The real-world effectiveness of an intranasal spray A8G6 antibody cocktail in the post-exposure prophylaxis of COVID-19 (preprint)

BackgroundDue to the continuous appearance of novel SARS-CoV-2 variants that are resistant to approved antibodies and leading to the epidemic rebound, several approved neutralizing antibodies have been paused for their usage against COVID-19. Previously, we identified A8G6, an antibody combination of two synergic SARS-CoV-2 neutralizing antibodies 55A8 and 58G6, that showed broad neutralizing activities against Omicron variants. When administrated by the nasal spray delivery system, A8G6 showed promising efficacy in COVID-19 animal models and also showed favorable safety profile in preclinical models as well as in a first-in-human trial. The aim of this study is to evaluate the real-world efficacy of A8G6 neutralizing antibody nasal spray in post-exposure prevention of COVID-19. MethodsFrom November 27, 2022 to January 31, 2023, an open-label, non-randomized, two-arm, blank-controlled, investigator-initiated trial was conducted in Chongqing, China. High-risk healthy participants (18-65 years) within 72 hours after close contact to SARS-CoV-2 infected individuals were recruited and received a three-dose (1.4 mg/dose) A8G6 nasal spray treatment daily or no treatment (blank control) for 7 consecutive days. The primary end points were 1) the occurrence of positive SARS-CoV-2 RT-PCR cases in A8G6 treated group vs blank control group at the end of day 7; 2) time to SARS-CoV-2 positive conversion at the end of day 7. The secondary end points were 1) viral load of SARS-CoV-2 when participants became SARS-CoV-2 positive; 2) the time from SARS-CoV-2 infection to negative COVID-19 conversion. Safety end point of the nasal spray AG86 was analyzed by recording adverse events during the whole course of this trial. This study was registered with Chictr.org (ChiCTR2200066416). FindingsOf 513 enrolled participants, 173 in the A8G6 treatment group and 340 in the blank-control group were included in the analysis. SARS-CoV-2 infection occurred in 151/340 (44.4%) subjects in the blank control group and 12/173 (6.9%) subjects with the A8G6 treatment group. The result indicates that the intranasal spray A8G6 reduces the risk of SARS-CoV-2 infection (HR=0.12, 95% CI, 0.07-0.22; p<0.001). The prevention efficacy of the A8G6 treatment within 72-hours exposure was calculated to be 84.4% (95% CI: 74.4%-90.4%). Moreover, compared to the blank-control group, the time from the SARS-CoV-2 negative to the positive COVID-19 conversion was significantly longer in the AG86 treatment group (mean time: 3.4 days in the A8G6 treatment group vs 2.6 days in the control group, p=0.019). In the secondary end-point analysis, the A8G6 nasal treatment had no effects on the viral load at baseline SARS-CoV-2 RT-PCR positivity and the time of the negative COVID-19 conversion (viral clearance). Finally, 5 participants (3.1%) in the treatment group reported general adverse effects. We did not observe any severe adverse effects related to the A8G6 treatment in this study. InterpretationIn this study, the intranasal spray AG86 antibody cocktail showed potent efficacy for prevention of SARS-CoV-2 infection in close contacts of COVID-19 patients. FundingChongqing Biomedical R&D Major Special Project, Project (No. CSTB2022TIAD-STX0013), Chongqing Science and Health Joint Medical High-end Talent Project (No. 2022GDRC012), Science and Technology Research Program of Chongqing Municipal Education Commission (No. KJZD-K202100402), CQMU Program for Youth Innovation in Future Medicine (No. W0073). Research in contextO_ST_ABSEvidence before the studyC_ST_ABSTwo potent neutralizing antibodies 55A8 and 58G6 against SARS-CoV-2 were identified from the plasma of COVID-19 convalescent patients. In our previous studies, the synergetic neutralization of the antibody combination of 55A8 and 58G6 (A8G6) had been shown in structural mechanism, as well as in vitro and in vivo. Pre-clinical evaluation of A8G6 nasal spray showed promising efficacy against Omicron BA.4/5 infection in golden syrian hamsters challenged with live virus. In a first-in-human trial, A8G6 also showed favorable safety profile and nasal concentration over IC90 of neutralization activity against Omicron BA.4/5. The preliminary data showed that the intranasal spray A8G6 had the excellent efficacy, safety and druggability to protect against COVID-19. Added value of this studyThis is the first human trial showing that a nasal spray of neutralizing antibody cocktail is efficacious in preventing SARS-CoV-2 infection but is not efficacious in the post-infection treatment of COVID-19. In the Omicron wave of the COVID-19 pandemic in China in November, 2022, COVID-19 close contacts receiving the A8G6 treatment in the designated quarantine hotels showed a significantly lower incidence of SARS-CoV-2 infection. Additionally, the A8G6 treatment delayed time from exposure to the diagnosis of the COVID-19 positivity (median time: 3.4 days in the treatment group vs 2.6 days in the control group). Furthermore, we analyzed the effects of the A8G6 treatment on the clinical status of close contacts who became infected with SARS-CoV-2. Results suggests that there were no significant differences in viral load of SARS-CoV-2 at the beginning of positive infection and the time of the viral clearance between A8G6 treatment and blank control groups. Overall, the trial result is consistent with the mechanism of action of nasal spray antibody cocktail for the prevention of SARS-CoV-2 infection. Finally, low safety risk of the nasal spray A8G6 was also shown in the trial. Implications of all the available evidenceWe observed the use of A8G6 to reduce the risk of SARS-CoV-2 infection. This study provided supporting evidences for the real-world effectiveness and safety of the nasal spray A8G6 among high-risk close contacts in the post-exposure prevention of COVID-19 during the Omicron BA.5.2 wave in China. This is the first proof of concept of using nasal spray neutralizing antibody for the prevention of viral infection. It implicates that the promising efficacy of the nasal spray A8G6 makes it possible for the fast-acting prevention in future COVID-19 waves.

