Safety and immunogenicity of a protein subunit COVID-19 vaccine (ZF2001) in healthy children and adolescents aged 3-17 years in China: a randomised, double-blind, placebo-controlled, phase 1 trial and an open-label, non-randomised, non-inferiority, phase 2 trial.

BACKGROUND: ZF2001 is a recombinant protein subunit vaccine against SARS-CoV-2 that has been approved for use in China, Colombia, Indonesia, and Uzbekistan in adults aged 18 years or older, but not yet in children and adolescents younger than 18 years. We aimed to evaluate the safety and immunogenicity of ZF2001 in children and adolescents aged 3-17 years in China. METHODS: The randomised, double-blind, placebo-controlled, phase 1 trial and the open-label, non-randomised, non-inferiority, phase 2 trial were done at the Xiangtan Center for Disease Control and Prevention (Hunan Province, China). Healthy children and adolescents aged 3-17 years, without a history of SARS-CoV-2 vaccination, without a history of COVID-19, without COVID-19 at the time of the study, and without contact with patients with confirmed or suspected COVID-19 were included in the phase 1 and phase 2 trials. In the phase 1 trial, participants were divided into three groups according to age (3-5 years, 6-11 years, and 12-17 years). Each group was randomly assigned (4:1), using block randomisation with five blocks, each with a block size of five, to receive three 25 µg doses of the vaccine, ZF2001, or placebo intramuscularly in the arm 30 days apart. The participants and investigators were masked to treatment allocation. In the phase 2 trial, participants received three 25 µg doses of ZF2001 30 days apart and remained stratified by age group. For phase 1, the primary endpoint was safety and the secondary endpoint was immunogenicity (humoral immune response on day 30 after the third vaccine dose: geometric mean titre [GMT] of prototype SARS-CoV-2 neutralising antibodies and seroconversion rate, and geometric mean concentration [GMC] of prototype SARS-CoV-2 receptor-binding domain [RBD]-binding IgG antibodies and seroconversion rate). For phase 2, the primary endpoint was the GMT of SARS-CoV-2 neutralising antibodies with seroconversion rate on day 14 after the third vaccine dose, and the secondary endpoints included the GMT of RBD-binding antibodies and seroconversion rate on day 14 after the third vaccine dose, the GMT of neutralising antibodies against the omicron BA.2 subvariant and seroconversion rate on day 14 after the third vaccine dose, and safety. Safety was analysed in participants who received at least one dose of the vaccine or placebo. Immunogenicity was analysed in the full-analysis set (ie, participants who received at least one dose and had antibody results) by intention to treat and in the per-protocol set (ie, participants who completed the whole vaccination course and had antibody results). Non-inferiority in the phase 2 trial (neutralising antibody titre of participants from this trial aged 3-17 years vs that of participants aged 18-59 years from a separate phase 3 trial) for clinical outcome assessment was based on the geometric mean ratio (GMR) and was considered met if the lower bound of the 95% CI for the GMR was 0·67 or greater. These trials are registered with ClinicalTrials.gov, NCT04961359 (phase 1) and NCT05109598 (phase 2). FINDINGS: Between July 10 and Sept 4, 2021, 75 children and adolescents were randomly assigned to receive ZF2001 (n=60) or placebo (n=15) in the phase 1 trial and were included in safety and immunogenicity analyses. Between Nov 5, 2021, and Feb 14, 2022, 400 participants (130 aged 3-7 years, 210 aged 6-11 years, and 60 aged 12-17 years) were included in the phase 2 trial and were included in the safety analysis; six participants were excluded from the immunogenicity analyses. 