Amino acids 484 and 494 of SARS-CoV-2 spike are hotspots of immune evasion affecting antibody but not ACE2 binding

Abstract

Understanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike. Mutations in spike impacting antibody and/or ACE2 binding are appearing worldwide, with the effect of mutation synergy still incompletely understood. We engineered 25 spike-pseudotyped lentiviruses containing individual and combined mutations, and confirmed that E484K evades antibody neutralization elicited by infection or vaccination, a capacity augmented when complemented by K417N and N501Y mutations. In silico analysis provided an explanation for E484K immune evasion. E484 frequently engages in interactions with antibodies but not with ACE2. Importantly, we identified a novel amino acid of concern, S494, which shares a similar pattern. Using the already circulating mutation S494P, we found that it reduces antibody neutralization of convalescent and post-immunization sera, particularly when combined with E484K and N501Y. Our analysis of synergic mutations provides a landscape for hotspots for immune evasion and for targets for therapies, vaccines and diagnostics.

Epistasis at the SARS-CoV-2 Receptor-Binding Domain Interface and the Propitiously Boring Implications for Vaccine Escape

“…Most prominently, mutations in site 494 and the surrounding neighborhood are likely to enhance vaccine escape in all variants. Indeed, 494P has been found in circulation and experimentally demonstrated to reduce antibody neutralization capacity of convalescent-phase sera ( 25 ).…”

Non-neutralizing secretory IgA and T cells targeting SARS-CoV-2 spike protein are transferred to the breastmilk upon BNT162b2 vaccination

“…1I). We next determined their virus-neutralizing activity through a microneutralization assay using spike pseudotyped lentivirus infection of ACE-2 receptor expressing 293T cell line 43 . At 10d post vaccine prime, milk IgA had no neutralizing activity (Fig.…”

“…Production of 293T cells stably expressing human ACE2 receptor was done as previously described 43 . Briefly, VSV-G pseudotyped lentiviruses encoding human ACE2, 293ET cells were transfected with pVSV-G, psPAX2 and pLEX-ACE2 using jetPRIME (Polyplus), according to manufacturer's instructions.…”

Saliva molecular testing bypassing RNA extraction is suitable for monitoring and diagnosing SARS-CoV-2 infection in children

“…The ELISA assay used to quantify saliva IgG, IgA, and IgM anti-full-length SARS-CoV-2 spike and its receptor-binding domain (RBD) was adapted from [ 22 ] as described in [ 23 , 24 ], with few modifications described here. Briefly, high binding 96‐well plates (Maxisorb) were coated with either RBD or spike as capture antigen at 0.5 μg/mL stored overnight at 4°C.…”

“…A second limitation of this study is that the number of samples from which we rescued infective viral particles in cultured cells does not allow a suitable statistical analysis to relate viral loads with symptoms and is not a formal demonstration of transmission from children. A third caveat is that the ELISA assay, despite being specific for sera and collected from infected people [ 23 ], was not established for saliva. In fact, we did not calibrate the ELISA with pre-pandemic saliva, and as children were not re-analyzed posteriorly for antibody development, there is a lack of saliva positive and negative controls, which also limits the conclusions we may draw from the data.…”