Extensive neutralization against SARS-CoV-2 variants elicited by Omicron-specific subunit vaccine as a heterologous booster

To overcome the increased risk of SARS-CoV-2 reinfection or post-vaccination infection caused by the Omicron variant, Omicron-specific vaccines were considered a potential strategy. We reported the increased magnitude and breadth of antibody response against VOCs elicited by post-vaccination Delta and Omicron infection, compared to WT infection without vaccination. Then, in mouse models, three doses of Omicron-RBD immunization elicited comparable neutralizing antibody (NAb) titers with three doses of WT-RBD immunization, but the neutralizing activity was not cross-active. By contrast, a heterologous Omicron-RBD booster following two doses of WT-RBD immunization increased the NAb titers against Omicron by 9 folds than the homologous WT-RBD booster. Moreover, it retains neutralization against both WT and current VOCs. Results suggest that Omicron-specific subunit booster shows its advantages in the immune protection from both WT and current VOCs and that SARS-CoV-2 vaccines including two or more virus lineages might improve the NAb response. Graphical

A cocktail containing two synergetic antibodies broadly neutralizes SARS-CoV-2 and its variants including Omicron BA.1 and BA.2 (preprint)

Neutralizing antibodies (NAbs) can prevent and treat infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, continuously emerging variants, such as Omicron, have significantly reduced the potency of most known NAbs. The selection of NAbs with broad neutralizing activities and the identification of conserved critical epitopes are still urgently needed. Here, we identified an extremely potent antibody (55A8) by single B-cell sorting from convalescent SARS-CoV-2-infected patients that recognized the receptor-binding domain (RBD) in the SARS-CoV-2 spike (S) protein. 55A8 could bind to wild-type SARS-CoV-2, Omicron BA.1 and Omicron BA.2 simultaneously with 58G6, a NAb previously identified by our group. Importantly, an antibody cocktail containing 55A8 and 58G6 (2-cocktail) showed synergetic neutralizing activity with a half-maximal inhibitory concentration (IC50) in the picomolar range in vitro and prophylactic efficacy in hamsters challenged with Omicron (BA.1) through intranasal delivery at an extraordinarily low dosage (25 g of each antibody daily) at 3 days post-infection. Structural analysis by cryo-electron microscopy (cryo-EM) revealed that 55A8 is a Class III NAb that recognizes a highly conserved epitope. It could block angiotensin-converting enzyme 2 (ACE2) binding to the RBD in the S protein trimer via steric hindrance. The epitopes in the RBD recognized by 55A8 and 58G6 were found to be different and complementary, which could explain the synergetic mechanism of these two NAbs. Our findings not only provide a potential antibody cocktail for clinical use against infection with current SARS-CoV-2 strains and future variants but also identify critical epitope information for the development of better antiviral agents.