25 (42%) of 60 participants in the ZF2001 group and seven (47%) of 15 participants in the placebo group in phase 1, and 179 (45%) of 400 participants in phase 2, had adverse events within 30 days after the third vaccination, without a significant difference between groups in phase 1. Most adverse events were grade 1 or 2 (73 [97%] of 75 in the phase 1 trial, and 391 [98%] of 400 in the phase 2 trial). One participant in the phase 1 trial and three in the phase 2 trial who received ZF2001 had serious adverse events. One serious adverse event (acute allergic dermatitis) in the phase 2 trial was possibly related to the vaccine. In the phase 1 trial, on day 30 after the third dose, in the ZF2001 group, seroconversion of neutralising antibodies against SARS-CoV-2 was observed in 56 (93%; 95% CI 84-98) of 60 participants, with a GMT of 176·5 (95% CI 118·6-262·8), and seroconversion of RBD-binding antibodies was observed in all 60 (100%; 95% CI 94-100) participants, with a GMC of 47·7 IU/mL (95% CI 40·1-56·6). In the phase 2 trial, on day 14 after the third dose, seroconversion of neutralising antibodies against SARS-CoV-2 was seen in 392 (99%; 95% CI 98-100) participants, with a GMT of 245·4 (95% CI 220·0-273·7), and seroconversion of RBD-binding antibodies was observed in all 394 (100%; 99-100) participants, with a GMT of 8021 (7366-8734). On day 14 after the third dose, seroconversion of neutralising antibodies against the omicron subvariant BA.2 was observed in 375 (95%; 95% CI 93-97) of 394 participants, with a GMT of 42·9 (95% CI 37·9-48·5). For the non-inferiority comparison of participants aged 3-17 years with those aged 18-59 years for SARS-CoV-2 neutralising antibodies, the adjusted GMR was 8·6 (95% CI 7·0-10·4), with the lower bound of the GMR greater than 0·67. INTERPRETATION: ZF2001 is safe, well tolerated, and immunogenic in children and adolescents aged 3-17 years. Vaccine-elicited sera can neutralise the omicron BA.2 subvariant, but with reduced activity. The results support further studies of ZF2001 in children and adolescents. FUNDING: Anhui Zhifei Longcom Biopharmaceutical and the Excellent Young Scientist Program from National Natural Science Foundation of China. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Molecular Basis of Mink ACE2 Binding to SARS-CoV-2 and Its Mink-Derived Variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is transmitted between humans and minks, and some mutations in the spike (S) protein, especially in the receptor-binding domain (RBD), have been identified in mink-derived viruses. Here, we examined binding of the mink angiotensin-converting enzyme 2 (ACE2) receptor to mink-derived and important human-originating variants, and we demonstrated that most of the RBD variants increased the binding affinities to mink ACE2 (mkACE2). Cryo-electron microscopy structures of the mkACE2-RBD Y453F (with a Y-to-F change at position 453) and mkACE2-RBD F486L complexes helped identify the key residues that facilitate changes in mkACE2 binding affinity. Additionally, the data indicated that the Y453F and F486L mutations reduced the binding affinities to some human monoclonal antibodies, and human vaccinated sera efficiently prevented infection of human cells by pseudoviruses expressing Y453F, F486L, or N501T RBD. Our findings provide an important molecular mechanism for the rapid adaptation of SARS-CoV-2 in minks and highlight the potential influence of the main mink-originating variants for humans. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a broad range of hosts. Mink-derived SARS-CoV-2 can transmit back to humans. There is an urgent need to understand the binding mechanism of mink-derived SARS-CoV-2 variants to mink receptor. In this study, we identified all mutations in the receptor-binding domain (RBD) of spike (S) protein from mink-derived SARS-CoV-2, and we demonstrated the enhanced binding affinity of mink angiotensin-converting enzyme 2 (ACE2) to most of the mink-derived RBD variants as well as important human-originating RBD variants. Cryo-electron microscopy structures revealed that the Y453F and F486L mutations enhanced the binding forces in the interaction interface. In addition, Y453F and F486L mutations reduced the binding affinities to some human monoclonal antibodies, and the SARS-CoV-2 pseudoviruses with Y453F, F486L, or N501T mutations were neutralized by human vaccinated sera. Therefore, our results provide valuable information for understanding the cross-species transmission mechanism of SARS-CoV-2.

Novel Insights Into the Sulfated Glucuronic Acid-Based Anti-SARS-CoV-2 Mechanism of Exopolysaccharides From Halophilic Archaeon Haloarcula hispanica.

