Immunomics-guided discovery of serum and urine antibodies for diagnosing urogenital schistosomiasis:A biomarker identification study

Background: Sensitive diagnostics are needed for effective management and surveillance of schistosomiasis so that current transmission interruption goals set by WHO can be achieved. We aimed to screen the Schistosoma haematobium secretome to find antibody biomarkers of schistosome infection, validate their diagnostic performance in samples from endemic populations, and evaluate their utility as point of care immunochromatographic tests (POC-ICTs) to diagnose urogenital schistosomiasis in the field. Methods: We did a biomarker identification study, in which we constructed a proteome array containing 992 validated and predicted proteins from S haematobium and screened it with serum and urine antibodies from endemic populations in Gabon, Tanzania, and Zimbabwe. Arrayed antigens that were IgG-reactive and a select group of antigens from the worm extracellular vesicle proteome, predicted to be diagnostically informative, were then evaluated by ELISA using the same samples used to probe arrays, and samples from individuals residing in a low-endemicity setting (ie, Pemba and Unguja islands, Zanzibar, Tanzania). The two most sensitive and specific antigens were incorporated into POC-ICTs to assess their ability to diagnose S haematobium infection from serum in a field-deployable format. Findings: From array probing, in individuals who were infected, 208 antigens were the targets of significantly elevated IgG responses in serum and 45 antigens were the targets of significantly elevated IgG responses in urine. Of the five proteins that were validated by ELISA, Sh-TSP-2 (area under the curve [AUC]serum=0·98 [95% CI 0·95-1·00]; AUCurine=0·96 [0·93-0·99]), and MS3_01370 (AUCserum=0·93 [0·89-0·97]; AUCurine=0·81 [0·72-0·89]) displayed the highest overall diagnostic performance in each biofluid and exceeded that of S haematobium-soluble egg antigen in urine (AUC=0·79 [0·69-0·90]). When incorporated into separate POC-ICTs, Sh-TSP-2 showed absolute specificity and a sensitivity of 75% and MS3_01370 showed absolute specificity and a sensitivity of 89%. Interpretation: We identified numerous biomarkers of urogenital schistosomiasis that could form the basis of novel antibody diagnostics for this disease. Two of these antigens, Sh-TSP-2 and MS3_01370, could be used as sensitive, specific, and field-deployable diagnostics to support schistosomiasis control and elimination initiatives, with particular focus on post-elimination surveillance. Funding: Australian Trade and Investment Commission and Merck Global Health Institute

A next-generation proteome array for Schistosoma mansoni

A proteome microarray consisting of 992 Schistosoma mansoni proteins was produced and screened with sera to determine antibody signatures indicative of the clinical stages of schistosomiasis and the identification of subunit vaccine candidates. Herein, we describe the methods used to derive the gene list for this array (representing approximately 10% of the predicted S. mansoni proteome). We also probed a pilot version of the microarray with sera from individuals either acutely or chronically infected with S. mansoni from endemic areas in Brazil and sera from individuals resident outside the endemic area (USA) to determine if the array is functional and informative

Distinct SARS-CoV-2 Antibody Responses Elicited by Natural Infection and mRNA Vaccination

We analyzed data from two ongoing COVID-19 longitudinal serological surveys in Orange County, CA., between April 2020 and March 2021. A total of 8,476 finger stick blood specimens were collected before and after an aggressive mRNA vaccination campaign. IgG levels were determined using a multiplex antigen microarray containing 10 SARS-CoV-2 antigens, 4 SARS, 3 MERS, 12 Common CoV, and 8 Influenza antigens. Twenty-six percent of 3,347 specimens from unvaccinated Orange County residents in December 2020 were SARS-CoV-2 seropositive. The Ab response was predominantly against nucleocapsid (NP), full length spike and the spike S2 domain. Anti-receptor binding domain (RBD) reactivity was low and there was no cross-reactivity against SARS S1 or SARS RBD. An aggressive mRNA vaccination campaign at the UCI Medical Center started on December 16, 2020 and 6,724 healthcare workers were vaccinated within 3 weeks. Seroprevalence increased from 13% in December to 79% in January, 93% in February and 99% in March. mRNA vaccination induced much higher Ab levels especially against the RBD domain and significant cross-reactivity against SARS RBD and S1 was also observed. Nucleocapsid protein Abs can be used to distinguish individuals in a population of vaccinees to classify those who have been previously infected and those who have not, because nucleocapsid is not in the vaccine. Previously infected individuals developed higher Ab titers to the vaccine than those who have not been previously exposed. These results indicate that mRNA vaccination rapidly induces a much stronger and broader Ab response than SARS-CoV-2 infection

Plasma and Mucosal Immunoglobulin M, Immunoglobulin A, and Immunoglobulin G Responses to the Vibrio cholerae O1 Protein Immunome in Adults With Cholera in Bangladesh

