Highly versatile antibody binding assay for the detection of SARS-CoV-2 infection and vaccination.

Monitoring the burden and spread of infection with the new coronavirus SARS-CoV-2, whether within small communities or in large geographical settings, is of paramount importance for public health purposes. Serology, which detects the host antibody response to the infection, is the most appropriate tool for this task, since virus-derived markers are most reliably detected during the acute phase of infection. Here we show that our ELISA protocol, which is based on antibody binding to the Receptor Binding Domain (RBD) of the S1 subunit of the viral Spike protein expressed as a novel fusion protein, detects antibody responses to SARS-CoV-2 infection and vaccination. We also show that our ELISA is accurate and versatile. It compares favorably with commercial assays widely used in clinical practice to determine exposure to SARS-CoV-2. Moreover, our protocol accommodates use of various blood- and non-blood-derived biospecimens, such as breast milk, as well as dried blood obtained with microsampling cartridges that are appropriate for remote collection. As a result, our RBD-based ELISA protocols are well suited for seroepidemiology and other large-scale studies requiring parsimonious sample collection outside of healthcare settings.

Highly versatile antibody binding assay for the detection of SARS-CoV-2 infection.

Monitoring the burden and spread of infection with the new coronavirus SARS-CoV-2, whether within small communities or in large geographical settings, is of paramount importance for public health purposes. Serology, which detects the host antibody response to the infection, is the most appropriate tool for this task, since virus-derived markers are most reliably detected during the acute phase of infection. Here we show that our ELISA protocol, which is based on antibody binding to the Receptor Binding Domain (RBD) of the S1 subunit of the viral Spike protein expressed as a novel fusion protein, detects antibody responses to SARS-CoV-2 infection and COVID-19 vaccination. We also show that our ELISA is accurate and versatile. It compares favorably with commercial assays widely used in clinical practice to determine exposure to SARS-CoV-2. Moreover, our protocol accommodates use of various blood- and non-blood-derived biospecimens, such as breast milk, as well as dried blood obtained with microsampling cartridges that are appropriate for remote collection. As a result, our RBD-based ELISA protocols are well suited for seroepidemiology and other large-scale studies requiring parsimonious sample collection outside of healthcare settings.

Senescence-associated ß-galactosidase reveals the abundance of senescent CD8+ T cells in aging humans.

Aging leads to a progressive functional decline of the immune system, rendering the elderly increasingly susceptible to disease and infection. The degree to which immune cell senescence contributes to this decline remains unclear, however, since markers that label immune cells with classical features of cellular senescence accurately and comprehensively have not been identified. Using a second-generation fluorogenic substrate for ß-galactosidase and multi-parameter flow cytometry, we demonstrate here that peripheral blood mononuclear cells (PBMCs) isolated from healthy humans increasingly display cells with high senescence-associated ß-galactosidase (SA-ßGal) activity with advancing donor age. The greatest age-associated increases were observed in CD8+ T-cell populations, in which the fraction of cells with high SA-ßGal activity reached average levels of 64% in donors in their 60s. CD8+ T cells with high SA-ßGal activity, but not those with low SA-ßGal activity, were found to exhibit features of telomere dysfunction-induced senescence and p16-mediated senescence, were impaired in their ability to proliferate, developed in various T-cell differentiation states, and had a gene expression signature consistent with the senescence state previously observed in human fibroblasts. Based on these results, we propose that senescent CD8+ T cells with classical features of cellular senescence accumulate to levels that are significantly higher than previously reported and additionally provide a simple yet robust method for the isolation and characterization of senescent CD8+ T cells with predictive potential for biological age.

Regulation of Transcription Factor E2-2 in Human Plasmacytoid Dendritic Cells by Monocyte-Derived TNFα.

Plasmacytoid dendritic cells (pDCs) are innate immune cells and potent producers of interferon alpha (IFNα). Regulation of pDCs is crucial for prevention of aberrant IFN production. Transcription factor E2-2 (TCF4) regulates pDC development and function, but mechanisms of E2-2 control have not been investigated. We used freshly-isolated human peripheral blood mononuclear cells stimulated with toll-like receptor 7, 9, and 4 agonists to determine which factors regulate E2-2. After activation, pDCs decreased E2-2 expression. E2-2 downregulation occurred during the upregulation of costimulatory markers, after maximal IFN production. In congruence with previous reports in mice, we found that primary human pDCs that maintained high E2-2 levels produced more IFN, and had less expression of costimulatory markers. Stimulation of purified pDCs did not lead to E2-2 downregulation; therefore, we investigated if cytokine signaling regulates E2-2 expression. We found that tumor necrosis factor alpha (TNFα) produced by monocytes caused decreased E2-2 expression. All together, we established that primary human pDCs decrease E2-2 in response to TNFα and E2-2 low pDCs produce less IFN but exhibit more costimulatory molecules. Altered expression of E2-2 may represent a mechanism to attenuate IFN production and increase activation of the adaptive immune compartment.