Pre-pandemic antibodies screening against SARS-CoV-2 and virus detection among children diagnosed with eruptive fevers.

OBJECTIVES: This study aims to assess the seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG antibodies against the spike (S) and nucleocapsid (NP) proteins, as well as neutralizing antibodies against the receptor-binding domain (RBD). Additionally, it aims to detect viral RNA of SARS-CoV-2 in pre-pandemic archival pediatric specimens collected before the announcement of the COVID-19 pandemic spread on March 20th, 2020, in Morocco. The objective is to investigate the existence of pre-pandemic immunity to SARS-CoV-2. METHODS: We conducted a cross-sectional study, to analyze IgG antibody levels in a cohort of 106 pre-pandemic pediatric participants. Using an indirect enzyme-linked immunosorbent assay (ELISA), we measured the IgG levels against the S and NP proteins of SARS-CoV-2. Additionally, we staged a competitive ELISA assay to evaluate the neutralizing capability of these antibodies. We used reverse transcription polymerase chain reaction (rRT-PCR) to detect viral NP and ORF1ab genes of SARS-CoV-2 in oropharyngeal swabs. Moreover, we conducted on the same specimens a multiplexed RT-PCR to detect RNA of the most common 27 pathogens involved in lower respiratory tract infections. RESULTS: Among the 106 serum samples, 13% (nn = =14) tested positive for SARS-CoV-2 IgG antibodies using ELISA. Temporal analysis indicated varying IgG positivity levels across 2019. Neutralizing antibodies were found in 21% of the 28 samples analyzed, including two with high inhibition rates (93%). The SARS-CoV-2 RNA was detected using rRT-PCR in 14 samples. None of the samples tested positive for the other 27 pathogens associated with lower respiratory tract infections, using multiplexed RT-PCR. CONCLUSION: Our study addresses the possibility, that COVID-19 infections occurred in Morocco before the recognized outbreak. On the other hand, some of the cases might reflect cross-reactivity with other coronaviruses or be influenced by previous viral exposures or vaccinations. Understanding these factors is crucial to comprehending pediatric immune responses to newly emerging infectious diseases.

Mechanisms of antibody mediated immunity - Distinct in early life.

Immune responses in early life are characterized by a failure to robustly generate long-lasting protective responses against many common pathogens or upon vaccination. This is associated with a reduced ability to generate T-cell dependent high affinity antibodies. This review highlights the differences in T-cell dependent antibody responses observed between infants and adults, in particular focussing on the alterations in immune cell function that lead to reduced T follicular helper cell-B cell crosstalk within germinal centres in early life. Understanding the distinct functional characteristics of early life humoral immunity, and how these are regulated, will be critical in guiding age-appropriate immunological interventions in the very young.

Unravelling humoral immunity in SARS-CoV-2: Insights from infection and vaccination.

Following infection and vaccination against SARS-CoV-2, humoral components of the adaptive immune system play a key role in protecting the host. Specifically, B cells generate high-affinity antibodies against various antigens of the virus. In this review, we discuss the mechanisms of immunity initiation through both natural infection and vaccination, shedding light on the activation of B cell subsets in response to SARS-CoV-2 infection and vaccination. The innate immune system serves as the initial line of primary and nonspecific defence against viruses. However, within several days following infection or a vaccine dose, a virus-specific immune response is initiated, primarily by B cells that produce antibodies. These antibodies contribute to the resolution of the disease. Subsequently, these B cells transition into memory B cells, which play a crucial role in providing long-term immunity against the virus. CD4+ T helper cells initiate a cascade, leading to B cell somatic hypermutation, germinal center memory B cells, and the production of neutralizing antibodies. B-cell dysfunction can worsen disease severity and reduce vaccine efficacy. Notably, individuals with B cell immunodeficiency show lower IL-6 production. Furthermore, this review delves into several aspects of immune responses, such as hybrid immunity, which has shown promise in boosting broad-spectrum protection. Cross-reactive immunity is under scrutiny as well, as pre-existing antibodies can offer protection against the disease. We also decipher breakthrough infection mechanisms, especially with the novel variants of the virus. Finally, we discuss some potential therapeutic solutions regarding B cells including convalescent plasma therapy, B-1 cells, B regulatory cell (Breg) modulation, and the use of neutralizing monoclonal antibodies in combating the infection. Ongoing research is crucial to grasp population immunity trends and assess the potential need for booster doses in maintaining effective immune responses against potential viral threats.

