Leptospirosis Vaccine Research Articles

Identification of Immunodominant B- and T-Cell Combined Epitopes in Outer Membrane Lipoproteins LipL32 and LipL21 of Leptospira interrogans

Leptospirosis is a serious infectious disease caused by pathogenic Leptospira. B- and T-cell-mediated immune responses contribute to the mechanisms of Leptospira interrogans infection and immune intervention. LipL32 and LipL21 are the conserved outer membrane lipoproteins of L. interrogans and are considered vaccine candidates. In this study, we identified B- and T-cell combined epitopes within LipL32 and LipL21 to further develop a novel vaccine. By using a computer prediction algorithm, two B- and T-cell combined epitopes of LipL21 and four of LipL32 were predicted. All of the predicted epitopes were expressed in a phage display system. Four epitopes, LipL21 residues 97 to 112 and 176 to 184 (LipL21(97-112) and LipL21(176-184), respectively) and LipL32(133-160) and LipL32(221-247) of LipL32 were selected as antigens by Western blotting and enzyme-linked immunosorbent assay. These selected epitopes were also recognized by CD4(+) T lymphocytes derived from LipL21- or LipL32-immunized BALB/c (H-2(d)) mice and mainly polarized the immune response toward a Th1 phenotype. The identification of epitopes that have both B- and T-cell immune reactivities is of value for studying the immune mechanisms in response to leptospiral infection and for designing an effective vaccine for leptospirosis.

English

Leptospirosis continues to have major impacts on people of developing countries with inestimable morbidity and mortality. As current leptospirosis vaccination is relatively unsuccessful, development of efficacious vaccines through novel means became utmost priority for prevention of frequent outbreaks of the disease. Herein, MHC class II restricted peptide epitopes were identified from pathogenic leptospiral membrane proteins to suggest T-cell epitope driven leptospirosis vaccines through reverse vaccinology. The proteome of Leptospira interrogans serovar Lai was screened to select 137 membrane proteins. Jemboss Antigenic server was employed to find antigenic peptides, followed by ProPred analysis to predict 30 short peptides having strong binding affinity with HLA DRB1*0101, DRB1*0401 and DRB1*1501 alleles. Predictions were revised to pick antigenic peptides with same core region binding with the selected HLA- DRB1 alleles and ten such peptides were found from seven proteins. Further, to confirm the interaction at structure level, Leptospira excinuclease ABC subunit B protein having short T-cell antigenic peptide (133- REDVVIVSSVSCIYGLG-149) was modeled using Modeller9v7. The model reliability was evaluated through Procheck, ProSA and ProQ. Docking calculations for selected HLA DRB1 alleles (receptor) and modeled Leptospira excinuclease ABC subunit B protein (ligand) were carried out using Hex 5.1. The protein -protein interaction revealed that the core region (136-VVIVSSVSC-144) of the antigenic peptide (133- REDVVIVSSVSCIYGLG-149) was well interacting with the receptors. Through the reverse vaccinology approach and docking studies, the short antigenic peptide 133-REDVVIVSSVSCIYGLG-149 of Leptospira excinuclease ABC subunit B is proposed as a potential novel T-cell driven subunit vaccine against

Onset and duration of protective immunity against clinical disease and renal carriage in dogs provided by a bi-valent inactivated leptospirosis vaccine

