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SARS-CoV-2 mutational variants evade humoral immune responses elicited by vaccines and current monoclonal antibody (mAb) therapies. Novel antibody-based treatments will thus need to exhibit broad neutralization against different variants. Bispecific antibodies (bsAbs) combine the specificities of two distinct antibodies into one antibody taking advantage of the avidity, synergy and cooperativity provided by targeting two different epitopes. Here we used controlled Fab-arm exchange (cFAE), a versatile and straightforward method, to produce bsAbs that neutralize SARS-CoV and SARS-CoV-2 variants, including Omicron and its subvariants, by combining potent SARS-CoV-2-specific neutralizing antibodies with broader but less potent antibodies that also neutralize SARS-CoV. We demonstrate that the parental IgGs rely on avidity for their neutralizing activity by comparing their potency to bsAbs containing one irrelevant "dead" Fab arm. We used single particle mass photometry to measure formation of antibody:spike complexes, and determined that bsAbs increase binding stoichiometry compared to corresponding cocktails, without a loss of binding affinity. The heterogeneous binding pattern of bsAbs to spike (S), observed by negative-stain electron microscopy and mass photometry provided evidence for both intra- and inter-spike crosslinking. This study highlights the utility of cross-neutralizing antibodies for designing bivalent or multivalent agents to provide a robust activity against circulating variants, as well as future SARS-like coronaviruses.
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The worldwide pandemic caused by SARS-CoV-2 has remained a human medical threat due to the continued evolution of multiple variants that acquire resistance to vaccines and prior infection. Therefore, it is imperative to discover monoclonal antibodies (mAbs) that neutralize a broad range of SARS-CoV-2 variants for therapeutic and prophylactic use. A stabilized autologous SARS-CoV-2 spike glycoprotein was used to enrich antigen-specific B cells from an individual with a primary Gamma variant infection. Five mAbs selected from those B cells showed considerable neutralizing potency against multiple variants of concern, with COVA309-35 being the most potent against the autologous virus, as well as against Omicron BA.1 and BA.2. When combining the COVA309 mAbs as cocktails or bispecific antibody formats, the breadth and potency was significantly improved against all tested variants. In addition, the mechanism of cross-neutralization of the COVA309 mAbs was elucidated by structural analysis. Altogether these data indicate that a Gamma-infected individual can develop broadly neutralizing antibodies.
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We investigate differences in protection from previous infection and/or vaccination against infection with Omicron BA.4/5 or BA.2. We observed a higher percentage of registered previous SARS-CoV-2 infections among 19836 persons infected with Omicron BA.4/5 compared to 7052 persons infected with BA.2 (31.3% vs. 20.0%) between 2 May and 24 July 2022 (adjusted odds ratio (aOR) for testing week, age group and sex: 1.4 (95%CI: 1.3-1.5)). No difference was observed in the distribution of vaccination status between BA.2 and BA.4/5 cases (aOR: 1.1 for primary and booster vaccination). Among reinfections, those newly infected with BA4/5 had a shorter interval between infections and the previous infection was more often caused by BA.1, compared to those newly infected with BA.2 (aOR: 1.9 (1.5-2.6). This suggests immunity induced by BA.1 is less effective against a BA.4/5 infection than against a BA.2 infection.
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OBJECTIVEUsing data from European prospective household studies, we systematically compared the symptom burden of the wild-type and Alpha variant infected individuals versus the Omicron BA.1 and BA.2 infected individuals across paediatric and adult age-groups. In addition, we measured the impact of COVID-19 vaccination on the Omicron symptom burden. METHODSThe household transmission studies were conducted during the wild-type and Alpha period (April 2020 to April 2021) and the early Omicron BA.1 and BA.2 dominant period (January to March 2022). All three studies used similar protocols. Households were prospectively followed from detection of the first SARS-CoV-2 index case until at least day 21 including (repeated) PCR testing, paired serology and daily symptom reporting for all household members. To avoid possible index-case ascertainment bias, we restricted analyses to secondary household cases. Age-stratified SARS-CoV-2 symptom burden was compared for wild-type/Alpha versus Omicron infections and for primary versus primary plus booster series vaccinated adult cases. FINDINGSIn total 216 secondary cases from wild-type/Alpha, and 130 from the Omicron period were included. Unvaccinated children <12 years experienced more symptoms and higher maximum and cumulative severity scores during the Omicron compared to the wild-type/Alpha period (p=0.004, p=0.011 and p=0.075, respectively). In adults, disease duration and maximum and cumulative severity scores were reduced during the Omicron period. Adjusted for age, gender and prior immunity Omicron was associated with lower odds for loss of smell or taste (Odds Ratio [OR]: 0.14; 95%CI 0.03-0.50), and higher, but non-significant odds for upper respiratory symptoms, fever and fatigue (ORs varying between 1.85-2.23). Comparing primary versus primary plus booster vaccinated adult cases during the Omicron period no differences were observed in disease severity or duration (p[≥]0.12). INTERPRETATIONIn children, the Omicron variant causes higher symptom burden compared to the wild-type/Alpha. Adults experienced a lower symptom burden possibly due to prior vaccination. A shift in most frequently reported symptoms occurred with a marked reduction in loss of smell or taste during the Omicron period. An additional effect of booster vaccination on symptom severity in infected adults compared to primary series only, could not be demonstrated.
