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In the present study we report the functional and structural characterization of 17T2, a new highly potent pan-neutralizing SARS-CoV-2 human monoclonal antibody (mAb) isolated from a convalescent COVID-19 individual infected during the first wave of the COVID-19 pandemic. 17T2 is a class 1 VH1-58/κ3-20 antibody, derived from a receptor binding domain (RBD)-specific IgA memory B cell and developed as a human recombinant IgG1. Functional characterization revealed that 17T2 mAb has a high and exceptionally broad neutralizing activity against all SARS-CoV-2 spike variants tested, including BQ.1.1. Moreover, 17T2 mAb has in vivo prophylactic activity against Omicron BA.1.1 infection in K18-hACE2 transgenic mice. 3D reconstruction from cryogenic-electron microscopy (cryo-EM) showed that 17T2 binds the Omicron BA.1 spike protein with the RBD domains in up position and recognizes an epitope overlapping with the receptor binding motif, as it is the case for other structurally similar neutralizing mAbs, including S2E12. Yet, unlike S2E12, 17T2 retains its high neutralizing activity against all Omicron sublineages tested, probably due to a larger contact area with the RBD, which could confer a higher resilience to spike mutations. These results highlight the impact of small structural antibody changes on neutralizing performance and identify 17T2 mAb as a potential candidate for future therapeutic and prophylactic interventions., We acknowledge access to the cryo-EM CNB-CSIC facility in the context of the CRIOMECORR project (ESFRI-2019-01-CSIC-16) and we thank the staff of the Protein Technology Unity (CRG) for the help in protein production. This study was supported by the COVID-19 call grant from Generalitat de Catalunya, Department of Health (to GM), grant Miguel Servet research program (to GM), and partially funded by the crowdfunding initiative #joemcorono and the Fundació Glòria Soler (to JB). A.P-G. was supported by a predoctoral grant from Generalitat de Catalunya and Fons Social Europeu (2022 FI_B 00698).

COVID-19 patients elicit strong responses to the nucleocapsid (N) protein of SARS-CoV-2 but binding antibodies are also detected in prepandemic individuals, indicating potential crossreactivity with common cold human coronaviruses (HCoV) and questioning its utility in seroprevalence studies. We investigated the immunogenicity of the full-length and shorter fragments of the SARS-CoV-2 N protein, and the crossreactivity of antibodies with HCoV. We identified a C-terminus region in SARS-CoV2 N of minimal sequence homology with HCoV that was more specific for SARS-CoV-2 and highly immunogenic. IgGs to the full-length SARS-CoV-2 N also recognized N229E N, and IgGs to HKU1 N recognized SARS-CoV-2 N. Crossreactivity with SARS-CoV-2 was stronger for alpha- rather than beta-HCoV despite having less sequence identity, revealing the importance of conformational recognition. Higher preexisting IgG to OC43 N correlated with lower IgG to SARS-CoV-2 N in rRT-PCR negative individuals, reflecting less exposure and indicating a potential protective association. Antibodies to SARS-CoV-2 N were higher in patients with more severe and longer duration of symptoms and in females. IgGs remained stable for at least 3 months, while IgAs and IgMs declined faster. In conclusion, N protein is a primary target of SARS-CoV-2-specific and HCoV crossreactive antibodies, both of which may affect the acquisition of immunity to COVID-19.

Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.g., that various (not fully understood) effects are contributing to the signal, that the technique is licensed to only a single instrument developer, NanoTemper Technology, and that its reliability and reproducibility have never been tested independently and systematically. Thus, a working group of ARBRE-MOBIEU has set up a benchmark study on MST/TRIC to assess this technique as a method to characterize biomolecular interactions. Here we present the results of this study involving 32 scientific groups within Europe and two groups from the US, carrying out experiments on 40 Monolith instruments, employing a standard operation procedure and centrally prepared samples. A protein-small molecule interaction, a newly developed protein-protein interaction system and a pure dye were used as test systems. We characterized the instrument properties and evaluated instrument performance, reproducibility, the effect of different analysis tools, the influence of the experimenter during data analysis, and thus the overall reliability of this method. All authors acknowledge the COST Action project ARBRE-MOBIEU CA15126 under the auspices of whose Working Group 4 this study was carried out. ARBRE-MOBIEU COST Action CA15126 funded meetings in the Center for Protein Research, University of Copenhagen (ECOST-MEETING-CA15126-280618-098884) to organize this study and in the Vienna Biocenter Core Facilities GmbH (ECOST-MEETING-CA15126-121119-111544) to discuss the results of the benchmark. Ondřej Vaněk acknowledges support from the Ministry of Education, Youth and Sports of the Czech Republic (LTC17065 in frame of the COST Action CA15126). Jitka Holková and Josef Houser acknowledge CF Biomolecular Interactions and Crystallization of CIISB, Instruct-CZ Centre, supported by MEYS CR (LM2018127). Jasmina Rokov-Plavec acknowledges support from the Croatian Science Foundation (IP-2016-06-6272). Alexander K. Buell thanks the Novo Nordisk Foundation (grant number NNFSA170028392) for funding. Li Peng Lundgren acknowledges for preliminary MST/TRIC experiments (STMS 37745 in the frame of the COST Action CA15126) and the COST Action (CA15126) for a STMS (STMS 44783) for the data analysis. The Novo Nordisk Foundation Center for Protein Research is supported financially by the Novo Nordisk Foundation (Grant agreement NNF14CC0001). Katja Pirc and Marjetka Podobnik acknowledge the grant by the Slovenian Research Agency P1-0391 (Title: Molecular Interactions). This research was funded by Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III and European Union (ERDF/ESF, “Investing in your future” PI18/00349 and Diputación General de Aragón Digestive Pathology Group B25_17R to O.A. Spanish Ministry of Science, Innovation and Universities, FPI Predoctoral Research Contract BES-2017-080739 to D.O.A. This research was funded by project no 2015/17/B/NZ2/01160 (granted to MA)

We evaluated the kinetics of antibody responses to Two years into the COVID-19 pandemic and 1 year after the start of vaccination rollout, the world faced a peak of cases associated with the highly contagious Omicron variant of concern (VoC) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) and nucleocapsid (N) antigens over five cross-sectional visits (January-November 2021), and the determinants of pre-booster immunoglobulin levels, in a prospective cohort of vaccinated primary health care workers in Catalonia, Spain. Antibodies against S antigens after a full primary vaccination course, mostly with BNT162b2, decreased steadily over time and were higher in pre-exposed (n = 247) than naïve (n = 200) individuals, but seropositivity was maintained at 100% (100% IgG, 95.5% IgA, 30.6% IgM) up to 319 days after the first dose. Antibody binding to variants of concern was highly maintained for IgG compared to wild type but significantly reduced for IgA and IgM, particularly for Beta and Gamma. Factors significantly associated with longer-term antibodies included age, sex, occupation, smoking, adverse reaction to vaccination, levels of pre-vaccination SARS-CoV-2 antibodies, interval between disease onset and vaccination, hospitalization, oxygen supply, post COVID and symptomatology. Earlier morning vaccination hours were associated with higher IgG responses in pre-exposed participants. Symptomatic breakthroughs occurred in 9/447 (2.01%) individuals, all among naïve (9/200, 4.5%) and generally boosted antibody responses. Additionally, an increase in IgA and/or IgM seropositivity to variants, and N seroconversion at later time points (6.54%), indicated asymptomatic breakthrough infections, even among pre-exposed. Seropositivity remained highly stable over almost a year after vaccination. However, gradually waning of anti-S IgGs that correlate with neutralizing activity, coupled to evidence of an increase in breakthrough infections during the Delta and Omicron predominance, provides a rationale for booster immunization.

Additional file 1: Table S1. Association of baseline variables and comorbidities with levels of SARS-CoV-2 antibodies at time point 4 (January-February 2021) prior to the massive rollout of vaccination. Table S2. Seropositivity against the SARS-CoV-2 receptor-binding domain antigen from variants of concern. Table S3. Reinfections. Table S4. Significant associations of baseline variables with differential antibody rate of change. Fig. S1. Overall distribution of antibody responses for each isotype and antigen pair of the 128 pre-pandemic samples (negative controls) along with the cutoffs.

