Distinct antibody responses of patients with mild and severe leptospirosis determined by whole proteome microarray analysis

Abstract

Background: Leptospirosis is an important zoonotic disease worldwide. Humans usually present a mild non-specific febrile illness, but a proportion of them develop more severe outcomes, such as multi-organ failure, lung hemorrhage and death. Such complications are thought to depend on several factors, including the host immunity. Protective immunity is associated with humoral immune response, but little is known about the immune response mounted during naturally-acquired Leptospira infection.

Methods and principal findings: Here, we used protein microarray chip to profile the antibody responses of patients with severe and mild leptospirosis against the complete Leptospira interrogans serovar Copenhageni predicted ORFeome. We discovered a limited number of immunodominant antigens, with 36 antigens specific to patients, of which 11 were potential serodiagnostic antigens, identified at acute phase, and 33 were potential subunit vaccine targets, detected after recovery. Moreover, we found distinct antibody profiles in patients with different clinical outcomes: in the severe group, overall IgM responses do not change and IgG responses increase over time, while both IgM and IgG responses remain stable in the mild patient group. Analyses of individual patients' responses showed that >74% of patients in the severe group had significant IgG increases over time compared to 29% of patients in the mild group. Additionally, 90% of IgM responses did not change over time in the mild group, compared to ~51% in the severe group.

Conclusions: In the present study, we detected antibody profiles associated with disease severity and speculate that patients with mild disease were protected from severe outcomes due to pre-existing antibodies, while patients with severe leptospirosis demonstrated an antibody profile typical of first exposure. Our findings represent a significant advance in the understanding of the humoral immune response to Leptospira infection, and we have identified new targets for the development of subunit vaccines and diagnostic tests.

Fig 1. Overall IgM and IgG antibody recognition of leptospiral proteins.

(A) Venn diagrams of IgM and IgG leptospiral proteins recognized by humans. Overlap of IgM (B) or IgG (C) sero-reactive antigens identified in patients with mild and severe leptospirosis.

Fig 2. Serodiagnostic antigens identified for patients with mild and severe leptospirosis.

Histograms plot the average normalized intensity (Y axis) of each antigen (X axis) for hyperendemic controls (dark gray bars) and patients with mild (A) or severe (B) disease (light gray bars), with the frequency of responsive individuals (black line, secondary axis). Error bars indicate S.E. Single or multi-antigens ROC curves of the identified serodiagnostic antigens for mild (A) or severe (B) groups are shown with sensitivity and specificity rates.

Fig 3. Validation of microarray results by MAPIA.

(A) MAPIA strips probed for specific IgG in leptospirosis patients and endemic controls. Strips are grouped by disease severity, in acute and convalescent phases. (B) SDS PAGE of the 6 purified recombinant proteins that were applied to MAPIA strips. (C) ROC curves of the combination of all 6 antigens are shown for mild and severe groups at both acute and convalescent phases.

Fig 4. IgM and IgG antibody kinetics in patients with mild and severe leptospirosis.

Percentage of patients that showed increase, decrease or unchanged IgG (A) and IgM (B) levels from acute to convalescent phases. Patients with severe disease are shown on the left and patients with mild form, on the right. (C) Boxplot shows the IgG fold-change (y-axis) of mild (dark gray) and severe (light gray) groups for each of the antigens in the x-axis. Significant differences are marked with star (*p<0.01; **p<0.001).