Region Liga
Pamula Harrison
The leptospiral LigA protein consists of 13 bacterial immunoglobulin-like (Big) domains and is the only purified recombinant subunit vaccine that has been demonstrated to protect against lethal challenge by a clinical isolate of Leptospira interrogans in the hamster model of leptospirosis. We determined the minimum number and location of LigA domains required for immunoprotection. Immunization with domains 11 and 12 was found to be required but insufficient for protection. Inclusion of a third domain, either 10 or 13, was required for 100% survival after intraperitoneal challenge with Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130. As in previous studies, survivors had renal colonization; here, we quantitated the leptospiral burden by qPCR to be 1.2103 to 8105 copies of leptospiral DNA per microgram of kidney DNA. Although renal histopathology in survivors revealed tubulointerstitial changes indicating an inflammatory response to the infection, blood chemistry analysis indicated that renal function was normal. These studies define the Big domains of LigA that account for its vaccine efficacy and highlight the need for additional strategies to achieve sterilizing immunity to protect the mammalian host from leptospiral infection and its consequences.
Leptospirosis is the most widespread bacterial infection transmitted to humans from host animals that harbor the bacteria in their kidneys. Human infections caused by the bacterium, Leptospira interrogans, frequently result in a life-threatening illness characterized by jaundice and kidney failure. Vaccines are urgently needed to prevent leptospirosis in populations at risk. The leptospiral protein, LigA, is a promising vaccine candidate because it is the first purified protein to be shown to protect animals from fatal leptospirosis. The goal of this study was to determine which of LigA's 13 domains are required for the protective effect. Immunization with domains 11 and 12 was found to be required, but was insufficient, for protection. A third domain, either 10 or 13, was required for 100% survival. As in previous studies, residual bacteria were cultured from the kidneys of survivors. However, in contrast to previous studies, we determined the amount of bacterial DNA in the kidneys as a measure of vaccine efficacy. We also examined the kidneys microscopically for signs of damage and measured blood chemistries to assess kidney function. These are important steps towards developing vaccines that provide protection from kidney damage and infection.
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Funding: This study was supported by Veterans Affiars Medical Research Funds (to D.A.H. and J.M.) and Public Health Service grant AI-034431 (to D.A.H.) from the National Institute of Allergy and Infectious Diseases. M.L.C. received support from Global Infectious Diseases Research Training Program Award 7D43TW000919. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Recombinant surface-exposed outer membrane proteins (OMPs) are attractive subunit vaccine candidates because in contrast to the lipopolysacchride, leptospiral OMPs are relatively well conserved and those that are surface-exposed represent potential targets for immune-mediated defense mechanisms. We have developed a suite of complementary approaches for determining which leptospiral OMPs are surface-exposed, including surface immunofluorescence, surface biotinylation, surface proteolysis, surface immunoprecipitation, and surface ELISA [12], [13], [14], [15]. Using these approaches, a number of transmembrane OMPs and surface lipoproteins have been identified [16], [17]. Despite the rapid increase in knowledge about leptospiral OMPs, progress in understanding their vaccine potential has been slow. Although LipL32 is the most abundant pathogenic leptospiral OMP [18], purified, recombinant LipL32 has no detectable vaccine efficacy [19]. Nevertheless, hamsters immunized with recombinant bacillus Calmette-Guerin expressing LipL32 were partially protected from lethal challenge [20] and there is evidence for immunoprotection employing lipL32-containing viral or DNA-based vectors [21], [22]. Synergistic immunoprotection has been observed using a combination of leptospiral OMPs, OmpL1 and lipidated LipL41, expressed as membrane proteins in E. coli [23].
Leptospiral immunoglobulin-like (Lig) proteins are of great interest as mediators of leptospiral pathogenetic mechanisms, as serodiagnostic antigens, and as effective recombinant vaccinogens [24], [25], [26], [27], [28]. At least two of the three members of the Lig protein family are outer membrane lipoproteins containing a tandem series of bacterial immunoglobulin-like (Big) domains [29]. Lig protein expression is associated with virulence and is strongly and rapidly induced by increasing the osmolarity of the culture medium to physiologic levels found in the mammalian host, suggesting that they may be involved in the initial stages of host tissue colonization [30], [31]. LigA consists of 13 Big domains, the first six of which are nearly identical in sequence to those in LigB, while the last seven are unique to LigA [32] and mediate interactions with host extracellular matrix proteins and fibrinogen [24], [33]. One study has found that the region shared by LigA and LigB was not immunoprotective [27], while another study reported that this region conferred some immunoprotective activity [34]. In contrast, several groups have reported that immunization with the LigA-unique region induced protection from lethal infection either in a mouse model [28] or in the hamster model [27], [35] of leptospirosis. Although hamsters surviving leptospiral challenge were found to have sublethal kidney infection, both the extent of infection and its effects on the kidney, the key target organ in leptospirosis, were not well understood. In this study, we determined which LigA domains are most strongly associated with immunoprotection and the effect of LigA immunization on the burden of infection and the histopathology in the kidney. Our results show that protection from lethal infection required immunization with domains 11 and 12 along with a third domain, either 10 or 13.