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BackgroundIn fall 2020 when schools in the Netherlands operated under a limited set of COVID-19 measures, we conducted outbreaks studies in four secondary schools to gain insight in the level of school transmission and the role of SARS-CoV-2 transmission via air and surfaces. MethodsOutbreak studies were performed between 11 November and 15 December 2020 when the wild-type variant of SARS-CoV-2 was dominant. Clusters of SARS-CoV-2 infections within schools were identified through a prospective school surveillance study. All school contacts of cluster cases, irrespective of symptoms, were invited for PCR testing twice within 48 hrs and 4-7 days later. Combined NTS and saliva samples were collected at each time point along with data on recent exposure and symptoms. Surface and active air samples were collected in the school environment. All samples were PCR-tested and sequenced when possible. ResultsOut of 263 sampled school contacts, 24 tested SARS-CoV-2 positive (secondary attack rate 9.1%), of which 62% remained asymptomatic and 42% had a weakly positive test result. Phylogenetic analysis on 12 subjects from 2 schools indicated a cluster of 8 and 2 secondary cases, respectively, but also other distinct strains within outbreaks. Of 51 collected air and 53 surface samples, none were SARS-CoV-2 positive. ConclusionOur study confirmed within school SARS-CoV-2 transmission and substantial silent circulation, but also multiple introductions in some cases. Absence of air or surface contamination suggests environmental contamination is not widespread during school outbreaks.
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Variants of concern (VOCs) of SARS-CoV-2 have caused resurging waves of infections worldwide. In the Netherlands, Alpha, Beta, Gamma and Delta variants circulated widely between September 2020 and August 2021. To understand how various control measures had impacted the spread of these VOCs, we analyzed 39,844 SARS-CoV-2 genomes collected under the Dutch national surveillance program. We found that all four VOCs were introduced before targeted flight restrictions were imposed on countries where the VOCs first emerged. Importantly, foreign introductions, predominantly from other European countries, continued during these restrictions. Our findings show that flight restrictions had limited effectiveness in deterring VOC introductions due to the strength of regional land travel importation risks. We also found that the Alpha and Delta variants largely circulated more populous regions with international connections after their respective introduction before asymmetric bidirectional transmissions occurred with the rest of the country and the variant dominated infections in the Netherlands. As countries consider scaling down SARS-CoV-2 surveillance efforts in the post-crisis phase of the pandemic, our results highlight that robust surveillance in regions of early spread is important for providing timely information for variant detection and outbreak control.
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Using a recently introduced efficient mass spectrometry-based approach we monitored individual donors IgG1 clonal responses in molecular detail, examining SARS-CoV-2 spike-protein-specific IgG1 repertoires. We monitored the plasma clonal IgG1 profiles of 8 donors (4 male and 4 female) who had recently experienced an infection by either the wild type Wuhan Hu-1 virus or one of 3 VOCs (Alpha, Beta and Gamma). In these donors we charted the full plasma IgG1 repertoires as well as the IgG1 repertoires targeting the SARS-CoV-2 spike protein trimer as antigen. We observed that shortly after infection in between <0.1% to almost 10% of all IgG1 antibody molecules present in plasma did bind to the spike protein. Each donor displayed a unique plasma IgG1 repertoire, but also each donor displayed a unique and polyclonal antibody response against the SARS-CoV-2 spike-protein variants. Our analyses revealed that certain clones exhibit (alike) binding affinity towards all four tested spike-protein variants, whereas other clones displayed strong unique mutant-specific affinity. We conclude that each infected person generates a unique polyclonal response following infection, whereby some of these clones can bind multiple viral variants, whereas other clones do not display such cross-reactivity. In general, by assessing IgG1 repertoires following infection it becomes possible to identify and select fully matured human plasma antibodies that target specific antigens, and display either high specificity or cross-reactivity versus mutated versions of the antigen, which will aid in selecting antibodies that may be developed into biotherapeutics.
