Rapid evaluation of heterologous chimeric RBD-dimer mRNA vaccine for currently-epidemic Omicron sub-variants as booster shot after inactivated vaccine.

With continuous mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the severe immune escape of Omicron sub-variants urges the development of next-generation broad-spectrum vaccines, especially as booster jabs after high-level vaccination coverage of inactivated vaccines in China and many other countries. Previously, we developed a coronavirus disease 2019 (COVID-19) protein subunit vaccine ZF2001® based on the tandem homo-prototype receptor-binding domain (RBD)-dimer of the SARS-CoV-2 spike protein. We upgraded the antigen into a hetero-chimeric prototype (PT)-Beta or Delta-BA.1 RBD-dimer to broaden the cross-protection efficacy and prove its efficiency with protein subunit and mRNA vaccine platforms. Herein, we further explored the hetero-chimeric RBD-dimer mRNA vaccines and evaluated their broad-spectrum activities as booster jabs following two doses of inactivated vaccine (IV) in mice. Our data demonstrated that the chimeric vaccines significantly boosted neutralizing antibody levels and specific T-cell responses against the variants, and PT-Beta was superior to Delta-BA.1 RBD as a booster in mice, shedding light on the antigen design for the next-generation COVID-19 vaccines.

Mpox multi-antigen mRNA vaccine candidates by a simplified manufacturing strategy afford efficient protection against lethal orthopoxvirus challenge.

Current unprecedented mpox outbreaks in non-endemic regions represent a global public health concern. Although two live-attenuated vaccinia virus (VACV)-based vaccines have been urgently approved for people at high risk for mpox, a safer and more effective vaccine that can be available for the general public is desperately needed. By utilizing a simplified manufacturing strategy of mixing DNA plasmids before transcription, we developed two multi-antigen mRNA vaccine candidates, which encode four (M1, A29, B6, A35, termed as Rmix4) or six (M1, H3, A29, E8, B6, A35, termed as Rmix6) mpox virus antigens. We demonstrated that those mpox multi-antigen mRNA vaccine candidates elicited similar potent cross-neutralizing immune responses against VACV, and compared to Rmix4, Rmix6 elicited significantly stronger cellular immune responses. Moreover, immunization with both vaccine candidates protected mice from the lethal VACV challenge. Investigation of B-cell receptor (BCR) repertoire elicited by mpox individual antigen demonstrated that the M1 antigen efficiently induced neutralizing antibody responses, and all neutralizing antibodies among the top 20 frequent antibodies appeared to target the same conformational epitope as 7D11, revealing potential vulnerability to viral immune evasion. Our findings suggest that Rmix4 and Rmix6 from a simplified manufacturing process are promising candidates to combat mpox.

Novel (Z)/(E)-1,2,4-triazole derivatives containing oxime ether moiety as potential ergosterol biosynthesis inhibitors: design, preparation, antifungal evaluation, and molecular docking.

Inspired by the highly effective and broad-spectrum antifungal activity of ergosterol biosynthesis inhibitions, a series of novel 1,2,4-triazole derivatives containing oxime ether moiety were constructed for screening the bioactivity against phytopathogenic fungi. The (Z)- and (E)-isomers of target compounds were successfully separated and identified by the spectroscopy and single crystal X-ray diffraction analyses. The bioassay results showed that the (Z)-isomers of target compounds possessed higher antifungal activity than the (E)-isomers. Strikingly, the compound (Z)-5o exhibited excellent antifungal activity against Rhizoctonia solani with the EC50 value of 0.41 µg/mL in vitro and preventive effect of 94.58% in vivo at 200 µg/mL, which was comparable to the positive control tebuconazole. The scanning electron microscopy observation indicated that the compound (Z)-5o caused the mycelial morphology to become wizened and wrinkled. The molecular docking modes of (Z)-5o and (E)-5o with the potential target protein RsCYP51 were especially compared. And the main interactions between ligands and amino acid residues were carefully analyzed to preliminarily explain the mechanism leading to the difference of activity between two isomers. The study provided a new lead molecular skeleton for developing novel triazole fungicides targeting ergosterol biosynthesis.

A neutralizing-protective supersite of human monoclonal antibodies for yellow fever virus.