L. interrogans serovar Copenhageni strain Fiocruz L1-130 was maintained in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium [36] supplemented with 1% rabbit serum (Rockland Immunochemicals, Gilbertsville, PA) and 100 g/ml 5-fluorouracil at 30C in a shaker incubator. Organisms were passaged no more than five times prior to hamster challenge. Hamster tissues were cultured in semi-solid EMJH or semi-solid Probumin Vaccine Grade Solution (Millipore, Billirica, MA) containing 0.2% Bacto agar (BD, Franklin Lakes, NJ) and 100 g/ml 5-fluorouracil in a stationary incubator at 30C and were examined for leptospiral growth for up to two months.
PCR primers were designed to amplify gene fragments encoding various immunoglobulin-like domains from ligA of L. interrogans serovar Copenhageni strain Fiocruz L1-130 (Table 1). DNA amplicons, which included Nde I and Xho I restriction endonuclease sites, were ligated into pET-20b(+) (Novagen), providing a carboxy-terminal His6 tag, and used to transform Escherichia coli BLR(DE3)pLysS (Novagen). Protein expression was induced with isopropyl-β-D-thiogalactopyranoside at 30C and soluble proteins were released with BugBuster (Novagen) and purified with nickel-affinity chromatography as previously described [25]. All proteins were stored at 4C after dialysis in PBS.
Ninety-six-well ELISA microtiter plates (Immulon 4HBX,Thermo Fisher, Waltham, MA) were coated either with 100 L of 10 g/mL of recombinant LigA protein or 1109 heat-inactivated leptospires/mL diluted in PBS, pH7.2 (Invitrogen, Carlsbad, CA), by overnight incubation at 4C. The plates were allowed to warm to room temperature (RT), washed once with 200 L of PBS, and blocked with Protein-Free Blocking Buffer (PFBB, Thermo Fisher, Rockford, IL) for 1 to 2 h at RT. Wells were washed with PBS, sera diluted with PFBB were added in a volume of 100 L, and plates were incubated for 1 h at 37C. Non-binding antibodies were removed with three PBS washes, and Horseradish peroxidase (HRP)-conjugated anti-Syrian hamster immunoglobulin secondary antibody (Jackson ImmunoResearch, West Grove, PA) 1:5000 was incubated for 30 min at RT. Following three washes with PBS, 100 L of 1-Step Turbo TMB HRP substrate (Thermo Fisher) was added and incubated for 30 min at RT with shaking. The reaction was stopped by the addition of 50 L of 2 M H2SO4, and plates were immediately read in a Bio-Rad 550 Microplate Reader at 450 nm. End-point titers were defined as the highest titer that yielded a reading two standard deviations above the result with sera from PBS-immunized hamsters. Geometric mean end-point titers were calculated as previously described [37].
Sera collected at euthanasia were examined at a 1:50 dilution by MAT as previously described [38] with live L. interrogans serovar Copenhageni strain Fiocruz L1-130. Briefly, heat-inactivated serum, diluted in physiologically buffered water, pH7.6, was incubated overnight at 4C with 2 to 4108 leptospires/mL and examined under dark-field microscopy for >50% reduction in the number of free leptospires when compared with serum from uninfected animals.
Eight clones were designed to express recombinant proteins corresponding to various LigA domains from the second half of domain 7 to domain 13 (Table 1) of L. interrogans serovar Copenhageni. All proteins were expressed and purified as soluble proteins and found to be stable at 4C after dialysis in sterile PBS. These proteins were employed as hamster immunogens in two independent experiments (#1 and #2) and as antigens in an indirect ELISA to measure the corresponding antibody response. As shown in Figure 1, hamsters had higher antibody titers after the third immunization than after one or two immunizations (one-way ANOVA with test for linear trend, P0.05).
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