Extensive neutralization against SARS-CoV-2 variants elicited by Omicron-specific subunit vaccine booster (preprint)

With the development of COVID-19, even though increased global vaccination coverage, a super variant of SARS-CoV-2, Omicron, carrying a great number of mutations, has been verified its strong capacity of immune escape. An increased risk of SARS-CoV-2 reinfection or breakthrough infection should be concerned. We analyzed the humoral immune response of Omicron breakthrough infection and found its cross-neutralization against VOCs. We established mouse models to verify whether Omicron-specific RBD subunit boost immune response by immunizing Omicron-RBD recombinant proteins. The results suggest that an additional boost vaccination with Omicron-RBD protein could increase humoral immune response against both WT and current VOCs.

Reduced infectivity but increased immune escape of the new SARS-CoV-2 variant of concern Omicron (preprint)

A new detected SARS-CoV-2 variant Omicron (B.1.1.529) had reported from more than 80 countries. In the past few weeks, a new wave of infection driven by Omicron is in progress. Omicron Spike (S) protein pseudotyped virus was used to determine the effect of S mutations on its capacity of infectivity and immune evasion. Our results showed the lower entry efficiency and less cleavage ability of Omicron than D614G variant. Pseudotype-based neutralizing assay was performed to analyze neutralizing antibodies elicited by previously infection or the RBD-based protein subunit vaccine ZF2001 against the Omicron variant. Sera sampled at around one month after symptom onset from 12 convalescents who were previously infected by SARS-CoV-2 original strain shows a more than 20-fold decrease of neutralizing activity against Omicron variant, when compared to D614G variant. Among 12 individuals vaccinated by RBD subunit vaccine, 58.3% (7/12) sera sampled at 15-60 days after 3rd-dose vaccination did not neutralize Omicron. Geometric mean titers (GMTs, 50% inhibitory dose [ID50]) of these sera against Omicron were 9.4-fold lower than against D614G. These results suggested a higher risk of Omicron breakthrough infections and reduced efficiency of the protective immunity elicited by existing vaccines. There are important implications about the modification and optimization of the current epidemic prevention and control including vaccine strategies and therapeutic antibodies against Omicron variant.

Reduced neutralization of SARS-CoV-2 B.1.617 variant by inactivated and RBD-subunit vaccine (preprint)

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The Spike protein that mediates coronavirus entry into host cells is a major target for COVID-19 vaccines and antibody therapeutics. However, multiple variants of SARS-CoV-2 have emerged, which may potentially compromise vaccine effectiveness. Using a pseudovirus-based assay, we evaluated SARS-CoV-2 cell entry mediated by the viral Spike B.1.617 and B.1.1.7 variants. We also compared the neutralization ability of monoclonal antibodies from convalescent sera and neutralizing antibodies (NAbs) elicited by CoronaVac (inactivated vaccine) and ZF2001 (RBD-subunit vaccine) against B.1.617 and B.1.1.7 variants. Our results showed that, compared to D614G and B.1.1.7 variants, B.1.617 shows enhanced viral entry and membrane fusion, as well as more resistant to antibody neutralization. These findings have important implications for understanding viral infectivity and for immunization policy against SARS-CoV-2 variants.

Coevolving Dynamics between Epidemic and Information Spreading considering the Dependence between Vigilance and Awareness Prevalence

It has been demonstrated that the propagation of information and awareness regarding a disease can assist in containing the outbreak of epidemics. Previous models for this coevolving usually introduced the dependence between these two processes by setting a lower but time-independent infection rate for individuals with awareness. However, a realistic scenario can be more complicated, as individual vigilance and the adopted protective measures may depend on the extent of the discussion on the disease, whereas individuals may be irrational or lack relevant knowledge, leading to improper measures being taken. These can introduce a time-varying dependence between epidemic dynamics and awareness prevalence and may weaken the effect of spreading awareness in containing a pandemic. To better understand this effect, we introduce a nonlinear dependence of the epidemic infection rate on awareness prevalence, focusing on the effect of different forms of dependence on the coevolving dynamics. We demonstrate that a positive correlation between vigilance and awareness prevalence can enhance the effect of information spreading in suppressing epidemics. However, this enhancement can be weakened if some individuals are irrational. Our results demonstrate the importance of rational behavior in the strategy of containing epidemics by propagation of disease information.