Protection against clinical disease and prevention of the renal carrier state remain the key objectives of vaccination against leptospirosis in the dog. In the present paper, groups of dogs were vaccinated twice with a commercial bacterin (EURICAN ® L) containing Leptospira interrogans serovars icterohaemorrhagiae and canicola and challenged with heterologous representatives of both serovars at 2 weeks (onset of immunity) or 14 months (duration of immunity) after the second vaccination. Control dogs were not vaccinated against leptospirosis and kept with the vaccinated dogs. The challenges, irrespective of the serovar, reliably produced clinical signs consistent with Leptospira infection in the control pups with up to 60% mortality. As expected clinical disease in the adult controls was less severe, but we were able to induce morbidity and mortality as well. Under these extreme challenge conditions, clinical signs in the vaccinated dogs were rare, and when observed, mild and transient in nature. Following experimental infection, 100% of the control pups and 83% of the adult controls became renal carriers. Despite the heavy challenges, none of the 18 vaccinated puppies (onset of immunity studies) and only 2 out of the 16 vaccinated adult dogs (duration of immunity studies) developed a renal carrier state. These results show that a primary course of two doses of EURICAN ® L provided quick onset and long-term protection against both clinical leptospirosis and the renal carrier stage. This vaccine should provide veterinarians with a powerful tool to prevent clinical disease in dogs and zoonotic transmission of leptospirosis to humans.

Monoclonal Antibodies to LipL32 Protect Against HeterologousLeptospiraspp. Challenge

A non-culture-based leptospirosis vaccine that cross-protects against infection caused by heterologous Leptospira spp. should replace the currently available products, which are qualitatively and quantitatively inadequate. With that in mind, two murine hybridomas secreting monoclonal antibodies (MAb) binding only to homogenates of pathogenic Leptospira spp., and not of the saprophytic L. biflexa, serogroup Patoc, serovar Patoc, were produced. The MAbs of both clones neutralized Leptospira-mediated human red blood cell lysis in vitro and rescued hamsters from lethal infection with heterologous Leptospira spp. The orthologous Leptospira spp. protein carrying the MAb epitope(s) was identified by two-dimensional gel electrophoresis (2DE)-based proteomics and database search. The epitopes of the MAbs were located on the major outer membrane protein LipL32 of the pathogenic Leptospira spp. The MAbs in their humanized version are potential leptospirosis immunotherapeutics. They are also suitable as detection reagents in antigen-based assays for the rapid diagnosis of leptospirosis. Recombinant LipL32 is a good candidate for a broad spectrum, non-culture-based leptospirosis vaccine.

Characterization of the ompL1 gene of pathogenic Leptospira species in China and cross-immunogenicity of the OmpL1 protein

BackgroundThe usefulness of available vaccine and serological tests for leptospirosis is limited by the low cross-reactivity of antigens from numerous serovars of pathogenic Leptospira spp. Identification of genus-specific protein antigens (GP-Ag) of Leptospira would be important for development of universal vaccines and serodiagnostic methods. OmpL1, a transmembrane porin of pathogenic leptospires, was identified as a possible GP-Ag, but its sequence diversity and immune cross-reactivity among different serovars of pathogenic leptospires remains largely unknown.ResultsPCR analysis demonstrated that the ompL1 gene existed in all 15 official Chinese standard strains as well as 163 clinical strains of pathogenic leptospires isolated in China. In the standard strains, the ompL1 gene could be divided into three groups (ompL1/1, ompL1/2 and ompL1/3) according to their sequence identities. Immune electron microscopy demonstrated that all products of the different gene types of ompL1 are located on the surface of leptospires. The microscopic agglutination test revealed extensive yet distinct cross-immunoagglutination among the antisera against recombinant OmpL1 (rOmpL1) and leptospiral strains belonging to different ompL1 gene types. These cross-immunoreactions were further verified by ELISAs using the OmpL1 proteins as the coated antigens in serum samples from 385 leptospirosis patients. All the antisera against rOmpL1 proteins could inhibit L. interrogans strain Lai from adhering to J774A.1 cells. Furthermore, immunization of guinea pigs with each of the rOmpL1 proteins could cause cross-immunoprotection against lethal challenge with leptospires from different ompL1 gene types.ConclusionThree types of the ompL1 gene are present in pathogenic leptospires in China. OmpL1 is an immunoprotective GP-Ag which should be considered in the design of new universal vaccines and serodiagnostic methods against leptospirosis.

