Comprehensive structural analysis reveals broad-spectrum neutralizing antibodies against SARS-CoV-2 Omicron variants.

The pandemic of COVID-19 caused by SARS-CoV-2 continues to spread around the world. Mutant strains of SARS-CoV-2 are constantly emerging. At present, Omicron variants have become mainstream. In this work, we carried out a systematic and comprehensive analysis of the reported spike protein antibodies, counting the epitopes and genotypes of these antibodies. We further comprehensively analyzed the impact of Omicron mutations on antibody epitopes and classified these antibodies according to their binding patterns. We found that the epitopes of the H-RBD class antibodies were significantly less affected by Omicron mutations than other classes. Binding and virus neutralization experiments showed that such antibodies could effectively inhibit the immune escape of Omicron. Cryo-EM results showed that this class of antibodies utilized a conserved mechanism to neutralize SARS-CoV-2. Our results greatly help us deeply understand the impact of Omicron mutations. Meanwhile, it also provides guidance and insights for developing Omicron antibodies and vaccines.

A bispecific nanobody dimer broadly neutralizes SARS-CoV-1 & 2 variants of concern and offers substantial protection against Omicron via low-dose intranasal administration.

Current SARS-CoV-2 Omicron subvariants impose a heavy burden on global health systems by evading immunity from most developed neutralizing antibodies and vaccines. Here, we identified a nanobody (aSA3) that strongly cross-reacts with the receptor binding domain (RBD) of both SARS-CoV-1 and wild-type (WT) SARS-CoV-2. The dimeric construct of aSA3 (aSA3-Fc) tightly binds and potently neutralizes both SARS-CoV-1 and WT SARS-CoV-2. Based on X-ray crystallography, we engineered a bispecific nanobody dimer (2-3-Fc) by fusing aSA3-Fc to aRBD-2, a previously identified broad-spectrum nanobody targeting an RBD epitope distinct from aSA3. 2-3-Fc exhibits single-digit ng/mL neutralizing potency against all major variants of concerns including BA.5. In hamsters, a single systemic dose of 2-3-Fc at 10 mg/kg conferred substantial efficacy against Omicron infection. More importantly, even at three low doses of 0.5 mg/kg, 2-3-Fc prophylactically administered through the intranasal route drastically reduced viral RNA loads and completely eliminated infectious Omicron particles in the trachea and lungs. Finally, we discovered that 2(Y29G)-3-Fc containing a Y29G substitution in aRBD-2 showed better activity than 2-3-Fc in neutralizing BA.2.75, a recent Omicron subvariant that emerged in India. This study expands the arsenal against SARS-CoV-1, provides potential therapeutic and prophylactic candidates that fully cover major SARS-CoV-2 variants, and may offer a simple preventive approach against Omicron and its subvariants.

Developing Pseudovirus-Based Neutralization Assay against Omicron-Included SARS-CoV-2 Variants.

The global spread of SARS-CoV-2 and its variants poses a serious threat to human health worldwide. Recently, the emergence of Omicron has presented a new challenge to the prevention and control of the COVID-19 pandemic. A convenient and reliable in vitro neutralization assay is an important method for validating the efficiency of antibodies, vaccines, and other potential drugs. Here, we established an effective assay based on a pseudovirus carrying a full-length spike (S) protein of SARS-CoV-2 variants in the HIV-1 backbone, with a luciferase reporter gene inserted into the non-replicate pseudovirus genome. The key parameters for packaging the pseudovirus were optimized, including the ratio of the S protein expression plasmids to the HIV backbone plasmids and the collection time for the Alpha, Beta, Gamma, Kappa, and Omicron pseudovirus particles. The pseudovirus neutralization assay was validated using several approved or developed monoclonal antibodies, underscoring that Omicron can escape some neutralizing antibodies, such as REGN10987 and REGN10933, while S309 and ADG-2 still function with reduced neutralization capability. The neutralizing capacity of convalescent plasma from COVID-19 convalescent patients in Wuhan was tested against these pseudoviruses, revealing the immune evasion of Omicron. Our work established a practical pseudovirus-based neutralization assay for SARS-CoV-2 variants, which can be conducted safely under biosafety level-2 (BSL-2) conditions, and this assay will be a promising tool for studying and characterizing vaccines and therapeutic candidates against Omicron-included SARS-CoV-2 variants.

