ABSTRACT
Continuous evolution of Omicron has led to a rapid and simultaneous emergence of numerous variants that display growth advantages over BA. 5. Despite their divergent evolutionary courses, mutations on their receptor-binding domain (RBD) converge on several hotspots. The driving force and destination of such convergent evolution and its impact on humoral immunity remain unclear. Here, we demonstrate that these convergent mutations can cause striking evasion of neutralizing antibody (NAb) drugs and convalescent plasma, including those from BA.5 breakthrough infection, while maintaining sufficient ACE2 binding capability. BQ.1.1.10, BA.4.6.3, XBB, and CH. 1.1 are the most antibody-evasive strain tested, even exceeding SARS-CoV-1 level. To delineate the origin of the convergent evolution, we determined the escape mutation profiles and neutralization activity of monoclonal antibodies (mAbs) isolated from BA.2 and BA.5 breakthrough-infection convalescents. Importantly, due to humoral immune imprinting, BA.2 and especially BA.5 breakthrough infection caused significant reductions in the epitope diversity of NAbs and increased proportion of non-neutralizing mAbs, which in turn concentrated humoral immune pressure and promoted convergent evolution. Moreover, we showed that the convergent RBD mutations could be accurately inferred by integrated deep mutational scanning (DMS) profiles, and the evolution trends of BA.2.75/BA.5 subvariants could be well-simulated through constructed convergent pseudovirus mutants. Together, our results suggest current herd immunity and BA.5 vaccine boosters may not provide good protection against infection. Broad-spectrum SARS-CoV-2 vaccines and NAb drugs development should be highly prioritized, and the constructed mutants could help to examine their effectiveness in advance.
ABSTRACT
Multiple BA.4 and BA.5 subvariants with R346 mutations on the spike glycoprotein have been identified in various countries, such as BA.4.6/BF.7 harboring R346T, BA.4.7 harboring R346S, and BA.5.9 harboring R346I. These subvariants, especially BA.4.6, exhibit substantial growth advantages compared to BA.4/BA.5. In this study, we showed that BA.4.6, BA.4.7, and BA.5.9 displayed higher humoral immunity evasion capability than BA.4/BA.5, causing 1.5 to 1.9-fold decrease in NT50 of the plasma from BA.1 and BA.2 breakthrough-infection convalescents compared to BA.4/BA.5. Importantly, plasma from BA.5 breakthrough-infection convalescents also exhibits significant neutralization activity decrease against BA.4.6, BA.4.7, and BA.5.9 than BA.4/BA.5, showing on average 2.4 to 2.6-fold decrease in NT50. For neutralizing antibody drugs, Bebtelovimab remains potent, while Evusheld is completely escaped by these subvariants. Together, our results rationalize the prevailing advantages of the R346 mutated BA.4/BA.5 subvariants and urge the close monitoring of these mutants, which could lead to the next wave of the pandemic.
ABSTRACT
SARS-CoV-2 Omicron sublineages have escaped most RBD-targeting therapeutic neutralizing antibodies (NAbs), which proves the previous NAb drug screening strategies deficient against the fast-evolving SARS-CoV-2. Better broad NAb drug candidate selection methods are needed. Here, we describe a rational approach for identifying RBD-targeting broad SARS-CoV-2 NAb cocktails. Based on high-throughput epitope determination, we propose that broad NAb drugs should target non-immunodominant RBD epitopes to avoid herd immunity-directed escape mutations. Also, their interacting antigen residues should focus on sarbecovirus conserved sites and associate with critical viral functions, making the antibody-escaping mutations less likely to appear. Following the criteria, a featured non-competing antibody cocktail, SA55+SA58, is identified from a large collection of broad sarbecovirus NAbs isolated from SARS convalescents. SA55+SA58 potently neutralizes ACE2-utilizing sarbecoviruses, including circulating Omicron variants, and could serve as broad SARS-CoV-2 prophylactics to offer long-term protection. Our screening strategy can also be applied to identify broad-spectrum NAb drugs against other fast-evolving viruses, such as influenza viruses.
