Proteomic Features Predict Seroreactivity against Leptospiral Antigens in Leptospirosis Patients

With increasing efficiency, accuracy, and speed we can access complete genome sequences from thousands of infectious microorganisms; however, the ability to predict antigenic targets of the immune system based on amino acid sequence alone is still needed. Here we use a <i>Leptospira interrogans</i> microarray expressing 91% (3359) of all leptospiral predicted ORFs (3667) and make an empirical accounting of all antibody reactive antigens recognized in sera from naturally infected humans; 191 antigens elicited an IgM or IgG response, representing 5% of the whole proteome. We classified the reactive antigens into 26 annotated COGs (clusters of orthologous groups), 26 JCVI Mainrole annotations, and 11 computationally predicted proteomic features. Altogether, 14 significantly enriched categories were identified, which are associated with immune recognition including mass spectrometry evidence of in vitro expression and in vivo mRNA up-regulation. Together, this group of 14 enriched categories accounts for just 25% of the leptospiral proteome but contains 50% of the immunoreactive antigens. These findings are consistent with our previous studies of other Gram-negative bacteria. This genome-wide approach provides an empirical basis to predict and classify antibody reactive antigens based on structural, physical–chemical, and functional proteomic features and a framework for understanding the breadth and specificity of the immune response to <i>L. interrogans</i>

Pathological findings in the hamster model.

<p>Animals vaccinated with rLigB(131–645) (A, C, E and G) or the PBS control group (B, D, F and H) were euthanized 10 days PC and tissue samples were collected. Vaccinated animals showed no gross pulmonary lesions (A) or microscopic pulmonary lesions (C). Liver (E) and kidney samples (G) showed no evidence of microscopic abnormalities. Unvaccinated animals showed gross pulmonary haemorrhaging (B) and they were confirmed to be alveolar haemorrhages by microscopic analysis (D). Dystrabeculaton (loss of cohesion) of hepatocytes (F) and swelling of kidney tubular epithelial cells (H) were prominent features. (C-F, haematoxylin-eosin, 100× magnification and G-H, haematoxylin-eosin, 200× magnification).</p

Protection conferred by immunization with rLigB(131–645) against lethal challenge in the hamster model of leptospirosis.

<p>Protection conferred by immunization with rLigB(131–645) against lethal challenge in the hamster model of leptospirosis.</p

Schematic of the Lig proteins, expression and purification of rLigB(131–645).

<p>A) The full length amino acid sequences for LigA (1224 amino acids, 128.1 kDa) and LigB (1890 amino acids, 200.8 kDa) are indicated (black line), the square boxes indicate the BIDs and the LigB C-terminal domain is shown (rectangle). The recombinant proteins used as vaccine candidates are indicated: LigB(131–645) (green boxes) includes amino acids 131–645 (53.5 kDa) and is highly identical (97.9% pairwise identity) to the same region in LigA; the LigA(631–1224), also known as LigANI, (red boxes, amino acids 631–1224, 62.8 kDa) and LigB(625–1259), also known as LigBNI, (blues boxes, amino acids 625–1259, 66.2 kDa) fragments are not highly conserved (38.1% pairwise identity). B) Expression and purification of rLigB(131–645) analysed by 10% SDS-PAGE and Coomassie staining. Lanes 1: molecular mass marker (kDa); Expression of rLigB(131–645) in an <i>E</i>. <i>coli</i>(pLigB(131–645)) clone, lane 2: supernatant (soluble) fraction and lane 3: insoluble fraction; lane 4: IMAC purified rLigB(131–645), expected molecular mass of 57.2 kDa. C) Immunoblot analysis of rLigB(131–645), following transfer the nitrocellulose membrane was probed with an anti-His-HRP antibody, lane 1: molecular mass marker (kDa); lane 2: purified rLigB(131–645).</p

Accuracy of a Dual Path Platform (DPP) Assay for the Rapid Point-of-Care Diagnosis of Human Leptospirosis