Study of a Leptospirosis Outbreak in Honduras Following Hurricane Mitch and Prophylactic Protection of the vax-SPIRAL® Vaccine

Honduras was one of the Central American countries most severely hit by Hurricane Mitch. Torrential rains and heavy flooding created conditions conducive to a leptospirosis outbreak in the country. A group of Cuban scientists studied 68 patients from the Department of Cort�s - one of the country's hardest hit areas - presenting clinical and epidemiological profiles indicative of leptospirosis. Blood and serum samples were taken from all subjects. A microscopic agglutination test (MAT) was used to identify Leptospira strains and to assess protection conferred by vax-SPIRAL® (Cuban leptospirosis vaccine) against the isolated strain. Prevalence of leptospires in the kidneys and liver was also verified. A male predominance was found in the group aged 15-49 years. Municipalities in this Department with the largest number of cases were San Pedro Sula, La Lima, and Chamelec�n. The most frequent symptoms included fever, headache, myalgia, and generalized discomfort. Over 80% of subjects reported presence of rodents in their homes, as well as contact with stagnant water and domestic animals. The strain isolated from positive blood cultures was from the Icterohaemorrhagiae serogroup, which was highly virulent in the animal model used. Protection was 100% in hamsters inoculated with vax-SPIRAL® and subsequently challenged with the Honduran strain. Additionally, macroscopic analysis of organs from immunized animals that survived the challenge showed no signs of leptospirosis infection.

Canine parvovirus type 2 vaccine protects against virulent challenge with type 2c virus

The ability of dogs vaccinated with a live attenuated CPV type 2 (Nobivac Intervet) vaccine to resist challenge with a current CPV2c isolate was investigated. Six SPF beagle dogs were given the minimum recommended course of vaccination, comprising a single inoculation of vaccine (Nobivac Lepto + Nobivac Pi) at 8–10 weeks of age followed 3 weeks later with a parvovirus vaccine in combination with distemper, adenovirus and parainfluenza virus (Nobivac DHPPi) and a repeat leptospirosis vaccine. Six control dogs were kept unvaccinated. All animals were challenged orally with a type 2c isolate of CPV and monitored for clinical signs, virus shedding, white blood cell fluctuations and serological responses. All vaccinated dogs were fully protected; showing no clinical signs nor shedding challenge virus in the faeces, in contrast to control animals, which displayed all the typical signs of infection with pathogenic CPV and shed challenge virus in the faeces.

Immunogenicity of the recombinant leptospiral putative outer membrane proteins as vaccine candidates

Leptospiral putative outer membrane proteins (OMPs) are likely to be essential components of more effective vaccines. Recently completed genomic sequences of Leptospira allowed us to target putative OMPs for the development of recombinant vaccines. We focused on 12 putative OMPs that had no homology with other organisms listed in the NCBI database except MceI and MceII of Leptospira, which are ∼25% homologous to MceI of Mycobacterium tuberculosis. All putative OMPs were cloned, expressed and purified as glutathione-S-transferase (GST) fusion proteins. Primary screening for immunoprotective potential was performed in hamsters challenged with an LD50 inoculum of low passage serovar Pomona. Out of these 12 OMPs three fusion proteins viz. rLp1454, rLp1118 and rMceII were found to be protective in a hamster model of leptospirosis. The protective efficacy was evaluated on the basis of survival, histopathological lesions in vital organs and antibody responses against each antigen. All the recombinant proteins were able to enhance the survival and reduce the histopathological lesions. In contrast, control animals immunized with rGST demonstrated low survival and had significant lesions. Further, these three proteins were evaluated for synergistic protective efficacy as compared to LigA, which has already been established as a protective antigen. Our results indicate that rLp1454, rLp1118, and rMceII showed protection individually and synergistically against serovar Pomona infection, which may help us to develop a multicomponent vaccine for leptospirosis.