Additional file 1: Table S1. Characteristics of participants with breakthrough infections, post-vaccination. Table S2. Allocation of vaccinated and non-vaccinated participants with evidence of infection according to the criterion of infection fulfilled. Table S3. Fold change (FC) (95% CI) in antibody levels within one year after infection estimated using two repeated samples among decayers. Estimates are based on linear mixed-effects models. Table S4. Spearman correlations for RBD antigen. All participants, n=1,076. Darker red=stronger association. Table S5. Cross tabulation between serostatus to RBD of Wuhan variant with the RBD of Alpha, Beta Gamma and Delta variants. Table S6. Characteristics of non-responders (seronagetive or with an undetermined status) to vaccination. Table S7. Association (fold change FC and 95% CI and p-values) between each determinant with log10 antibody leves in vaccinated people after adjusting each model for time since last vaccination and number of doses. Participants with any vaccination excluding Janssen (n=923). Table S8. P-values for comparisons related to Figs. 3 and 5 and Figure S6. Figure S1. Dates and density of positive viral detection tests, sampling in 2020 (1st serological assessment) and 2021 (2nd serological assessment) and receipt of 1st vaccine dose in the study population (n=1,076). Figure S2. Venn diagram illustrating overlap between sustainer groups of IgA or IgG antibodies against nucleoprotein and spike antigens, among all infected unvaccinated participants (n=64). Figure S3. Differences in IgG antibody responses against RBD between Wuhan, Alpha, Beta, Gamma and Delta variant among vaccinated people. All differences were statistically significant apart from Delta vs Wuhan (p=0.861) and Alpha vs Wuhan (p=0.051). Figure S4. Generalized additive models for associations of days since vaccination with antibody responses to the six isotype-antigen combinations in infected (red) and naïve (blue) participants after first or second dose in people vaccinated by Vaxzevria (a), Comirnaty (b) or Spikevax (c). Fitted lines after adjustment for participant’s age. Plus symbols (+) represent measured responses for a specific participant. Figure S5. Differences in antibody responses by infection and/or vaccination and number of doses in people vaccinated with Comirnaty (a), Spikevarx (b), Vaxzevria (c) of Janssen COVID-19 vaccine (d). Figure S6. Differences in IgM responses by infection and/or vaccination and number of doses. Table S8 presents corresponding p-values.

BackgroundTwo doses of mRNA vaccination have shown >94% efficacy at preventing COVID-19 mostly in naive adults, but it is not clear if the second dose is needed to maximize effectiveness in those previously exposed to SARS-CoV-2 and what other factors affect responsiveness.MethodsWe measured IgA, IgG and IgM levels against SARS-CoV-2 spike (S) and nucleocapsid (N) antigens from the wild-type and S from the Alpha, Beta and Gamma variants of concern, after BNT162b2 (Pfizer/BioNTech) or mRNA-1273 (Moderna) vaccination in a cohort of health care workers (N=578). Neutralizing capacity and antibody avidity were evaluated. Data were analyzed in relation to COVID-19 history, comorbidities, vaccine doses, brand and adverse events.FindingsVaccination induced robust IgA and IgG levels against all S antigens. Neutralization capacity and S IgA and IgG levels were higher in mRNA-1273 vaccinees, previously SARS-CoV-2 exposed, particularly if symptomatic, and in those experiencing systemic adverse effects. A second dose in pre-exposed did not increase antibody levels. Smoking and comorbidities were associated with lower neutralization and antibody levels. Among fully vaccinated, 6.3% breakthroughs were detected up to 189 days post-vaccination. Among pre-exposed non-vaccinated, 90% were IgG seropositive more than 300 days post-infection.InterpretationOur data support administering a single-dose in pre-exposed healthy individuals. However, heterogeneity of responses suggests that personalized recommendations may be necessary depending on COVID-19 history and life-style. Higher mRNA-1273 immunogenicity would be beneficial for those expected to respond worse to vaccination. Persistence of antibody levels in pre-exposed unvaccinated indicates maintenance of immunity up to one year.FundingThis work was supported by Institut de Salut Global de Barcelona (ISGlobal) internal funds, in-kind contributions from Hospital Clínic de Barcelona, the Fundació Privada Daniel Bravo Andreu, and European Institute of Innovation and Technology (EIT) Health (grant number 20877), supported by the European Institute of Innovation and Technology, a body of the European Union receiving support from the H2020 Research and Innovation Programme. We acknowledge support from the Spanish Ministry of Science and Innovation and State Research Agency through the “Centro de Excelencia Severo Ochoa 2019-2023” Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. L. I. work was supported by PID2019-110810RB-I00 grant from the Spanish Ministry of Science & Innovation. Development of SARS-CoV-2 reagents was partially supported by the National Institute of Allergy and Infectious Diseases Centers of Excellence for Influenza Research and Surveillance (contract number HHSN272201400008C). The funders had no role in study design, data collection and analysis, the decision to publish, or the preparation of the manuscript.