ABSTRACT
Recently emerged SARS-CoV-2 Omicron subvariant, BA.2.75, displayed a local growth advantage over BA.2.38, BA.2.76 and BA.5 in India. The underlying mechanism of BA.2.75s enhanced infectivity, especially compared to BA.5, remains unclear. Here, we show that BA.2.75 exhibits substantially higher ACE2-binding affinity than BA.5. Also, BA.2.75 spike shows decreased thermostability and increased "up" RBD conformation in acidic conditions, suggesting enhanced low-pH-endosomal cell-entry pathway utilization. BA.2.75 is less humoral immune evasive than BA.4/BA.5 in BA.1/BA.2 breakthrough-infection convalescents; however, BA.2.75 shows heavier neutralization evasion in Delta breakthrough-infection convalescents. Importantly, plasma from BA.5 breakthrough infection exhibit significantly weaker neutralization against BA.2.75 than BA.5, mainly due to BA.2.75s distinct RBD and NTD-targeting antibody escaping pattern from BA.4/BA.5. Additionally, Evusheld and Bebtelovimab remain effective against BA.2.75, and Sotrovimab recovered RBD-binding affinity. Together, our results suggest BA.2.75 may prevail after the global BA.4/BA.5 wave, and its increased receptor-binding capability could allow further incorporation of immune-evasive mutations.
ABSTRACT
Recent studies found that Omicron variant escapes vaccine-elicited immunity. Interestingly, potent cross-clade pan-sarbecovirus neutralizing antibodies were found in survivors of the infection by SARS-CoV-1 after BNT162b2 mRNA vaccination (N Engl J Med. 2021 Oct 7;385(15):1401-1406). These pan-sarbecovirus neutralizing antibodies were observed to efficiently neutralize the infection driven by the S protein from both SARS-CoV and multiple SARS-CoV-2 variants of concern (VOC) including B.1.1.7 (Alpha), B.1.351 (Beta), and B.1.617.2 (Delta). However, whether these cross-reactive antibodies could neutralize the Omicron variant is still unknown. Based on the data collected from a cohort of SARS-CoV-1 survivors received 3-dose of immunization, our studies reported herein showed that a high level of neutralizing antibodies against both SARS-CoV-1 and SARS-CoV-2 were elicited by a 3rd-dose of booster vaccination of protein subunit vaccine ZF2001. However, a dramatically reduced neutralization of SARS-CoV-2 Omicron Variant (B.1.1.529) is observed in sera from these SARS-CoV-1 survivors received 3-dose of Vaccination. Our results indicates that the rapid development of pan-variant adapted vaccines is warranted.
ABSTRACT
SARS-CoV-2 Omicron sublineages BA.2.12.1, BA.4 and BA.5 exhibit higher transmissibility over BA.21. The new variants receptor binding and immune evasion capability require immediate investigation. Here, coupled with Spike structural comparisons, we show that BA.2.12.1 and BA.4/BA.5 exhibit comparable ACE2-binding affinities to BA.2. Importantly, BA.2.12.1 and BA.4/BA.5 display stronger neutralization evasion than BA.2 against the plasma from 3-dose vaccination and, most strikingly, from post-vaccination BA.1 infections. To delineate the underlying antibody evasion mechanism, we determined the escaping mutation profiles2, epitope distribution3 and Omicron neutralization efficacy of 1640 RBD-directed neutralizing antibodies (NAbs), including 614 isolated from BA.1 convalescents. Interestingly, post-vaccination BA.1 infection mainly recalls wildtype-induced humoral memory. The resulting elicited antibodies could neutralize both wildtype and BA.1 and are enriched on non-ACE2-competing epitopes. However, most of these cross-reactive NAbs are heavily escaped by L452Q, L452R and F486V. BA.1 infection can also induce new clones of BA.1-specific antibodies that potently neutralize BA.1; nevertheless, these NAbs are largely escaped by BA.2/BA.4/BA.5 due to D405N and F486V, and react weakly to pre-Omicron variants, exhibiting poor neutralization breadths. As for therapeutic NAbs, Bebtelovimab4 and Cilgavimab5 can effectively neutralize BA.2.12.1 and BA.4/BA.5, while the S371F, D405N and R408S mutations would undermine most broad sarbecovirus NAbs. Together, our results indicate that Omicron may evolve mutations to evade the humoral immunity elicited by BA.1 infection, suggesting that BA.1-derived vaccine boosters may not achieve broad-spectrum protection against new Omicron variants.
