°C overnight. The complete inactivation was assessed by innocuity test as specified by OIE (OIE, 2014). The inactivated culture of each serovar was washed three times in 0.85% salineby centrifugation at 9000 rpm at 4°C. A final concentration of 2×10⁹ Leptospires of each serovar per ml was used as antigen for vaccine production. The multivalent vaccines were prepared by mixing antigen cocktail with either Montanide ISA61 VG, Montanide ISA 71 VG, Montanide ISA 201 VG (M/s Seppic, France, 1998) which were received from M/s Seppic, India or aluminium hydroxide gel (10%) (Banihashemi et al., 2013) to get the final concentration of 2×10⁹ of each serogroups per ml of vaccine. The mixtures were homogenised using hand- held low-shear homogenizer. Quality tests of sterility and safety were performed as per OIE guidelines (OIE, 2014). Viscosity was measured (Mahboob et al., 1997).
 
Animal experiments
 
The animal experiments were carried out as per the CPCSEA regulations and the experimental procedures were approved by the Institutional Animal Ethical Committee of Tamil Nadu Veterinary and Animal Sciences University (1614/DFBS/B/2014). For the in vivo immunogenicity study, Male or Female Dunkin-Hartley Guinea pigs (3-4months) procured from BCG vaccine Laboratory, Chennai were grouped into five groups of eight numbers in each. Each group was immunized subcutaneously using 0.5 ml of vaccine prototype-I, vaccine- prototype II, vaccine-III prototype, vaccine prototype -IV respectively and group-V was kept as unvaccinated control. A booster vaccine dose was administrated 15 days after primary vaccination. Blood samples were collected on days 0, 15, 30, 60, 90, 120, 150 and 180 to assess the immunogenicity of vaccines.
 
Assessment of immune responses
 
The humoral immune response was assessed by measuring antibodies against leptospires using Microscopic Agglutination Test (MAT). MAT assay was done as per OIE (2014) in U bottom microtitre plate. The plates were viewed under dark-field microscope for presence of agglutination and or reduction of 50% free cells. The titre of anti leptospiral antibody is reported as the dilution of serum that showed 50% agglutination, leaving 50% free cells compared with the antigen control.
       
The cell mediated immune (CMI) response was assessed by measuring lymphocyte proliferation. Peripheral blood mononuclear cells (PBMC) from both vaccinated and control animals were isolated by Ficoll-Paque Plus density gradients method (Naiman et al., 2001). Two hundred microlitre of 5×10⁵ PBMC was aliquoted into 96-well flat-bottom microtitre plate in triplicate.  Sonicated Hardjo antigen (0.5 µg of protein ml^-1) was added. ConA (200 µg) was used as positive control. The plates were incubated at 37^oC in a 5% CO₂ atmosphere for 5 days. Appropriate assay controls were included. On 5^th day, 20 μl of MTT (2,5-di-phenyl- tetrazolium bromide) reagent (5 mg/ml) was added into all wells and the plates were incubated for three hours at 37°C. After incubation, the plates were removed, the supernatant medium pipetted out and 100 μl of solubilisation solution (M/s Himedia, India) was added to each well. The plates were gently shacked for 10 minutes to enhance dissolution. The absorbance was read on a ELISA plate reader at 570 nm with a reference wavelength of 650 nm. The cell proliferation was calculated as stimulation index (He et al., 2008). The results are expressed as mean stimulation index±SD.
       
For protection against challenge study, fifty numbers of Guinea pigs were grouped into five groups I, II, III, IV and V and were immunized subcutaneously with 0.5 ml (2×10⁹ organisms/ml) of vaccine I, II, III, IV  respectively and the group V animals were used as control. On 15^th day post vaccination a booster vaccine was given to the respective groups. After thirty days animals from each group was challenged separately by injecting 0.5 ml of 10⁴ ID₅₀ virulent culture of serovar Australis, Hardjo, Hebdomadis, Javanica and Pomona serovars intraperitoneally. The Guinea pigs were observed for 28 days for clinical signs. The serum samples were collected from live animals on 28^th day for demonstration of antiserovar antibodies by MAT. The tissues and urine from dead and live animals respectively were collected for detection of leptospires by dark-field microscopy (DFM), isolation of organism by culture.

