The SARS-CoV-2 B.1.1.529 (Omicron) variant contains 15 mutations of the receptor-binding domain (RBD). How Omicron evades RBD-targeted neutralizing antibodies requires immediate investigation. Here ...we use high-throughput yeast display screening
to determine the profiles of RBD escaping mutations for 247 human anti-RBD neutralizing antibodies and show that the neutralizing antibodies can be classified by unsupervised clustering into six epitope groups (A-F)-a grouping that is highly concordant with knowledge-based structural classifications
. Various single mutations of Omicron can impair neutralizing antibodies of different epitope groups. Specifically, neutralizing antibodies in groups A-D, the epitopes of which overlap with the ACE2-binding motif, are largely escaped by K417N, G446S, E484A and Q493R. Antibodies in group E (for example, S309)
and group F (for example, CR3022)
, which often exhibit broad sarbecovirus neutralizing activity, are less affected by Omicron, but a subset of neutralizing antibodies are still escaped by G339D, N440K and S371L. Furthermore, Omicron pseudovirus neutralization showed that neutralizing antibodies that sustained single mutations could also be escaped, owing to multiple synergetic mutations on their epitopes. In total, over 85% of the tested neutralizing antibodies were escaped by Omicron. With regard to neutralizing-antibody-based drugs, the neutralization potency of LY-CoV016, LY-CoV555, REGN10933, REGN10987, AZD1061, AZD8895 and BRII-196 was greatly undermined by Omicron, whereas VIR-7831 and DXP-604 still functioned at a reduced efficacy. Together, our data suggest that infection with Omicron would result in considerable humoral immune evasion, and that neutralizing antibodies targeting the sarbecovirus conserved region will remain most effective. Our results inform the development of antibody-based drugs and vaccines against Omicron and future variants.
The spike protein of SARS-CoV-2 has been undergoing mutations and is highly glycosylated. It is critically important to investigate the biological significance of these mutations. Here, we ...investigated 80 variants and 26 glycosylation site modifications for the infectivity and reactivity to a panel of neutralizing antibodies and sera from convalescent patients. D614G, along with several variants containing both D614G and another amino acid change, were significantly more infectious. Most variants with amino acid change at receptor binding domain were less infectious, but variants including A475V, L452R, V483A, and F490L became resistant to some neutralizing antibodies. Moreover, the majority of glycosylation deletions were less infectious, whereas deletion of both N331 and N343 glycosylation drastically reduced infectivity, revealing the importance of glycosylation for viral infectivity. Interestingly, N234Q was markedly resistant to neutralizing antibodies, whereas N165Q became more sensitive. These findings could be of value in the development of vaccine and therapeutic antibodies.
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•Over 100 mutations were selected for analyses on their infectivity and antigenicity•The dominant D614G itself and combined with other mutations are more infectious•Ablation of both N331 and N343 glycosylation at RBD drastically reduced infectivity•Ten mutations such as N234Q, L452R, A475V, and V483A was markedly resistant to some mAbs
Eighty natural variants and 26 glycosylation spike mutants of SARS-CoV-2 were analyzed in terms of infectivity and antigenicity using high throughput pseudovirus assay in conjunction with neutralizing antibodies.
Considering that the L455 is predominantly located at the epitope of receptor binding domain Class 1 antibodies, as indicated by earlier research, our study further examined the evasion capabilities ...of JN.1 in response to eight XBB.1·5-neutralising class 1 monoclonal antibodies.7 Pseudovirus neutralisation assays showed that the addition of the L455S mutation enhanced JN.1's ability to evade class 1 antibodies (figure D). In summary, JN.1, by inheriting BA.2.86's antigenic diversity and acquisition of L455S, rapidly achieved extensive resistance across receptor binding domain class 1, 2, and 3 antibodies,1 and showed higher immune evasion compared with BA.2.86 and other resistant strains like HV.1 and JD.1·1, at the expense of reduced human ACE2 binding. ...strains could survive and transmit at low levels since their antigenic difference would allow them to target distinct populations compared with dominant strains and have the potential to quickly accumulate highly immune-evasive mutations at the cost of human ACE2 binding capabilities.
Summary
Emerging and reemerging infectious diseases have a strong negative impact on public health. However, because many of these pathogens must be handled in biosafety level, 3 or 4 containment ...laboratories, research and development of antivirals or vaccines against these diseases are often impeded. Alternative approaches to address this issue have been vigorously pursued, particularly the use of pseudoviruses in place of wild‐type viruses. As pseudoviruses have been deprived of certain gene sequences of the virulent virus, they can be handled in biosafety level 2 laboratories. Importantly, the envelopes of these viral particles may have similar conformational structures to those of the wild‐type viruses, making it feasible to conduct mechanistic investigation on viral entry and to evaluate potential neutralizing antibodies. However, a variety of challenging issues remain, including the production of a sufficient pseudovirus yield and the inability to produce an appropriate pseudotype of certain viruses. This review discusses current progress in the development of pseudoviruses and dissects the factors that contribute to low viral yields.