We assessed the duration and baseline determinants of antibody responses to SARS-CoV-2 spike antigens and the occurrence of reinfections in a prospective cohort of 173 Spanish primary health care worker patients followed initially for 9 months and subsequently up to 12.5 months after COVID-19 symptoms onset. Seropositivity to SARS-CoV-2 spike and receptor-binding domain antigens up to 149–270 days was 92.49% (90.17% IgG, 76.3% IgA, 60.69% IgM). In a subset of 64 health care workers who had not yet been vaccinated by April 2021, seropositivity was 96.88% (95.31% IgG, 82.81% IgA) up to 322–379 days post symptoms onset. Four suspected reinfections were detected by passive case detection, two among seronegative individuals (5 and 7 months after the first episode), and one low antibody responder. Antibody levels significantly correlated with fever, hospitalization, anosmia/hypogeusia, allergies, smoking, and occupation. Stable sustainment of IgG responses raises hope for long-lasting COVID-19 vaccine immunity. Supplementary Information The online version contains supplementary material available at 10.1186/s12916-021-02032-2.

mRNA-based vaccines effectively induce protective neutralizing antibody responses against SARS-CoV-2, the etiological agent of COVID-19. The specific compositional patterns of these responses remain largely unknown. We found that SARS-CoV-2-naïve individuals receiving the first dose of an mRNA vaccine developed a SARS-CoV-2-specific antibody response with a subclass profile comparable to that induced by the natural infection, except IgA2, which did not increase. SARS-CoV-2-naïve subjects also mounted a robust virus-specific recall response after receiving the second dose. This response increased all IgG subclasses, but boosted neither IgM nor IgA1 and IgA2 subclasses. In contrast, individuals recovered from COVID-19 mounted peak virus-specific antibody responses upon primary immunization and did not further augment such responses following secondary immunization. Remarkably, compared to SARS-CoV-2-naïve subjects, individuals with pre-existing immunity showed increased levels of all virus-specific antibodies but IgG3 following primary vaccination. By dissecting the heterogeneity of mRNA vaccine-induced humoral responses to SARS-CoV-2, our findings indicate that the induction of optimal immune protection may require the development of personalized vaccination programs.

The surge of SARS‐CoV‐2 has challenged health systems worldwide and efficient tests to detect viral particles, as well as antibodies generated against them, are needed. Specificity, sensitivity, promptness or scalability are the main parameters to estimate the final performance, but rarely all of them match in a single test. We have developed SCOVAM, a protein microarray with several viral antigens (spike, nucleocapsid, main protease Nsp5) as capturing probes in a fluorescence immunoassay for COVID‐19 serological testing. SCOVAM depicts IgG and IgM antibody responses against each of these proteins of 22 individuals in a single microscope slide. It detects specific IgM (0.094 μg ml‐1) and IgG (~0.017 μg ml‐1) and is scalable and cost‐effective. We validated SCOVAM by comparing with a widely used chemiluminescent commercial serological test (n = 742). SCOVAM showed twice the sensitivity and allowed following seroconversion in a single assay. By analysing the prevalence 4 months later in a subset of 76 positive sera, we still detected 93.42% of positives, almost doubling the detection of the commercial assay. The higher sensitivity of SCOVAM is especially relevant to screen sera for convalescent plasma‐based treatments, high‐throughput antibody response monitoring after vaccination or evaluation of vaccine efficiency., This work has been funded by INTA’s internal budget and the Spanish Ministry of Science and Innovation grant nos. RTI2018‐094368‐B‐I00, and MDM‐2017‐0737 (Excelencia ‘María de Maeztu’ to Centro de Astrobiología). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. DRG is funded by the ‘Programa de Atracción de Talento’ of local Government of Madrid. Centro Superior de Investigaciones Cientificas: project number 202020E079.

