Immunoprotection of recombinant leptospiral immunoglobulin-like protein A against Leptospira interrogans serovar Pomona infection

Abstract

We previously reported the cloning and characterization of leptospiral immunoglobulin-like proteins LigA and LigB of Leptospira interrogans. LigA and LigB are conserved at the amino-terminal region but are variable at the carboxyl-terminal region. Here, we evaluate the potential of recombinant LigA (rLigA) as a vaccine candidate against infection by L. interrogans serovar Pomona in a hamster model. rLigA was truncated into conserved (rLigAcon) and variable (rLigAvar) regions and expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (rLigA). Golden Syrian hamsters were immunized at 3 and 6 weeks of age with rLigA (rLigAcon and rLigAvar) with aluminum hydroxide as an adjuvant. Hamsters given recombinant glutathione-S-transferase (rGST)-adjuvant and phosphate-buffered saline-adjuvant served as nonvaccinated controls. Three weeks after the last vaccination, all animals were challenged intraperitoneally with 10(8) L. interrogans serovar Pomona bacteria (NVSL 1427-35-093002). All hamsters immunized with recombinant LigA survived after challenge and had no significant histopathological changes. In contrast, nonimmunized and rGST-immunized hamsters were subjected to lethal doses, and the hamsters that survived showed severe tubulointerstitial nephritis. All vaccinated animals showed a rise in antibody titers against rLigA. Results from this study indicate that rLigA is a potential vaccine candidate against L. interrogans serovar Pomona infection.

FIG. 1.

Expression and purification of conserved and variable regions of LigA. The conserved and variable regions of LigA were cloned into pGEX4T-2 and expressed as GST fusion proteins as described in Materials and Methods. GST fusion protein was purified by affinity chromatography and subjected to SDS-PAGE followed by Coomassie blue staining. (A) Expression of the conserved region of LigA. (B) Expression of the variable region of LigA. Lane 1, E. coli with vector and pGEX4T-2 only (control); lane 2, uninduced E. coli with recombinant construct; lane 3, IPTG-induced E. coli with recombinant construct; lane 4, affinity chromatography-purified GST fusion proteins (∼1.5 μg). M represents the molecular size marker in kilodaltons (Bio-Rad).

FIG. 2.

Serum IgG responses in hamsters after two subcutaneous injections at 3-week intervals with recombinant proteins. Sera from hamsters immunized with 50 μg of recombinant protein (rCon-GST and rVarA-GST), rGST, or PBS were diluted 1:100 and subjected to KELA with rCon-GST (A), rVarA-GST (B), and rGST. The reactivity of hamsters to rLigA was determined by subtracting the reactivity of rGST. Day 0 (D0) represents preimmune sera, D21 and D42 indicate serum samples after the first (D21) and second (D42) immunizations, and D71 represents serum samples after challenge (29 days postinfection). Expt, experiment.

FIG. 3.

Histopathological analysis of hamster tissues. (A) Kidney from hamster vaccinated with rLigA and challenged with 10⁸ L. interrogans serovar Pomona organisms by intraperitoneal injection. Note the normal renal architecture. Bar = 200 μm. (B) High-magnification view of cortex from panel A. Bar = 10 μm. (C) Kidney from hamster vaccinated with rGST and challenged with 10⁸ L. interrogans serovar Pomona organisms by intraperitoneal injection. The renal profile is distorted due to atrophy of proximal convoluted tubules and interstitial fibrosis. Bar = 200 μm. (D) High-magnification view of cortex from panel C. The interstitium is infiltrated by small numbers of lymphocytes and plasma cells (long arrow). Occasionally, tubules are dilated and lined by a moderately attenuated epithelium (short arrow). Bowman's capsules of glomeruli are dilated, and the uriniferous spaces are filled with a faintly eosinophilic lacy material (upper right corner). Bar = 10 μm. All tissues were stained with hematoxylin and eosin.