Cow, Farm, and Management Factors During the Dry Period that Determine the Rate of Clinical Mastitis After Calving

The purpose of the research was to investigate cow characteristics, farm facilities, and herd management strategies during the dry period to examine their joint influence on the rate of clinical mastitis after calving. Data were collected over a 2-yr period from 52 commercial dairy farms throughout England and Wales. Cows were separated for analysis into those housed for the dry period (8,710 cow-dry periods) and those at pasture (9,964 cow-dry periods). Multilevel models were used within a Bayesian framework with 2 response variables, the occurrence of a first case of clinical mastitis within the first 30 d of lactation and time to the first case of clinical mastitis during lactation. A variety of cow and herd management factors were identified as being associated with an increased rate of clinical mastitis and these were found to occur throughout the dry period. Significant cow factors were increased parity and at least one somatic cell count ≥200,000 cells/mL in the 90 d before drying off. A number of management factors related to hygiene were significantly associated with an increased rate of clinical mastitis. These included measures linked to the administration of dry-cow treatments and management of the early and late dry-period accommodation and calving areas. Other farm factors associated with a reduced rate of clinical mastitis were vaccination with a leptospirosis vaccine, selection of dry-cow treatments for individual cows within a herd rather than for the herd as a whole, routine body condition scoring of cows at drying off, and a pasture rotation policy of grazing dry cows for a maximum of 2 wk before allowing the pasture to remain nongrazed for a period of 4 wk. Models demonstrated a good ability to predict the farm incidence rate of clinical mastitis in a given year, with model predictions explaining over 85% of the variability in the observed data. The research indicates that specific dry-period management strategies have an important influence on the rate of clinical mastitis during the next lactation.

Leptospirosis vaccines

Leptospirosis is a serious infection disease caused by pathogenic strains of the Leptospira spirochetes, which affects not only humans but also animals. It has long been expected to find an effective vaccine to prevent leptospirosis through immunization of high risk humans or animals. Although some leptospirosis vaccines have been obtained, the vaccination is relatively unsuccessful in clinical application despite decades of research and millions of dollars spent. In this review, the recent advancements of recombinant outer membrane protein (OMP) vaccines, lipopolysaccharide (LPS) vaccines, inactivated vaccines, attenuated vaccines and DNA vaccines against leptospirosis are reviewed. A comparison of these vaccines may lead to development of new potential methods to combat leptospirosis and facilitate the leptospirosis vaccine research. Moreover, a vaccine ontology database was built for the scientists working on the leptospirosis vaccines as a starting tool.

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Duration of immunity in dogs vaccinated against leptospirosis with a bivalent inactivated vaccine

Duration of immunity in dogs induced with current commercial inactivated leptospirosis vaccines and evaluated against experimental infection, to date, has hardly been documented. The purpose of the present work was to assess the duration of immunity in dogs that is attainable with a commercial inactivated bivalent leptospirosis vaccine. For this purpose, young dogs were vaccinated twice followed by challenge with either Leptospira interrogans serovar canicola or L. interrogans serovar icterohaemorrhagiae 5 weeks, 27 weeks or 56 weeks after the second vaccination. For assessment of the duration of immunity, titres of agglutinating serum antibodies were measured before and after challenge, and the effects of challenge on a variety of parameters were determined including reisolation of challenge organisms from blood, urine and kidney. Both challenge strains induced a generalised infection in control dogs, the canicola strain being most virulent. From the results with different parameters it appeared that the two vaccinations induced a high rate of protection from generalised infection with canicola and icterohaemorrhagiae at 5, 27 and 56 weeks after the second vaccination. In addition, after 56 weeks, still a high level of immunity against renal infection with sv. canicola and, as a consequence, urinary shedding of sv. canicola bacteria, was demonstrated. It was, therefore, concluded that with this vaccine, using this vaccination schedule, a duration of immunity of 1 year can be attained against infection with both serovars.

