The expression of CD45RA is generally associated with naive T cells. However, a subset of effector memory T cells re-expresses CD45RA (termed TEMRA) after antigenic stimulation with unknown molecular ...characteristics and functions. CD4 TEMRA cells have been implicated in protective immunity against pathogens such as dengue virus (DENV). Here we show that not only the frequency but also the phenotype of CD4 TEMRA cells are heterogeneous between individuals. These cells can be subdivided into two major subsets based on the expression of the adhesion G protein-coupled receptor GPR56, and GPR56
TEMRA cells display a transcriptional and proteomic program with cytotoxic features that is distinct from effector memory T cells. Moreover, GPR56
TEMRA cells have higher levels of clonal expansion and contain the majority of virus-specific TEMRA cells. Overall, this study reveals the heterogeneity of CD4 TEMRA cells and provides insights into T-cell responses against DENV and other viral pathogens.
Our results highlight for the first time that a significant proportion of cell doublets in flow cytometry, previously believed to be the result of technical artifacts and thus ignored in data ...acquisition and analysis, are the result of biological interaction between immune cells. In particular, we show that cell:cell doublets pairing a T cell and a monocyte can be directly isolated from human blood, and high resolution microscopy shows polarized distribution of LFA1/ICAM1 in many doublets, suggesting in vivo formation. Intriguingly, T cell-monocyte complex frequency and phenotype fluctuate with the onset of immune perturbations such as infection or immunization, reflecting expected polarization of immune responses. Overall these data suggest that cell doublets reflecting T cell-monocyte in vivo immune interactions can be detected in human blood and that the common approach in flow cytometry to avoid studying cell:cell complexes should be re-visited.
The four dengue virus (DENV) serotypes are mosquito-borne flaviviruses of humans. The interactions between DENVs and the human host that lead to asymptomatic, mild, or severe disease are poorly ...understood, in part, because laboratory models are poor surrogates for human DENV disease. Virologists are interested in how the properties of DENVs replicating in people compare with virions propagated on laboratory cell lines, which are widely used for research and vaccine development. Using clinical samples from a DENV type 1 epidemic in Sri Lanka and new ultrasensitive assays, we compared the properties of DENVs in human plasma and after one passage on laboratory cell lines. DENVs in plasma were 50- to 700-fold more infectious than cell culture-grown viruses. DENVs produced by laboratory cell lines were structurally immature and hypersensitive to neutralization by human antibodies compared with DENVs circulating in people. Human plasma and cell culture-derived virions had identical genome sequences, indicating that these phenotypic differences were due to the mature state of plasma virions. Several dengue vaccines are under development. Recent studies indicate that vaccine-induced antibodies that neutralized DENVs in cell culture assays were not sufficient for protecting people from DENV infections. Our results about structural differences between DENVs produced in humans versus cell lines may be key to understanding vaccine failure and developing better models for vaccine evaluation.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Leptospirosis is diagnosed on clinical grounds, and confirmed by microscopic agglutination test (MAT). IgM-ELISA (Serion-Virion) and immunochromatography test (Leptocheck-WB) are two immunodiagnostic ...assays for leptospirosis. Their sensitivity, specificity and applicability in Sri Lanka have not been systematically evaluated.
Clinically diagnosed leptospirosis patients (n = 919) were recruited from three hospitals in the Western Province of Sri Lanka, during June 2012 to December 2013. MAT, IgM-ELISA and Leptocheck-WB were performed on all patient sera. MAT titer of ≥ 400 in single sample, four-fold rise or seroconversion ≥ 100 in paired samples were considered as positive for MAT. For diagnostic confirmation, MAT was performed during both acute and convalescent phases. Anti-leptospiral IgM ≥ 20 IU/ml and appearance of a band in the test window were considered as positive for IgM-ELISA and Leptocheck-WB test respectively. Patients with an alternative diagnosis (n = 31) were excluded. Data analysis was performed using two methods, i) considering MAT as reference standard and ii) using Bayesian latent class model analysis (BLCM) which considers each test as imperfect.
MAT, IgM-ELISA and Leptocheck-WB positivity were 39.8%, 45.8% and 38.7% respectively during the acute phase. Acute-phase MAT had specificity and sensitivity of 95.7% and 55.3% respectively, when compared to overall MAT positivity. IgM-ELISA and Leptocheck-WB had similar diagnostic sensitivity when compared with acute-phase MAT as the gold standard, although IgM-ELISA showed higher specificity (84.5%) than Leptocheck-WB (73.3%). BLCM analysis showed that IgM-ELISA and Leptocheck-WB had similar sensitivities (86.0% and 87.4%), while acute-phase MAT had the lowest sensitivity (77.4%). However, acute-phase MAT had high specificity (97.6%), while IgM-ELISA and Leptocheck-WB showed similar but lower specificity (84.5% and 82.9%).
