Natural immunogenic properties of bioinformatically predicted linear B-cell epitopes of dengue envelope and pre-membrane proteins.

BACKGROUND: The natural antibody responses to B-cell epitopes from dengue structural proteins were assessed using immune sera from people having well-defined past dengue infections with one of the four serotypes. METHOD: Based on an immune-computational analysis previously conducted, nineteen epitopes from the envelope (E) and eight epitopes from pre-membrane (prM), which were more than 50% conserved across all the four DENV serotypes, were selected. Peptides to represent these B-cell epitopes were obtained from commercially available arrays, and were subjected to enzyme linked immunosorbent assay with sera obtained from dengue seropositive healthy volunteers (DENV1 n = 12: DENV2 n = 12: DENV3 n = 12 and DENV4 n = 12), and 10 dengue seronegative healthy volunteers from Sri Lanka. The cut-off value for the positive antibody response was set by taking the mean response of a peptide to the negative sera plus three standard deviations. The peptides (N = 7) showing the broad immune responses were used to generate antibodies in three mice (Balb/c) batches. The mice antisera were then subjected to microneutralization assays against all the four DENV serotypes. An EC50 viral neutralization ≥ 40 times the serum dilution was considered as neutralizing. RESULTS: Five of the E-peptide and two prM peptides were recognised by most individuls exposed to infections with each of the four serotypes, showing a serotype cross-reactive broad antibody response. The mice immune sera against the peptides representing the five E protein epitopes neutralized all the four DENV serotypes. Two of these five epitopes are from the Domain II, whereas one of them includes the whole bc-loop region. CONCLUSION: The antibody responses of highly conserved epitopes across the serotypes, were broadly responsive with sera of all four DENV serotypes collected from individuals infected with only one DENV serotype. Weakly conserved epitopes showed rather specific antibody responses dominated by one or few serotypes.

Prevalence of cutaneous leishmaniasis infection and clinico-epidemiological patterns among military personnel in Mullaitivu and Kilinochchi districts of the Northern Province, early war-torn areas in Sri Lanka.

BACKGROUND: The 30-year-old armed conflict in Sri Lanka resulted in a general breakdown of civil administration in the Northern and Eastern provinces, leading to mobilisation of many armed forces personnel to assist with reconstruction and resettlement. This occupational group has been identified as a priority risk group for leishmaniasis. METHODS: Individuals enlisted at all military establishments in Mullaitivu and Kilinochchi districts, Northern Province of Sri Lanka were included. Five thousand individuals were screened for skin lesions between September 2018 and August 2019. Persons with lesions suspected as cutaneous leishmaniasis (CL) were further investigated. Information on sociodemographic/other potential risk factors was obtained through an interviewer-administered structured questionnaire. The diagnosis was confirmed by microscopic visualization of parasitic stages from different samples obtained (skin scraping, lesion aspirate and tissue impression smears), histopathology and polymerase chain reaction DNA amplification. RESULTS: Among 5000 individuals screened, 74 individuals were suspected of having CL. Of these, 67.6% (n = 50) patients were confirmed for CL by microscopy. Around two third of both males (67.6%; n = 48) and females (66.6%; n = 2) were positive for Leishmania. The soldiers belonging to 26-35-year age group reported the highest susceptibility (83.3%; OR: 4.83, 95% CI: 3.49-6.20%). Of the sociodemographic factors, age, wearing short-sleeved upper body clothing as the uniform and non-use of insect repellents were identified as significant risk factors. Most of the CL patients had a single lesion (86.0%; n = 43) of an ulcerative type (34.0%; n = 17), mostly on their upper limb (67.9%; n = 34). Lesions were mostly 5-10 mm diameter (59.9%; n = 30) in size with poorly defined margins (72.0%; n = 36). Amongst the diagnostic techniques, microscopic examination of slit skin smear and tissue impression smear were able to discriminate the majority of patients (92.1%; n = 46) for CL. CONCLUSIONS: In order to highlight the true burden of leishmaniasis in the military personnel, cases of leishmaniasis from military institutes should be recognized as a different entity per say and be included in the national figures so as to depict the real magnitude of the disease burden amongst this high-risk group.

A surveillance system for lymphatic filariasis after its elimination in Sri Lanka.