Surveillance tools to estimate infection rates in young populations are essential to guide recommendations for school reopening and management during viral epidemics. Ideally, field-deployable non-invasive, sensitive techniques are required to detect low viral load exposures among asymptomatic children. We determined SARS-CoV-2 antibody conversion by high-throughput Luminex assays in saliva samples collected weekly in 1,509 children and 396 adults in 22 Summer schools and 2 pre-schools in 27 venues in Barcelona, Spain, from June 29thto July 31st2020, between the first and second COVID-19 pandemic waves. Saliva antibody conversion defined as ≥4-fold increase in IgM, IgA and/or IgG levels to SARS-CoV-2 antigens between two visits over a 5-week period was 3.22% (49/1518), or 2.36% if accounting for potentially cross-reactive antibodies, six times higher than the cumulative infection rate (0.53%) by weekly saliva RT-PCR screening. IgG conversion was higher in adults (2.94%, 11/374) than children (1.31%, 15/1144) (p=0.035), IgG and IgA levels moderately increased with age, and antibodies were higher in females. Most antibody converters increased both IgG and IgA antibodies but some augmented either IgG or IgA, with a faster decay over time for IgA than IgG. Nucleocapsid rather than spike was the main antigen target. Anti-spike antibodies were significantly higher in individuals not reporting symptoms than symptomatic individuals, suggesting a protective role against COVID-19. To conclude, saliva antibody profiling including three isotypes and multiplexing antigens is a useful and more user-friendly tool for screening pediatric populations to determine SARS-CoV-2 exposure and guide public health policies during pandemics.

COVID-19 affects children to a lesser extent than adults but they can still get infected and transmit SARS-CoV-2 to their contacts. Field deployable non-invasive sensitive diagnostic techniques are needed to evaluate the infectivity dynamics of the coronavirus in pediatric populations and guide public health interventions.We evaluated the utility of high-throughput Luminex-based assays applied to saliva samples to quantify IgM, IgA and IgG antibodies against five SARS-CoV-2 spike (S) and nucleocapsid (N) antigens in the context of a contacts and infectivity longitudinal study. We compared the antibody levels obtained in saliva versus serum/plasma samples from a group of children and adults tested weekly by RT-PCR over 35 days and diagnosed as positive (n=58), and a group of children and adults who consistently tested negative over the follow up period (n=61), in the Summer of 2020 in Barcelona, Spain.Antibody levels in saliva samples from individuals with confirmed RT-PCR diagnosis of SARS-CoV-2 infection were significantly higher than in negative individuals and correlated with those measured in sera/plasmas. Higher levels of anti-S IgG were found in asymptomatic individuals that could indicate protection against disease in infected individuals. Higher anti-S IgG and IgM levels in serum/plasma and saliva, respectively, in infected children compared to infected adults could also be related to stronger clinical immunity in them. Among infected children, males had higher levels of saliva IgG to N and RBD than females. Despite overall correlation, individual clustering analysis suggested that responses that may not be detected in blood could be patent in saliva, and vice versa, and therefore that both measurements are complementary.In addition to serum/plasma, measurement of SARS-CoV-2-specific saliva antibodies should be considered as a complementary non-invasive assay to better estimate the percentage of individuals who have experienced coronavirus infection. Saliva antibody detection could allow determining COVID-19 prevalence in pediatric populations, alternative to bleeding or nasal swab, and serological diagnosis following vaccination.