Ultrapotent SARS-CoV-2 neutralizing antibodies with protective efficacy against newly emerged mutational variants (preprint)

Accumulating mutations in the SARS-CoV-2 Spike (S) protein can increase the possibility of immune escape, challenging the present COVID-19 prophylaxis and clinical interventions. Here, 3 receptor binding domain (RBD) specific monoclonal antibodies (mAbs), 58G6, 510A5 and 13G9, with high neutralizing potency blocking authentic SARS-CoV-2 virus displayed remarkable efficacy against authentic B.1.351 virus. Each of these 3 mAbs in combination with one neutralizing Ab recognizing non-competing epitope exhibited synergistic effect against authentic SARS-CoV-2 virus. Surprisingly, structural analysis revealed that 58G6 and 13G9, encoded by the IGHV1-58 and the IGKV3-20 germline genes, both recognized the steric region S470-495 on the RBD, overlapping the E484K mutation presented in B.1.351. Also, 58G6 directly bound to another region S450-458 in the RBD. Significantly, 58G6 and 510A5 both demonstrated prophylactic efficacy against authentic SARS-CoV-2 and B.1.351 viruses in the transgenic mice expressing human ACE2 (hACE2), protecting weight loss and reducing virus loads. These 2 ultrapotent neutralizing Abs can be promising candidates to fulfill the urgent needs for the prolonged COVID-19 pandemic.

Ultrapotent SARS-CoV-2 neutralizing antibodies effectively block new-emerging mutational variants (preprint)

Accumulating mutations on SARS-CoV-2 Spike (S) protein may increase the possibility of immune escape, challenging the present COVID-19 prophylaxis and clinical interventions. Here, in a panel of receptor binding domain (S-RBD) specific monoclonal antibodies (mAbs) with high neutralizing potency against authentic SARS-CoV-2, at least 6 of them were found to efficiently block the pseudovirus of 501Y.V2, a highly transmissible SARS-CoV-2 variant with escape mutations. The top 3 neutralizing Abs (13G9, 58G6 and 510A5) exhibited comparative ultrapotency as those being actively pursued for clinical development. Interestingly, the antigenic sites for the majority of our neutralizing Abs overlapped with a single epitope (13G9e) on S-RBD. Further, the 3-dimensional structures of 2 ultrapotent neutralizing Abs 13G9 or 58G6 in complex with SARS-CoV-2 S trimer demonstrated that both Abs bound to a steric region within S472–490. Moreover, a specific linear region (S450–457) was identified as an additional target for 58G6. Importantly, our cryo-electron microscopy (cryo-EM) analysis revealed a unique phenomenon that the S-RBDs interacting with the fragments of antigen binding (Fabs) of 13G9 or 58G6 encoded by the IGHV1-58 and the IGKV3-20 gene segments were universally in the ‘up’ conformation in all observed particles. The potent neutralizing Abs presented in the current study may be promising candidates to fulfill the urgent needs for the current pandemic of SARS-CoV-2, and may of fundamental value for the next-generation vaccine development.

CCR1 regulatory variants linked to pulmonary macrophage recruitment in severe COVID-19 (preprint)

Genome-wide association studies have identified 3p21.31 as the main risk locus for severe symptoms and hospitalization in COVID-19 patients. To elucidate the mechanistic basis of this genetic association, we performed a comprehensive epigenomic dissection of the 3p21.31 locus. Our analyses pinpoint activating variants in regulatory regions of the chemokine receptor-encoding CCR1 gene as potentially pathogenic by enhancing infiltration of monocytes and macrophages into the lungs of patients with severe COVID-19.