The pandemic caused by SARS-CoV-2 is the most widely spread disease in the 21st century. Due to the continuous emergence of variants across the world, it is necessary to expand our understanding of host-virus interactions and explore new agents against SARS-CoV-2. In this study, it was found exopolysaccharides (EPSs) from halophilic archaeon Haloarcula hispanica ATCC33960 can bind to the spike protein of SARS-CoV-2 with the binding constant KD of 2.23 nM, block the binding of spike protein to Vero E6 and bronchial epithelial BEAS-2B cells, and inhibit pseudovirus infection. However, EPSs from the gene deletion mutant △HAH_1206 almost completely lost the antiviral activity against SARS-CoV-2. A significant reduction of glucuronic acid (GlcA) and the sulfation level in EPSs of △HAH_1206 was clearly observed. Our results indicated that sulfated GlcA in EPSs is possible for a main structural unit in their inhibition of binding of SARS-CoV-2 to host cells, which would provide a novel antiviral mechanism and a guide for designing new agents against SARS-CoV-2.

Broader-species receptor binding and structural bases of Omicron SARS-CoV-2 to both mouse and palm-civet ACE2s.

The Omicron variant of SARS-CoV-2 carries multiple unusual mutations, particularly in the receptor-binding domain (RBD) of the spike (S) protein. Moreover, host-adapting mutations, such as residues 493, 498, and 501, were also observed in the Omicron RBD, which indicates that it is necessary to evaluate the interspecies transmission risk of the Omicron variant. Herein, we evaluated the interspecies recognition of the Omicron BA.1 and Delta RBDs by 27 ACE2 orthologs, including humans. We found that Omicron BA.1 expanded its receptor binding spectra to palm-civet, rodents, more bats (least horseshoe bat and greater horseshoe bat) and lesser hedgehog tenrec. Additionally, we determined the cryo-electron microscopy (cryo-EM) structure of the Omicron BA.1 S protein complexed with mouse ACE2 (mACE2) and the crystal structure of Omicron RBD complexed with palm-civet ACE2 (cvACE2). Several key residues for the host range have been identified. These results suggest that surveillance should be enhanced on the Omicron variant for its broader-species receptor binding to prevent spillover and expansion of reservoir hosts for a prolonged pandemic.

Binding and structural basis of equine ACE2 to RBDs from SARS-CoV, SARS-CoV-2 and related coronaviruses.

The origin and host range of SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), are important scientific questions as they might provide insight into understanding of the potential future spillover to infect humans. Here, we tested the binding between equine angiotensin converting enzyme 2 (eqACE2) and the receptor binding domains (RBDs) of SARS-CoV, SARS-CoV-2 prototype (PT) and variant of concerns (VOCs), as well as their close relatives bat-origin coronavirus (CoV) RaTG13 and pangolin-origin CoVs GX/P2V/2017 and GD/1/2019. We also determined the crystal structures of eqACE2/RaTG13-RBD, eqACE2/SARS-CoV-2 PT-RBD and eqACE2/Omicron BA.1-RBD. We identified S494 of SARS-COV-2 PT-RBD as an important residue in the eqACE2/SARS-COV-2 PT-RBD interaction and found that N501Y, the commonly recognized enhancing mutation, attenuated the binding affinity with eqACE2. Our work demonstrates that horses are potential targets for SARS-CoV-2 and highlights the importance of continuous surveillance on SARS-CoV-2 and related CoVs to prevent spillover events.

