Flow Cytometry Cells were incubated in 50 L of FACS buffer containing a cocktail of antibodies for 30 min on ice prior to washing and analysis on a FACS Aria (BD)

Flow Cytometry Cells were incubated in 50 L of FACS buffer containing a cocktail of antibodies for 30 min on ice prior to washing and analysis on a FACS Aria (BD). OC2 monoclonal antibody and of human plasma. These assays could prove useful for studying humoral responses to OC43 and cross-neutralization with other medically important betacoronaviruses. Keywords: human coronavirus, OC43, neutralization assays, neutralizing antibodies 1. Introduction OC43 is one of four human coronaviruses (HCoVs) known to circulate endemically and is a major cause of upper and lower respiratory tract infections in adults and children [1]. Lower respiratory tract infection with HCoVs is associated with significant mortality in hematopoietic stem cell transplant recipients and in three recent case series OC43 was the most commonly identified of the four HCoVs in this population [2,3,4]. OC43 was first isolated in 1967 from a respiratory specimen obtained from a human adult male with a coldlike illness [5]. The viral isolate was initially cultured on human tracheal organ cultures, grown in mouse brain, and then adapted for tissue culture by passaging in primary rhesus monkey kidney cells and then BSC-1 cells [5,6,7]. OC43 belongs to the same genus Betacoronavirus as SARS-CoV-2; however, it is classified into a separate subgenus, Embecovirus. Based on phylogenetic sequence analyses, OC43 is thought to have originated from ancestral rodent-associated coronaviruses and bovine coronaviruses L-Hydroxyproline followed by zoonotic acquisition by humans from ungulate livestock leading to a pandemic in the late 19th century [1,8,9,10]. Since this spillover event over a century ago, the seroprevalence of OC43 in the general population is now close to 100% [11]. Neutralizing antibodies translocated from the serum into the respiratory tract or produced locally at the mucosa play a significant role in protection against many respiratory viral infections [12,13,14,15]. With the recent emergence of SARS-CoV-2, the causative agent of the COVID-19 pandemic, neutralization assays have been rapidly developed and deployed to study the antibody response and correlates of protection in animal models and humans to SARS-CoV-2 [12,16]. To identify antibodies that could cross-neutralize SARS-CoV-2 and the HCoVs, pseudotyped viral neutralization assays have been reported for OC43 [17,18]. However, the production of the pseudotyped vesicular stomatitis virus requires the simultaneous expression of bovine coronavirus hemagglutinin esterase to allow proper maturation of the OC43 spike protein. Live virus neutralization assays are considered a gold standard for assessing virus neutralizing potency of serum or monoclonal antibodies. To L-Hydroxyproline date, live virus neutralization assays using authentic OC43 have not yet been reported in the literature. We report here the development of two in vitro methods for measuring OC43 neutralizing antibodies, one using cytopathic effect and another using an ELISA of infected cells. Both methods are readily accessible with commercially available reagents, and the ELISA-based method can be scaled up into a high throughput format. We also report the isolation of an OC43 neutralizing monoclonal antibody (mAb) L-Hydroxyproline that can be used as a positive control in these assays. 2. Materials and Methods 2.1. Cell Lines HCT-8 cells (ATCC, cat#CCL-244) were cultured in RPMI (Gibco, Waltham, MA, USA, cat#22400089) supplemented with 10% horse serum (Gibco, cat#26050-088) and 100 U/mL penicillin plus 100 g/mL streptomycin (Gibco, cat#15140122). We cultured 293 F cells (Thermo Fisher, Waltham, MA, USA, cat#”type”:”entrez-nucleotide”,”attrs”:”text”:”R79007″,”term_id”:”855288″,”term_text”:”R79007″R79007) in Freestyle 293 media (Thermo Fisher, cat#12338026). We cultured 3T3 CD40L/IL-2/IL-21 feeder cells in DMEM supplemented with 10% fetal calf serum (Peak, Wellington, CO, USA, cat#PS-FB2), penicillin and streptomycin, plus 0.4 mg/mL geneticin as described [19]. Irradiation was performed with 5000 rads. 2.2. Viruses OC43 was obtained from BEI Resources, NIAID, NIH Rabbit Polyclonal to ACAD10 (Bethesda, MD, USA, cat#Human Coronavirus, OC43, NR-52725). HCT-8 cells were inoculated with the OC43 stock at a MOI = 0.1 and incubated at 33 C for 5 days. The supernatant was collected and frozen at ?80 C to generate viral working pools. 2.3. Human Specimens Informed consent was obtained from all human subjects, including blood donors, involved in the study. Studies involving human spleens were deemed nonhuman subjects research since tissue was de-identified, otherwise discarded, and originated from deceased individuals. Tissue fragments were passed through a basket screen, centrifuged at 300 for 7 min, incubated with ACK lysis buffer (Thermo Fisher, cat#A1049201) for 3.5 min, resuspended in RPMI (Gibco, cat#11875093), and passed through a stacked 500 m and 70 m cell strainer. Cells were resuspended in 10% dimethylsulfoxide in heat-inactivated fetal calf serum (Gibco, cat#16000044) and cryopreserved in liquid nitrogen before use. Plasma samples from SARS-CoV-2 seronegative individuals were obtained from a Fred Hutchinson Cancer Center repository assembled from a COVID-19 seroepidemiology study conducted in a single county in the western US [20]. This study was approved by the Fred Hutchinson Cancer Research Center institutional review board (#10453). 2.4. Immunofluorescence HCT-8 cells were seeded into 96-well plates at.