Exploring the natural origins of SARS-CoV-2 (preprint)

The lack of an identifiable intermediate host species for the proximal animal ancestor of SARS-CoV-2 and the distance (~1500 km) from Wuhan to Yunnan province, where the closest evolutionary related coronaviruses circulating in horseshoe bats have been identified, is fueling speculation on the natural origins of SARS-CoV-2. Here we analyse SARS-CoV-2's related horseshoe bat and pangolin Sarbecoviruses and confirm Rhinolophus affinis continues to be the likely reservoir species as its host range extends across Central and Southern China. This would explain the bat Sarbecovirus recombinants in the West and East China, trafficked pangolin infections and bat Sarbecovirus recombinants linked to Southern China. Recent ecological disturbances as a result of changes in meat consumption could then explain SARS-CoV-2 transmission to humans through direct or indirect contact with the reservoir wildlife, and subsequent emergence towards Hubei in Central China. The only way, however, of finding the animal progenitor of SARS-CoV-2 as well as the whereabouts of its close relatives, very likely capable of posing a similar threat of emergence in the human population and other animals, will be by increasing the intensity of our sampling.

Emerging SARS-CoV-2 variants reduce neutralization sensitivity to convalescent sera and monoclonal antibodies (preprint)

SARS-CoV-2 Spike-specific antibodies contribute the majority of the neutralizing activity in most convalescent human sera. Two SARS-CoV-2 variants, N501Y.V1 (also known as B.1.1.7 lineage or VOC-202012/01) and N501Y.V2 (B.1.351 lineage), reported from the United Kingdom and South Africa, contain several mutations in the receptor binding domain of Spike and are of particular concern. To address the infectivity and neutralization escape phenotypes potentially caused by these mutations, we used SARS-CoV-2 pseudovirus system to compare the viral infectivity, as well as the neutralization activities of convalescent sera and monoclonal antibodies (mAbs) against SARS-CoV-2 variants. Our results showed that N501Y Variant 1 and Variant 2 increase viral infectivity compared to the reference strain (wild-type, WT) in vitro. At 8 months after symptom onset, 17 serum samples of 20 participants (85%) retaining titers of ID50 >40 against WT pseudovirus, whereas the NAb titers of 8 samples (40%) and 18 samples (90%) decreased below the threshold against N501Y.V1 and N501Y.V2, respectively. In addition, both N501Y Variant 1 and Variant 2 reduced neutralization sensitivity to most (6/8) mAbs tested, while N501Y.V2 even abrogated neutralizing activity of two mAbs. Taken together the results suggest that N501Y.V1 and N501Y.V2 reduce neutralization sensitivity to some convalescent sera and mAbs.

Identification of bis-benzylisoquinoline alkaloids as SARS-CoV-2 entry inhibitors from a library of natural products in vitro (preprint)

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major public health issue. To screen for antiviral drugs for COVID-19 treatment, we constructed a SARS-CoV-2 spike (S) pseudovirus system using an HIV-1-based lentiviral vector with a luciferase reporter gene to screen 188 small potential antiviral compounds. Using this system, we identified nine compounds, specifically, bis-benzylisoquinoline alkaloids, that potently inhibited SARS-CoV-2 pseudovirus entry, with EC50 values of 0.1-10 M. Mechanistic studies showed that these compounds, reported as calcium channel blockers (CCBs), inhibited Ca2+-mediated membrane fusion and consequently suppressed coronavirus entry. These candidate drugs showed broad-spectrum efficacy against the entry of several coronavirus pseudotypes (SARS-CoV, MERS-CoV, SARS-CoV-2 [S-D614 and S-G614]) in different cell lines (293T, Calu-3, and A549). Antiviral tests using native SARS-CoV-2 in Vero E6 cells confirmed that four of the drugs (SC9/cepharanthine, SC161/hernandezine, SC171, and SC185/neferine) reduced cytopathic effect and supernatant viral RNA load. Among them, cepharanthine showed the strongest anti-SARS-CoV-2 activity. Collectively, this study offers new lead compounds for coronavirus antiviral drug discovery.

Changes of Humoral Immunity Response in SARS-CoV-2 Convalescent Patients over 8 months (preprint)

Many countries around the world have all seen a sharp rise in COVID-19 cases as the second wave since the beginning of October 2020. Decline of antibodies response to severe acute respiratory syndrome coronavirus (SARS-CoV-2) that was reported exclusively in the early month increases the risk of reinfection for convalescent individuals. There is a current need to follow the maintenance of special antibodies against SARS-CoV-2. Here, we reported changes of antibodies against SARS-CoV-2 in convalescent patients over 8 months. Antibodies of all 20 participants targeting SARS-CoV-2 spike receptor binding-domain (RBD) had decreased from a mean OD450 value 1.78 to 0.38 over 8 months. The neutralizing antibody (NAb) titers decreased from the mean ID50 value 836 to 170. The NAb titers were significantly correlated with IgG level during 8 months (P<0.001). Furthermore, while RBD-specific IgG existence of 25% (5/20) convalescent plasma was undetectable, the NAb titers of 15% (3/20) convalescent plasma decreased below the threshold. In addition, compared to wild-type SARS-CoV-2 (S-D614), lower titers of neutralizing antibodies against its G614 variant were shown at 8 months after symptom onset. This study has important implications when considering antibody protection against SARS-CoV-2 reinfection.