Leptospirosis

Leptospirosis is a globally important zoonotic disease that affects humans on all continents, in both urban and rural contexts, and in temperate and tropical climes. Leptospirosis is a disease of the environment; transmission depends on interactions between humans and mammalian reservoir hosts. A variety of infectious diseases that present as undifferentiated febrile syndromes, such as malaria, dengue and influenza, as well as viral hemorrhagic fevers can mimic leptospirosis. The importance of pulmonary hemorrhage as a lethal complication of leptospirosis has become more widely recognized. In contrast to textbook dogma, population-based studies indicate that there is a poor correlation between infecting leptospiral strain and clinical expression of disease. Genetic transformation of a Leptospira sp. has now been reported, which should allow for detailed analysis of a variety of leptospiral genes. Publication of the whole Leptospira genome is eagerly awaited. Following recent reports of a new, highly effective conjugate typhoid vaccine, new efforts to find leptospirosis vaccines should include the manufacture and testing of conjugate leptospiral lipopolysaccharide vaccines. Recent advances, particularly in epidemiology, molecular genetics and pathogenesis, are placing leptospirosis at the cutting edge of biomedical science.

Serologic response of llamas to a commercially prepared leptospirosis vaccine

Leptospirosis has been reported in llamas living in North America. Llamas in some locations are routinely vaccinated against leptospirosis in an attempt to prevent the disease. In order to determine if llamas respond to a leptospirosis vaccine, 14 llamas were immunized twice at 2 week intervals with a commercially available five-way leptospirosis vaccine. Blood was then collected for serum titer evaluation using a microagglutination technique for 5 months. Serologic response to the vaccine was low and of short duration to all five serovars, regardless of prevaccination titer. By 12 weeks postvaccination, only animals with serum titers ⩾ 1:200 to Leptospira pomona prevaccination still had serum titers ⩾ 1:200. Seven references are cited.

Leptospirosis vaccination in beef cattle: Use of decision tree analysis

Decision tree analysis was used to evaluate the economic benefits of leptospirosis vaccination in beef cattle. The analysis used estimated values for the cost of vaccination, the probability of calf losses from leptospirosis and the economic losses resulting from calf mortality to calculate the expected value of vaccination. A computer spreadsheet was used to calculate the expected value of the vaccination programme over a wide range of estimated costs and probabilities and to determine the break-even point at which vaccination becomes economic. A sensitivity analysis demonstrated that the outcome of the analysis was most dependent on calf value and the probability of calf loss due to leptospirosis. The analysis was relatively insensitive to changes in vaccine cost, vaccine efficacy and herd fertility.

Protein and antigen profiles of prevalent serovars of Leptospira interrogans

Whole-cell and detergent-soluble proteins, enriched for outer membrane antigens, of the Leptospira interrogans serovars present in commercially available pentavalent vaccines (hardjo, pomona, icterohaemorrhagiae, grippotyphosa, and canicola) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting). Protein and antigenic profiles of these serovars, representing several serogroups, were compared with similar profiles of the most common North American bovine pathogen, serovar hardjo type hardjo bovis. The reference strain of serovar balcanica and a hardjoprajitno bovine field isolate (serovar hardjo) were also assayed. Coomassie blue-stained gels revealed extensive protein similarities, and Western blots demonstrated antigenic relatedness throughout the low- and high-molecular-weight regions. Possible quantitative differences in protein expression among the strains were noted, as were similarities in the protein profiles of type hardjo bovis and the balcanica reference strain. A cocktail composed of these homologous antigens may serve as an efficacious subunit vaccine for leptospirosis.

Serological reactions againstLeptospira interrogansserovars in alpacas after vaccination

Abstract New Zealand Veterinary Journal 39, 32–3, 1991 In the past few years, increasing numbers of alpacas have been imported into New Zealand from South America, mainly Chile. The Ministry of Agriculture and Fisheries brought a group of alpacas to Flock House Agricultural Centre in February 1990. As part of routine animal health procedures the alpaca were drenched with oral ivermectin (Ivomec oral solution for cattle, Merck, Sharpe and Dohme (NZ) Ltd) and vaccinated against clostridial diseases (Tasvax Covax 5, Coopers Animal Health NZ Ltd) and leptospirosis. A serological survey was undertaken to determine the response of the alpacas to leptospirosis vaccination.