The yellow fever virus (YFV) is a life-threatening human pathogen. Owing to the lack of available therapeutics, non-vaccinated individuals are at risk. Here, we isolated eight human monoclonal antibodies that neutralize YFV infection. Five recognized overlapping epitopes and exhibited potent neutralizing activity. Two (YD6 and YD73) were ultra-potent and conferred complete protection against the lethal challenge of YFV as both prophylactics and therapeutics in a mouse model. Crystal structures revealed that YD6 engaged the YFV envelope protein in both pre- and post-fusion states, suggesting viral inhibition by a "double-lock" mechanism. The recognition determinants for YD6 and YD73 are clustered at the premembrane (prM)-binding site. Notably, antibodies targeting this site were present in minute traces in YFV-infected individuals but contributed significantly to neutralization, suggesting a vulnerable supersite of YFV. We provide two promising candidates for immunotherapy against YFV, and the supersite represents an ideal target for epitope-based vaccine design.

A combination vaccine against SARS-CoV-2 and H1N1 influenza based on receptor binding domain trimerized by six-helix bundle fusion core.

BACKGROUND: Increasing severe morbidity and mortality by simultaneous or sequential infections with SARS-CoV-2 and influenza A viruses (IAV), especially in the elderly and obese patients, highlight the urgency of developing a combination vaccine against COVID-19 and influenza. METHODS: Self-assembling SARS-CoV-2 RBD-trimer and Influenza H1N1 HA1-trimer antigens were constructed, upon the stable fusion core in post-fusion conformation. Immunogenicity of SARS-CoV-2 RBD-trimer vaccine and H1N1 HA1-trimer antigens candidates were evaluated in mice. Protection efficacy of a combination vaccine candidate against SARS-CoV-2 and IAV challenge was identified using the K18-hACE2 mouse model. FINDINGS: Both the resultant RBD-trimer for SARS-CoV-2 and HA1-trimer for H1N1 influenza fully exposed receptor-binding motifs (RBM) or receptor-binding site (RBS). Two-dose RBD-trimer induced significantly higher binding and neutralizing antibody titers, and also a strong Th1/Th2 balanced cellular immune response in mice. Similarly, the HA1-trimer vaccine was confirmed to exhibit potent immunogenicity in mice. A combination vaccine candidate, composed of RBD-trimer and HA1-trimer, afforded high protection efficacy in mouse models against stringent lethal SARS-CoV-2 and homogenous H1N1 influenza co-infection, characterized by 100% survival rate. INTERPRETATION: Our results represent a proof of concept for a combined vaccine candidate based on trimerized receptor binding domain against co-epidemics of COVID-19 and influenza. FUNDING: This project was funded by the Strategic Priority Research Program of CAS (XDB29040201), the National Natural Science Foundation of China (81830050, 81901680, and 32070569) and China Postdoctoral Science Foundation (2021M703450).

Structure-based neutralizing mechanisms for SARS-CoV-2 antibodies.

The devastating economic and public health consequences caused by the COVID-19 pandemic have prompted outstanding efforts from the scientific community and pharmaceutical companies to develop antibody-based therapeutics against SARS-CoV-2. Those efforts are encouraging and fruitful. An unprecedentedly large number of monoclonal antibodies (mAbs) targeting a large spectrum of epitopes on the spike protein has been developed in the last two years. The development of structural biology, especially the cryo-EM technology, provides structural insights into the molecular neutralizing mechanisms of those mAbs. Moreover, neutralizing antibodies are essential in protecting host from infection. Therefore, understanding the antibody neutralizing mechanism is critical for optimizing effective antibody-based therapeutics and developing next-generation pan-coronavirus vaccines. This review summarizes the latest understanding of antibody neutralizing mechanisms against SARS-CoV-2 at the molecular and structural levels.

Anti-diabetic effects of linarin from Chrysanthemi Indici Flos via AMPK activation.