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Given the emergence of the SARS-CoV-2 Omicron BA.1 and BA.2 variants and the roll-out of booster COVID-19 vaccination, evidence is needed on protection conferred by primary vaccination, booster vaccination and previous SARS-CoV-2 infection by variant. We employed a test-negative design and used multinomial logistic regression on data from community PCR testing in the Netherlands. S-gene target failure (SGTF) was used as proxy to discern Delta, Omicron BA.1 and Omicron BA.2 infections. Two cohorts were defined to assess protection from vaccination and previous infection by variant: Delta-Omicron BA.1 cohort including data from 22 November 2021 to 7 January 2022 (n = 354,653) and Omicron BA.1-BA.2 cohort including data from 26 January to 31 March 2022 (n = 317,110). In the Delta-Omicron BA.1 cohort, including 39,889 Delta and 13,915 Omicron BA.1 infections, previous infection, primary vaccination or both protected well against Delta infection (76%, 71%, 92%, respectively, at 7+ months after infection or vaccination). Protection against Omicron BA.1 was much lower compared to Delta infections, but BA.1 estimates were imprecise. In the Omicron BA.1-BA.2 cohort, including 67,887 BA.1 and 41,670 BA.2 infections, protection was similar against Omicron BA.1 compared to BA.2 infection for previous infection (34 and 38% at 7+ months post-infection), primary (39 and 32% at 7+ months post-vaccination) and booster vaccination (68 and 63% at 1 month post-vaccination). Higher protection was observed against all variants in individuals with both vaccination and previous infection compared with either one. Protection against all variants by either vaccination or infection decreased over time since last vaccination or infection. Primary vaccination with current COVID-19 vaccines and previous SARS-CoV-2 infections offer low protection against Omicron BA.1 and BA.2 infection. Booster vaccination considerably increases protection against Omicron infection, but decreases rapidly after vaccination.
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BackgroundDuring the first two years of the COVID-19 pandemic, the circulation of seasonal influenza viruses was unprecedentedly low. This led to concerns that the lack of immune stimulation to influenza viruses combined with waning antibody titres could lead to increased susceptibility to influenza in subsequent seasons, resulting in larger and more severe epidemics. MethodsWe analyzed historical influenza virus epidemiological data from 2003-2019 to assess the historical frequency of near-absence of seasonal influenza virus circulation and its impact on the size and severity of subsequent epidemics. Additionally, we measured haemagglutination inhibition-based antibody titres against seasonal influenza viruses using longitudinal serum samples from 165 healthy adults, collected before and during the COVID-19 pandemic, and estimated how antibody titres against seasonal influenza waned during the first two years of the pandemic. FindingsLow country-level prevalence of influenza virus (sub)types over one or more years occurred frequently before the COVID-19 pandemic and had relatively small impacts on subsequent epidemic size and severity. Additionally, antibody titres against seasonal influenza viruses waned negligibly during the first two years of the pandemic. InterpretationThe commonly held notion that lulls in influenza virus circulation, as observed during the COVID-19 pandemic, will lead to larger and/or more severe subsequent epidemics might not be fully warranted, and it is likely that post-lull seasons will be similar in size and severity to pre-lull seasons. FundingEuropean Research Council, Netherlands Organization for Scientific Research, Royal Dutch Academy of Sciences, Public Health Service of Amsterdam. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSDuring the first years of the COVID-19 pandemic, the incidence of seasonal influenza was unusually low, leading to widespread concerns of exceptionally large and/or severe influenza epidemics in the coming years. We searched PubMed and Google Scholar using a combination of search terms (i.e., "seasonal influenza", "SARS-CoV-2", "COVID-19", "low incidence", "waning rates", "immune protection") and critically considered published articles and preprints that studied or reviewed the low incidence of seasonal influenza viruses since the start of the COVID-19 pandemic and its potential impact on future seasonal influenza epidemics. We found a substantial body of work describing how influenza virus circulation was reduced during the COVID-19 pandemic, and a number of studies projecting the size of future epidemics, each positing that post-pandemic epidemics are likely to be larger than those observed pre-pandemic. However, it remains unclear to what extent the assumed relationship between accumulated susceptibility and subsequent epidemic size holds, and it remains unknown to what extent antibody levels have waned during the COVID-19 pandemic. Both are potentially crucial for accurate prediction of post-pandemic epidemic sizes. Added value of this studyWe find that the relationship between epidemic size and severity and the magnitude of circulation in the preceding season(s) is decidedly more complex than assumed, with the magnitude of influenza circulation in preceding seasons having only limited effects on subsequent epidemic size and severity. Rather, epidemic size and severity are dominated by season-specific effects unrelated to the magnitude of circulation in the preceding season(s). Similarly, we find that antibody levels waned only modestly during the COVID-19 pandemic. Implications of all the available evidenceThe lack of changes observed in the patterns of measured antibody titres against seasonal influenza viruses in adults and nearly two decades of epidemiological data suggest that post-pandemic epidemic sizes will likely be similar to those observed pre-pandemic, and challenge the commonly held notion that the widespread concern that the near-absence of seasonal influenza virus circulation during the COVID-19 pandemic, or potential future lulls, are likely to result in larger influenza epidemics in subsequent years.