Algorithms for testing COVID-19 focused on use of RT-PCR and high-affinity serological testing: A consensus statement from a panel of Latin American experts.

The COVID-19 pandemic has caused an unprecedented public health, social, and economic crisis. Improving understanding on available tests for detecting COVID-19 is critical for effective management of the pandemic. We proposed that a multidisciplinary expert panel can establish recommendations on ideal use of diagnostic tools, with a focus on RT-PCR and serological high-affinity antibodies (both IgM and IgG) tests for the Latin America region. STUDY DESIGN: A collaborative multidisciplinary panel of 5 recognized experts in Latin America (an infectious disease specialist, three pathologists, and an immunologist) was convened and supported by Roche Diagnostics to develop standard guidelines and an evidence-based document of best practices on the use of diagnostic tools for COVID-19. RESULTS: The authors reached consensus on the applicability of diagnostic tools to provide testing algorithms for the use of RT-PCR and serological high-affinity antibodies (both IgM and IgG) tests in three settings: 1) For asymptomatic subjects exposed to a SARS-CoV-2 infected person; 2) For epidemiological purposes and; 3) For symptomatic subjects. CONCLUSION: The serological high-affinity SARS-CoV-2 antibodies (both IgM and IgG) tests play a key role in COVID-19 diagnosis. These tests can be applied for suspected false-negative RT-PCR results and for individual determination of response. The use of these tests can also contribute greatly to public health strategies, such as population screening and supporting vaccination planning. Serological status for high-affinity antibodies (both IgM and IgG) should be performed ideally 21 days after potential infectious contact, given that the majority of exposed individuals will have seroconverted.

Rapid and fully-automated detection of Clostridium difficile Toxin B via magnetic-particle-based chemiluminescent immunoassay.

Clostridium difficile colitis is caused by a cytotoxin produced by the anaerobic bacteria C. difficile on the epithelial cells of the large intestine, particularly C. difficile toxin B (Tcd B). Current C. difficile toxin assays have proven to be insensitive and have thus been ruled out from diagnostic purposes. Therefore, Tcd B detection via sandwich-type chemiluminescent immunoassay was proposed as a straightforward approach with potential diagnostic applicability. Here, two high-affinity anti-Tcd B monoclonal antibodies were successfully identified and implemented in a fully-automated magnetic-particle-based chemiluminescent immunoassay (CLEIA). In this test, toxin B was sandwiched between the anti-toxin B antibody-coated magnetic particles and alkaline phosphate-labeled anti-toxin B antibodies. Compared with traditional techniques, the proposed immunoassay demonstrated high sensitivity for toxin B identification and was further optimized to achieve a linear response ranging from 0.12 to 150 ng/mL with a limit of detection (LOD) of 0.47 ng/mL. Importantly, the entire process could be completed in less than 30 minutes. The proposed assay was used to detect toxin B in 104 randomly-selected human stool samples and delivered similar results to those of a commercial ELISA kit, highlighting its great potential for rapid and efficient toxin B determination in human stool specimens.

Quantitative and Qualitative Analysis of Humoral Immunity Reveals Continued and Personalized Evolution in Chronic Viral Infection.

