Introduction: Recently, volumetric absorptive microsampling (VAMS) has been used for accurate sampling of a fixed peripheral blood volume (10 µL) on a volumetric swab, and long-term sample storage. The mPlex-Flu assay is a novel, high-throughput assay that simultaneously measures the concentration of antibodies against the hemagglutinin (HA) proteins from multiple influenza virus strains with ≤5 µL of serum. Here we describe combining these two methods to measure multidimensional anti-influenza IgG activity in whole blood samples collected by a finger stick and VAMS, with correction for serum volume based on simultaneous hemoglobin measurement. Methods: We compared capillary blood samples obtained from a finger stick using a VAMS device with serum samples collected by traditional phlebotomy from 20 subjects, with the influenza antibody profiles measured by the mPlex-Flu assay. Results: We found that results with the two sampling methods were highly correlated within subjects and across all influenza strains (mean R2 = 0.9470). Adjustment for serum volume, based on hemaglobin measurement, was used to estimate serum volume of samples and improved the accuracy. IgG measurements were stable over 3 weeks when VAMS samples were stored at room temperature or transported using a variety of shipping methods. Additionally, when volunteers performed finger-stick VAMS at-home by themselves, the comparison results of anti-HA antibody concentrations were highly consistent with sampling performed by study personnel on-site (R2 = 0.9496). Conclusions: This novel approach can provide a simple, accurate, and low-cost means for monitoring the IgG anti-influenza HA antibody responses in large population studies and clinical trials.
The COVID-19 pandemic is caused by SARS-CoV-2, a novel zoonotic coronavirus. Emerging evidence indicates that preexisting humoral immunity against other seasonal human coronaviruses (HCoVs) plays a critical role in the specific antibody response to SARS-CoV-2. However, current work to assess the effects of preexisting and cross-reactive anti-HCoVs antibodies has been limited. To address this issue, we have adapted our previously reported multiplex assay to simultaneously and quantitatively measure anti-HCoV antibodies. The full mPlex-CoV panel covers the spike (S) and nucleocapsid (N) proteins of three highly pathogenic HCoVs (SARS-CoV-1, SARS-CoV-2, MERS) and four human seasonal strains (OC43, HKU1, NL63, 229E). Combining this assay with volumetric absorptive microsampling (VAMS), we measured the anti-HCoV IgG, IgA, and IgM antibodies in fingerstick blood samples. The results demonstrate that the mPlex-CoV assay has high specificity and sensitivity. It can detect strain-specific anti-HCoV antibodies down to 0.1 ng/ml with 4 log assay range and with low intra- and inter-assay coefficients of variation (%CV). We also estimate multiple strain HCoVs IgG, IgA and IgM concentration in VAMS samples in three categories of subjects: pre-COVID-19 (n=21), post-COVID-19 convalescents (n=19), and COVID-19 vaccine recipients (n=14). Using metric multidimensional scaling (MDS) analysis, HCoVs IgG concentrations in fingerstick blood samples were well separated between the pre-COVID-19, post-COVID-19 convalescents, and COVID-19 vaccine recipients. In addition, we demonstrate how multi-dimensional scaling analysis can be used to visualize IgG mediated antibody immunity against multiple human coronaviruses. We conclude that the combination of VAMS and the mPlex-Cov assay is well suited to performing remote study sample collection under pandemic conditions to monitor HCoVs antibody responses in population studies.
Background A protective SARS-COV-2 (SARS2) antibody response is crucial to decrease morbidity and mortality from severe COVID-19 disease and for vaccine efficacy. The effects of pre-existing anti-human coronavirus (HCoV) antibodies on the SARS2-specific IgG responses and severity of disease are currently unclear. Methods We profiled anti-spike (S), S1, S2, RBD IgG antibodies against SARS2 and six HCoVs using a multiplex assay (mPLEX-CoV) with serum samples from SARS2 infection (155 patients) and pre-COVID-19 (188 subjects) cohorts. Results Anti-S SARS2 IgG levels were significantly increased and highly correlated with IgG antibodies that recognized OC43 and HKU1 S proteins in COVID-19 patients. However, OC43 and HKU1 anti-S antibodies in sera collected pre-COVID-19 did not cross-react to SARS2 S. Moreover, these ”uni-directional” cross-reactive antibodies elicited by the SARS2 infection were distinct from the ”bi-directional” cross-reactive antibodies that recognized the homologous antigen strains, RaTG13 and SARS-CoV-1 (SARS1). Notably, high OC43 and anti-S2 antibodies were associated with a rapid and robust anti-SARS2 antibody response and increased disease severity. In addition, a higher ratio of S2/S1-reactive antibodies developed over time in severe ICU patients. Conclusions Our study suggested that early and rapid OC43 S- and S2-reactive antibodies emerging after SARS2 infection may correlate with COVID-19 disease severity.
The antigenic shift and draft of hemagglutinin (HA) in influenza viruses is accepted as one of the major reasons for immune evasion. The analysis of B cell immune responses to influenza infection and vaccination is complicated by the impact of exposure history and antibody cross-reactions between antigenically similar influenza strains.
The human antibody response to influenza virus infection or vaccination is as complicated as it is essential for protection against flu. The constant antigenic changes of the virus to escape human herd immunity hinder the yearly selection of vaccine strains since it is hard to predict which virus strains will circulate for the coming flu season. A “universal” influenza vaccine that could induce broad cross-influenza subtype protection would help to address this issue. However, the human antibody response is intricate and often obscure, with factors such as antigenic seniority or original antigenic sin (OAS), and back-boosting ensuring that each person mounts a unique immune response to infection or vaccination with any new influenza virus strain. Notably, the effects of existing antibodies on cross-protective immunity after repeated vaccinations are unclear. More research is needed to characterize the mechanisms at play, but traditional assays such as hemagglutinin inhibition (HAI) and microneutralization (MN) are excessively limited in scope and too resource-intensive to effectively meet this challenge. In the past ten years, new multiple dimensional assays (MDAs) have been developed to help overcome these problems by simultaneously measuring antibodies against a large panel of influenza hemagglutinin (HA) proteins with a minimal amount of sample in a high throughput way. MDAs will likely be a powerful tool for accelerating the study of the humoral immune response to influenza vaccination and the development of a universal influenza vaccine.
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