Both IgM-ELISA and Leptocheck-WB shows similar sensitivities and specificities. IgM-ELISA may be superior to MAT during the acute phase and suitable for early diagnosis of leptospirosis. Leptocheck-WB is suitable as a rapid immunodiagnostic screening test for resource limited settings.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Bats are described as the natural reservoir host for a wide range of viruses. Although an increasing number of bat-associated, potentially human pathogenic viruses were discovered in the past, the ...full picture of the bat viromes is not explored yet. In this study, the virome composition of Miniopterus phillipsi bats (formerly known as Miniopterus fuliginosus bats in Sri Lanka) inhabiting the Wavul Galge cave, Sri Lanka, was analyzed. To assess different possible excretion routes, oral swabs, feces and urine were collected and analyzed individually by using metagenomic NGS. The data obtained was further evaluated by using phylogenetic reconstructions, whereby a special focus was set on RNA viruses that are typically associated with bats. Two different alphacoronavirus strains were detected in feces and urine samples. Furthermore, a paramyxovirus was detected in urine samples. Sequences related to Picornaviridae, Iflaviridae, unclassified Riboviria and Astroviridae were identified in feces samples and further sequences related to Astroviridae in urine samples. No viruses were detected in oral swab samples. The comparative virome analysis in this study revealed a diversity in the virome composition between the collected sample types which also represent different potential shedding routes for the detected viruses. At the same time, several novel viruses represent first reports of these pathogens from bats in Sri Lanka. The detection of two different coronaviruses in the samples indicates the potential general persistence of this virus species in M. phillipsi bats. Based on phylogenetics, the identified viruses are closely related to bat-associated viruses with comparably low estimation of human pathogenic potential. In further studies, the seasonal variation of the virome will be analyzed to identify possible shedding patterns for particular viruses.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Dengue virus (DENV) can cause diseases ranging from dengue fever (DF) to more severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Whether antiviral T cells contribute to the protection ...against or pathogenesis of severe disease is not well defined. Here, we identified antigen-specific IL-10+IFN-γ+ double-positive (DP) CD4 T cells during acute DENV infection. While the transcriptomic signatures of DP cells partially overlapped with those of cytotoxic and type 1 regulatory CD4 T cells, the majority of them were non-cytotoxic/Tr1 and included IL21, IL22, CD109, and CCR1. Although we observed a higher frequency of DP cells in DHF, the transcriptomic profile of DP cells was similar in DF and DHF, suggesting that DHF is not associated with the altered phenotypic or functional attributes of DP cells. Overall, this study revealed a DENV-specific DP cell subset in patients with acute dengue disease and argues against altered DP cells as a determinant of DHF.
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•DENV-specific IL-10+IFN-γ+ DP CD4 T cells are prominent during acute disease•Most DP cell DE genes are non-cytotoxic/Tr1 and include IL21, IL22, CD109, and CCR1•DP cells have similar gene expression in DF and DHF, despite higher frequency in DHF•Disease severity is not associated with altered DP cell phenotype or functionality
Tian et al. identify and characterize antigen-specific IL-10+IFN-γ+ double-positive (DP) CD4 T cells in acute dengue patients. DP cells display similar transcriptomic profiles in mild DF and severe DHF, despite their increased frequency in DHF, suggesting that DHF is not associated with the altered phenotype or functionality of DP cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Sri Lanka after eliminating malaria in 2012, is in the prevention of re-establishment (POR) phase. Being a tropical country with high malariogenic potential, maintaining vigilance is important. All ...malaria cases are investigated epidemiologically and followed up by integrated drug efficacy surveillance (iDES). Occasionally, that alone is not adequate to differentiate Plasmodium falciparum reinfections from recrudescences. This study evaluated the World Health Organization and Medicines for Malaria Venture (MMV) recommended genotyping protocol for the merozoite surface proteins (msp1, msp2) and the glutamate-rich protein (glurp) to discriminate P. falciparum recrudescence from reinfection in POR phase.
All P. falciparum patients detected from April 2014 to December 2019 were included in this study. Patients were treated and followed up by iDES up to 28 days and were advised to get tested if they develop fever at any time over the following year. Basic socio-demographic information including history of travel was obtained. Details of the malariogenic potential and reactive entomological and parasitological surveillance carried out by the Anti Malaria Campaign to exclude the possibility of local transmission were also collected. The msp1, msp2, and glurp genotyping was performed for initial and any recurrent infections. Classification of recurrent infections as recrudescence or reinfection was done based on epidemiological findings and was compared with the genotyping outcome.