Pseudotyped viruses are useful virological tools because of their safety and versatility. On the basis of a vesicular stomatitis virus (VSV) pseudotyped virus production system, we developed a ...pseudotyped virus-based neutralization assay against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in biosafety level 2 facilities. Compared with the binding antibody test, the neutralization assay could discriminate the protective agents from the antibody family. This protocol includes production and titration of the SARS-CoV-2 S pseudotyped virus and the neutralization assay based on it. Various types of samples targeting virus attachment and entry could be evaluated for their potency, including serum samples derived from animals and humans, monoclonal antibodies and fusion inhibitors (peptides or small molecules). If the pseudotyped virus stock has been prepared in advance, it will take 2 days to get the potency data for the candidate samples. Experience in handling cells is needed before implementing this protocol.
The 501Y.V2 variants of SARS-CoV-2 containing multiple mutations in spike are now dominant in South Africa and are rapidly spreading to other countries. Here, experiments with 18 pseudotyped viruses ...showed that the 501Y.V2 variants do not confer increased infectivity in multiple cell types except for murine ACE2-overexpressing cells, where a substantial increase in infectivity was observed. Notably, the susceptibility of the 501Y.V2 variants to 12 of 17 neutralizing monoclonal antibodies was substantially diminished, and the neutralization ability of the sera from convalescent patients and immunized mice was also reduced for these variants. The neutralization resistance was mainly caused by E484K and N501Y mutations in the receptor-binding domain of spike. The enhanced infectivity in murine ACE2-overexpressing cells suggests the possibility of spillover of the 501Y.V2 variants to mice. Moreover, the neutralization resistance we detected for the 501Y.V2 variants suggests the potential for compromised efficacy of monoclonal antibodies and vaccines.
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•501Y.V2 showed no higher infectivity in cells with hACE2 comparing to 614G variant•501Y.V2 showed increased infectivity in cells with mACE2 compared to 614G variant•501Y.V2 escaped neutralization by most of neutralizing monoclonal antibodies•501Y.V2 significantly compromised the inhibitory effects of polyclonal antibodies
Experiments with pseudotyped viruses show that the 501Y.V2 variant of SARS-CoV-2 exhibits resistance to neutralization from monoclonal antibodies and sera from convalescent as well as immunized individuals, predominantly due to the E484K and N501Y mutations in the receptor-binding domain of the viral spike protein.
The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented public health crisis. There are no approved ...vaccines or therapeutics for treating COVID-19. Here we report a humanized monoclonal antibody, H014, that efficiently neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses as well as authentic SARS-CoV-2 at nanomolar concentrations by engaging the spike (S) receptor binding domain (RBD). H014 administration reduced SARS-CoV-2 titers in infected lungs and prevented pulmonary pathology in a human angiotensin-converting enzyme 2 mouse model. Cryo-electron microscopy characterization of the SARS-CoV-2 S trimer in complex with the H014 Fab fragment unveiled a previously uncharacterized conformational epitope, which was only accessible when the RBD was in an open conformation. Biochemical, cellular, virological, and structural studies demonstrated that H014 prevents attachment of SARS-CoV-2 to its host cell receptors. Epitope analysis of available neutralizing antibodies against SARS-CoV and SARS-CoV-2 uncovered broad cross-protective epitopes. Our results highlight a key role for antibody-based therapeutic interventions in the treatment of COVID-19.
Omicron (B.1.1.529), the most heavily mutated SARS-CoV-2 variant so far, is highly resistant to neutralizing antibodies, raising concerns about the effectiveness of antibody therapies and vaccines
. ...Here we examined whether sera from individuals who received two or three doses of inactivated SARS-CoV-2 vaccine could neutralize authentic Omicron. The seroconversion rates of neutralizing antibodies were 3.3% (2 out of 60) and 95% (57 out of 60) for individuals who had received 2 and 3 doses of vaccine, respectively. For recipients of three vaccine doses, the geometric mean neutralization antibody titre for Omicron was 16.5-fold lower than for the ancestral virus (254). We isolated 323 human monoclonal antibodies derived from memory B cells in triple vaccinees, half of which recognized the receptor-binding domain, and showed that a subset (24 out of 163) potently neutralized all SARS-CoV-2 variants of concern, including Omicron. Therapeutic treatments with representative broadly neutralizing monoclonal antibodies were highly protective against infection of mice with SARS-CoV-2 Beta (B.1.351) and Omicron. Atomic structures of the Omicron spike protein in complex with three classes of antibodies that were active against all five variants of concern defined the binding and neutralizing determinants and revealed a key antibody escape site, G446S, that confers greater resistance to a class of antibodies that bind on the right shoulder of the receptor-binding domain by altering local conformation at the binding interface. Our results rationalize the use of three-dose immunization regimens and suggest that the fundamental epitopes revealed by these broadly ultrapotent antibodies are rational targets for a universal sarbecovirus vaccine.
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