Lymphatic filariasis (LF) has been declared eliminated in Sri Lanka in September 2016. To maintain elimination status, a surveillance system to detect hidden endemic foci or LF resurgence is of highest priority. In this paper, we have reported an investigation of LF transmission in Trincomalee district where a surveillance program was not carried out due to 30 years of civil unrest. Proposed surveillance system included, measurement of anti-filarial IgG4 in urine of schoolchildren in areas where LF transmission could exist and assessment of circulating filarial antigen (CFA) and microfilaria (mf) in all urine antibody positive schoolchildren, their family members and 10-15 neighbours of each urine antibody positive household. Spatial distribution of the anti-filarial antibody titers in urine in a high antibody suspected area was analyzed using GPS logger data. Among 2301 school children from 11 schools studied, 41 (1.8%) urine antibody positives were found. The antibody positive rates of the schools ranged between 0 and 4.0%. Nine of the 630 (1.4%) examined became positive for CFA but were negative for mf. Although there were no mf positives, positive CFA and antibody results indicated the existence of Wuchereria bancrofti in Trincomalee. Highest antibody titres in an area correlated with the prevalences of urine antibodies and CFA. Spatial analysis showed LF transmission foci. Therefore, a combination of the non-invasive methods, urine ELISA and GPS mapping, will be a new effective surveillance system to identify hidden LF transmission foci.

Natural antibody responses to the capsid protein in sera of Dengue infected patients from Sri Lanka.

This study aims to characterize the antigenicity of the Capsid (C) protein and the human antibody responses to C protein from the four dengue virus (DENV) serotypes. Parker hydrophilicity prediction, Emini surface accessibility prediction and Karplus & Schulz flexibility predictions were used to bioinformatically characterize antigenicity. The human antibody response to C protein was assessed by ELISA using immune sera and an array of overlapping DENV2 C peptides. DENV2 C protein peptides P1 (located on C protein at 2-18 a.a), P11 (79-95 a.a) and P12 (86-101 a.a) were recognized by most individuals exposed to infections with only one of the 4 DENV serotypes as well as people exposed to infections with two serotypes. These conserved peptide epitopes are located on the amino (1-40 a.a) and carboxy (70-100 a.a) terminal regions of C protein, which were predicted to be antigenic using different bioinformatic tools. DENV2 C peptide P6 (39-56 a.a) was recognized by all individuals exposed to DENV2 infections, some individuals exposed to DENV4 infections and none of the individuals exposed to DENV1 or 3 infections. Thus, unlike C peptides P1, P11 and P12, which contain epitopes, recognized by DENV serotype cross-reactive antibodies, DENV2 peptide P6 contains an epitope that is preferentially recognized by antibodies in people exposed to this serotype compared to other serotypes. We discuss our results in the context of the known structure of C protein and recent work on the human B-cell response to DENV infection.

Systematic Bioinformatic Approach for Prediction of Linear B-Cell Epitopes on Dengue E and prM Protein.

B-cell epitopes on the envelope (E) and premembrane (prM) proteins of dengue virus (DENV) were predicted using bioinformatics tools, BepiPred, Ellipro, and SVMTriP. Predicted epitopes, 32 and 17 for E and prM proteins, respectively, were then characterized for their level of conservations. The epitopes, EP4/E (48-55), epitope number 4 of E protein at amino acids 48-55, EP9/E (165-182), EP11/E (218-233), EP20/E (322-349), EP21/E (326-353), EP23/E (356-365), and EP25/E (380-386), showed a high intraserotype conservancy with very low pan-serotype conservancy, demonstrating a potential target as serotype specific diagnostic markers. EP3 (30-41) located in domain-I and EP26/E (393-409), EP27/E (416-435), EP28/E (417-430) located in the stem region of E protein, and EP8/prM (93-112) from the prM protein have a pan-serotype conservancy higher than 70%. These epitopes indicate a potential use as universal vaccine candidates, subjected to verification of their potential in viral neutralization. EP2/E (16-21), EP5/E (62-123), EP6/E (63-89), EP19/E (310-329), and EP24/E (371-402), which have more than 50% pan-serotype conservancies, were found on E protein regions that are important in host cell attachment. Previous studies further show evidence for some of these epitopes to generate cross-reactive neutralizing antibodies, indicating their importance in antiviral strategies for DENV. This study suggests that bioinformatic approaches are attractive first line of screening for identification of linear B-cell epitopes.

A comparison of two tests for filarial antigenemia in areas in Sri Lanka and Indonesia with low-level persistence of lymphatic filariasis following mass drug administration.