Adaptation of leptospires in low protein medium
 
Leptospires were adapted for culture in low protein medium to reduce the adverse vaccine reactions caused by residual extraneous proteins. Specific set of five serovars of leptospira were choosen for vaccine formulation, based on the epidemiological data of bovine leptospirosis in different agroclimatic zones of Tamil Nadu from 2012 to 2015 (Senthilkumar, 2016). A few experimental attempts to develop leptospiral vaccines using a cocktail of serovars were made previously in India. However the choice of serovars in these study was not based on the extensive epidemiological data (Seshagiri Rao and Keshavamurthy, 1985; Venugopal and Ratnam, 1992; Balakrishnan and Roy, 2014; Rani Prameela et al., 2015). The time taken to reach the concentration of 2×10⁸ organisms per ml for each serovar of leptospires in EMJH medium with 1% BSA was 6-7 days became 12-14 days in EMJH medium with 0.5% BSA. The serovars Hardjo, Hebdomadis, Javanica grow faster when compared to Australis and Pomona. Bey and Johnson (1986) reported that culturing of Leptospires in low protein medium has been shown not to alter their antigenicity and immunogenicity. This concentration of BSA (0.5%) was sufficient to render the fatty acids (Tween 80) in medium to nontoxic. Culturing leptospires in medium with BSA levels below 0.5% required additional treatment with activated charcoal for detoxification of tween 80 and hence has not been used for vaccine stock production.

Formulation and quality testing of vaccine
 
Three different vaccine formulations were made using Montanide ISA61 VG, ISA71 VG, (water in oil) and Montanide ISA201 VG (water-in-oil-water) and compared with conventional Aluminium hydroxide gel (suspension). Aluminium hydroxide gel was the most commonly used adjuvant in previous leptospirosis vaccine trails in India (Seshagiri Rao and Keshavamurthy, 1985; Venugopal and Ratnam, 1992) and USA (Bolin and Alt, 2001). However water-in-oil emulsion vaccines have been shown to stimulate long term immunity and enhance cellular immune response by inducing higher IgG2 antibody level and cytotoxic T cell response (Aucouturier et al., 2001). The leptospires inactivated using formalin (0.4 per cent, v/v) when inoculated in EMJH medium did not show any growth until 60 days. The viscosity of vaccine-I, II, III prototypes were found to be 0.53 sec, 0.8 sec and 0.47 sec respectively. The prototype vaccines I, II, III, IV did not show any growth of microorganisms in Nutrient agar, Thioglycollate broth and Sabouraud’s Dextrose agar during the incubation period of 14 days showing freedom from extraneous bacterial contamination. The prototype vaccine injected guinea pigs did not show any adverse reaction such as raise in body temperature, reddening of skin at the site or any other abnormal clinical signs during the observation period of   seven days indicated the vaccines were safe.
 
Assessment of immune response
 
The humoral immune response was expressed in geometric mean MAT titre (log₂). The Guinea pigs vaccinated with four types of pentavalent leptospiral vaccine showed seroconversion to all serovars in vaccine, whereas the unvaccinated control animals did not show any detectable seroconversion throughout study period of six month. The details of MAT titres for serovars at different time period are presented (Table 1 and 2; Fig 1).  The MAT titre was ranging from 2⁵ to 2¹², Geometric mean antibody (MAT) titre of more than 6.64 log₂ (≥1:100) was considered as protective. The protective antibody titre (>2^6.64) with the vaccine-I, II, III for serovar Australis was seen as early on 15^th day post-vaccination (dpv) and for other serovars on 30 day post vaccination. The vaccine IV that received Aluminium hydroxide gel adjuvant vaccine showed protective titre (>2^6.64) only for serovar Australis (30 dpv) and for serovars Hardjo, Hebdomadis, Javanica and Pomona able to induce titre of more than 24 but did not reach the protective threshold. Between vaccines-I, II, III formulation, vaccines showed only marginal protection for serovar Javanica; however for all other serovars it showed MAT titre of more than 2^6.64. In vaccine formulation I, II, III the immunogenicity was persisted for 180 days with higher titre of above 2^6.64 for serovars Australis, Hardjo, Hebdomadis and Pomona except serovar Javanica, which showed only up to 150 days. An increased antigenic concentration of serovar Javanica may perhaps to produce longer duration of immunity, similar to that of other serovars.The humoral immune response noticed   between vaccines are highly significant. The vaccine I formulation elicited an early immune response and high antibody titres when compared to other vaccine formulations II, III, IV. The differences in immune response induced by serovars Australis, Hardjo, Hebdomadis and Javanica in between vaccine formulations are highly significant but there is no significant differences in immune responses induced by serovar Pomona in different vaccine formulations (F value 1.6^NS). This shows that Montanide adjuvants are better in inducing anti-leptospiral antibodies than aluminium hydroxide gel adjuvant vaccine.
 