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The SARS-CoV-2 Omicron variant has a growth advantage over the Delta variant, due to higher transmissibility, immune evasion, or a shorter serial interval. Using S-gene target failure (SGTF) as indication for Omicron BA.1, we identify 908 SGTF and 1621 non-SGTF serial intervals in the same period. Within households, we find that the mean serial interval for SGTF cases is 0.2-0.6 days shorter than for non-SGTF cases. This suggests that the growth advantage of Omicron is partly due to a shorter serial interval.
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Large-scale vaccination campaigns have prevented countless hospitalizations and deaths due to COVID-19. However, the emergence of SARS-CoV-2 variants that escape from immunity challenges the effectiveness of current vaccines. Given this continuing evolution, an important question is when and how to update SARS-CoV-2 vaccines to antigenically match circulating variants, similar to seasonal influenza viruses where antigenic drift necessitates periodic vaccine updates. Here, we studied SARS-CoV-2 antigenic drift by assessing neutralizing activity against variants-of-concern (VOCs) of a unique set of sera from patients infected with a range of VOCs. Infections with D614G or Alpha strains induced the broadest immunity, while individuals infected with other VOCs had more strain-specific responses. Omicron BA.1 and BA.2 were substantially resistant to neutralization by sera elicited by all other variants. Antigenic cartography revealed that Omicron BA.1 and BA.2 are antigenically most distinct from D614G, associated with immune escape and likely requiring vaccine updates to ensure vaccine effectiveness.
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Infections by the Omicron SARS-CoV-2 variant are rapidly increasing worldwide. Among 70,983 infected individuals (age [≥] 12 years), we observed an increased risk of S-gene target failure, predictive of the Omicron variant, in fully vaccinated (odds ratio: 5.0; 95% confidence interval: 4.0-6.1) and previously infected individuals (OR: 4.9: 95% CI: 3.1-7.7) compared with infected naive individuals. This suggests a substantial decrease in protection from vaccine- or infection-induced immunity against SARS-CoV-2 infections caused by the Omicron variant compared with the Delta variant.
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AIMThe CoKids study aimed to estimate the community incidence of symptomatic and asymptomatic SARS-CoV-2 in children and parents and to assess the symptomatology of SARS-COV-2 infections relative to SARS-CoV-2 negative respiratory episodes. METHODSIn this prospective study, households with at least one child <18 years were recruited from three existing Dutch cohorts. Participation included SARS-CoV-2 screening at 4-6 weeks intervals for all household members during 23 weeks of follow-up and active reporting of new onset respiratory symptoms until July 1st 2021. Follow-up was temporarily intensified following new onset respiratory symptoms in a household member or a SARS-CoV-2 positive screening test and included daily symptom recording, repeated PCR testing (nose-throat, saliva and fecal samples) and SARS-CoV-2 antibody measurement (paired dried blood spots) in all household members. Age-stratified incidence rates for SARS-CoV-2 positive and negative episodes were calculated. Symptomatology and disease burden of respiratory episodes were compared by SARS-CoV-2 status and age. RESULTSIn total 307 households were enrolled including 1209 subjects. We detected 64 SARS-CoV-2 positive and 118 SARS-CoV-2 negative respiratory outbreaks. The highest incidence rate was found in children <12 years for SARS-CoV-2 negative episodes (0.93/ person-year (PY); 95%CI: 0.88-0.96). The SARS-CoV-2 incidence in this age-group was 0.21/PY for confirmed only, and 0.41/PY if probable cases were included. SARS-CoV-2 incidence did not differ by age group (p>0.27). Nasal congestion/runny nose, with or without cough and fatigue were the three most prevalent symptom clusters for both SARS-CoV-2 positive and negative respiratory episodes. Among children, no differences were observed in the symptomatology and severity of SARS-CoV-2 positive versus negative respiratory episodes, whereas among adults, SARS-CoV-2 positive episodes had a higher number and severity of symptoms and with a longer duration p<0.001). CONCLUSIONUsing active, longitudinal household follow up, we detected a high incidence rate of SARS-CoV-2 infections in children that was similar to adults. The findings suggest that after 20 months of COVID-19 pandemic, up to 2/3 of Dutch children < 12 years have been infected with SARS-CoV-2. Symptomatology and disease severity of SARS-CoV-2 in children is similar to respiratory illness from other causes. In adults, SARS-COV-2 positive episodes are characterized by more and prolonged symptoms, and higher severity. These findings may assist decisions on COVID-19 policies targeting children.