ABSTRACT
Omicron sub-lineage BA.2 has rapidly surged globally, accounting for over 60% of recent SARS-CoV-2 infections. Newly acquired RBD mutations and high transmission advantage over BA.1 urge the investigation of BA.2s immune evasion capability. Here, we show that BA.2 causes strong neutralization resistance, comparable to BA.1, in vaccinated individuals plasma. However, BA.2 displays more severe antibody evasion in BA.1 convalescents, and most prominently, in vaccinated SARS convalescents plasma, suggesting a substantial antigenicity difference between BA.2 and BA.1. To specify, we determined the escaping mutation profiles1,2 of 714 SARS-CoV-2 RBD neutralizing antibodies, including 241 broad sarbecovirus neutralizing antibodies isolated from SARS convalescents, and measured their neutralization efficacy against BA.1, BA.1.1, BA.2. Importantly, BA.2 specifically induces large-scale escape of BA.1/BA.1.1-effective broad sarbecovirus neutralizing antibodies via novel mutations T376A, D405N, and R408S. These sites were highly conserved across sarbecoviruses, suggesting that Omicron BA.2 arose from immune pressure selection instead of zoonotic spillover. Moreover, BA.2 reduces the efficacy of S309 (Sotrovimab)3,4 and broad sarbecovirus neutralizing antibodies targeting the similar epitope region, including BD55-5840. Structural comparisons of BD55-5840 in complexes with BA.1 and BA.2 spike suggest that BA.2 could hinder antibody binding through S371F-induced N343-glycan displacement. Intriguingly, the absence of G446S mutation in BA.2 enabled a proportion of 440-449 linear epitope targeting antibodies to retain neutralizing efficacy, including COV2-2130 (Cilgavimab)5. Together, we showed that BA.2 exhibits distinct antigenicity compared to BA.1 and provided a comprehensive profile of SARS-CoV-2 antibody escaping mutations. Our study offers critical insights into the humoral immune evading mechanism of current and future variants.
ABSTRACT
Since the first report on November 24, 2021, the Omicron SARS-CoV-2 variant is now overwhelmingly spreading across the world. Two SARS-CoV-2 inactivated vaccines (IAVs), one recombinant protein subunit vaccine (PRV), and one adenovirus-vectored vaccine (AdV) have been widely administrated in many countries including China to pursue herd immunity. Here we investigated cross-neutralizing activities in 341 human serum specimens elicited by full-course vaccinations with IAV, PRV and AdV, and by various vaccine boosters following prime IAV and AdV vaccinations. We found that all types of vaccines induced significantly lower neutralizing antibody titers against the Omicron variant than against the prototype strain. For prime vaccinations with IAV and AdV, heterologous boosters with AdV and PRV, respectively, elevated serum Omicron-neutralizing activities to the highest degrees. In a mouse model, we further demonstrated that among a series of variant-derived RBD-encoding mRNA vaccine boosters, it is only the Omicron booster that significantly enhanced Omicron neutralizing antibody titers compared with the prototype booster following a prime immunization with a prototype S-encoding mRNA vaccine candidate. In summary, our systematical investigations of various vaccine boosters inform potential booster administrations in the future to combat the Omicron variant.
ABSTRACT
The SARS-CoV-2 B.1.1.529 variant (Omicron) contains 15 mutations on the receptor-binding domain (RBD). How Omicron would evade RBD neutralizing antibodies (NAbs) requires immediate investigation. Here, we used high-throughput yeast display screening1,2 to determine the RBD escaping mutation profiles for 247 human anti-RBD NAbs and showed that the NAbs could be unsupervised clustered into six epitope groups (A-F), which is highly concordant with knowledge-based structural classifications3-5. Strikingly, various single mutations of Omicron could impair NAbs of different epitope groups. Specifically, NAbs in Group A-D, whose epitope overlap with ACE2-binding motif, are largely escaped by K417N, G446S, E484A, and Q493R. Group E (S309 site)6 and F (CR3022 site)7 NAbs, which often exhibit broad sarbecovirus neutralizing activity, are less affected by Omicron, but still, a subset of NAbs are escaped by G339D, N440K, and S371L. Furthermore, Omicron pseudovirus neutralization showed that single mutation tolerating NAbs could also be escaped due to multiple synergetic mutations on their epitopes. In total, over 85% of the tested NAbs are escaped by Omicron. Regarding NAb drugs, the neutralization potency of LY-CoV016/LY-CoV555, REGN10933/REGN10987, AZD1061/AZD8895, and BRII-196 were greatly reduced by Omicron, while VIR-7831 and DXP-604 still function at reduced efficacy. Together, data suggest Omicron would cause significant humoral immune evasion, while NAbs targeting the sarbecovirus conserved region remain most effective. Our results offer instructions for developing NAb drugs and vaccines against Omicron and future variants.
