Kalimuthusamy Natarajaseenivasan

The emergence of >300 serovars ofLeptospiraconfounded 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 usingin vivoexpressed, 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 pathogenicLeptospiraspp., (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.

Introduction: Leptospirosis is a zoonotic disease caused by the spirochete of genus Leptospira with widespread distribution in tropical, subtropical and temperate zones. Leptospirosis is often confused with other febrile illnesses including jaundice, dengue, and malaria. Generally, the disease is often underdiagnosed or misdiagnosed. Though leptospirosis is curable with antibiotic treatment, the laboratory diagnosis of the disease is specialized and open to interpretation with multiple kits available to detect the different serological markers of Leptospira. Moreover, when leptospirosis is misdiagnosed, the disease can lead to multi-organ failure and may have fatal effects. There is a need for strategies to develop vaccines and prevent leptospirosis. In the present study, the immunogenic potential of leptospiral recombinant protein LipL21 (rLipL21) and its truncated form I-LipL21 (rI-LipL21) was evaluated. Methodology: The recombinant proteins were established in cyclophosphamide treated BALB/c mice model infected with L. interrogans serovar Autumnalis strain N2. Results: The vaccination study showed 66% and 83% survivability among mice immunized with rLipL21 and rI-LipL21 respectively and post-challenge with leptospiral strain N2 compared to control groups that showed 100% lethality. Additionally, a significant increase in antibody levels and cytokine levels (TNF-a, IFN-γ and IL-10) was observed evidencing a marked stimulation of both humoral and cell-mediated immune response in mice immunized with rLipL21/rI-LipL21 compared to whole cell leptospiral lysate (WCL). Conclusions: This study evidenced protective immunization against leptospirosis with rLipL21 and rI-LipL21 recombinant proteins and are potential candidates for the development of leptospiral vaccine.

Leptospirosis is underdiagnosed due to low sensitivity, need of specialised equipment, and expensive reagents for serological and molecular diagnosis respectively. Considering the sensitivity, rapidity, inexpensive reagents and collection of clinical samples, the monoclonal antibody based antigen detection method from urine samples has been developed and evaluated. LigA (LK90) based B-cell specific epitopes were predicted and synthesised as peptides for the production of monoclonal antibody. LK90₅₄₃: SNAQKNQGNA (amino acids: 543 to 552), and LK90₁₁₁₀: DHHTQSSYTP (amino acids: 1110 to 1119) with VaxiJen score of 1.3719 and 1.2215, respectively were used. Thirty two and 28 urine samples from confirmed and seronegative healthy human subjects, respectively were included for the evaluation of MAb-based dot blot ELISA. The specificity of the evaluated MAbs, P1B1 and P4W2 were found to be in the range of ~93–96%. Moreover, the MAbs did not show cross-reactivity with other bacterial antigens as confirmed by IgG ELISA, further validating its specificity for leptospiral antigens. These findings suggest that the developed MAb based dot blot ELISA is a simple, rapid performed in less than 8 h, inexpensive with a ICER of $8.7/QALY, and affordable in developing countries and area where laboratory facilities are limited.

Leptospirosis is mainly considered an occupational disease, prevalent among agriculture, sewage works, forestry, and animal slaughtering populations. However, putative risk to miners and their inclusion in the high-risk leptospirosis group remain in need of rigorous analysis. Therefore, a study was conducted with the objective to assess the leptospirosis seroprevalence among miners of two districts of Tamil Nadu, India. A total of 244 sera samples from Pudukkottai miners (124) and Karur miners (120) were analyzed by microscopic agglutination test. Antibodies to leptospires were detected in 94 samples giving an overall seroprevalence of 38.5%. The seroprevalence was higher among Pudukkottai miners (65.3%) when compared with Karur miners (10.8%). Seroprevalence among control population (13%) was significantly less than that of the Pudukkottai miners marking a possible high-risk population group distinction. Subject sera most commonly reacted with organisms of the serogroup Autumnalis, and the pattern was similar in carrier animals of the study areas. Two leptospires were isolated from kidney samples of rats. The prevalence of Autumnalis among rodents and humans source tracked human leptospirosis among the miners. The study also determined that Pudukkottai miners are subjected to high-risk challenges such as exposure to water bodies on the way to the mines (odds ratio [OR] = 10.6), wet mine areas (OR = 10.6), rat infestation (OR = 4.6), and cattle rearing (OR = 10.4) and are thus frequently exposed to leptospirosis compared with Karur miners. Hence, control strategies targeting these populations will likely to prove to be effective remediation strategies benefiting Pudukkottai miners and workers in similar environments across occupations.

