Antibodies utilizing VL6-57 light chains target a convergent cryptic epitope on SARS-CoV-2 spike protein driving the genesis of Omicron variants (preprint)

Continued evolution of SARS-CoV-2 generates variants to challenge antibody immunity established by infection and vaccination. A connection between population immunity and genesis of virus variants has long been suggested but its molecular basis remains poorly understood. Here, we identify a class of SARS-CoV-2 neutralising public antibodies defined by their shared usage of VL6-57 light chains. Although heavy chains of diverse genotypes are utilized, convergent HCDR3 rearrangements have been observed among these public antibodies to cooperate with germline VL6-57 LCDRs to target a convergent epitope defined by RBD residues S371-S373-S375. Antibody repertoire analysis identifies that this class of VL6-57 antibodies is present in SARS-CoV-2-naive individuals and is clonally expanded in most COVID-19 patients. We confirm that Omicron specific substitutions at S371, S373 and S375 mediate escape of antibodies of the VL6-57 class. These findings support that this class of public antibodies constitutes immune pressure promoting the introduction of S371L/F-S373P-S375F in Omicron variants. The results provide further molecular evidences to support that antigenic evolution of SARS-CoV-2 is driven by antibody mediated population immunity.

Inhibition mechanism and antiviral activity of an α-ketoamide based SARS-CoV-2 main protease inhibitor (preprint)

SARS-CoV-2 has demonstrated extraordinary ability to evade antibody immunity by antigenic drift. Small molecule drugs may provide effective therapy while being part of a solution to circumvent SARS-CoV-2 immune escape. In this study we report an alpha-ketoamide based peptidomimetic inhibitor of SARS-CoV-2 main protease (Mpro), RAY1216. Enzyme inhibition kinetic analysis established that RAY1216 is a slow-tight inhibitor with a Ki of 8.6 nM; RAY1216 has a drug-target residence time of 104 min compared to 9 min of PF-07321332 (nirmatrelvir), the antiviral component in Paxlovid, suggesting that RAY1216 is approximately 12 times slower to dissociate from the protease-inhibitor complex compared to PF-07321332. Crystal structure of SARS-CoV-2 Mpro:RAY1216 complex demonstrates that RAY1216 is covalently attached to the catalytic Cys145 through the alpha-ketoamide warhead; more extensive interactions are identified between bound RAY1216 and Mpro active site compared to PF-07321332, consistent with a more stable acyl-enzyme inhibition complex for RAY1216. In cell culture and human ACE2 transgenic mouse models, RAY1216 demonstrates comparable antiviral activities towards different SARS-CoV-2 virus variants compared to PF-07321332. Improvement in pharmacokinetics has been observed for RAY1216 over PF-07321332 in various animal models, which may allow RAY1216 to be used without ritonavir. RAY1216 is currently undergoing phase III clinical trials (https://clinicaltrials.gov/ct2/show/NCT05620160) to test real-world therapeutic efficacy against COVID-19.

Disulfide stabilization reveals conserved dynamic features between SARS-CoV-1 and SARS-CoV-2 spikes. (preprint)

SARS-CoV-2 spike protein (S) is structurally dynamic and has been observed by cryo-EM to adopt a variety of prefusion conformations that can be categorized as locked, closed and open. The locked conformations feature tightly packed trimers with structural elements incompatible with RBD in "up" position. For SARS-CoV-2 S, it has been shown that the locked conformations are transient under neutral pH. Probably due to their transience, locked conformations remain largely uncharacterized for SARS-CoV-1 S. Intriguingly, locked conformations were the only conformations captured for S proteins of bat and pangolin origin SARS-related coronaviruses. In this study, we introduced x1, x2, and x3 disulfides into SARS-CoV-1 S. Some of these disulfides have been shown to preserve rare locked conformations when introduced to SARS-CoV-2 S. Introduction of these disulfides allowed us to image a variety of locked and other rare conformations for SARS-CoV-1 S by cryo-EM. We identified bound cofactors and structural features that are associated with SARS-CoV-1 S locked conformations. We compare newly determined structures to other available spike structures of Sarbecoviruses to identify conserved features and discuss their possible functions.