 

 

       
The cell mediated immune response to serovar Hardjo was carried out by measuring the lymphocyte proliferation by MTT test and calculating stimulation index since the protection against serovar Hardjo require cell mediated immune response irrespective of the serum antibody titre. Development of an antigen-specific Th1 response appears to be critical for limiting renal colonisation and urinary shedding of leptospires (Ellis et al., 2000; Naiman et al., 2001; Brown et al., 2003). The stimulation index for vaccine-I, vaccine-II, vaccine-III and vaccine-IV and control group are detailed (Table 3 and Fig 2). The PBMC collected vaccinated groups, stimulated with Concanovalin A showed significant lymphocyte proliferation compared to unvaccinated group. A high lympho proliferation response was noticed in vaccine group I, II, III for serovar Hardjo antigen from 7th day to 180 day. The CMI response elicited by Montanide adjuvants (vaccine I, II, III) are significantly higher when compared to vaccine adjuvanted with Aluminium hydroxide gel (F value 12.16** P<0.05). but  there was no significant differences among the Montanide ISA adjuvant vaccine groups in eliciting cell mediated immune response (F value 3.23^NS). This reveals the characteristics of Montanide adjuvants in inducing CMI response.
 

 

 
Protection against challenge
 
The control animals challenged with serovars Australis, Hebdomadis, Javanica and Pomona showed rise in temperature, dull, depressed and succumbed to the disease following intraperitoneal challenge and animals challenged with serovar Hardjo survived but showed increase MAT titre. For serovars, that are not lethal to guinea pig or hamster such as Hardjo, the potency is measured by induction of suitable antibody titre (OIE, 2014). The induction of high antibody titres is essential to qualify the vaccine as potent. All unvaccinated control animals showed presence of virulent Leptospires in tissues and urine samples. The guinea pigs that received Montanide adjuvant vaccines showed complete protection against challenge with serovars (Australis, Hardjo, Hebdomadis Javanica and Pomona), whereas in the group vaccinated with Aluminum hydroxide gel adjuvant vaccine, all animals were protected except for the one challenged with serovar Javanica. It is interesting to note that the animals in Aluminum hydroxide gel adjuvant vaccine group were protected inspite of their antibody titres against Leptospires (< 2^6.64). Urine samples collected from all vaccinated and surviving animals did not show presence of live Leptospires on DFM and no isolation of leptospires on culture, indicating the efficiency of the formulations to prevent renal colonization.

An inactivated pentavalent oil emulsion based vaccine, incorporating the prevalent serovars namely L.interrogans serogroup Australis serovar Australis, serogroup Sejore serovar Hardjo, serogroup Hebdomadis serovar Hebdomadis, serogroup Javanica serovar Poi and serogroup Pomona serovar Pomona along with Montanide adjuvant was developed and compared with the aluminium hydroxide gel adjuvant vaccine formulation. The vaccine blended with oil adjuvants resulted in higher protective antibody titres of more than 6.64 log₂ (≥1:100) and lasted upto 180 days, except for serogroup Javanica (150 days). The Montanide adjuvants were also able to produce cell mediated immune response, necessary for protection against serovar Hardjo. Vaccination and challenge study in Guinea pigs showed complete protection following vaccination using pentavalent vaccine blended with Montanide adjuvants, while the aluminium hydroxide adjuvant was able to confer only partial protection. The Montanide blended vaccines were also able to prevent renal colonisation of all five serogroups This Montanide adjuvant vaccine was found to be superior in inducing long lasting protective humoral and cell mediated immune responses in guinea pig model. The potential of Montanide adjuvant inducing high humoral immune and cell mediated immune response, makes for its use in the largescale vaccination programmes against bovine leptospirosis.

The authors were thankful to Indian Council of Agricultural Research, New Delhi, India for funding under the scheme on Outreach programme on zoonotic diseases.

No conflict of interest declared.

Senthilkumar and Ravikumar designed the research work and conducted the animals experiment. Aravindhbabu has coordinated for the statistical analysis. All authors contributed to the writing and preparation of the manuscript.