Control of established chronic lymphocytic choriomeningitis virus (LCMV) infection requires the production of neutralizing antibodies, but it remains unknown how the ensemble of antibodies evolves during ongoing infection. Here, we analyze the evolution of antibody responses during acute or chronic LCMV infection, combining quantitative functional assays and time-resolved antibody repertoire sequencing. We establish that antibody responses initially converge in both infection types on a functional and repertoire level, but diverge later during chronic infection, showing increased clonal diversity, the appearance of mouse-specific persistent clones, and distinct phylogenetic signatures. Chronic infection is characterized by a longer-lasting germinal center reaction and a continuous differentiation of plasma cells, resulting in the emergence of higher-affinity plasma cells exhibiting increased antibody secretion rates. Taken together, our findings reveal the emergence of a personalized antibody response in chronic infection and support the concept that maintaining B cell diversity throughout chronic LCMV infection correlates with the development of infection-resolving antibodies.

A sensitive and rapid chemiluminescence immunoassay for point-of-care testing (POCT) of copeptin in serum based on high-affinity monoclonal antibodies via cytokine-assisted immunization.

Purpose: Antibodies are key reagents in the development of immunoassay. We attempted to develop high-performance CPP immunoassays using high-affinity monoclonal antibodies prepared via cytokine-assisted immunization. Methods: We used fetal liver tyrosine kinase 3 ligand (Flt3L), CC subtype chemokine ligand 20 (CCL20), and granulocyte-macrophage colony-stimulating factor (GM-CSF) to assist traditional subcutaneous immunization of preparing high-affinity monoclonal antibodies, and further to develop high-performance immunoassay methods for CPP. Results: This novel immune strategy significantly enhanced immune response against CPP. Six anti-CPP monoclonal antibodies (mAbs) with high affinity were successfully screened and selected for application in a fully automated magnetic chemiluminescence immunoassay (CLIA). This robust and rapid assay can efficiently detect CPP in the range of 1.2-1250 pmol L-1 with a detection limit of 6.25 pmol L-1. Significantly, the whole incubation process can be completed in 30 min as compared to about 4.5 hr for the control ELISA kit. Furthermore, this assay exhibited high sensitivity and specificity, low intra-assay and inter-assay coefficients of variation (CVs < 15%). The developed assay was applied in the detection of CPP in 115 random serum samples and results showed a high correlation with data obtained using a commercially available ELISA kit (correlation coefficient, 0.9737). Conclusion: Our assay could be applied in the point-of-care testing of CPP in the serum samples, and also the method developed in this study could be adopted to explore the detection and diagnosis of other biomarkers for various diseases.

Development of anti-Müllerian hormone immunoassay based on biolayer interferometry technology.

Anti-Müllerian hormone (AMH) is a biomarker for the assessment of female fertility. The accurate measurement of the concentration of AMH is relevant for the success of assisted reproductive therapies and diagnosis of clinical cases. In this study, we show that cytokines such as fetal liver tyrosine kinase 3 ligand (Flt3L), CC subtype chemokine ligand 20 (CCL20), granulocyte-macrophage colony-stimulating factor (GM-CSF), and ß2-microglobulin (ß2M) significantly enhance the immune response against AMH. Two anti-AMH monoclonal antibodies (mAbs) with high affinity were selected by biolayer interferometry (BLI) technology for application in a fully automated magnetic chemiluminescence immunoassay (CLIA). This robust and rapid assay can efficiently detect AMH in the range of 0.125~20 ng mL-1 with a detection limit of 0.099 ng mL-1. This immunoassay showed high specificity with no cross-reaction with structurally related proteins and some of the other members of the TGF-ß super family, such as inhibin A, activin A, follicle-stimulating hormone, and luteinizing hormone. The average recovery rates of three different batches were 100.19%, 102.72%, and 103.59%, respectively, with coefficients of variation of less than 12%. The developed assay was applied in the detection of AMH in 69 serum samples from randomly selected patients. Our data showed a high correlation with those obtained using commercially available ELISA kits (correlation coefficient, 0.9831). Hence, we suggest that this immunoassay could find application in the development of POCT for the diagnosis of AMH in clinical samples. Graphical abstract.