Host metabolite-cytokine correlation landscape in SARS-CoV-2 infection (preprint)

The systemic cytokine release syndrome (CRS) is a major cause of the multi-organ injury and fatal outcome induced by SARS-CoV-2 infection in severe COVID-19 patients. It has been well-known that metabolism plays a role in modulating the immune responses in infectious diseases. Yet, how the host metabolism correlates with CRS in COVID-19 patients and how the perturbed metabolites affect the cytokine release remains unclear. Here, we performed both metabolomics and cytokine/chemokine profiling on serum samples from the same cohort of healthy controls, mild and severe COVID-19 patients and delineated the global metabolic and immune response landscape along disease progression. Intriguingly, the correlation analysis revealed the tight link between metabolites and proinflammatory cytokines and chemokines, such as IL-6, M-CSF, IL-1α, IL-1β, implying the potential regulatory role of arginine metabolism, tryptophan metabolism, and purine metabolism in hyperinflammation. Importantly, we demonstrated that targeting metabolism markedly modulated the proinflammatory cytokines release by PBMCs isolated from SARS-CoV-2-infected rhesus macaques ex vivo. Beyond providing a comprehensive resource of metabolism and immunology data of SARS-CoV-2 infection, our study showed that metabolic alterations can be potentially exploited to develop novel strategy for the treatment of fatal CRS in COVID-19.

A rapid and efficient screening system for neutralizing antibodies and its application for SARS-CoV-2 (preprint)

After the epidemic of COVID-19, neutralizing antibodies (NAbs) against SARS-CoV-2 has been developed for the preventative and therapeutic purposes. However, few methodologies are reported in detail on how to rapidly and efficiently generate NAbs of interest. Here, we present a strategically optimized screening method for NAbs, which has enabled us to obtain SARS-CoV-2 receptor-binding domain (RBD) specific monoclonal Abs within 4 days, followed by additional 2 days to evaluate their neutralizing activities. Using this method, we obtained 198 specific Abs against SARS-CoV-2 RBD from the blood samples of COVID-19 convalescent patients, and 96 of them showed neutralizing activity. At least 20% of these NAbs exhibited high neutralizing potency. The top 2 NAbs showed the half-maximal inhibitory concentration (IC50) to block authentic SARS-CoV-2 at 9.88 and 11.13 ng/ml, respectively. Altogether, our study provides a fundamental methodology for discovering NAbs with potential preventative and therapeutic value for emerging infectious diseases.

A key linear epitope for a potent neutralizing antibody to SARS-CoV-2 S-RBD (preprint)

The spread of SARS-CoV-2 confers a serious threat to the public health without effective intervention strategies1-3. Its variant carrying mutated Spike (S) protein D614G (SD614G) has become the most prevalent form in the current global pandemic4,5. We have identified a large panel of potential neutralizing antibodies (NAbs) targeting the receptor-binding domain (RBD) of SARS-CoV-2 S6. Here, we focused on the top 20 potential NAbs for the mechanism study. Of them, the top 4 NAbs could individually neutralize both authentic SARS-CoV-2 and SD614G pseudovirus efficiently. Our epitope mapping revealed that 16/20 potent NAbs overlapped the same steric epitope. Excitingly, we found that one of these potent NAbs (58G6) exclusively bound to a linear epitope on S-RBD (termed as 58G6e), and the interaction of 58G6e and the recombinant ACE2 could be blocked by 58G6. We confirmed that 58G6e represented a key site of vulnerability on S-RBD and it could positively react with COVID-19 convalescent patients plasma. We are the first, as far as we know, to provide direct evidences of a linear epitope that can be recognized by a potent NAb against SARS-CoV-2 S-RBD. This study paves the way for the applications of these NAbs and the potential safe and effective vaccine design.