<div><h3>Background</h3><p>Diagnosis of leptospirosis by the gold standard serologic assay, the microscopic agglutination test (MAT), requires paired sera and is not widely available. We developed a rapid assay using immunodominant <em>Leptospira</em> immunoglobulin-like (Lig) proteins in a Dual Path Platform (DPP). This study aimed to evaluate the assay's diagnostic performance in the setting of urban transmission.</p> <h3>Methodology</h3><p>We determined test sensitivity using 446 acute and convalescent sera from MAT-confirmed case-patients with severe or mild leptospirosis in Brazil. We assessed test specificity using 677 sera from the following groups: healthy residents of a Brazilian slum with endemic transmission, febrile outpatients from the same slum, healthy blood donors, and patients with dengue, hepatitis A, and syphilis. Three operators independently interpreted visual results without knowing specimen status.</p> <h3>Results</h3><p>The overall sensitivity for paired sera was 100% and 73% for severe and mild disease, respectively. In the acute phase, the assay achieved a sensitivity of 85% and 64% for severe and mild leptospirosis, respectively. Within seven days of illness onset, the assay achieved a sensitivity of 77% for severe disease and 60% for mild leptospirosis. Sensitivity of the DPP assay was similar to that for IgM-ELISA and increased with both duration of symptoms (chi-square regression P = 0.002) and agglutinating titer (Spearman ρ = 0.24, <em>P</em><0.001). Specificity was ≥93% for dengue, hepatitis A, syphilis, febrile outpatients, and blood donors, while it was 86% for healthy slum residents. Inter-operator agreement ranged from very good to excellent (kappa: 0.82–0.94) and test-to-test reproducibility was also high (kappa: 0.89).</p> <h3>Conclusions</h3><p>The DPP assay performed acceptably well for diagnosis of severe acute clinical leptospirosis and can be easily implemented in hospitals and health posts where leptospirosis is a major public health problem. However, test accuracy may need improvement for mild disease and early stage leptospirosis, particularly in regions with high transmission.</p> </div

Specificity of the DPP assay and IgM-ELISA for diagnosing human leptospirosis.

<p><b>NOTE.</b> DPP = Dual Path Platform; ELISA = enzyme-linked immunosorbent assay; ND = not determined.</p>*<p>Samples obtained from individuals in Salvador, Brazil.</p>†<p>Outpatients with acute fever (microagglutination titer <1∶100 in both acute and convalescent samples).</p

DPP, IgM-ELISA, and 1∶100 MAT sensitivity and mean MAT titer for acute-phase sera of human leptospirosis.

<p>Acute-phase sera from (A) 282 MAT-confirmed severe leptospirosis case-patients (259 from Salvador and 23 from Recife, Brazil) and from (B) 28 MAT-confirmed mild leptospirosis case-patients from Salvador. 95% confidence intervals calculated for point estimates of sensitivity and the geometric mean (± geometric standard deviation) calculated for reciprocal MAT titers. DPP = Dual Path Platform; MAT = microagglutination test; ELISA = enzyme-linked immunosorbant assay; ND = not determined because no case-patients were identified within the corresponding time interval.</p

Representative non-reactive, strongly reactive, and weakly reactive DPP assay results.

<p>Demonstration of (A) non-reactive, (B) strongly reactive, and (C) weakly reactive visual interpretations for the DPP assay. Coloration of the test line (“T” on rapid test cartridge) indicates the presence of anti-rLig antibodies in the biological sample while coloration of the control line (“C” on rapid test cartridge) indicates the presence of non-specific antibodies and denotes a valid test result. DPP = Dual Path Platform.</p

Sensitivity of the DPP assay and IgM-ELISA for diagnosing human leptospirosis.

<p><b>NOTE.</b> DPP = Dual Path Platform assay; ELISA = enzyme-linked immunosorbent assay.</p>*<p>Random samples of acute and convalescent specimens from severe disease case-patients from Salvador were selected independently, after which 42 serum pairs from individual case-patients were identified.</p>†<p>Represents 5 case-patients with paired sera as convalescent samples from severe cases from Recife were generally not available for testing.</p>‡<p>Represents 26 mild disease case-patients with paired sera.</p