A LigA three-domain region protects hamsters from lethal infection by Leptospira interrogans

Abstract

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 1.2×10(3) to 8×10(5) 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.

Figure 1. Antibody response in hamsters immunized with recombinant LigA proteins or heat-killed leptospires (HKL).

Total hamster immunoglobulin responses to immunogens were measured by ELISA. Geometric mean end-point titers and standard deviations (n = 4) are shown after the first (gray), second (blue), and third (black) immunizations. In all cases, pre-immune sera were negative. Recombinant LigA proteins are represented by their domain numbers. Asterisks indicate proteins that provided 100% protection against lethal challenge.

Figure 2. Hamster weight as an end-point for leptospiral infection.

Animals were weighed at the time of challenge and daily thereafter for 28 days. Data are shown for experiment #2. A. Animals immunized with recombinant LigA7′-13 progressively gained weight (lines represent individual animals). B. Control animals sham-immunized with phosphate-buffered saline (PBS) had stable or increasing weights until day 8 or 9 after challenge, after which they lost weight and met the end-point criterion of a 10% weight decrease (lines represent individual animals).

Figure 3. Mapping of the immunoprotective segment of LigA.

Recombinant LigA proteins were tested for protective efficacy. The number of animals surviving (survivors/total) and days to endpoint after challenge are shown. Surviving animals were observed for up to 28 days. Ig-like domains of fully protective proteins are represented by dark symbols with green numbers while Ig-like domains of partially protective and non-protective proteins are represented by white symbols with red numbers.

Figure 4. Percentage weight gain in hamsters immunized with protective immunogens.

Mean and standard deviation (n = 4) of percent weight gain from challenge to 28 days in groups that had 100% survival, including hamsters immunized with recombinant LigA proteins (represented by their domain numbers), heat-killed leptospires (HKL) and a non-immunized and unchallenged control (CTRL) group.

Figure 5. Leptospiral burden in kidney tissue.

Kidney tissue was subjected to DNA extraction and real-time PCR to measure the leptospiral burden per microgram of tissue DNA. Means are depicted as bold horizontal bars along with standard deviations. Animals that survived to 28 days (blue diamonds) had higher leptospiral burdens than those that met end-point criterion early (red circles). Of the groups with 100% survival to 28 days, animals immunized with heat-killed leptospires (HKL) had lower bacterial burdens than those immunized with LigA fragments. LigA-immunized groups that survived up to 28 days were used for statistical comparisons (one-way ANOVA, P<0.05).

Figure 6. Renal histopathology showing tubulointerstitial changes.

Representative PAS-stained kidney sections obtained from hamsters 28 days after leptospiral challenge showing, A. Normal tubular epithelium (40x); B. Moderate tubular damage (40x); C. Severe tubular atrophy (40x); D. Interstitial inflammation (arrow, 40x); E. Tubular scarring with depressed renal capsule (arrow, 4x); and F. Tubular deposition of intensely PAS-positive material consistent with Tamm-Horsfall glycoprotein (arrowheads, 40x).