On time 5, antibodies were purified using MabSelectTM PrismA (Cytiva, Marlborough, MA, USA, 17549801) affinity chromatography

On time 5, antibodies were purified using MabSelectTM PrismA (Cytiva, Marlborough, MA, USA, 17549801) affinity chromatography. 2.4. ADE activity noticed on FcR-expressing cells in vitro might not reflect the problem in vivo necessarily; therefore, pet and scientific data ought to be included for ADE evaluation. Keywords: antibody-dependent improvement, COVID-19, SARS-CoV-2, vaccination serum, convalescent serum 1. Launch The COVID-19 pandemic, due to severe severe respiratory symptoms coronavirus-2 (SARS-CoV-2), provides pass on W-2429 to Rabbit polyclonal to ZGPAT over 200 countries, leading to wide-spread mortality and morbidity, aswell as massive financial loss [1,2]. To control the pandemic and prevent the recurrence of SARS-CoV-2, several vaccines designed by different platforms have been developed and approved, in addition to other immediate treatments, such as antibody-based therapies [3,4]. However, one of the biggest safety concerns with vaccines and antibody-based therapeutics is the W-2429 antibody-dependent enhancement (ADE) of viral infections [5,6,7,8,9]. SARS-CoV-2 relies on angiotensin-converting enzyme 2 (ACE2) as its primary cell surface receptor to enter host cells. Neutralizing antibodies can effectively block the entry of the virus by inhibiting the binding of the SARS-CoV-2 Spike (S) to ACE2 [10,11]. However, certain antibodies could bind to Fc receptors (FcRs) on immune cells and be internalized, leading to an enhancement in virus entry [12]. This ADE phenomenon has been observed in the past with SARS-CoV, MERS-CoV, dengue virus (DENV), respiratory syncytial virus (RSV), and measles [13,14,15,16,17,18], raising concerns W-2429 about the risk of ADE for SARS-CoV-2 vaccines and antibody-based therapies. Due to the great similarity between several bat coronaviruses and SARS-CoV-2, previous exposure to such viruses may lead to ADE of SARS-CoV-2 [19,20]. Higher antibody titers in patients with SARS-CoV-2 infection have been reported to associate with more severe disease, suggesting a possible link with ADE [21]. Several in vitro studies have demonstrated that the ADE infection of SARS-CoV-2 [22,23,24] and the emergence of new SARS-CoV-2 variants may increase the likelihood of ADE [25]. However, it remains unclear if in vitro ADE of infection can accurately predict enhanced infection in vivo. Using FcR-expressing B cells, W-2429 we also observed enhanced viral infections induced by vaccination/convalescent sera and monoclonal antibodies (mAbs) in vitro. However, it is important to note that this ADE phenomenon could be eliminated through the addition of human serum/IgG or the introduction of mutations in the antibodys Fc region. These results suggest that the observed in vitro ADE may not be a true predictor of ADE in real-life scenarios in the complex in vivo environment. 2. Materials and Methods 2.1. Cell Lines Expi293F cells (Thermo Fisher, Waltham, MA, USA, Cat# A14527) were cultured in the serum-free SMM 293-TI medium (Sino Biological Inc., Beijing, China) at 37 C with 8% CO2 on an orbital shaker platform. HEK293T cells (Cat# CRL-3216) and Vero-E6 cells (cat# CRL-1586) were acquired from ATCC and cultured in 10% Fetal Bovine Serum (FBS, GIBCO cat# 16140071) supplemented with Dulbeccos Modified Eagle Medium (DMEM, ATCC cat# 30-2002) at 37 C, 5% CO2. Raji cells (Cat# CCL-86) and THP-1 cells (cat# TIB-202) were acquired from ATCC and cultured in 10% FBS supplemented with Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher, W-2429 cat# 31870-082) at 37 C, 5% CO2. I1 mouse hybridoma cells (ATCC, cat# CRL-2700) were cultured in Eagles Minimum Essential Medium (EMEM, ATCC cat# 30-2003) with 20% FBS. 2.2. Serum Samples Sera from 7 individuals who received three doses of inactivated vaccine, or 7 individuals who were infected with the BA.5 variant after receiving three doses of inactivated vaccine, were recruited at the Nanjing Hospital of Chinese Medicine. For all COVID-19 participants, the clinical diagnosis criteria were based on the ninth National COVID-19 guidelines. The SARS-CoV-2 infection of all the subjects was confirmed by polymerase chain reaction (PCR) and sequencing. All participants involved in this study showed mild symptoms, or were asymptomatic. Two healthy individuals with no history of vaccination or infection were enrolled before the onset of the COVID-19 pandemic as controls at Huashan Hospital, Fudan University. 2.3. Monoclonal Antibodies Monoclonal antibodies tested in this study were constructed and produced at Fudan University. For each antibody, variable genes were optimized for human cell expression and synthesized by HuaGeneTM (Shanghai, China). VH and VL were inserted separately into plasmids (gWiz or pcDNA3.4) that encode the constant region for the H chain and L chain. Monoclonal antibodies were expressed in Expi293F (Thermo Fisher, A14527) by co-transfection of the H chain and L chain expressing plasmids using polyethylenimine and cultured at 37 C with shaking at 125 rpm and 8% CO2. On day 5, antibodies were purified using MabSelectTM PrismA (Cytiva, Marlborough, MA, USA, 17549801) affinity chromatography. 2.4. Construction and Production of Variant Pseudoviruses Plasmids encoded with the WT (D614G) SARS-CoV-2 spike.