Among 106 P. falciparum patients, six had recurrent infections. All the initial infections were imported, with a history of travel to malaria endemic countries. In all instances, the reactive entomological and parasitological surveillance had no evidence for local transmission. Five recurrences occurred within 28 days of follow-up and were classified as recrudescence. They have not travelled to malaria endemic countries between the initial and recurrent infections. The other had a recurrent infection after 105 days. It was assumed a reinfection, as he had travelled to the same malaria endemic country in between the two malaria attacks. Genotyping confirmed the recrudescence and the reinfection.
The msp1, msp2 and glurp genotyping method accurately differentiated reinfections from recrudescence. Since reinfection without a history of travel to a malaria endemic country would mean local transmission, combining genotyping outcome with epidemiological findings will assist classifying malaria cases without any ambiguity.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Dengue viral infection is an ongoing epidemic in Sri Lanka, causing significant mortality and morbidity. A descriptive-analytical study was carried out using serologically confirmed Dengue patients ...during a 6 month period. The relationship between the elevation of hepatic enzymes and severity of Dengue was assessed after stratifying recorded maximum AST/ALT (SGOT/SGPT) values 2-15 times elevated and by the phases of the illness. Sensitivity, specificity, predictive values, and ROC curves were assessed using maximum values for AST and ALT.
Out of 255 patients, 107(42%) were females. The majority (52.9%) were in the 20-39 year age group. Only 19.6% had DHF. No statistically significant difference was noticed in the values of maximum transaminases during the febrile phase among DF and DHF patients. Higher sensitivity and low specificity with the 1-5 times elevation range was noticed, and a higher cut-off level of more than 5 times elevation showed low sensitivity and higher specificity. The combination of both transaminases cut-offs with age and sex also does not show clinically significant predictability of severe disease. The AST and ALT elevations are not showing discriminatory predictive value on dengue severity. As different serotypes cause different epidemics, it is important to carry out large-scale specific studies considering the serotypes.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Leptospirosis is a zoonotic disease caused by Leptospira species. Variations in lipopolysaccharide (LPS) structure in Leptospira are known to be associated with the serovar diversity and ...antigenicity. Development of immunodiagnostics for early detection of leptospirosis based on immune responses against different pathogenic antigens as well as development of vaccines are important. Hence, this study has assessed the immune response generated against leptospiral LPS and whole antigen preparations of pathogenic and saprophytic Leptospira and specific changes in peritoneal cells was also studied to elucidate the cellular responses associated with immune response of Wistar rats. During the study, immune response induced by two types of Leptospira antigen preparations of two selected serovars was compared. Changes in the specific peritoneal cell subpopulations following immunizations of rats were analyzed using flow cytometry. Of the two antigen preparations tested, the LPS extract induced a higher IgM immune response as opposed to the sonicated antigen preparation. Of the two serovars tested, L. interrogans serovar Pyrogenes had induced a higher IgM response compared to that by L. biflexa serovar Patoc. Considering the IgG titers, equivalent responses were observed with all four antigen preparations. Significant increases in lymphocytes were observed following immunization with LPS of both serovars. Interestingly, the B2 cell percentages increased significantly during the immunization period. Further, significant correlations were observed with both IgM and IgG responses and percentage of B2 cells in the peritoneal cavity (PC). LPS extract of L. interrogans serovar Pyrogenes induced higher IgM response while the IgG response was equivalent among the four antigen preparations tested. Significant increase of B2 cell percentage in the peritoneal cavity during the immunization reflects the accumulation of B2 cells in the PC which may play considerable role in generating humoral response against Leptospira antigens.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
According to the WHO 2009 classification, dengue with warning signs is at the risk of developing severe form of dengue disease. One of the most important warning signs is plasma leakage, which can be ...a serious complication associated with higher morbidity and mortality. We report that the frequency of CD4
CD8
double-positive (DP) T cells is significantly increased in patients at risk of developing plasma leakage. Transcriptomic analysis demonstrated that CD4
CD8
DP cells were distinct from CD4
Single Positive (SP) T cells but co-clustered with CD8
SP cells, indicating a largely similar transcriptional profile. Twenty significant differentially expressed (DE) genes were identified between CD4
CD8
DP and CD8
SP cells. These genes encode OX40 and CCR4 proteins as well as other molecules associated with cell signaling on the cell surface (
,
, and
). While comparing the profile of gene expression in CD4
CD8
DP cells from patients with and without warning signs of plasma leakage, similar expression profile was observed, implying a role of CD4
CD8
DP cells in plasma leakage through a quantitative increase rather than functional alteration. This study provided novel insight into the host immune response during the acute febrile phase of DENV infection and the role of CD4
CD8
DP T cells in the pathogenesis of plasma leakage.
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