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The extent to which severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC) break through infection- or vaccine-induced immunity is not well understood. Here, we analyze 28,578 sequenced SARS-CoV-2 samples from individuals with known immune status obtained through national community testing in the Netherlands from March to August 2021. We find evidence for an increased risk of infection by the Beta (B.1.351), Gamma (P.1), or Delta (B.1.617.2) variants compared to the Alpha (B.1.1.7) variant after vaccination. No clear differences were found between vaccines. However, the effect was larger in the first 14-59 days after complete vaccination compared to 60 days and longer. In contrast to vaccine-induced immunity, no increased risk for reinfection with Beta, Gamma or Delta variants relative to Alpha variant was found in individuals with infection-induced immunity.
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This study investigated the dynamics of SARS-CoV-2 infection and diagnostics in household members of different ages and with different symptom severity after SARS-CoV-2 exposure during the early phase of the pandemic. Households with a SARS-CoV-2 confirmed positive case and at least one child in the Netherlands were followed for 6 weeks. Naso (NP)- and oropharyngeal (OP) swabs, oral fluid and feces specimens were analyzed for SARS-CoV-2 RNA and serum for SARS-CoV-2-specific antibodies. The dynamics of the presence of viral RNA and the serological response was modeled to determine the sampling time-frame and sample type with the highest sensitivity to confirm or reject a SARS-CoV-2 diagnosis. Transmission of SARS-CoV-2 between adults and children within a household was correlated with symptom severity of index cases. In children higher viral loads compared to adults were detected at symptom onset. Early in infection, higher viral loads were detected in NP and OP specimens, while RNA in especially feces were longer detectable. SARS-CoV-2-specific antibodies have a 90% probability of detection from 7 days (total Ig) and 18 days (IgG) since symptom onset. In conclusion this study has shown that on average, children carry higher loads of virus as compared to adults early after infection. For highest probability of detection in SARS-CoV-2 diagnostics early in infection, RT-PCR on NP and OP specimens are more sensitive than on oral fluid and feces. For SARS-CoV-2 diagnostics late after infection, RT-PCR on feces specimens and serology are more valuable.
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Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus and the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Efforts to identify inhibitors of SARS-CoV-2 replication enzymes and better understand the mechanisms underlying viral RNA synthesis have largely relied on biosafety level 3 (BSL3) laboratories, limiting throughput and accessibility. Recently, replicon systems have been proposed that involve ~30 kb RNA-based replicons or large plasmids that express the viral structural and non-structural proteins (nsp) in addition to a positive-sense reporter RNA. Unfortunately, these assays are not user-friendly due to plasmid instability or a poor signal to background ratio. We here present a simple mini-genome assay consisting of a ~2.5 kb-long negative-sense, nanoluciferase-encoding sub-genomic reporter RNA that is expressed from a plasmid, and amplified and transcribed by the SARS-CoV-2 RNA polymerase core proteins nsp7, nsp8 and nsp12. We show that expression of nsp7, 8 and 12 is sufficient to obtain robust positive- and negative-sense RNA synthesis in cell culture, that addition of other nsps modulates expression levels, and that replication of the reporter RNA can be inhibited by active site mutations in nsp12 or the SARS-CoV-2 replication inhibitor remdesivir. The mini-genome assay provides a signal that is 170-fold above background on average, providing excellent sensitivity for high-throughput screens, while the use of small plasmids facilitates site-directed mutagenesis for fundamental analyses of SARS-CoV-2 RNA synthesis. Importance statementThe impact of the COVID-19 pandemic has made it essential to better understand the basic biology of SARS-CoV-2, and to search for compounds that can block the activity of key SARS-CoV-2 replication enzymes. However, studies with live SARS-CoV-2 require biosafety level 3 facilities, while existing replicon systems depend on long positive-sense subgenomes that are often difficult to manipulate or produce a high background signal, limiting drug-screens and a rapid analysis of emerging SARS-CoV-2 mutations during the COVID-19 pandemic. To make it easier to study emerging SARS-CoV-2 mutants and screen for inhibitors, we developed a simple mini-replicon that produces a minimal background signal, that can be used in any tissue culture lab, and that only requires four small plasmids to work.
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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.
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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.