ABSTRACT
BackgroundBRII-196 and BRII-198 are two anti-SARS-CoV-2 monoclonal neutralizing antibodies with modified Fc region that extends half-life and are being developed as cocktail therapy for the treatment of COVID-19. Safety, tolerability, pharmacokinetics, and immunogenicity of BRII-196 and BRII-198 were investigated in healthy adults. MethodsSingle ascending doses of BRII-196 and BRII-198 were evaluated in parallel in the first-in-human, placebo-controlled phase 1 studies. A total of 32 healthy adults were randomized and received a single intravenous infusion of 750, 1500, and 3000 mg of BRII-196 (n=12), BRII-198 (n=12), or placebo (n=8) and were followed for 180 days. ResultsAll infusions were well tolerated at infusion rates between 0.5 mL/min to 4 mL/min with no dose-limiting adverse events, deaths, serious adverse events, or any systemic or local infusion reactions. Most treatment-emergent adverse events were isolated asymptomatic laboratory abnormalities of Grade 1-2 in severity. Each mAb displayed pharmacokinetics expected of Fc-engineered human IgG1 with mean terminal half-lives of approximately 46 days and 76 days, respectively, with no evidence of significant anti-drug antibody development. ConclusionsBRII-196 and BRII-198 were well-tolerated. Clinical results support further development as therapeutic or prophylactic options for SARS-CoV-2 infection.
ABSTRACT
Developing a high-throughput screening method is of great importance for directed evolution of atrazine chlorohydrolase. A mutagenesis library of atzA from Pseudomonas sp. ADP and Arthrobacter sp. AD1 was constructed using error-prone PCR and DNA shuffling. Candidate mutants were screened through Haematococcus pluvialis expression system, using atrazine as selection pressure. Sequence analysis showed that mutations in the obtained 12 mutants with enhanced activity were all point-substitutions and scattered throughout the gene. Enzymatic activity analysis showed that the mutants all had higher activities than that of the wild type. The activities were 1.8-3.6 fold of the wild-type enzyme when cultured in BBM medium with 1 mg/L atrazine, whereas 1.8-2.6 fold with 2 mg/L atrazine. These results indicated that Haematococcus pluvialis expression system is an ideal high throughput screening system for directed evolution of atrazine chlorohydrolase.
Subject(s)
Amidohydrolases , Genetics , Atrazine , Metabolism , Bacterial Proteins , Genetics , Biodegradation, Environmental , Chlorophyta , Genetics , Metabolism , Herbicides , Metabolism , High-Throughput Screening Assays , Hydrolases , Genetics , Mutagenesis, Insertional , Pseudomonas , GeneticsABSTRACT
Objective To express nonstructural protein 2 transactivated protein (NS2TP) of hepatitis C virus (HCV) in the prokaryotic expression system and prepare polyclonal antibody,and to study the expressions in different liver tissues.Methods NS2TP gene was amplified by polymerase chain reaction (PCR) technique and cloned into the prokaryotic expression vector pET-32a(+),which was transformed into E.coli BL21.The protein was induced by isopropyl thiogalactose (IPTG) and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed by Western blotting assay.The recombinant protein were expressed and purified in a large amount.The rabbit was immunized with the purified protein to prepare polyelonal antibody.The liver tissues of patients with chronic HCV infection and healthy controls were detected by immunohistochemistry method.Results The recombinant NS2TP protein (relative molecular mass:33×103 ) and polyclonal antibody with high titer and specificity were successfully prepared.NS2TP expressions in the liver of patients with chronic HCV infection were higher than those of healthy controls,and were mainly distributed in the nucleus of hepatocytes.Conclusions The NS2TP expression level and intracellular location in liver tissue of patients with chronic HCV infection are understanded,which could bring new clues for further study of the biological function of NS2TP and the pathogenesis of HCV infection.
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AIM: To determine the content of emodin and physcion in rat plasma by nonaqueous RP-HPLC. METHODS: After hydrolysis and extraction, the content of emodin in the plasma was determined by nonaqueous RP-HPLC. The separation was performed on Kromasil C 18 column (250 mm?4.6 mm, 5.0 ?m) with the mobile phase comprised of methanol-acetic acid (99.9∶0.1). The flow rate was 1.0 mL?min -1 and the detection wavelength was at 254 nm. RESULTS: The linear ranges for emodin and physcion were in the range of 0.0425-2.8 ?g?mL -1 and 0.0491-3.14 ?g?mL -1 , respectively. The average recoveries of emodin and physcion were 95.7%-100.1% and 96.2%-99.8%, with corresponding RSD of 1.3% and 1.6% respectively. CONCLUSION: This method is simple, rapid, accurate and reproducible with RP-HPLC to detect rhein in plasma.