SARS-CoV-2 breakthrough infections induce somatically hypermutated broadly neutralizing antibodies against heterologous variants (preprint)

Currently circulating SARS-CoV-2 Omicron variants feature highly mutated spike proteins with extraordinary abilities in evading acute-infection-induced germline antibodies isolated earlier in the pandemic. We identified that memory B cells from Delta variant breakthrough-infection patients expressed antibodies with more extensive somatic hypermutations (SHMs) allowing isolation of a number of broadly neutralizing antibodies with activities against heterologous variants of concerns (VOCs) including Omicron variant. Structural studies identified that SHM introduced altered amino acids and highly unusual HCDR2 insertions respectively in two representative broadly neutralizing antibodies - YB9-258 and YB13-292. Previously, insertion/deletion were rarely reported for antiviral antibodies except for those induced by HIV-1 chronic infections. Identified SHMs involved heavily in epitope recognition, they broadened neutralization breadth by rendering antibodies resistant to VOC mutations highly detrimental to previously isolated antibodies targeting similar epitopes. These data provide molecular mechanisms for enhanced immunity to heterologous SARS-CoV-2 variants after repeated antigen exposures with implications for future vaccination strategy.

A population antibody response against SARS-CoV-2 with differential neutralization activities towards the Delta and Omicron variants (preprint)

Population antibody response is believed to be important in selection of new variant viruses. We identified that SARS-CoV-2 infections elicit a population immune response mediated by a lineage of VH1-69 germline antibodies. The representative antibody R1-32 targets a novel semi-cryptic epitope defining a new class of RBD targeting antibodies. Binding to this non-ACE2 competing epitope leading to spike destruction impairing virus entry. Based on epitope location, neutralization mechanism and analysis of antibody binding to spike variants we propose that recurrent substitutions at 452 and 490 are associated with immune evasion of this population antibody response. These substitutions, including L452R found in the Delta variant, disrupt interaction mediated by the VH1-69 specific hydrophobic HCDR2 to impair antibody-antigen association allowing variants to escape. Lacking 452/490 substitutions, the Omicron variant is sensitive to this class of antibodies. Our results provide new insights into SARS-CoV-2 variant genesis and immune evasion.

Structures and function of locked conformations of SARS-CoV-2 spike (preprint)

The spike protein (S) of SARS-CoV-2 has been observed in three distinct pre-fusion conformations: locked, closed and open. Of these, the locked conformation was not previously observed for SARS-CoV-1 S and its function remains poorly understood. Here we engineered a SARS-CoV-2 S protein construct "S-R/x3" to arrest SARS-CoV-2 spikes in the locked conformation by a disulfide bond. Using this construct we determined high-resolution structures revealing two distinct locked states, with or without the D614G substitution that has become fixed in the globally circulating SARS-CoV-2 strains. The D614G mutation induces a structural change in domain D from locked-1 to locked-2 conformation to alter spike dynamics, promoting transition into the closed conformation from which opening of the receptor binding domain is permitted. The transition from locked to closed conformations is additionally promoted by a change from low to neutral pH. We propose that the locked conformations of S are present in the acidic cellular compartments where virus is assembled and egresses. In this model, release of the virion into the neutral pH extracellular space would favour transition to the closed form which itself can stochastically transition into the open form. The S-R/x3 construct provides a tool for the further structural and functional characterization of the locked conformations of S, as well as how sequence changes might alter S assembly and regulation of receptor binding domain dynamics.