BACKGROUND: Filarial antigen tests are key tools for mapping the distribution of bancroftian filariasis and for detecting areas with persistent infections following mass drug administration (MDA). A recent study showed that the new Alere Filariasis Test Strip (FTS) has better analytical sensitivity than the BinaxNOW Filariasis card test (Card Test) for detecting circulating filarial antigen, and the FTS detected more positive results than the Card Test in a field study performed in a highly endemic area in Liberia. METHODS: The present study compared the performance of the FTS and the Card Test in community surveys that were conducted in southern Sri Lanka and in Indonesia (Central Java) in areas with low-level persistence of LF following multiple rounds of MDA with diethylcarbamazine plus albendazole. The studies were performed in densely populated semi-urban areas where Wuchereria bancrofti is transmitted by Culex quinquefasciatus. RESULTS: Antigenemia rates by FTS were 138% higher in the Sri Lanka study (43/852 vs. 18/852) and 21% higher in the Indonesia study (50/778 vs. 41/778) than antigenemia rates by Card Test. Antigenemia rates were significantly higher in males than in females and higher in adults than in children in both study sites. Although overall antigenemia rates and test scores were significantly higher by FTS than by Card Test in both study areas, rates in young children were similar with both tests in both areas. CONCLUSIONS: These results extend the previously reported superior sensitivity of the FTS to areas with low residual infection rates following MDA, and this could affect mapping and post-MDA survey results in adults. However, our findings suggest that results of transmission assessment surveys (TAS) performed in school-aged children are likely to be similar with both tests.

Genetic diversity of Plasmodium vivax Duffy Binding Protein II (PvDBPII) under unstable transmission and low intensity malaria in Sri Lanka.

Elucidating the genetic diversity of the Duffy Binding Protein II (PvDBPII), a leading vaccine candidate for vivax malaria, in different geographical settings is vital. In Sri Lanka malaria transmission is unstable with low intensity. A relatively high level of allelic diversity, with 27 polymorphic nucleotides and 33 (aa) haplotypes was detected among the PvdbpII gene in 100 local Plasmodium vivax isolates collected from two hypoendemic areas, and from a non endemic area of the country. Mutations, recombination and balancing selection seem to maintain the observed local allelic diversity of PvdbpII. Lack of gene flow was evidenced by high Fst values between the two endemic study sites. Some of the aa polymorphisms may alter the binding and expression capacity of predicted T cell epitopes in PvDBPII. Of the 8 binding inhibitory linear B cell epitopes, 2 (H2 and M1) in the vicinity of the exact binding region of PvDBPII appeared to be highly conserved in Sri Lankan, Iran and Colombian isolates, while H3, M2, M3 and L3 neutralizing epitopes seem to be polymorphic globally, with H1 and L2 conserved in Colombian, South Korean and Iran isolates. In comparison to the reference Sal-1 strain, among 402 world-wide isolates (302 global and 100 local), 121 aa polymorphisms and 138 haplotypes were recorded of which 3 aa polymorphisms and 21 haplotypes seem to be unique to Sri Lanka. PvdbpII phylogeny suggests that local P. vivax parasites represent a sample of the global population. The ubiquitous presence of some PvDBPII aa haplotypes among both local and global P. vivax isolates may aid future vaccination strategies based on PvDBPII.

Genetic diversity and selection at the Plasmodium vivax apical membrane antigen-1 (PvAMA-1) locus in a Sri Lankan population.

Plasmodium vivax apical membrane antigen 1 (PvAMA-1) is an important malaria vaccine candidate. We present the first comprehensive analysis of nucleotide diversity across the entire PvAMA-1 gene using a single population sample from Sri Lanka. In contrast to what has been observed at the AMA-1 locus of Plasmodium falciparum, the signature of diversifying selection is seen most strongly in Domain II of PvAMA-1, indicating that the different domains in each species may be subject to varying selective pressures and functional constraints. We also find that recombination plays an important role in generating haplotype diversity at this locus, even in a region of low endemicity such as Sri Lanka. Mapping of diversity and recombination hotspots onto a 3-dimensional structural model of the protein indicates that one surface of the molecule may be particularly likely to bear epitopes for antibody recognition. Regions of this surface that show constrained variability may prove to be promising vaccine targets.

Natural human antibody responses to Plasmodium vivax apical membrane antigen 1 under low transmission and unstable malaria conditions in Sri Lanka.

Plasmodium vivax apical membrane antigen 1, an important malaria vaccine candidate, was immunogenic during natural malaria infections in Sri Lanka, where low transmission and unstable malaria conditions prevail. Antibody prevalence increased with exposure in areas where malaria was or was not endemic. A marked isotype switch to cytophilic (immunoglobulin G1 [IgG1]/IgG3) antibodies was evident with increasing exposure exclusively in residents from areas of endemicity.