Characterization of Enzyme-Linked Immunosorbent Assay (ELISA) for Quantification of Antibodies against Salmonella Typhimurium and Salmonella Enteritidis O-Antigens in Human Sera.

Nontyphoidal Salmonella (NTS) is a leading cause of morbidity and mortality caused by enteric pathogens worldwide in both children and adults, and vaccines are not yet available. The measurement of antigen-specific antibodies in the sera of vaccinated or convalescent individuals is crucial to understand the incidence of disease and the immunogenicity of vaccine candidates. A solid and standardized assay used to determine the level of specific anti-antigens IgG is therefore of paramount importance. In this work, we presented the characterization of a customized enzyme-linked immunosorbent assay (ELISA) with continuous readouts and a standardized definition of EU/mL. We assessed various performance parameters: standard curve accuracy, dilutional linearity, intermediate precision, specificity, limits of blanks, and quantification. The simplicity of the assay, its high sensitivity and specificity coupled with its low cost and the use of basic consumables and instruments without the need of high automation makes it suitable for transfer and application to different laboratories, including resource-limiting settings where the disease is endemic. This ELISA is, therefore, fit for purpose to be used for quantification of antibodies against Salmonella Typhimurium and Salmonella Enteritidis O-antigens in human samples, both for vaccine clinical trials and large sero-epidemiological studies.

Development and Characterization of a Luminescence-Based High-Throughput Serum Bactericidal Assay (L-SBA) to Assess Bactericidal Activity of Human Sera against Nontyphoidal Salmonella.

Salmonella Typhimurium and Salmonella Enteritidis are leading causative agents of invasive nontyphoidal Salmonella (iNTS) disease, which represents one of the major causes of death and morbidity in sub-Saharan Africa, still partially underestimated. Large sero-epidemiological studies are necessary to unravel the burden of disease and guide the introduction of vaccines that are not yet available. Even if no correlate of protection has been determined so far for iNTS, the evaluation of complement-mediated functionality of antibodies generated towards natural infection or elicited upon vaccination may represent a big step towards this achievement. Here we present the setup and the intra-laboratory characterization in terms of repeatability, intermediate precision, linearity, and specificity of a high-throughput luminescence-based serum bactericidal assay (L-SBA). This method could be useful to perform sero-epidemiological studies across iNTS endemic countries and for evaluation of antibodies raised against iNTS vaccine candidates in upcoming clinical trials.

Differential localization and limited cytotoxic potential of duodenal CD8+ T cells.

The duodenum is a major site of HIV persistence during suppressive antiretroviral therapy despite harboring abundant tissue-resident memory (Trm) CD8+ T cells. The role of duodenal Trm CD8+ T cells in viral control is still not well defined. We examined the spatial localization, phenotype, and function of CD8+ T cells in the human duodenal tissue from people living with HIV (PLHIV) and healthy controls. We found that Trm (CD69+CD103hi) cells were the predominant CD8+ T cell population in the duodenum. Immunofluorescence imaging of the duodenal tissue revealed that CD103+CD8+ T cells were localized in the intraepithelial region, while CD103-CD8+ T cells and CD4+ T cells were mostly localized in the lamina propria (LP). Furthermore, HIV-specific CD8+ T cells were enriched in the CD69+CD103-/lo population. However, the duodenal HIV-specific CD8+ Trm cells rarely expressed canonical molecules for potent cytolytic function (perforin and granzyme B) but were more polyfunctional than those from peripheral blood. Taken together, our results show that duodenal CD8+ Trm cells possess limited perforin-mediated cytolytic potential and are spatially separated from HIV-susceptible LP CD4+ T cells. This could contribute to HIV persistence in the duodenum and provides critical information for the design of cure therapies.

High SARS-CoV-2 seroprevalence in health care workers but relatively low numbers of deaths in urban Malawi.

