Four SARS-CoV-2 vaccines induce quantitatively different antibody responses against SARS-CoV-2 variants (preprint)

BackgroundEmerging and future SARS-CoV-2 variants may jeopardize the effectiveness of vaccination campaigns. Therefore, it is important to know how the different vaccines perform against diverse SARS-CoV-2 variants. MethodsIn a prospective cohort of 165 SARS-CoV-2 naive health care workers, vaccinated with either one of four vaccines (BNT162b2, mRNA-1273, AZD1222 or Ad26.COV2.S), we performed a head-to-head comparison of the ability of sera to recognize and neutralize SARS-CoV-2 variants of concern (VOCs; Alpha, Beta, Gamma, Delta and Omicron). Repeated serum sampling was performed 5 times during a year (from January 2021 till January 2022), including before and after booster vaccination with BNT162b2. FindingsFour weeks after completing the initial vaccination series, SARS-CoV-2 wild-type neutralizing antibody titers were highest in recipients of BNT162b2 and mRNA-1273 (geometric mean titers (GMT) of 197 [95% CI 149-260] and 313 [95% CI 218-448], respectively), and substantially lower in those vaccinated with the adenovirus vector-based vaccines AZD1222 and Ad26.COV2.S (GMT of 26 [95% CI 18-37] and 14 [95% CI 8-25] IU/ml, respectively). These findings were robust for adjustment to age and sex. VOCs neutralization was reduced in all vaccine groups, with the largest (9- to 80-fold) reduction in neutralization observed against the Omicron variant. The booster BNT162b2 vaccination increased neutralizing antibody titers for all groups with substantial improvement against the VOCs including the Omicron variant. Study limitations include the lack of cellular immunity data. ConclusionsOverall, this study shows that the mRNA vaccines appear superior to adenovirus vector-based vaccines in inducing neutralizing antibodies against VOCs four weeks after initial vaccination and after booster vaccination.

Cross-reactive antibodies after SARS-CoV-2 infection and vaccination (preprint)

Current SARS-CoV-2 vaccines are losing efficacy against emerging variants and may not protect against future novel coronavirus outbreaks, emphasizing the need for more broadly protective vaccines. To inform the development of a pan-coronavirus vaccine, we investigated the presence and specificity of cross-reactive antibodies against the spike (S) proteins of human coronaviruses (hCoV) after SARS-CoV-2 infection and vaccination. We found an 11 to 123-fold increase in antibodies binding to SARS-CoV and MERS-CoV as well as a 2 to 4-fold difference in antibodies binding to seasonal hCoVs in COVID-19 convalescent sera compared to pre-pandemic healthy donors, with the S2 subdomain of the S protein being the main target for cross-reactivity. In addition, we detected cross-reactive antibodies to all hCoV S proteins after SARS-CoV-2 S protein immunization in macaques, with higher responses for hCoV more closely related to SARS-CoV-2. These findings support the feasibility of and provide guidance for development of a pan-coronavirus vaccine.

Emerging SARS-CoV-2 variants of concern evade humoral immune responses from infection and vaccination (preprint)

Emerging SARS-CoV-2 variants pose a threat to human immunity induced by natural infection and vaccination. We assessed the recognition of three variants of concern (B.1.1.7, B.1.351 and P.1) in cohorts of COVID-19 patients ranging in disease severity (n = 69) and recipients of the Pfizer/BioNTech vaccine (n = 50). Spike binding and neutralization against all three VOC was substantially reduced in the majority of samples, with the largest 4-7-fold reduction in neutralization being observed against B.1.351. While hospitalized COVID-19 patients and vaccinees maintained sufficient neutralizing titers against all three VOC, 39% of non-hospitalized patients did not neutralize B.1.351. Moreover, monoclonal neutralizing antibodies (NAbs) show sharp reductions in their binding kinetics and neutralizing potential to B.1.351 and P.1, but not to B.1.1.7. These data have implications for the degree to which pre-existing immunity can protect against subsequent infection with VOC and informs policy makers of susceptibility to globally circulating SARS-CoV-2 VOC.

Single-dose SARS-CoV-2 vaccine in a prospective cohort of COVID-19 patients (preprint)

Background The urgent need for, but limited availability of, SARS-CoV-2 vaccines worldwide has led to widespread consideration of dose sparing strategies, particularly single vaccine dosing of individuals with prior SARS-CoV-2 infection. Methods We evaluated SARS-CoV-2 specific antibody responses following a single-dose of BNT162b2 (Pfizer-BioNTech) mRNA vaccine in 155 previously SARS-CoV-2-infected individuals participating in a population-based prospective cohort study of COVID-19 patients. Participants varied widely in age, comorbidities, COVID-19 severity and time since infection, ranging from 1 to 15 months. Serum antibody titers were determined at time of vaccination and one week after vaccination. Responses were compared to those in SARS-CoV-2-naive health care workers after two BNT162b2 mRNA vaccine doses. Results Within one week of vaccination, IgG antibody levels to virus spike and RBD proteins increased 27 to 29-fold and neutralizing antibody titers increased 12-fold, exceeding titers of fully vaccinated SARS-CoV-2-naive controls (95% credible interval (CrI): 0.56 to 0.67 v. control 95% CrI: -0.16 to -0.02). Pre-vaccination neutralizing antibody titers had the largest positive mean effect size on titers following vaccination (95% CrI (0.16 to 0.45)). COVID-19 severity, the presence of comorbidities and the time interval between infection and vaccination had no discernible impact on vaccine response. Conclusion A single dose of BNT162b2 mRNA vaccine up to 15 months after SARS-CoV-2 infection provides neutralizing titers exceeding two vaccine doses in previously uninfected individuals. These findings support wide implementation of a single-dose mRNA vaccine strategy after prior SARS-CoV-2 infection.