Cross-species recognition of SARS-CoV-2 to bat ACE2.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged as a major threat to global health. Although varied SARS-CoV-2-related coronaviruses have been isolated from bats and SARS-CoV-2 may infect bat, the structural basis for SARS-CoV-2 to utilize the human receptor counterpart bat angiotensin-converting enzyme 2 (bACE2) for virus infection remains less understood. Here, we report that the SARS-CoV-2 spike protein receptor binding domain (RBD) could bind to bACE2 from Rhinolophus macrotis (bACE2-Rm) with substantially lower affinity compared with that to the human ACE2 (hACE2), and its infectivity to host cells expressing bACE2-Rm was confirmed with pseudotyped SARS-CoV-2 virus and SARS-CoV-2 wild virus. The structure of the SARS-CoV-2 RBD with the bACE2-Rm complex was determined, revealing a binding mode similar to that of hACE2. The analysis of binding details between SARS-CoV-2 RBD and bACE2-Rm revealed that the interacting network involving Y41 and E42 of bACE2-Rm showed substantial differences with that to hACE2. Bats have extensive species diversity and the residues for RBD binding in bACE2 receptor varied substantially among different bat species. Notably, the Y41H mutant, which exists in many bats, attenuates the binding capacity of bACE2-Rm, indicating the central roles of Y41 in the interaction network. These findings would benefit our understanding of the potential infection of SARS-CoV-2 in varied species of bats.

Immune suppression in the early stage of COVID-19 disease.

The outbreak of COVID-19 has become a worldwide pandemic. The pathogenesis of this infectious disease and how it differs from other drivers of pneumonia is unclear. Here we analyze urine samples from COVID-19 infection cases, healthy donors and non-COVID-19 pneumonia cases using quantitative proteomics. The molecular changes suggest that immunosuppression and tight junction impairment occur in the early stage of COVID-19 infection. Further subgrouping of COVID-19 patients into moderate and severe types shows that an activated immune response emerges in severely affected patients. We propose a two-stage mechanism of pathogenesis for this unusual viral infection. Our data advance our understanding of the clinical features of COVID-19 infections and provide a resource for future mechanistic and therapeutics studies.

Excretion of SARS-CoV-2 through faecal specimens.

Coronavirus disease 2019 (COVID-19) has become a pandemic with increasing numbers of cases worldwide. SARS-CoV-2, the causative virus of COVID-19, is mainly transmitted through respiratory droplets or through direct and indirect contact with an infected person. The possibility of potential faecal-oral transmission was investigated in this study. We collected 258 faecal specimens from nine provinces in China and detected the nucleic acid of SARS-CoV-2 using real-time RT-PCR. Vero cells were used to isolate the virus from SARS-CoV-2 nucleic acid positive samples, after which sequencing of Spike gene in eight samples was performed. In all, 93 of 258 (36%) stool samples were positive for SARS-CoV-2 RNA. The positive rates of critical, severe, moderate, and mild patients were 54.4%, 56.1%, 30.8%, and 33.3%, respectively. The content of nucleic acid increased within 2 weeks after the onset of the disease. From the perspective of clinical typing, the nucleic acid can be detected in the faeces of critical patients within two weeks and until four to five weeks in the faeces of severe and mild patients. SARS-CoV-2 was isolated from stool specimens of two severe patients. Four non-synonymous mutations in Spike gene were newly detected in three stool samples. A small number of patients had strong faecal detoxification ability. The live virus in faeces could be an important source of contamination, which may lead to infection and further spread in areas with poor sanitary conditions. The findings of this study have public health significance and they should be considered when formulating disease control strategies.

Both Boceprevir and GC376 efficaciously inhibit SARS-CoV-2 by targeting its main protease.

COVID-19 was declared a pandemic on March 11 by WHO, due to its great threat to global public health. The coronavirus main protease (Mpro, also called 3CLpro) is essential for processing and maturation of the viral polyprotein, therefore recognized as an attractive drug target. Here we show that a clinically approved anti-HCV drug, Boceprevir, and a pre-clinical inhibitor against feline infectious peritonitis (corona) virus (FIPV), GC376, both efficaciously inhibit SARS-CoV-2 in Vero cells by targeting Mpro. Moreover, combined application of GC376 with Remdesivir, a nucleotide analogue that inhibits viral RNA dependent RNA polymerase (RdRp), results in sterilizing additive effect. Further structural analysis reveals binding of both inhibitors to the catalytically active side of SARS-CoV-2 protease Mpro as main mechanism of inhibition. Our findings may provide critical information for the optimization and design of more potent inhibitors against the emerging SARS-CoV-2 virus.