No abstract

We have developed a serology test platform for identifying individuals with prior exposure to specific viral infections and provide data to help reduce public health risks. The serology test composed of a pair of cell lines engineered to express either a viral envelop protein (Target Cell) or a receptor to recognize the Fc region of an antibody (Reporter Cell), that is, Diagnostic‐Cell‐Complex (DxCell‐Complex). The formation of an immune synapse, facilitated by the analyte antibody, resulted into a dual‐reporter protein expression by the Reporter Cell. We validated it with human serum with confirmed history of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection. No signal amplification steps were necessary. The DxCell‐Complex quantitatively detected the target‐specific immunoglobulin G (IgG) within 1 h. Validation with clinical human serum containing SARS‐CoV‐2 IgG antibodies confirmed 97.04% sensitivity and 93.33% specificity. The platform can be redirected against other antibodies. Self‐replication and activation‐induced cell signaling, two attributes of the cell, will enable rapid and cost‐effective manufacturing and its operation in healthcare facilities without requiring time‐consuming signal amplification steps.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the cause of the COVID-19 pandemic, is initiated by its binding to the ACE2 receptor and other co-receptors on mucosal epithelial cells. Variable outcomes of the infection and disease severity can be influenced by pre-existing risk factors. Human immunodeficiency virus (HIV), the cause of AIDS, targets the gut mucosal immune system and impairs epithelial barriers and mucosal immunity. We sought to determine the impact and mechanisms of pre-existing HIV infection increasing mucosal vulnerability to SARS-CoV-2 infection and disease. We investigated changes in the expression of ACE2 and other SARS-CoV-2 receptors and related pathways in virally inflamed gut by using the SIV infected rhesus macaque model of HIV/AIDS. Immunohistochemical analysis showed sustained/enhanced ACE2 expression in the gut epithelium of SIV infected animals compared to uninfected controls. Gut mucosal transcriptomic analysis demonstrated enhanced expression of host factors that support SARS-CoV-2 entry, replication, and infection. Metabolomic analysis of gut luminal contents revealed the impact of SIV infection as demonstrated by impaired mitochondrial function and decreased immune response, which render the host more vulnerable to other pathogens. In summary, SIV infection resulted in sustained or increased ACE2 expression in an inflamed and immune-impaired gut mucosal microenvironment. Collectively, these mucosal changes increase the susceptibility to SARS-CoV-2 infection and disease severity and result in ineffective viral clearance. Our study highlights the use of the SIV model of AIDS to fill the knowledge gap of the enteric mechanisms of co-infections as risk factors for poor disease outcomes, generation of new viral variants and immune escape in COVID-19.

Wildfire smoke contains inhalable toxic gaseous and particulate matter that is associated with detrimental health effects yet there is no quantifiable outcome of wildfire smoke inhalation on the immune system available. We hypothesized that wildfire smoke exposure activates a dendritic cell response. METHODS: Subject recruitment was coordinated by the clinical trial team of the UC Davis Lung Center. 23 age (18-65 years old) and sex matched healthy subjects were recruited from 2020 Aug 23rd-Sept 3rd (Visit 1), during the peak time of potential smoke exposures from multiple complex fires. Peripheral blood was collected at both visits and processed in an unidentified fashion. Mononuclear cells were isolated by density gradient centrifugation and stained for dendritic cell (DC) membrane markers with fluorescent conjugated antibodies. To study cell activation, multicolor characterization was performed using standard flow cytometric gating strategies (Figure 1 left panel). FACS data was analyzed by FlowJo®. Statistical analysis was performed using Wilcoxon matched-pairs signed rank test (Prism). RESULTS: The wildfires raging around the Sacramento area resulted in a mean AQI (air quality index) of 113 Between 2020 Aug 23rd and Sept 3rd. Although smoke exposure gradually decreased, during the time between visits (the end of Visit 1 and end of Visit 2) there was still an average AQI of 77. All volunteers completed visit 2 and remained healthy. There was no difference in total blood cell count between visits. However, subjects in Visit 1 had a significantly decreased proportion of plasmacytoid Dendritic Cells (pDC) [CD45+ HLA-DR+ CD11c-CD123+] in the circulation, compared to their Visit 2 counts (p=0.004). Meanwhile, the numbers of myeloid lineage conventional type 1 or type 2 Dendritic Cells (cDC1, cDC2) [CD45+ HLA-DR+ CD11c+ CD141+] [CD45+ HLA-DR+ CD11c+ cDC1+ CD16-] did not change significantly. However, wildfire smoke exposure (Visit 1) significantly increased CCR4 (p=0.0002) and CD40 (p=0.01), but not CCR7 expression on cDC2. CONCLUSION: The increase in expression of the chemokine receptor CCR4 suggest increased migration of cDC2 cells to the peripheral tissues. cDC2 cells play a potential role in increased sensitization to allergens and could promote the development of lung disease.

e15022 Background: Sequencing circulating tumor DNA is a promising method for monitoring cancer treatment response and detecting recurrence, but sensitivity at the low tumor fractions typical of early-stage, post-treatment, and early recurrent tumors is limited by the small number of variants targeted in commercially available assays. Methods: We developed a tumor-informed minimum residual disease (MRD) assay using tumor-normal whole-genome sequencing (WGS) followed by interrogation of select somatic variants in cell-free DNA (cfDNA). We describe theoretical limits on panel designability as a function of tumor purity and sequencing depth using simulations and a discovery cohort of 31 patient samples. cfDNA assay performance was tested by creating serial dilutions of patient plasma before somatic enrichment and sequencing. Simulations and in-silico downsampling of target sites were used to test sensitivity as a function of panel size. A bioinformatics pipeline incorporating somatic calling, copy number inference, error assessment, and tumor-fraction estimation was developed to analyze results. Results: Across a range of tissues, tumor-normal WGS identified from hundreds to tens of thousands of somatic variants to inform an MRD capture panel. 97% of samples tested returned at least 300 high-confidence somatic variants, and targeted sequencing demonstrated over 95% positive predictive value in somatic variant calls. In cfDNA dilution experiments, we observed > 99% sensitivity at a tumor fraction of 0.01%. Expanding the panel size from 16 to 300 target variants increased sensitivity at 0.005% tumor fraction from approximately 20% to > 95%. Our tumor fraction model returned accurate quantification down to the parts per hundred thousand (0.001%) range, even when only a single tumor molecule could be observed at each target site. Conclusions: Increasing the number of targeted variants in an MRD assay increases sensitivity at low tumor fractions, and WGS-driven panel design ensures a sufficient number of interrogated variants while minimizing the failure rate due to panel designability. High sensitivity MRD has the potential to enable earlier recurrence detection while also giving researchers and clinicians new tools for monitoring patient treatment responses.