Bovine Abortion

A farm in the Midwest is experiencing problems with abortion. This farm is milking around 80 cows, raises its own heifers at another place, and has a rolling herd average of 16,000 pounds. Heifers are bred with a bull at the other farm and brought to the milking farm 4 - 5 weeks before freshening. These springers are housed in the same area as the dry cows until approximately one week before freshening. All cows are vaccinated with a multivalent vaccine for Leptospirosis, Infectious Bovine Rhinotracheitis, and P13 at the time of dry off. Milking cows are fed a total mixed ration of corn silage, haylage, and high moisture corn. Dry cows and springers are fed corn silage with hay or green chop, depending on the time of year.
 On May 1 an aborted calf approximately 120 days gestational age was found in the freestall area. On June 1, one heifer aborted 2 weeks before her calving date. On June 10 another heifer aborted. The veterinarian was called out and he submitted serum samples from both heifers to the diagnostic lab, results were negative. A dry cow aborted on June 15. The fresh fetus and placenta were carried, by the producer, to the diagnostic lab. It will be 2 weeks before results are available. Another heifer and a cow had late term abortions on June 17 and 18. Another cow in the low group aborted twins on June 21. Immediate action is called for but no diagnosis can be made from the laboratory findings.

The efficacy of a leptospirosis vaccine in preventing leptospiruria in pigs

A commercially manufactured leptospirosis vaccine containing serovars pomona and hardjo and licensed for use in cattle and sheep was investigated to determine if it would prevent leptospiruria in pigs exposed to serovar pomona. Twenty piglets were each vaccinated twice at an interval of three weeks. Twenty other piglets were unvaccinated and served as controls. Three weeks after the second dose of vaccine all animals were exposed for 64 to 89 days to a natural infection with pomona. During the investigation blood samples were examined serologically and urine samples were examined by dark ground microscopy and cultured for the presence of leptospirae. Attempts were made to culture leptospirae from kidneys at slaughter. Kidneys were also examined histologically for evidence of leptospira infection. One vaccinated animal developed a respiratory disease. It was treated with antibiotics and removed from the trial. Leptosphuria was demonstrated in six of the remaining 19 vaccinated pigs and leptospirae were found in nine of 578 (1.5%) urine samples examined from these animals during the period of exposure. In contrast leptospiruria occurred in 19 of 20 unvaccinated pigs and leptospirae were found in 253 of 642 (39.4%) urine samples examined from these animals. Histopathological lesions consistent with leptospirosis were found in kidneys examined from two of 16 vaccinates and 17 of 18 non-vaccinates. Antibodies to serovar pomona were detected in 12 of 19 vaccinated pigs examined three weeks after the second dose of vaccine and before exposure to infection, and in all of 18 unvaccinated pigs examined after exposure to infection. It was concluded that use of this vaccine in pigs resulted in a significant degree of protection against leptospiruria.

Immunohistochemical investigations of leptospira-infected golden hamsters in potency tests

Golden hamsters taken from routine potency testing of leptospirosis vaccines were investigated for leptospirae after a challenge infection. Kidneys, livers, and spleens were examined immunohistochemically by the unlabelled antibody enzyme (PAP) method, and samples of these organs were additionally cultured for the evidence of living leptospirae. In non-vaccinated animals the recultivation method indicated leptospirae during the bacteraemic phase, beginning immediately after infection. Both methods resulted in positive reactions after organ manifestations. Intravascular evidence of leptospirae in the kidneys could be seen first on day 3 after infection. On day 7, there was intracellular and intratubu-lar evidence as well, combined with tubulo-interstitial alterations. In vaccinated animals the bacteraemia after infection was reduced. Organ manifestations were seen rarely; there were less severe or no pathohistological alterations.