No abstract

SARS-CoV-2 has infected millions of people and is on a trajectory to kill more than one million globally. Virus entry depends on the receptor-binding domain (RBD) of the spike protein. Although previous studies demonstrated anti-spike and -RBD antibodies as essential for protection and convalescent plasma as a promising therapeutic option, little is known about the immunoglobulin (Ig) isotypes capable of blocking virus entry. Here, we studied spike- and RBD-specific Ig isotypes in plasma/sera from two acutely infected and 29 convalescent individuals. Spike- and RBD-specific IgM, IgG1, and IgA1 antibodies were produced by all or nearly all subjects at varying levels and detected at 7-8 days post-disease onset. IgG2, IgG3, IgG4, and IgA2 were also present but at much lower levels. All samples also displayed neutralizing activity. IgM, IgG, and IgA were capable of mediating neutralization, but neutralization titers correlated better with binding levels of IgM and IgA1 than IgG.

Measles virus (MV), a member of the familyAlpha/beta interferons (IFN-␣/␤) play central roles in the host defense against viral infections (42). Pattern recognition receptors are the host molecules that detect pathogen-associated molecular patterns and activate the innate immune responses that operate at the early stage of infections (1). Tolllike receptor 3 (TLR3) and TLR7 recognize viral RNAs in the endosome and induce the production of various cytokines, including IFNs. In the cytoplasm of virus-infected cells, two RNA helicases, melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIG-I), are involved in virus recognition and IFN induction (21,58,59). Studies have shown that MDA5 recognizes long doublestranded RNAs (dsRNAs) (22), which are produced in cells infected with picornaviruses and reoviruses (23, 28), while RIG-I detects single-stranded RNAs with 5Ј-triphosphate (19, 37) and short dsRNAs (22), which are found in cells infected with a variety of RNA viruses. Therefore, it is generally believed that RIG-I plays a major role in the recognition of many RNA viruses, including paramyxoviruses (23, 28), whereas MDA5 only acts as an RNA sensor for certain RNA viruses.Measles is a febrile acute infectious disease that remains a major cause of child deaths worldwide, especially in developing countries (7). Measles virus (MV) is a member of the genus Morbillivirus in the family Paramyxoviridae. The MV genome possesses six genes, which encode N (nucleocapsid), P (phospho-), M (matrix), F (fusion), H (hemagglutinin), and L (large) proteins, respectively (16). The P gene encodes two additional proteins, the V and C proteins (5, 10). During transcription of the P gene, an additional guanine residue may be inserted at a specific site in the nascent transcript via the recognition of an editing motif, producing the V mRNA (10). Consequently, the V protein shares the N-terminal 231 amino acid residues with the P protein but has a unique C-terminal region with 68 amino acid residues. The C protein is translated from the P and V mRNAs using an alternative reading frame (5). Although the V and C proteins are dispensable for MV growth in some cultured cells (40, 45), they promote viral replication by circumventing the host innate immune responses (11,31,53) and are associated with MV virulence in vivo (12,35,53,54). The V protein blocks signal transduction in response to 13,14,32,34,41,51,57) and inhibits TLR7-and TLR9-mediated IFN-␣/␤ production in human plasmacytoid dendritic cells by binding to IB kinase ␣ and IFN regulatory factor (IRF) 7 (36). Furthermore, the V proteins of many paramyxoviruses, including MV, bind to MDA5 and inhibit its function (2). Therefore, the role of MDA5 in the recognition of paramyxoviruses may have been underestimated in previous studies.In the present study, we generated RIG-I and MDA5 knockdown human cells and a V protein-deficient recombinant MV (MV⌬V) and examined the roles of RIG-I and MDA5 in the detection of MV and the resulting IFN induction. Our res...

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected at least 180 million people since its identification as the cause of the current COVID-19 pandemic. The rapid pace of vaccine development has resulted in multiple vaccines already in use worldwide. The contemporaneous emergence of SARS-CoV-2 ‘variants of concern’ (VOC) across diverse geographic locales underscores the need to monitor the efficacy of vaccines being administered globally. All WHO designated VOC carry spike (S) polymorphisms thought to enable escape from neutralizing antibodies. Here, we characterize the neutralizing activity of post-Sputnik V vaccination sera against the ensemble of S mutations present in alpha (B.1.1.7) and beta (B.1.351) VOC. Using de novo generated replication-competent vesicular stomatitis virus expressing various SARS-CoV-2-S in place of VSV-G (rcVSV-CoV2-S), coupled with a clonal 293T-ACE2 + TMPRSS2 + cell line optimized for highly efficient S-mediated infection, we determine that only 1 out of 12 post-vaccination serum samples shows effective neutralization (IC90) of rcVSV-CoV2-S: B.1.351 at full serum strength. The same set of sera efficiently neutralize S from B.1.1.7 and exhibit only moderately reduced activity against S carrying the E484K substitution alone. Taken together, our data suggest that control of some emergent SARS-CoV-2 variants may benefit from updated vaccines.

The on-going coronavirus disease 2019 (COVID-19) pandemic has mobilized a global effort to develop vaccines and therapeutics that inhibit viral entry by inducing or transferring antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (CoV2-S). Phase I/II vaccine clinical trials, monoclonal antibodies, and convalescent sera have all shown promise. However, these efforts often require extensive screening with the live virus under onerous high biocontainment conditions (BSL-3). Virus neutralization assays (VNAs) remain the gold standard for evaluating the anti-viral potency of antibodies and entry inhibitors. The proliferation of pseudotyped virus systems that can be used in BSL-2 compatible VNAs is a positive development. Yet, there is marked variability between VNAs and how the findings are presented, making inter-group comparisons difficult. To address these limitations, we developed a standardized VNA using VSVdeltaG based CoV-2-S pseudotyped particles (CoV2pp) that can be robustly produced at scale. We used our CoV2pp to interrogate the role of exogenous and endogenous proteases in CoV-2-S mediated entry and standardized our VNA based on that understanding. Our CoV2pp VNA showed a strong positive correlation with CoV2-S ELISA and live virus neutralizations in a validated set of patient sera. Our system was subsequently validated by three independent groups as an out-of-the-box VNA. More than 120 patient sera were screened, and we report descriptive statistics for absolute (abs) IC50, IC80, and IC90 values from all positive patient sera. Lastly, we used our CoV2pp in a screen to identify ultrapermissive 293T clones that stably express ACE2 or ACE2+TMPRSS2. When used in combination with our CoV2pp, we can now produce CoV2pp sufficient for 150,000 standardized VNA/week.