Results 3.1. COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has spread to over 200 countries, causing widespread morbidity and mortality, as well as massive economic losses Tcf4 [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]. Crolibulin However, one of the biggest safety concerns with vaccines and antibody-based therapeutics is the 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 computer 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 computer virus entry [12]. This ADE phenomenon has been observed in the past with SARS-CoV, MERS-CoV, dengue computer virus (DENV), respiratory syncytial computer virus (RSV), and measles [13,14,15,16,17,18], raising concerns 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 contamination have been reported to associate with more severe disease, suggesting a possible link with ADE [21]. Several in vitro studies have demonstrated that this ADE contamination of SARS-CoV-2 [22,23,24] and the emergence of new SARS-CoV-2 variants may increase the likelihood Crolibulin of ADE [25]. However, it remains unclear if in vitro ADE of contamination can accurately predict enhanced contamination in vivo. Using FcR-expressing B cells, 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, Crolibulin 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, Crolibulin 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, 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 those COVID-19 participants, the clinical diagnosis criteria were based on the ninth National COVID-19 guidelines. The SARS-CoV-2 contamination of all the subjects was confirmed by polymerase chain reaction (PCR) and sequencing. All participants involved in this study showed moderate symptoms, or were asymptomatic. Two healthy individuals with no history of vaccination or contamination 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.