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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 1 , 2 . 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. Individual antibodies identified in the blood of people triple-vaccinated against SARS-CoV-2 predominantly bind spike protein and are highly effective at neutralizing SARS-CoV-2 variants, including Omicron (B.1.1.529).

Newcastle disease virus (NDV) belongs to Paramyxoviridae, which contains lethal human and animal pathogens. NDV RNA genome is replicated and transcribed by a multifunctional 250 kDa RNA-dependent RNA polymerase (L protein). To date, high-resolution structure of NDV L protein complexed with P protein remains to be elucidated, limiting our understanding of the molecular mechanisms of Paramyxoviridae replication/transcription. Here, we used cryo-EM and enzymatic assays to investigate the structure-function relationship of L-P complex. We found that C-terminal of CD-MTase-CTD module of the atomic-resolution L-P complex conformationally rearranges, and the priming/intrusion loops are likely in RNA elongation conformations different from previous structures. The P protein adopts a unique tetrameric organization and interacts with L protein. Our findings indicate that NDV L-P complex represents elongation state distinct from previous structures. Our work greatly advances the understanding of Paramyxoviridae RNA synthesis, revealing how initiation/elongation alternates, providing clues for identifying therapeutic targets against Paramyxoviridae. Newcastle disease virus (NDV) belongs to Paramyxoviridae and encodes a large protein (L) and phosphoprotein (P) for viral RNA synthesis. Here the authors present cryo-EM structures of the L-P complex, and propose a model of how RNA initiation/elongation alternates during viral RNA synthesis.

Echovirus 3 (E3), a serotype of human enterovirus B (HEV-B), causes severe diseases in infants. Here, we determined the structures of E3 with a monoclonal antibody (MAb) 6D10 by cryo-EM to comprehensively understand the specificities and the immunological characteristic of this serotype. The solved cryo-EM structures of the F-, A-, and E-particles of E3 bound with 6D10 revealed the structural features of the virus–antibody interface. Importantly, the structures of E-particles bound with 6D10 revealed for the first time the nature of the C-terminus of VP1 for HEV-Bs at the structural level. The highly immunogenic nature of this region in the E-particles provides new strategies for vaccine development for HEV-Bs.

Spike‐protein‐based pseudotyped viruses were used to evaluate vaccines during the COVID‐19 pandemic. However, they cannot be used to evaluate the envelope (E), membrane (M), and nucleocapsid (N) proteins. The first generation of virus‐like particle (VLP) pseudotyped viruses contains these four structural proteins, but their titers for wild‐type severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) are relatively low, even lower for the omicron variant, rendering them unsuitable for neutralizing antibody detection. By optimizing the spike glycoprotein signal peptide, substituting the complexed M and E proteins with SARS‐COV‐1, optimizing the N protein with specific mutations (P199L, S202R, and R203M), and truncating the packaging signal, PS9, we increased the titer of the wild‐type VLP pseudotyped virus over 100‐fold, and successfully packaged the omicron VLP pseudotyped virus. The SARS‐CoV‐2 VLP pseudotyped viruses maintained stable titers, even through 10 freeze–thaw cycles. The key neutralization assay parameters were optimized, including cell type, cell number, and viral inoculum. The assay demonstrated minimal variation in both intra‐ and interassay results, at 11.5% and 11.1%, respectively. The correlation between the VLP pseudotyped virus and the authentic virus was strong (r = 0.9). Suitable for high‐throughput detection of various mutant strains in clinical serum. In summary, we have developed a reliable neutralization assay for SARS‐CoV‐2 based on VLP pseudotyped virus. Through the optimization of the structural proteins of coronavirus virus‐like particle (VLP) pseudotyped virus, we successfully constructed high titer the VLP pseudotyped virus variant. We optimized the conditions for neutralizing antibodies and validated the methodology. We have established a detection method for neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) VLP pseudotyped virus, which shows good consistency with authentic viruses and suitable for detecting various variants.

Several human papillomavirus (HPV) L1-based virus-like particle (VLP) vaccines are in development to meet future global vaccination needs. Type-specific monoclonal antibodies with good reactivity to all types of vaccines are urgently needed to evaluate vaccine potency. In this study, binding activity, neutralizing activity, conformational sensitivity, immunodominance in human serum, and versatility were compared among antibodies. A broad-spectrum binding antibody (C4-F5-127) was selected as the capture antibody; four type-specific neutralizing antibodies (6-F5-77, 11-F5-187, 16-F5-196, and 18-F5-203) were selected as detection antibodies for HPV6, 11, 16, and 18, respectively. These antibodies formed a standardized and universal in vitro relative potency (IVRP) assay kit. High-resolution cryo-electron microscopy (cryo-EM) structures of HPV6-6-F5-77, HPV11-11-F5-187, HPV16-16-F5-196 and HPV18-18-F5-203 complexes define the location and nature of epitopes, revealing serotype specific binding modes and neutralization mechanisms. The IVRP results were correlated with potency data from mouse models, offering an efficient alternative to in vivo potency experiments.