SARS-CoV-2 spike protein arrested in the closed state induces potent neutralizing responses (preprint)

The majority of SARS-CoV-2 vaccines in use or in advanced clinical development are based on the viral spike protein (S) as their immunogen. S is present on virions as pre-fusion trimers in which the receptor binding domain (RBD) is stochastically open or closed. Neutralizing antibodies have been described that act against both open and closed conformations. The long-term success of vaccination strategies will depend upon inducing antibodies that provide long-lasting broad immunity against evolving, circulating SARS-CoV-2 strains, while avoiding the risk of antibody dependent enhancement as observed with other Coronavirus vaccines. Here we have assessed the results of immunization in a mouse model using an S protein trimer that is arrested in the closed state to prevent exposure of the receptor binding site and therefore interaction with the receptor. We compared this with a range of other modified S protein constructs, including representatives used in current vaccines. We found that all trimeric S proteins induce a long-lived, strongly neutralizing antibody response as well as T-cell responses. Notably, the protein binding properties of sera induced by the closed spike differed from those induced by standard S protein constructs. Closed S proteins induced more potent neutralising responses than expected based on the degree to which they inhibit interactions between the RBD and ACE2. These observations suggest that closed spikes recruit different, but equally potent, virus-inhibiting immune responses than open spikes, and that this is likely to include neutralizing antibodies against conformational epitopes present in the closed conformation. Together with their improved stability and storage properties we suggest that closed spikes may be a valuable component of refined, next-generation vaccines.

The Virucidal Efficacy of Oral Rinse Components Against SARS-CoV-2 In Vitro (preprint)

The ability of widely-available mouthwashes to inactivate SARS-CoV-2 in vitro was tested using a protocol capable of detecting a 5-log10 reduction in infectivity, under conditions mimicking the naso/oropharynx. During a 30 second exposure, two rinses containing cetylpyridinium chloride and a third with ethanol/ethyl lauroyl arginate eliminated live virus to EN14476 standards (>4-log10 reduction), while others with ethanol/essential oils and povidone-iodine (PVP-I) eliminated virus by 2-3-log10. Chlorhexidine or ethanol alone had little or no ability to inactivate virus in this assay. Studies are warranted to determine whether these formulations can inactivate virus in the human oropharynx in vivo, and whether this might impact transmission.

An evolutionary analysis of the SARS-CoV-2 genomes from the countries in the same meridian (preprint)

In the current study we analyzed the genomes of SARS-CoV-2 strains isolated from Italy, Sweden, Congo (countries in the same meridian) and Brazil, as outgroup country. Evolutionary analysis revealed codon 9628 under episodic selective pressure for all four countries, suggesting it as a key site for the virus evolution. Belonging to the P0DTD3 (Y14_SARS2) uncharacterized protein 14, further investigation has been conducted showing the codon mutation as responsible for the helical modification in the secondary structure. According to the predictions done, the codon is placed into the more ordered region of the gene (41-59) and close the area acting as transmembrane (54-67), suggesting its involvement into the attachment phase of the virus. The predicted structures of P0DTD3 mutated and not confirmed the importance of the codon to define the protein structure and the ontological analysis of the protein emphasized that the mutation enhances the binding probability.

Critical care workers have lower seroprevalence of SARS-CoV-2 IgG compared with non-patient facing staff in first wave of COVID19. (preprint)

With the first 2020 surge of the COVID-19 pandemic, many health care workers (HCW) were re-deployed to critical care environments to support intensive care teams to look after high numbers of patients with severe COVID-19. There was considerable anxiety of increased risk of COVID19 for staff working in these environments. Using a multiplex platform to assess serum IgG responses to SARS-CoV-2 N, S and RBD proteins, and detailed symptom reporting, we screened over 500 HCW (25% of the total workforce) in a quaternary level hospital to explore the relationship between workplace and evidence of exposure to SARS-CoV-2. Whilst 45% of the cohort reported symptoms that they consider may have represented COVID-19, overall seroprevalence was 14% with anosmia and fever being the most discriminating symptoms for seropositive status. There was a significant difference in seropositive status between staff working in clinical and non-clinical roles (9% patient facing critical care, 15% patient facing non-critical care, 22% nonpatient facing). In the seropositive cohort, symptom severity increased with age for men and not for women. In contrast, there was no relationship between symptom severity and age or sex in the seronegative cohort reporting possible COVID-19 symptoms. Of the 12 staff screened PCR positive (10 symptomatic), 3 showed no evidence of seroconversion in convalescence. Conclusion: The current approach to Personal Protective Equipment (PPE) appears highly effective in protecting staff from patient acquired infection in the critical care environment including protecting staff managing interhospital transfers of COVID-19 patients. The relationship between seroconversion and disease severity in different demographics warrants further investigation. Longitudinally paired virological and serological surveillance, with symptom reporting are urgently required to better understand the role of antibody in the outcome of HCW exposure during subsequent waves of COVID-19 in health care environments.

