Evaluation Of Recombinant Proteins Against Leptospira Interrogans And Comparison Of Their Phenotypic Characterization

Leptospirosis, a zoonosis of worldwide distribution and recognized as an emerging infectious disease, is caused by gram negative bacteria belonging to the genus Leptospira. Occupational exposure, recreational activity, flooding, and household environments in close contact with animals increase the risk of transmission to humans. The clinical syndromes of leptospirosis vary from a subclinical, self-limiting, anicteric, febrile illness to severe and potentially fatal disease. Leptospires are widely distributed in both urban and rural areas of tropical, sub-tropical and temperate regions. Over the past several decades, changes in epidemiological patterns underscore the importance of leptospirosis as a major public health problem. More than 500,000 cases of severe leptospirosis are reported each year, with case fatality rates exceeding 10%. The pathogenesis of leptospiral infection and establishment of infection in a particular host involve invasion of host cells with the help of various surface molecules (virulence factors), persistence or colonization, and immune evasion.Currently, the only commercial vaccine is whole cell vaccines, which are hobbled by several limitations like short term immunity, LPS reactogenicity, requirement of boosters and in ability to provide cross protection against the myriad of leptosprial serovars. Subunit vaccines based on outer membrane proteins (OMPs) are attractive alternatives to whole cell inactivated vaccines because of their antigenic conservation among serovars and high throughput technologies for vaccine production. Till todate, several OMPs of Leptospira, for example, OmpL1, LipL41, LigA, LigB, Lp0607, Lp1118, and Lp1454 have shown promise in the development of subunit vaccines for leptospirosis。Leptospira binds to host extracellular matrix (ECM) through surface exposed outer membrane proteins called adhesin in order to initiate infection. Of various adhesins present on the surface of the spirochete, Lig proteins and LipL32 are most abundant, widely distributed among pathogenic serovars and well characterized. Various fragments of Lig proteins (Lig-con4, LigCon4-7.5, LigBcen2) and C-terminus fragment of LipL32 all of that bind to host ECM were fused, expressed and purified in soluble form as fusion proteins. Moreover, we truncate N-terminus of LigC into LigC-J and LigC-K expressed and purified in soluble form as recombinant proteins. Four week hamsters were immunized subcutaneously with various fusion proteins emulsified in EMULSIGEN-D adjuvant and subsequently boosted at 3 weeks. The protective efficacy of these novel fusion proteins was evaluated against subsequent challenge with highly virulent L. interrogans serovar Pomona. Our results indicate that fusion protein based vaccine induced significant protection against acute infection with respect to PBS-adjuvant vaccinated controls as revealed by enhanced survival and reduced pulmonary hemorrhage. Moreover, the protection mediated by these fusion proteins and LigC-N-terminus (LigC-J and LigC-K) were higher than that of LigCon(established protective antigen) and correlated to the level of antibodies. LipL32 failed to impart significant protection, however fusing its immunogenic C-terminus domain to Lig fragments slightly delayed the morbidity of the infected animals. Moreover, the protection ability hasn't been promoted after LigA added to the mixtures (Lp1118, Lp1454 and Lp0607). This indicated that we can't relay on multi-antigens strategies to obtain an ideal protection against Leptospira interrogans. However, our results demonstrate that this novel strategy which fuses multiple attaching domains as a single antigen could be promising in developing effective subunit vaccine to combat this zoonotic infection.These vaccine candidates have immune protection, but the rate of protection still need to be further improved. To understand of the pathogenic mechanism of leptospirosis and prevention of the disease, we used phenotype microarray (PM) to the study of leptospirosis. PM technology provides a set of nearly 2000 culture conditions in which one can test the ability of a microorganism to respire and grow. The set includes about 200 C-sources, 400 N-sources, 100 P- and S-sources, 100 nutrient supplements, and a range of conditions varying the pH, ion and osmotic status of the culture environment. This enables scientists to see what stimulates growth and, equally important, what inhibits growth. The technology was first developed for Escherichia coli, but there are now protocols for testing of more than 1000 bacterial species, as well as most yeast and filamentous fungi. It was the first time to apply PM technology to spirochete, and we set up a new protocol for leptospira. This study conducted on comparative analysis the phenotypic differences of pathogenic Leptospira interrogans serovar Lai and non-pathogenic Leptospira (Leptospira interrogans serovar lai mutant Loa22 and Leptospira biflexa, a free-living saprophytic spirochete). The results showed that there is little difference phenotypes between Lai stain and its mutant Loa22 mutant on PH, osmotic and ion and chemical sensitive, while there are many phenotypes difference on C, N, S and P metabolism and biosynthesis pathways. We found that all aspects of these plates have several different phenotypes after comparative of Leptospira Biflexa and Lai strain and Loa22 mutant strain. All of the different phenotypes created a clue for searching the virulence related pathways. Meanwhile, the data of chemicals sensitive related phenotypes can be used for clinical application. The negative wells antibiotics can be used for treatment leptospirosis, while the positive antibiotics can be combined several components together for the clinical leptospira purification and culture.In conclusion, Since the diagnostic is limited, vaccine is still one of the best choices for controlling this disease. Recently, we fused these ECM high bonding domains of LigB and Lipl32, used them as vaccine candidates and get a better protection results. Meantime, the N terminus of LigC (LigC-J and LigC-K) also has protection. And the mechanism of the recombinant proteins of LigC-N-terminus need to be elucidate in the future. PM results showed the difference phenotypes between the pathogenic Lai strains and non-pathogenic strain. The differences phenotypes can aid us to find the virulence related signal pathways, which can explain why the Loa22 mutant is avirulence. To achieve this goal, we are working on the cDNA microarray experiment, which can explain it from transcriptional levels.