Evolution of COVID-19 symptoms during the first 9 months after illness onset (preprint)

BackgroundFew longitudinal data on COVID-19 symptoms across the full spectrum of disease severity are available. We evaluated symptom onset, severity and recovery up to nine months after illness onset. MethodsThe RECoVERED Study is a prospective cohort study based in Amsterdam, the Netherlands. Participants aged>18 years were recruited following SARS-CoV-2 diagnosis via the local Public Health Service and from hospitals. Standardised symptom questionnaires were completed at recruitment, at one week and month after recruitment, and monthly thereafter. Clinical severity was defined according to WHO criteria. Kaplan-Meier methods were used to compare time from illness onset to symptom recovery, by clinical severity. We examined determinants of time to recovery using multivariable Cox proportional hazards models. ResultsBetween 11 May 2020 and 31 January 2021, 301 COVID-19 patients (167[55%] male) were recruited, of whom 99/301(32.9%) had mild, 140/301(46.5%) moderate, 30/301(10.0%) severe and 32/301(10.6%) critical disease. The proportion of symptomatic participants who reported at least one persistent symptom at 12 weeks after illness onset was greater in those with severe/critical disease (81.7%[95%CI=68.7-89.7%]) compared to those with mild or moderate disease (33.0%[95%CI=23.0-43.3%] and 63.8%[95%CI=54.8-71.5%]). Even at nine months after illness onset, almost half of all participants (42.1%[95%CI=35.6-48.5]) overall continued to report [≥]1 symptom. Recovery was slower in participants with BMI[≥]30kg/m2 (HR 0.51[95%CI=0.30-0.87]) compared to those with BMI<25kg/m2, after adjusting for age, sex and number of comorbidities. ConclusionsCOVID-19 symptoms persisted for nine months after illness onset, even in those with mild disease. Obesity was the most important determinant of speed of recovery from symptoms.

Cryo-EM Structure of the N501Y SARS-CoV-2 Spike Protein in Complex with a Potent Neutralizing Antibody (preprint)

The recently reported ''UK variant'' of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. Here, we report cryo-EM structures of SARS-CoV-2 spike protein ectodomains with and without the N501Y mutation, in complex with the VH fragment of the potent neutralizing antibody, VH -Fc ab8. The mutation results in localized structural perturbations near Y501, but VH -Fc ab8 retains the ability to bind and neutralize pseudotyped viruses expressing the N501Y mutant with efficiencies comparable to that of unmutated viruses. Our results show that despite the higher affinity of ACE2 for the N501Y mutant, it can still be neutralized efficiently by an antibody that binds epitopes in the receptor binding domain of the SARS-CoV-2 spike protein.

Snake venom phospholipases A2 possess a strong virucidal activity against SARS-CoV-2 in vitro and block the cell fusion mediated by spike glycoprotein interaction with the ACE2 receptor (preprint)

A new coronavirus was recently discovered and named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In the absence of specific therapeutic and prophylactic agents, the virus has infected almost hundred million people, of whom nearly two million have died from the viral disease COVID-19. The ongoing COVID-19 pandemic is a global threat requiring new therapeutic strategies. Among them, antiviral studies based on natural molecules are a promising approach. The superfamily of phospholipases A2 (PLA2s) consists of a large number of members that catalyze the hydrolysis of phospholipids at a specific position. Here we show that secreted PLA2s from the venom of various snakes protect to varying degrees the Vero E6 cells widely used for the replication of viruses with evident cytopathic action, from SARS-CoV-2 infection PLA2s showed low cytotoxicity to Vero E6 cells and the high antiviral activity against SARS-CoV-2 with IC50 values ranged from 0.06 to 7.71 ug/ml. Dimeric PLA2 HDP-2 from the viper Vipera nikolskii, as well as its catalytic and inhibitory subunits, had potent virucidal (neutralizing) activity against SARS-CoV-2. Inactivation of the enzymatic activity of the catalytic subunit of dimeric PLA2 led to a significant decrease in antiviral activity. In addition, dimeric PLA2 inhibited cell-cell fusion mediated by SARS-CoV-2 spike glycoprotein. These results suggest that snake PLA2s, in particular dimeric ones, are promising candidates for the development of antiviral drugs that target lipid bilayers of the viral envelope and may be good tools to study the interaction of viruses with host cell membranes.