In Vitro Activity of Itraconazole Against SARS-CoV-2 (preprint)

Background: As long as there is no vaccine available, having access to inhibitors of SARS-CoV-2 will be of utmost importance. Antivirals against coronaviruses do not exist, hence global drug re-purposing efforts have been carried out to identify agents that may provide clinical benefit to patients with COVID-19. Itraconazole, an antifungal agent, has been reported to have potential activity against animal coronaviruses. Methods: Using cell-based phenotypic assays, the in vitro antiviral activity of itraconazole and 17-OH itraconazole was assessed against clinical isolates from a German and Belgian patient infected with SARS-CoV-2. Results: Itraconazole demonstrated antiviral activity in human Caco-2 cells (EC50 = 2.3 M; MTT assay). Similarly, its primary metabolite, 17-OH itraconazole, showed inhibition of SARS-CoV-2 activity (EC50 = 3.6 M). Remdesivir inhibited viral replication with an EC50 = 0.4 M. Itraconazole and 17-OH itraconazole resulted in a viral yield reduction in vitro of approximately 2-log10 and approximately 1-log10, as measured in both Caco-2 cells and VeroE6-eGFP cells, respectively. The viral yield reduction brought about by remdesivir or GS-441524 (parent nucleoside of the antiviral prodrug remdesivir; positive control) was more pronounced, with an approximately 3 log10 drop and >4 log10 drop in Caco-2 cells and VeroE6-eGFP cells, respectively. Discussion: Itraconazole and 17-OH itraconazole exert in vitro low micromolar activity against SARS-CoV-2. Despite the in vitro antiviral activity, itraconazole did not result in a beneficial effect in hospitalized COVID-19 patients in a clinical study (EudraCT Number: 2020-001243-15).

Structures, conformations and distributions of SARS-CoV-2 spike protein trimers on intact virions (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude. Heavily glycosylated S trimers bind the ACE2 receptor and mediate entry of virions into target cells. S exhibits extensive conformational flexibility: it modulates the exposure of its receptor binding site and later undergoes complete structural rearrangement to drive fusion of viral and cellular membranes. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy. The structure and distribution of S on the virion surface, however, has not been characterised. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions, determining the high-resolution structure, conformational flexibility and distributions of S trimers in situ on the virion surface. These results provide a basis for understanding the conformations of S present on the virion, and for studying their interactions with neutralizing antibodies.

Combined point of care nucleic acid and antibody testing for SARS-CoV-2: a prospective cohort study in suspected moderate to severe COVID-19 disease. (preprint)