Objective: The purpose of this study is to investigate the anti-diabetic effects of linarin, a flavonoid extracted from Chrysanthemi Indici Flos (CIF), and its potential mechanisms. Methods: The effects of linarin on cell viability and glucose consumption in HepG2 cells were measured. Meanwhile, monosodium glutamate (MSG) mouse model was constructed to monitor the changes of insulin tolerance, glucose tolerance, triglyceride and cholesterol. The protein expression levels of p-AMPK, p-ACC, PEPCK and p-GS were detected by Western blot. Results: Linarin could increase the relative glucose consumption of HepG2 cells, improve insulin tolerance and glucose tolerance, and decrease the levels of triglyceride and cholesterol of MSG mice. Simultaneously, the expression levels of p-AMPK and p-ACC in HepG2 cells and the liver tissue of MSG mice were increased, while the expression levels of PEPCK and p-GS were decreased after treatment with linarin. Conclusion: Insulin resistance could be ameliorated by linarin in type 2 diabetes, and its mechanism may be related to AMPK signaling pathway.

[Clinical progress of neutralizing antibodies against SARS-CoV-2].

Since the palivizumab for respiratory syncytial virus was approved in 1998, therapeutic antibodies against infectious diseases have been widely used in clinical treatment. Since the outbreak of COVID-19, plenty of neutralizing antibodies were developed and transferred into clinical trials, holding enormous promise for the treatment of COVID-19 under the context of emergency use authorization. This review summarizes the clinical progress of these drugs, in order to provide a reference for the research and development of neutralizing antibody drugs for the future.

Efficacy and Safety of the RBD-Dimer-Based Covid-19 Vaccine ZF2001 in Adults.

BACKGROUND: The ZF2001 vaccine, which contains a dimeric form of the receptor-binding domain of severe acute respiratory syndrome coronavirus 2 and aluminum hydroxide as an adjuvant, was shown to be safe, with an acceptable side-effect profile, and immunogenic in adults in phase 1 and 2 clinical trials. METHODS: We conducted a randomized, double-blind, placebo-controlled, phase 3 trial to investigate the efficacy and confirm the safety of ZF2001. The trial was performed at 31 clinical centers across Uzbekistan, Indonesia, Pakistan, and Ecuador; an additional center in China was included in the safety analysis only. Adult participants (≥18 years of age) were randomly assigned in a 1:1 ratio to receive a total of three 25-µg doses (30 days apart) of ZF2001 or placebo. The primary end point was the occurrence of symptomatic coronavirus disease 2019 (Covid-19), as confirmed on polymerase-chain-reaction assay, at least 7 days after receipt of the third dose. A key secondary efficacy end point was the occurrence of severe-to-critical Covid-19 (including Covid-19-related death) at least 7 days after receipt of the third dose. RESULTS: Between December 12, 2020, and December 15, 2021, a total of 28,873 participants received at least one dose of ZF2001 or placebo and were included in the safety analysis; 25,193 participants who had completed the three-dose regimen, for whom there were approximately 6 months of follow-up data, were included in the updated primary efficacy analysis that was conducted at the second data cutoff date of December 15, 2021. In the updated analysis, primary end-point cases were reported in 158 of 12,625 participants in the ZF2001 group and in 580 of 12,568 participants in the placebo group, for a vaccine efficacy of 75.7% (95% confidence interval [CI], 71.0 to 79.8). Severe-to-critical Covid-19 occurred in 6 participants in the ZF2001 group and in 43 in the placebo group, for a vaccine efficacy of 87.6% (95% CI, 70.6 to 95.7); Covid-19-related death occurred in 2 and 12 participants, respectively, for a vaccine efficacy of 86.5% (95% CI, 38.9 to 98.5). The incidence of adverse events and serious adverse events was balanced in the two groups, and there were no vaccine-related deaths. Most adverse reactions (98.5%) were of grade 1 or 2. CONCLUSIONS: In a large cohort of adults, the ZF2001 vaccine was shown to be safe and effective against symptomatic and severe-to-critical Covid-19 for at least 6 months after full vaccination. (Funded by the National Science and Technology Major Project and others; ClinicalTrials.gov number, NCT04646590.).

A tandem-repeat dimeric RBD protein-based covid-19 vaccine zf2001 protects mice and nonhuman primates.