Novel RT-ddPCR assays for determining the transcriptional profile of SARS-CoV-2 (preprint)

The exact mechanism of coronavirus replication and transcription is not fully understood; however, a hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from discontinuous transcription. Existing PCR-based diagnostic assays for SAR-CoV-2 are qualitative or semi-quantitative and do not provide the resolution needed to assess the complex transcription dynamics of SARS-CoV-2 over the course of infection. We developed and validated a novel panel of specially designed SARS-CoV-2 ddPCR-based assays to map the viral transcription profile. Application of these assays to clinically relevant samples will enhance our understanding of SARS-CoV-2 replication and transcription and may also inform the development of improved diagnostic tools and therapeutics.

Inhibitor Binding Modulates Protonation States in the Active Site of SARS-CoV-2 Main Protease (preprint)

The main protease (3CL Mpro) from SARS-CoV-2, the virus that causes COVID-19, is an essential enzyme for viral replication with no human counterpart, making it an attractive drug target. Although drugs have been developed to inhibit the proteases from HIV, hepatitis C and other viruses, no such therapeutic is available to inhibit the main protease of SARS-CoV-2. To directly observe the protonation states in SARS-CoV-2 Mpro and to elucidate their importance in inhibitor binding, we determined the structure of the enzyme in complex with the -ketoamide inhibitor telaprevir using neutron protein crystallography at near-physiological temperature. We compared protonation states in the inhibitor complex with those determined for a ligand-free neutron structure of Mpro. This comparison revealed that three active-site histidine residues (His41, His163 and His164) adapt to ligand binding, altering their protonation states to accommodate binding of telaprevir. We suggest that binding of other -ketoamide inhibitors can lead to the same protonation state changes of the active site histidine residues. Thus, by studying the role of active site protonation changes induced by inhibitors we provide crucial insights to help guide rational drug design, allowing precise tailoring of inhibitors to manipulate the electrostatic environment of SARS-CoV-2 Mpro.

Serologic Surveillance and Phylogenetic Analysis of SARS-CoV-2 Infection in Hospital Health Care Workers (preprint)

BACKGROUND It is unclear how, when and where health care workers (HCW) working in hospitals are infected with SARS-CoV-2. METHODS Prospective cohort study comprising 4-weekly measurement of SARS-CoV-2 specific antibodies and questionnaires from March to June 2020. We compared SARS-CoV-2 incidence between HCW working in Covid-19 patient care, HCW working in non-Covid-19 patient care and HCW not in patient care. Phylogenetic analyses of SARS-CoV-2 samples from patients and HCW were performed to identify potential transmission clusters. RESULTS We included 801 HCW: 439 in the Covid-19 patient care group, 164 in the non-Covid-19 patient care group and 198 in the no patient care group. SARS-CoV-2 incidence was highest in HCW working in Covid-19 patient care (13.2%), as compared with HCW in non-Covid-19 patient care (6.7%, hazard ratio [HR] 2.2, 95% confidence interval [CI] 1.2 to 4.3) and in HCW not working in patient care (3.6%, HR 3.9, 95% CI 1.8 to 8.6). Within the group of HCW caring for Covid-19 patients, SARS-CoV-2 cumulative incidence was highest in HCW working on Covid-19 wards (25.7%), as compared with HCW working on intensive care units (7.1%, HR 3.6, 95% CI 1.9 to 6.9), and HCW working in the emergency room (8.0%, HR 3.3, 95% CI 1.5 to 7.1). Phylogenetic analyses on Covid-19 wards identified multiple potential HCW-to-HCW transmission clusters while no patient-to-HCW transmission clusters were identified. CONCLUSIONS HCW working on Covid-19 wards are at increased risk for nosocomial SARS-CoV-2 infection, with an important role for HCW-to-HCW transmission.

Homologous and heterologous antibodies to coronavirus 229E, NL63, OC43, HKU1, SARS, MERS and SARS-CoV-2 antigens in an age stratified cross-sectional serosurvey in a large tertiary hospital in The Netherlands (preprint)

Understanding the coronavirus (CoV) antibody landscape in relation to disease and susceptibility is critical for modelling of steps in the next phase during the current covid-19 pandemic. In March 2020, during the first month of the epidemic in The Netherlands, we performed cross sectional studies at two time points amongst patients of the Erasmus Medical Centre in Rotterdam, to assess the presence of antibodies against seasonal human coronaviruses (OC43, 229E, NL63, HKU1), emerging zoonotic coronaviruses (SARS, MERS) and SARS-CoV-2 in nine different age groups. We observed minimal SARS-CoV-2 reactivity early March (0.7% of sera), increasing to 3.0%, four weeks later, suggesting probably undetected cases during this early phase of the epidemic. Antibody responses were mostly coronavirus species specific at young age, but possible cross-reactivity between human seasonal CoVs was observed with increasing age.