Molecular Characterization, Phylogenetic and Variation Analyzes of SARS-CoV-2 strains in Turkey (preprint)

IntroductionWe present the sequence analysis for 47 complete genomes for SARS-CoV-2 isolates on Turkish patients. To identify their genetic similarity, phylogenetic analysis was performed by comparing the worldwide SARS-CoV-2 sequences, selected from GISAID, to the complete genomes from Turkish isolates. In addition, we focused on the variation analysis to show the mutations on SARS-CoV-2 genomes. MethodsIllumina MiSeq platform was used for sequencing the libraries. The raw reads were aligned to the known SARS-CoV-2 genome (GenBank: MN908947.3) using the Burrows-Wheeler aligner (v.0.7.1). The phylogenetic tree was constructer using Phylip v.3.6 with Neighbor-Joining and composite likelihood method. The variants were detected by using Genome Analysis Toolkit-HaplotypeCaller v.3.8.0 and were inspected on GenomeBrowse v2.1.2. ResultsAll viral genome sequences of our isolates was located in lineage B under the different clusters such as B.1 (n=3), B.1.1 (n=28), and B.1.9 (n=16). According to the GISAID nomenclature, all our complete genomes were placed in G, GR and GH clades. Five hundred forty-nine total and 53 unique variants were detected. All 47 genomes exhibited different kinds of variants. The distinct variants consist of 274 missense, 225 synonymous, and 50 non-coding alleles. ConclusionThe results indicated that the SARS-CoV-2 sequences of our isolates have great similarity with all Turkish and European sequences. Further studies should be performed for better comparison of strains, after more complete genome sequences will be released. We also believe that collecting and sharing any data about SARS-CoV-2 virus and COVID-19 will be effective and may help the related studies.

A comparative survey of betacoronavirus strain molecular dynamics identifies key ACE2 binding sites (preprint)

Comparative functional analysis of the binding interactions between various betacoronavirus strains and their potential human target proteins, such as ACE1, ACE2 and CD26, is critical to our future understanding and combating of COVID-19. Here, employing large replicate sets of GPU accelerated molecular dynamics simulations, we statistically compare atom fluctuations of the known human target proteins in both the presence and absence of different strains of the viral receptor binding domain (RBD) of the S spike glycoprotein. We identify a common interaction site between the N-terminal helices of ACE2 and the viral RBD in all strains (hCoV-OC43, hCoV-HKU1, MERS-CoV, SARS-CoV1, and SARS-CoV-2) and a second more dynamically complex RBD interaction site involving the ACE2 amino acid sites K353, Q325, and a novel motif, AAQPFLL (386-392) in the more recent cross-species spillovers (i.e. absent in hCoV-OC43). We use computational mutagenesis to further confirm the functional relevance of these sites. We propose a "one touch/two touch" model of viral evolution potentially involved in functionally facilitating binding interactions in zoonotic spillovers. We also observe these two touch sites governing RBD binding activity in simulations on hybrid models of the suspected viral progenitor, batCoV-HKU4, interacting with both the human SARS target, ACE2, and the human MERS target, CD26. Lastly, we confirm that the presence of a common hypertension drug (lisinopril) within the target site of SARS-CoV-2 bound models of ACE1 and ACE2 acts to enhance the RBD interactions at the same key sites in our proposed model. In the near future, we recommend that our comparative computational analysis identifying these key viral RBD-ACE2 binding interactions be supplemented with comparative studies of site-directed mutagenesis in order to screen for current and future coronavirus strains at high risk of zoonotic transmission to humans. STATEMENT OF SIGNIFICANCEWe generated structural models of the spike glycoprotein receptor binding domain from recent and past betacoronavirus outbreak strains aligned to the angiotensin 1 converting enzyme 2 protein, the primary target protein of the SARS-CoV-2 virus causing COVID 19. We then statistically compared computer simulated molecular dynamics of viral bound and unbound versions of each model to identify locations where interactions with each viral strain have dampened the atom fluctuations during viral binding. We demonstrate that all known strains of betacoronavirus are strongly interactive with the N-terminal helix region of ACE2. We also identify a more complex viral interaction with three novel sites that associates with more recent and deadly SARS strains, and also a bat progenitor strain HKU4.