Safe, efficacious, and deployable vaccines are urgently needed to control COVID-19 in the large-scale vaccination campaigns. We report here the preclinical studies of an approved protein subunit vaccine against COVID-19, ZF2001, which contains tandem-repeat dimeric receptor-binding domain (RBD) protein with alum-based adjuvant. We assessed vaccine immunogenicity and efficacy in both mice and non-human primates (NHPs). ZF2001 induced high levels of RBD-binding and SARS-CoV-2 neutralizing antibody in both mice and non-human primates, and elicited balanced TH1/TH2 cellular responses in NHPs. Two doses of ZF2001 protected Ad-hACE2-transduced mice against SARS-CoV-2 infection, as detected by reduced viral RNA and relieved lung injuries. In NHPs, vaccination of either 25 µg or 50 µg ZF2001 prevented infection with SARS-CoV-2 in lung, trachea, and bronchi, with milder lung lesions. No evidence of disease enhancement was observed in both animal models. ZF2001 has been approved for emergency use in China, Uzbekistan, Indonesia, and Columbia. The high safety, immunogenicity, and protection efficacy in both mice and NHPs found in this preclinical study was consistent with the results in human clinical trials.

Etesevimab in combination with JS026 neutralizing SARS-CoV-2 and its variants.

The neutralizing antibody is a potential therapeutic for the ongoing COVID-19 pandemic. As an antiviral agent, numerous mAbs recognize the epitopes that overlap with ACE2-binding sites in the SARS-CoV-2-RBD. Some studies have shown that residual changes on the spike protein can significantly decrease the efficiency of neutralizing antibodies. To address this issue, a therapeutic cocktail could be an effective countermeasure. In the present study, we isolated a fully human neutralizing antibody, JS026, from a convalescent patient. The comparative analysis revealed that JS026 binding to SARS-CoV-2-RBD mainly located between epitopes for class 2 and class 3 mAbs as opposed to that of class 1 (etesevimab) antibodies. A cocktail of etesevimab and JS026 increased neutralizing efficacy against both wild-type SARS-CoV-2 and the recent emergence of Alpha, Beta, Gamma, and Delta variants. JS026 and the cocktail reduced virus titers in the infected lungs of hACE2 transgenic mice and relieved pathological changes. These findings would benefit antibody-based therapeutic countermeasures in the treatment of COVID-19.

Macrophage deletion of Noc4l triggers endosomal TLR4/TRIF signal and leads to insulin resistance.

In obesity, macrophages drive a low-grade systemic inflammation (LSI) and insulin resistance (IR). The ribosome biosynthesis protein NOC4 (NOC4) mediates 40 S ribosomal subunits synthesis in yeast. Hereby, we reported an unexpected location and function of NOC4L, which was preferentially expressed in human and mouse macrophages. NOC4L was decreased in both obese human and mice. The macrophage-specific deletion of Noc4l in mice displayed IR and LSI. Conversely, Noc4l overexpression by lentivirus treatment and transgenic mouse model improved glucose metabolism in mice. Importantly, we found that Noc4l can interact with TLR4 to inhibit its endocytosis and block the TRIF pathway, thereafter ameliorated LSI and IR in mice.

Molecular insights into receptor binding of recent emerging SARS-CoV-2 variants.

Multiple SARS-CoV-2 variants of concern (VOCs) have been emerging and some have been linked to an increase in case numbers globally. However, there is yet a lack of understanding of the molecular basis for the interactions between the human ACE2 (hACE2) receptor and these VOCs. Here we examined several VOCs including Alpha, Beta, and Gamma, and demonstrate that five variants receptor-binding domain (RBD) increased binding affinity for hACE2, and four variants pseudoviruses increased entry into susceptible cells. Crystal structures of hACE2-RBD complexes help identify the key residues facilitating changes in hACE2 binding affinity. Additionally, soluble hACE2 protein efficiently prevent most of the variants pseudoviruses. Our findings provide important molecular information and may help the development of novel therapeutic and prophylactic agents targeting these emerging mutants.

A broadly neutralizing humanized ACE2-targeting antibody against SARS-CoV-2 variants.

