Immunoprotective Activity Induced by Leptospiral Outer Membrane Proteins in Hamster Model of Acute Leptospirosis

Abstract

Leptospirosis is a zoonotic disease of worldwide distribution, affecting both humans and animals. The development of an effective vaccine against leptospirosis has long been pursued but without success. Humans are contaminated after direct contact with the urine of infected animals or indirectly by contaminated water or soil. The vaccines available consist of inactivated whole-bacterial cells, and the active immunoprotective antigen is the lipopolysaccharide moiety, which is also the basis for serovar classification. However, these vaccines are short-lasting, and protection is only against serovars contained in the preparation. The search for prevalent antigens, present in pathogenic species of Leptospira, represents the most cost-effective strategy for prevention of leptospirosis. Thus, the identification of these antigens is a priority. In this study, we examined the immunoprotective effect of eight leptospiral recombinant proteins using hamster as the challenge model. Animals received subcutaneously two doses of vaccine containing 50 μg of each recombinant protein adsorbed on alum adjuvant. Two weeks after the booster, animals were challenged with virulent leptospires and monitored for 21 days. All proteins were able to induce a specific immune response, although significant protective effects on survival rate were observed only for the proteins Lsa14, rLIC13259, and rLIC11711. Of these, only rLIC13259 and rLIC11711 were found to be highly prospective in promoting renal clearance. The sterilizing potential of both proteins will be further investigated to elucidate the immunoprotective mechanisms involved in leptospirosis control. These are the first proteins involved with human complement components with the capacity to protect against virulent challenge and to eliminate the bacteria from the host.

Keywords: Leptospira; immunogenicity; immunoprotection; leptospirosis; recombinant proteins; vaccine.

Figure 1

Scheme of predicted coding sequences and purification analysis of each recombinant protein. (A) Left panel: the diagram represents the linear sequence of the proteins and its respective conserved domains and/or features, predicted by amino acids sequence analysis. The presence signal peptide sequence shown in red is representative, all genes were cloned without this portion. Right panel: the diagram represents the extracellular matrix and plasma molecules that bound to the respective recombinant proteins, experimentally characterized in vitro. (B) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified recombinant proteins and immunoblotting probed with anti-his tag antibody. Lane 1: molecular mass protein marker; lane 2: Lsa25.6; lane 3: Lsa16; lane 4: LipL46; lane 5: Lsa14; lane 6: Lsa19; lane 7: Lsa24.9; lane 8: rLIC11711 and lane 9: rLIC13259.

Figure 2

Total IgG production after animal immunization. Hamsters were immunized subcutaneously with emulsions of recombinant protein plus Alhydrogel, twice at 2 weeks intervals. Serum samples were obtained after 2 weeks of each immunization and IgG levels evaluated by ELISA. For experimental control, animals were immunized with phosphate-buffered saline (PBS) in Alhydrogel (control) and with heat-killed whole-leptospires (bacterin). Unpaired t-test was used to determine statistically significant difference between proteins and PBS. All of them showed significance < 0.05.

Figure 3

Evaluation of IgG isotypes produced after immunization with each recombinant protein. ELISA plates were coated with each recombinant protein and incubated with increasing concentrations of hamsters’ sera, obtained after immunizations with the respective recombinant proteins. The detection of IgG isotypes was obtained by incubation with HRP-conjugated anti-hamster IgG1 or anti-hamster IgG2/IgG3 (1:5,000) and readings were taken at 492 nm. One-way ANOVA was performed to analyze the difference between IgG1 or IgG2/3 and protein groups. For both cases significance was <0.05.

Figure 4

Survival curves of immunized hamsters after challenge with Leptospira interrogans. Virulent leptospires (10⁴) were inoculated intraperitoneally in hamsters, previously immunized with recombinant proteins, and monitored daily for 21 days, inspecting the clinical signs of leptospirosis. Animal groups immunized with PBS (control) and bacterin were used as negative and positive controls, respectively. The graphs illustrate the survival curves obtained for Lsa25.6, Lsa16, LipL46, and Lsa14 (A) and Lsa19, rLIC11711, Lsa24.9, and rLIC13259 (B) in two independent experiments (left and middle) and the average result (right).

Figure 5

Protective effect elicited in hamster by recombinant proteins assessing the presence or absence of leptospires in animal kidneys after the challenge. Kidneys were removed from euthanized hamsters in 21 days-post challenges with Leptospira interrogans and the resulting macerate was incubated in liquid Ellinghausen-McCullough-Johnson-Harris (EMJH) medium and monitored for 30 days. The presence or absence of leptospires in cultures was determined as positive and negative culture, respectively. The graph represents the percentages obtained of two experiments.