Developing Pseudovirus-Based Neutralization Assay against Omicron-Included SARS-CoV-2 Variants

The global spread of SARS-CoV-2 and its variants poses a serious threat to human health worldwide. Recently, the emergence of Omicron has presented a new challenge to the prevention and control of the COVID-19 pandemic. A convenient and reliable in vitro neutralization assay is an important method for validating the efficiency of antibodies, vaccines, and other potential drugs. Here, we established an effective assay based on a pseudovirus carrying a full-length spike (S) protein of SARS-CoV-2 variants in the HIV-1 backbone, with a luciferase reporter gene inserted into the non-replicate pseudovirus genome. The key parameters for packaging the pseudovirus were optimized, including the ratio of the S protein expression plasmids to the HIV backbone plasmids and the collection time for the Alpha, Beta, Gamma, Kappa, and Omicron pseudovirus particles. The pseudovirus neutralization assay was validated using several approved or developed monoclonal antibodies, underscoring that Omicron can escape some neutralizing antibodies, such as REGN10987 and REGN10933, while S309 and ADG-2 still function with reduced neutralization capability. The neutralizing capacity of convalescent plasma from COVID-19 convalescent patients in Wuhan was tested against these pseudoviruses, revealing the immune evasion of Omicron. Our work established a practical pseudovirus-based neutralization assay for SARS-CoV-2 variants, which can be conducted safely under biosafety level-2 (BSL-2) conditions, and this assay will be a promising tool for studying and characterizing vaccines and therapeutic candidates against Omicron-included SARS-CoV-2 variants.

Broadly neutralizing antibodies against Omicron-included SARS-CoV-2 variants induced by vaccination.

The SARS-CoV-2 Omicron variant shows substantial resistance to neutralization by infection- and vaccination-induced antibodies, highlighting the demands for research on the continuing discovery of broadly neutralizing antibodies (bnAbs). Here, we developed a panel of bnAbs against Omicron and other variants of concern (VOCs) elicited by vaccination of adenovirus-vectored COVID-19 vaccine (Ad5-nCoV). We also investigated the human longitudinal antibody responses following vaccination and demonstrated how the bnAbs evolved over time. A monoclonal antibody (mAb), named ZWD12, exhibited potent and broad neutralization against SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa, Delta, and Omicron by blocking the spike protein binding to the angiotensin-converting enzyme 2 (ACE2) and provided complete protection in the challenged prophylactic and therapeutic K18-hACE2 transgenic mouse model. We defined the ZWD12 epitope by determining its structure in complex with the spike (S) protein via cryo-electron microscopy. This study affords the potential to develop broadly therapeutic mAb drugs and suggests that the RBD epitope bound by ZWD12 is a rational target for the design of a broad spectrum of vaccines.

Integrated single-cell analysis revealed immune dynamics during Ad5-nCoV immunization.

Coronavirus disease 2019 (COVID-19), driven by SARS-CoV-2, is a severe infectious disease that has become a global health threat. Vaccines are among the most effective public health tools for combating COVID-19. Immune status is critical for evaluating the safety and response to the vaccine, however, the evolution of the immune response during immunization remains poorly understood. Single-cell RNA sequencing (scRNA-seq) represents a powerful tool for dissecting multicellular behavior and discovering therapeutic antibodies. Herein, by performing scRNA/V(D)J-seq on peripheral blood mononuclear cells from four COVID-19 vaccine trial participants longitudinally during immunization, we revealed enhanced cellular immunity with concerted and cell type-specific IFN responses as well as boosted humoral immunity with SARS-CoV-2-specific antibodies. Based on the CDR3 sequence and germline enrichment, we were able to identify several potential binding antibodies. We synthesized, expressed and tested 21 clones from the identified lineages. Among them, one monoclonal antibody (P3V6-1) exhibited relatively high affinity with the extracellular domain of Spike protein, which might be a promising therapeutic reagent for COVID-19. Overall, our findings provide insights for assessing vaccine through the novel scRNA/V(D)J-seq approach, which might facilitate the development of more potent, durable and safe prophylactic vaccines.

A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2.

Developing therapeutics against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could be guided by the distribution of epitopes, not only on the receptor binding domain (RBD) of the Spike (S) protein but also across the full Spike (S) protein. We isolated and characterized monoclonal antibodies (mAbs) from 10 convalescent COVID-19 patients. Three mAbs showed neutralizing activities against authentic SARS-CoV-2. One mAb, named 4A8, exhibits high neutralization potency against both authentic and pseudotyped SARS-CoV-2 but does not bind the RBD. We defined the epitope of 4A8 as the N-terminal domain (NTD) of the S protein by determining with cryo-eletron microscopy its structure in complex with the S protein to an overall resolution of 3.1 angstroms and local resolution of 3.3 angstroms for the 4A8-NTD interface. This points to the NTD as a promising target for therapeutic mAbs against COVID-19.

Antibodies in the breast milk of a maternal woman with COVID-19.

A maternal woman was positive for SARS-CoV-2 tested in throat swabs but negative tested in other body fluids, and she had IgG and IgA detected in breast milk. Her infant negative for SARS-CoV-2 at birth had elevated IgG in serum but quickly decayed. These findings suggest that breastfeeding might have the potential benefit to the neonates.

Aerosol and Surface Distribution of Severe Acute Respiratory Syndrome Coronavirus 2 in Hospital Wards, Wuhan, China, 2020.

To determine distribution of severe acute respiratory syndrome coronavirus 2 in hospital wards in Wuhan, China, we tested air and surface samples. Contamination was greater in intensive care units than general wards. Virus was widely distributed on floors, computer mice, trash cans, and sickbed handrails and was detected in air ≈4 m from patients.