BACKGROUND: In low-income countries, like Malawi, important public health measures including social distancing or a lockdown have been challenging to implement owing to socioeconomic constraints, leading to predictions that the COVID-19 pandemic would progress rapidly. However, due to limited capacity to test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, there are no reliable estimates of the true burden of infection and death. We, therefore, conducted a SARS-CoV-2 serosurvey amongst health care workers (HCWs) in Blantyre city to estimate the cumulative incidence of SARS-CoV-2 infection in urban Malawi. METHODS: We recruited 500 otherwise asymptomatic HCWs from Blantyre City (Malawi) from 22nd May 2020 to 19th June 2020 and serum samples were collected from all participants. A commercial ELISA was used to measure SARS-CoV-2 IgG antibodies in serum. RESULTS: A total of 84 participants tested positive for SARS-CoV-2 antibodies. The HCWs with positive SARS-CoV-2 antibody results came from different parts of the city. The adjusted seroprevalence of SARS-CoV-2 antibodies was 12.3% [CI 8.2 - 16.5]. Using age-stratified infection fatality estimates reported from elsewhere, we found that at the observed adjusted seroprevalence, the number of predicted deaths was eight times the number of reported deaths. CONCLUSIONS: The high seroprevalence of SARS-CoV-2 antibodies among HCWs and the discrepancy in the predicted versus reported deaths suggests that there was early exposure but slow progression of COVID-19 epidemic in urban Malawi. This highlights the urgent need for development of locally parameterised mathematical models to more accurately predict the trajectory of the epidemic in sub-Saharan Africa for better evidence-based policy decisions and public health response planning.

Evasion of MAIT cell recognition by the African Salmonella Typhimurium ST313 pathovar that causes invasive disease.

Mucosal-associated invariant T (MAIT) cells are innate T lymphocytes activated by bacteria that produce vitamin B2 metabolites. Mouse models of infection have demonstrated a role for MAIT cells in antimicrobial defense. However, proposed protective roles of MAIT cells in human infections remain unproven and clinical conditions associated with selective absence of MAIT cells have not been identified. We report that typhoidal and nontyphoidal Salmonella enterica strains activate MAIT cells. However, S. Typhimurium sequence type 313 (ST313) lineage 2 strains, which are responsible for the burden of multidrug-resistant nontyphoidal invasive disease in Africa, escape MAIT cell recognition through overexpression of ribB This bacterial gene encodes the 4-dihydroxy-2-butanone-4-phosphate synthase enzyme of the riboflavin biosynthetic pathway. The MAIT cell-specific phenotype did not extend to other innate lymphocytes. We propose that ribB overexpression is an evolved trait that facilitates evasion from immune recognition by MAIT cells and contributes to the invasive pathogenesis of S. Typhimurium ST313 lineage 2.

Plasma cell-free DNA predicts pediatric cerebral malaria severity.

BACKGROUNDPrediction of adverse outcomes in cerebral malaria (CM) is difficult. We hypothesized that cell-free DNA (cfDNA) levels would facilitate identification of severe and potentially fatal CM cases.METHODSIn this retrospective study, plasma from Malawian children with CM (n = 134), uncomplicated malaria (UM, n = 77), and healthy controls (HC, n = 60) was assayed for cfDNA using a fluorescence assay. Host and parasite cfDNA was measured by quantitative PCR. Immune markers were determined by ELISA, Luminex, or cytometric bead array.RESULTSTotal cfDNA increased with malaria severity (HC versus UM, P < 0.001; HC versus CM, P < 0.0001; UM versus CM, P < 0.0001), was elevated in retinopathy-positive (Ret+) CM relative to Ret- CM (7.66 versus 5.47 ng/µL, P = 0.027), and differentiated Ret+ fatal cases from survivors (AUC 0.779; P < 0.001). cfDNA levels in patients with non-malarial febrile illness (NMF, P = 0.25) and non-malarial coma (NMC, P = 0.99) were comparable with UM. Host DNA, rather than parasite DNA, was the major cfDNA contributor (UM, 268 versus 67 pg/µL; CM, 2824 versus 463 pg/µL). Host and parasite cfDNA distinguished CM by retinopathy (host, AUC 0.715, P = 0.0001; parasite, AUC 0.745, P = 0.0001), but only host cfDNA distinguished fatal cases (AUC 0.715, P = 0.0001). Total cfDNA correlated with neutrophil markers IL-8 (rs = 0.433, P < 0.0001) and myeloperoxidase (rs = 0.683, P < 0.0001).CONCLUSIONQuantifying plasma cfDNA is a simple assay useful in identifying children at risk for fatal outcome and has promise as a point-of-care assay. Elevated cfDNA suggests a link with host inflammatory pathways in fatal CM.FUNDINGNIH NCATS (AK), Burroughs-Wellcome (AK), and National Health and Medical Research Council of Australia (SJR).