BackgroundRapid COVID-19 diagnosis in hospital is essential for patient management and identification of infectious patients to limit the potential for nosocomial transmission. The diagnosis of infection is complicated by 30-50% of COVID-19 hospital admissions with nose/throat swabs testing negative for SARS-CoV-2 nucleic acid, frequently after the first week of illness when SARS-CoV-2 antibody responses become detectable. We assessed the diagnostic accuracy of combined rapid antibody point of care (POC) and nucleic acid assays for suspected COVID-19 disease in the emergency department. MethodsWe developed (i) an in vitro neutralization assay using a lentivirus expressing a genome encoding luciferase and pseudotyped with spike (S) protein and (ii) an ELISA test to detect IgG antibodies to nucleocapsid (N) and S proteins from SARS-CoV-2. We tested two lateral flow rapid fingerprick tests with bands for IgG and IgM. We then prospectively recruited participants with suspected moderate to severe COVID-19 and tested for SARS-CoV-2 nucleic acid in a combined nasal/throat swab using the standard laboratory RT-PCR and a validated rapid POC nucleic acid amplification (NAAT) test. Additionally, serum collected at admission was retrospectively tested by in vitro neutralisation, ELISA and the candidate POC antibody tests. We evaluated the performance of the individual and combined rapid POC diagnostic tests against a composite reference standard of neutralisation and standard laboratory based RT-PCR. Results45 participants had specimens tested for nucleic acid in nose/throat swabs as well as stored sera for antibodies. Using the composite reference standard, prevalence of COVID-19 disease was 53.3% (24/45). Median age was 73.5 (IQR 54.0-86.5) years in those with COVID-19 disease by our reference standard and 63.0 (IQR 41.0-72.0) years in those without disease. The overall detection rate by rapid NAAT was 79.2% (95CI 57.8-92.9%), decreasing from 100% (95% CI 65.3-98.6%) in days 1-4 to 50.0% (95% CI 11.8-88.2) for days 9-28 post symptom onset. Correct identification of COVID-19 with combined rapid POC diagnostic tests was 100% (95CI 85.8-100%) with a false positive rate of 5.3-14.3%, driven by POC LFA antibody tests. ConclusionsCombined POC tests have the potential to transform our management of COVID-19, including inflammatory manifestations later in disease where nucleic acid test results are negative. A rapid combined approach will also aid recruitment into clinical trials and in prescribing therapeutics, particularly where potentially harmful immune modulators (including steroids) are used.

A thermostable, closed, SARS-CoV-2 spike protein trimer. (preprint)

The spike (S) protein of SARS-CoV-2 mediates receptor binding and cell entry and is the dominant target of the immune system. S exhibits substantial conformational flexibility. It transitions from closed to open conformations to expose its receptor binding site, and subsequently from prefusion to postfusion conformations to mediate fusion of viral and cellular membranes. S protein derivatives are components of vaccine candidates and diagnostic assays, as well as tools for research into the biology and immunology of SARS-CoV-2. Here we have designed mutations in S which allow production of thermostable, crosslinked, S protein trimers that are trapped in the closed, pre-fusion, state. We have determined the structures of crosslinked and non-crosslinked proteins, identifying two distinct closed conformations of the S trimer. We demonstrate that the designed, thermostable, closed S trimer can be used in serological assays. This protein has potential applications as a reagent for serology, virology and as an immunogen.

Are COVID-19 infected children with gastrointestinal symptoms different from those without symptoms? A comparative study of the clinical characteristics and epidemiological trend of 244 pediatric cases from Wuhan (preprint)

Objective: COVID-19 patients presenting with gastrointestinal (GI) symptoms occur in both adults and children. To date, however, no large sample size study focusing on gastrointestinal symptoms in pediatric cases has been published. We analyzed COVID-19 infected children in Wuhan who presented with initial GI symptoms to determine the GI characteristics and epidemiological trend of the disease. Design: We retrospectively analyzed 244 children patients confirmed with COVID-19 at Wuhan Children's Hospital from 21 Jan to 20 Mar 2020. Symptomatic cases were divided into two groups according to whether the patients presented with or without GI symptoms on admission. Demographic, epidemiological, symptoms, and laboratory data were compared. We also analyzed the respective trends of case number changes of GI cases and asymptomatic cases. Results: 34 out of 193 symptomatic children had GI symptoms. They had lower median age and weight, a higher rate of fever, a longer length of stay and more hematological and biochemical abnormalities than patients without GI symptoms. There was no significant difference in chest CT findings or stool SARS-CoV-2 test positive percentages between the two groups. The number of patients admitted with GI symptoms showed an overall downward trend with time. At the time of writing, 242 patients were discharged, one died, and one critically ill patient was still in the intensive care unit. Conclusion: COVID-19 infected children with GI symptoms are prone to presenting with more clinical and laboratory abnormalities than patients without GI symptoms. More attention and timely hospital admission are needed for these patients.