The successive emergences and accelerating spread of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages and evolved resistance to some ongoing clinical therapeutics increase the risks associated with the coronavirus disease 2019 (COVID-19) pandemic. An urgent intervention for broadly effective therapies to limit the morbidity and mortality of COVID-19 and future transmission events from SARS-related coronaviruses (SARSr-CoVs) is needed. Here, we isolate and humanize an angiotensin-converting enzyme-2 (ACE2)-blocking monoclonal antibody (MAb), named h11B11, which exhibits potent inhibitory activity against SARS-CoV and circulating global SARS-CoV-2 lineages. When administered therapeutically or prophylactically in the hACE2 mouse model, h11B11 alleviates and prevents SARS-CoV-2 replication and virus-induced pathological syndromes. No significant changes in blood pressure and hematology chemistry toxicology were observed after injections of multiple high dosages of h11B11 in cynomolgus monkeys. Analysis of the structures of the h11B11/ACE2 and receptor-binding domain (RBD)/ACE2 complexes shows hindrance and epitope competition of the MAb and RBD for the receptor. Together, these results suggest h11B11 as a potential therapeutic countermeasure against SARS-CoV, SARS-CoV-2, and escape variants.

Protective Zika vaccines engineered to eliminate enhancement of dengue infection via immunodominance switch.

Antibody-dependent enhancement (ADE) is an important safety concern for vaccine development against dengue virus (DENV) and its antigenically related Zika virus (ZIKV) because vaccine may prime deleterious antibodies to enhance natural infections. Cross-reactive antibodies targeting the conserved fusion loop epitope (FLE) are known as the main sources of ADE. We design ZIKV immunogens engineered to change the FLE conformation but preserve neutralizing epitopes. Single vaccination conferred sterilizing immunity against ZIKV without ADE of DENV-serotype 1-4 infections and abrogated maternal-neonatal transmission in mice. Unlike the wild-type-based vaccine inducing predominately cross-reactive ADE-prone antibodies, B cell profiling revealed that the engineered vaccines switched immunodominance to dispersed patterns without DENV enhancement. The crystal structure of the engineered immunogen showed the dimeric conformation of the envelope protein with FLE disruption. We provide vaccine candidates that will prevent both ZIKV infection and infection-/vaccination-induced DENV ADE.

Tolerability, Safety, Pharmacokinetics, and Immunogenicity of a Novel SARS-CoV-2 Neutralizing Antibody, Etesevimab, in Chinese Healthy Adults: a Randomized, Double-Blind, Placebo-Controlled, First-in-Human Phase 1 Study.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread rapidly worldwide. This study is the first to report the tolerability, safety, pharmacokinetics (PK), and immunogenicity of a recombinant human anti-SARS-CoV-2 monoclonal antibody, etesevimab (CB6, JS016, LY3832479, or LY-CoV016), in healthy adults. This paper describes a randomized, double-blind, placebo-controlled, phase 1 study. A total of 40 participants were enrolled to receive a single intravenous dose of either etesevimab or placebo in one of four sequential ascending intravenous dose cohorts. All 40 participants completed the study. Seventeen (42.5%) participants experienced 22 treatment emergent adverse events (TEAEs) that were drug-related, and the rates of these TEAEs among different dose cohorts were numerically comparable. No difference was observed between the combined etesevimab group and the placebo group. The exposure after etesevimab infusion increased in an approximately proportional manner as the dose increased from 2.5 to 50 mg/kg. The elimination half-life (t1/2) value did not differ among different dose cohorts and was estimated to be around 4 weeks. Etesevimab was well tolerated after administration of a single dose at a range of 2.5 mg/kg to 50 mg/kg in healthy Chinese adults. The PK profiles of etesevimab in healthy volunteers showed typical monoclonal antibody distribution and elimination characteristics. (This study has been registered at ClinicalTrials.gov under identifier NCT04441918.).

COVID-19 mRNA vaccines.

The ongoing COVID-19 pandemic and its unprecedented global societal and economic disruptive impact highlight the urgent need for safe and effective vaccines. Taking substantial advantages of versatility and rapid development, two mRNA vaccines against COVID-19 have completed late-stage clinical assessment at an unprecedented speed and reported positive results. In this review, we outline keynotes in mRNA vaccine development, discuss recently published data on COVID-19 mRNA vaccine candidates, focusing on those in clinical trials and analyze future potential challenges.