Leptospirosis is a serious zoonosis that is underdiagnosed because of limited access to laboratory facilities in Southeast Asia, Central and South America, and Oceania. Timely diagnosis of locally distributed serovars of high virulence is crucial for successful care and outbreak management. Using pooled patient sera, an expression gene library of a virulent Leptospira interrogans serovar Autumnalis strain N2 isolated in South India was screened. The identified genes were characterized, and the purified recombinant proteins were used as antigens in IgM enzyme-linked immunosorbent assay (ELISA) either singly or in combination. Sera ( n = 118) from cases of acute leptospirosis along with sera ( n = 58) from healthy subjects were tested for reactivity with the identified proteins in an ELISA designed to detect specific IgM responses. We have identified nine immunoreactive proteins, ArgC, RecA, GlpF, FliD, TrmD, RplS, RnhB, Lp28.6, and Lrr44.9, which were found to be highly conserved among pathogenic leptospires. Apparently, the proteins ArgC, RecA, GlpF, FliD, TrmD, and Lrr44.9 are expressed during natural infection of the host and undetectable in in vitro cultures. Among all the recombinant proteins used as antigens in IgM ELISA, ArgC had the highest sensitivity and specificity, 89.8% and 95.5%, respectively, for the conclusive diagnosis of leptospirosis. The use of ArgC and RecA in combination for IgM ELISA increased the sensitivity and specificity to 95.7% and 94.9%, respectively. ArgC and RecA thus elicited specific IgM responses and were therefore effective in laboratory confirmation of Leptospira infection.

Leptospirosis is a re-emerging infectious disease that is under-recognized due to low-sensitivity and cumbersome serological tests. MAT is the gold standard test and it is the only serogroup specific test used till date. Rapid reliable alternative serogroup specific tests are needed for surveillance studies to identify locally circulating serogroups in the study area. In the present investigation the serological specificity of leptospiral lipopolysaccharides (LPS) was evaluated by enzyme linked immunosorbent assay (ELISA), dot blot assay and rapid immunochromatography based lateral flow assay (ICG-LFA). Sera samples from 120 MAT positive cases, 174 cases with febrile illness other than leptospirosis, and 121 seronegative healthy controls were evaluated for the diagnostic sensitivity and specificity of the developed assays. LPS was extracted from five locally predominant circulating serogroups including: Australis (27.5%), Autumnalis (11.7%), Ballum (25.8%), Grippotyphosa (12.5%), Pomona (10%) and were used as antigens in the diagnostics to detect IgM antibodies in patients’ sera. The sensitivity observed by IgM ELISA and dot blot assay using various leptospiral LPS was >90% for homologous sera. Except for Ballum LPS, no other LPS showed cross-reactivity to heterologous sera. An attempt was made to develop LPS based ICG-LFA for rapid and sensitive serogroup specific diagnostics of leptospirosis. The developed ICG-LFA showed sensitivity in the range between 93 and 100% for homologous sera. The Wilcoxon analysis showed LPS based ICG-LFA did not differ significantly from the gold standard MAT (P>0.05). The application of single array of LPS for serogroup specific diagnosis is first of its kind. The developed assay could potentially be evaluated and employed for as MAT alternative.