Host Cell Entry and Neutralization Sensitivity of SARS-CoV-2 Lineages B.1.620 and R.1.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) facilitates viral entry into host cells and is the key target for neutralizing antibodies. The SARS-CoV-2 lineage B.1.620 carries fifteen mutations in the S protein and is spread in Africa, the US and Europe, while lineage R.1 harbors four mutations in S and infections were observed in several countries, particularly Japan and the US. However, the impact of the mutations in B.1.620 and R.1 S proteins on antibody-mediated neutralization and host cell entry are largely unknown. Here, we report that these mutations are compatible with robust ACE2 binding and entry into cell lines, and they markedly reduce neutralization by vaccine-induced antibodies. Our results reveal evasion of neutralizing antibodies by B.1.620 and R.1, which might have contributed to the spread of these lineages.

Augmented neutralization of SARS-CoV-2 Omicron variant by boost vaccination and monoclonal antibodies.

Effective vaccines and monoclonal antibodies have been developed against coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the appearance of virus variants with higher transmissibility and pathogenicity is a major concern because of their potential to escape vaccines and clinically approved SARS-CoV-2- antibodies. Here, we use flow cytometry-based binding and pseudotyped SARS-CoV-2 neutralization assays to determine the efficacy of boost immunization and therapeutic antibodies to neutralize the dominant Omicron variant. We provide compelling evidence that the third vaccination with BNT162b2 increases the amount of neutralizing serum antibodies against Delta and Omicron variants, albeit to a lower degree when compared to the parental Wuhan strain. Therefore, a third vaccination is warranted to increase titers of protective serum antibodies, especially in the case of the Omicron variant. We also found that most clinically approved and otherwise potent therapeutic antibodies against the Delta variant failed to recognize and neutralize the Omicron variant. In contrast, some antibodies under preclinical development potentially neutralized the Omicron variant. Our studies also support using a flow cytometry-based antibody binding assay to rapidly monitor therapeutic candidates and serum titers against emerging SARS-CoV-2 variants.

A pair of noncompeting neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model.

TRIANNI mice carry an entire set of human immunoglobulin V region gene segments and are a powerful tool to rapidly isolate human monoclonal antibodies. After immunizing these mice with DNA encoding the spike protein of SARS-CoV-2 and boosting with spike protein, we identified 29 hybridoma antibodies that reacted with the SARS-CoV-2 spike protein. Nine antibodies neutralize SARS-CoV-2 infection at IC50 values in the subnanomolar range. ELISA-binding studies and DNA sequence analyses revealed one cluster of three clonally related neutralizing antibodies that target the receptor-binding domain and compete with the cellular receptor hACE2. A second cluster of six clonally related neutralizing antibodies bind to the N-terminal domain of the spike protein without competing with the binding of hACE2 or cluster 1 antibodies. SARS-CoV-2 mutants selected for resistance to an antibody from one cluster are still neutralized by an antibody from the other cluster. Antibodies from both clusters markedly reduced viral spread in mice transgenic for human ACE2 and protected the animals from SARS-CoV-2-induced weight loss. The two clusters of potent noncompeting SARS-CoV-2 neutralizing antibodies provide potential candidates for therapy and prophylaxis of COVID-19. The study further supports transgenic animals with a human immunoglobulin gene repertoire as a powerful platform in pandemic preparedness initiatives.

Immunizations with diverse sarbecovirus receptor-binding domains elicit SARS-CoV-2 neutralizing antibodies against a conserved site of vulnerability.

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.

Immunizations with diverse sarbecovirus receptor binding domains elicit SARS-CoV-2 neutralizing antibodies against a conserved site of vulnerability

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Burnett et al. immunized humanized mice with different diverse sarbecovirus RBDs to elicit antibodies targeting conserved sites. Non-neutralizing cross-reactive antibodies targeting the conserved class 4 epitope were readily elicited. Neutralizing ability was reserved only for antibodies binding this conserved supersite through an elongated CDRH3 that obstructed ACE2-RBD interactions.

A surrogate cell-based SARS-CoV-2 spike blocking assay.

To monitor infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and successful vaccination against coronavirus disease 2019 (COVID-19), the kinetics of neutralizing or blocking anti-SARS-CoV-2 antibody titers need to be assessed. Here, we report the development of a quick and inexpensive surrogate SARS-CoV-2 blocking assay (SUBA) using immobilized recombinant human angiotensin-converting enzyme 2 (hACE2) and human cells expressing the native form of surface SARS-CoV-2 spike protein. Spike protein-expressing cells bound to hACE2 in the absence or presence of blocking antibodies were quantified by measuring the optical density of cell-associated crystal violet in a spectrophotometer. The advantages are that SUBA is a fast and inexpensive assay, which does not require biosafety level 2- or 3-approved laboratories. Most importantly, SUBA detects blocking antibodies against the native trimeric cell-bound SARS-CoV-2 spike protein and can be rapidly adjusted to quickly pre-screen already approved therapeutic antibodies or sera from vaccinated individuals for their ACE2 blocking activities against any emerging SARS-CoV-2 variants.