Abstract Background. Cholera is a severe dehydrating illness of humans caused by toxigenic strains of Vibrio cholerae O1 or O139. Identification of immunogenic V. cholerae antigens could lead to a better understanding of protective immunity in human cholera. Methods. We probed microarrays containing 3652 V. cholerae antigens with plasma and antibody-in-lymphocyte supernatant (ALS, a surrogate marker of mucosal immune responses) from patients with severe cholera caused by V. cholerae O1 in Bangladesh and age-, sex-, and ABO-matched Bangladeshi controls. We validated a subset of identified antigens using enzyme-linked immunosorbent assay. Results. Overall, we identified 608 immunoreactive V. cholerae antigens in our screening, 59 of which had higher immunoreactivity in convalescent compared with acute-stage or healthy control samples (34 in plasma, 39 in mucosal ALS; 13 in both sample sets). Identified antigens included cholera toxin B and A subunits, V. cholerae O–specific polysaccharide and lipopolysaccharide, toxin coregulated pilus A, sialidase, hemolysin A, flagellins (FlaB, FlaC, and FlaD), phosphoenolpyruvate-protein phosphotransferase, and diaminobutyrate–2-oxoglutarate aminotransferase. Conclusions. This study is the first antibody profiling of the mucosal and systemic antibody responses to the nearly complete V. cholerae O1 protein immunome; it has identified antigens that may aid in the development of an improved cholera vaccine

Immunoreactivity of current schistosomiasis vaccine antigens printed on the microarray.

<p>Signal intensity values depicting isotype/subclass-specific antibody responses of individual subjects in each cohort to proteins corresponding to known schistosomiasis vaccine antigens and other select RTS proteins that were spotted on the microarray - <i>Sm-</i>TSP-2, <i>Sm-</i>14, <i>Sj</i>-23 and <i>Sm</i>p80. Three new RTS antigens from <i>S. mansoni</i> were also included: Smp_139970, Smp_050270 and Smp_008310. Only groups from the schistosomiasis endemic area are represented: Putative Resistant (PR - blue), <i>S. mansoni</i> chronically infected with low (CI-Light – orange), moderate (CI-Mod - purple) and high (CI-Heavy - gray) intensity infections. The red line is the cut-off calculated as one standard deviation of the no-DNA control spots printed on the array and probed for each specific antibody isotype/subclass.</p

Immunoreactivity of <i>Schistosoma</i> proteins by multi-dimensional cluster analysis.

<p>(A) Multi-dimensional clustered distribution of all proteins according to the antibody isotype/subclass responses in distinct cohorts. To identify clusters containing proteins with the same antibody reactivity profiles, a distance matrix estimated from the pairwise Euclidian distance of log transformed signal intensity (SI) was generated for each antigen based on the cut-off values for each antibody isotype/subclass in the different cohorts. Proteins formed 7 clusters, defined by the following colors: cluster 1 - black (4 proteins); cluster 2 – blue (11 proteins); cluster 3 – grey (5 proteins); cluster 4 – green (31 proteins); cluster 5 – magenta (47 proteins); cluster 6 – orange (11 proteins) and cluster 7 – red (106 proteins). (B) Two-dimensional depiction of the average signal intensity for each clustered interaction separated by antibody isotype/subclass and cohort. The dotted line represents the cut-off based on the no-DNA control spots. Proteins with signal intensity below the cut-off were set to zero to decrease background noise. Identities of proteins within clusters are provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004033#ppat.1004033.s007" target="_blank">Tables S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004033#ppat.1004033.s008" target="_blank">S2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004033#ppat.1004033.s005" target="_blank">Figure S5</a>.</p

IgG1 reactivity profiles of resistant and susceptible human cohorts to <i>Schistosoma</i> proteins printed on a proteome microarray.

<p>Heatmap showing IgG1 responses of individual subjects (columns) in each cohort to 45 recombinant antigens (rows) printed on the microarray. Green represents no immunoreactivity through to red symbolizing strong immunoreactivity. The bar graph depicts the average signal intensity with mean standard deviation of each cohort. Proteins are ordered based on SI mean, highest to lowest, in the groups. Human cohorts represented: non-endemic Brazilian (n = 12), Putative Resistant (n = 20), CI-Light (n = 30), CI-Mod (n = 18) and CI-Heavy (n = 17). The red line is the cut-off for reactivity, calculated as one standard deviation of the mean of the no-DNA control spots printed on the array and probed with anti-IgG1. NS represents no significant differences between endemic groups calculated by Kruskall Wallis with Dunn's multiple comparison test. All other proteins showed significant differences between at least two of the exposed groups (PR, CI-Light, CI-Mod and CI-Heavy) (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004033#ppat.1004033.s010" target="_blank">Table S4</a>).</p