Unraveling the long-term kinetics of antibodies to SARS-CoV-2 and its determinants, including the impact of pre-existing antibodies to human coronaviruses causing common cold (HCoVs), is essential to understand protective immunity to COVID-19 and devise effective surveillance strategies. IgM, IgA and IgG levels against six SARS-CoV-2 antigens and the nucleocapsid antigen of the four HCoV (229E,NL63, OC43 and HKU1) were quantified by Luminex, and antibody neutralization capacity was assessed by flow cytometry, in a cohort of health care workers followed-up for 6 months (N = 578). Seroprevalence increased over time from 13.5% (month 0) and 15.6% (month 1) to 16.4% (month 6). Levels of antibodies, including those with neutralizing capacity, were stable over time, except IgG to nucleocapsid antigen and IgM levels that waned. After the peak response, anti-spike antibody levels increased from ∼150 days post-symptom onset in all individuals (73% for IgG), in the absence of any evidence of re-exposure. Pre-existing antibodies to alpha-HCoV were lower in individuals who subsequently seroconverted for SARS-CoV-2. IgG and IgA to HCoV were significantly higher in asymptomatic than symptomatic seropositive individuals. Thus, pre-existing cross-reactive HCoVs antibodies could have a protective effect against SARS-CoV-2 infection and COVID-19 disease.

Reliable serological tests are required to determine the prevalence of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to characterize immunity to the disease in order to address key knowledge gaps in the coronavirus disease 2019 (COVID-19) pandemic. Quantitative suspension array technology (qSAT) assays based on the xMAP Luminex platform overcome the limitations of rapid diagnostic tests and enzyme-linked immunosorbent assays (ELISAs) with their higher precision, dynamic range, throughput, miniaturization, cost-efficiency, and multiplexing capacity., Reliable serological tests are required to determine the prevalence of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to characterize immunity to the disease in order to address key knowledge gaps in the coronavirus disease 2019 (COVID-19) pandemic. Quantitative suspension array technology (qSAT) assays based on the xMAP Luminex platform overcome the limitations of rapid diagnostic tests and enzyme-linked immunosorbent assays (ELISAs) with their higher precision, dynamic range, throughput, miniaturization, cost-efficiency, and multiplexing capacity. We developed three qSAT assays for IgM, IgA, and IgG against a panel of eight SARS-CoV-2 antigens, including spike protein (S), nucleocapsid protein (N), and membrane protein (M) constructs. The assays were optimized to minimize the processing time and maximize the signal-to-noise ratio. We evaluated their performances using 128 prepandemic plasma samples (negative controls) and 104 plasma samples from individuals with SARS-CoV-2 diagnosis (positive controls), of whom 5 were asymptomatic, 51 had mild symptoms, and 48 were hospitalized. Preexisting IgG antibodies recognizing N, M, and S proteins were detected in negative controls, which is suggestive of cross-reactivity to common-cold coronaviruses. The best-performing antibody/antigen signatures had specificities of 100% and sensitivities of 95.78% at ≥14 days and 95.65% at ≥21 days since the onset of symptoms, with areas under the curve (AUCs) of 0.977 and 0.999, respectively. Combining multiple markers as assessed by qSAT assays has the highest efficiency, breadth, and versatility to accurately detect low-level antibody responses for obtaining reliable data on the prevalence of exposure to novel pathogens in a population. Our assays will allow gaining insights into antibody correlates of immunity and their kinetics, required for vaccine development to combat the COVID-19 pandemic.

We determined the duration and baseline determinants of antibody responses to SARS-CoV-2 up to nine months after COVID-19 symptoms onset in 173 primary health care worker patients from Spain. Seropositivity to SARS-CoV-2 spike and RBD antigens was 92.49% (60.69% IgM, 76.3% IgA, 90.17% IgG), with four suspected reinfection cases. Antibody levels significantly correlated with fever, hospitalization, anosmia/hypogeusia, allergies, smoking and occupation, and persisted 149-270 days in this cohort of patients

COVID-19 patients elicit strong responses to the nucleocapsid (N) protein of SARS-CoV-2 but binding antibodies are also detected in prepandemic individuals, indicating potential crossreactivity with common cold human coronaviruses (HCoV) and questioning its utility in seroprevalence studies. We investigated the immunogenicity of the full-length and shorter fragments of the SARS-CoV-2 N protein, and the crossreactivity of antibodies with HCoV. We indentified a C-terminus region in SARS-CoV2 N of minimal sequence homology with HCoV that was more specific and highly immunogenic. IgGs to the full-length SARS-CoV-2 N also recognised N229E N, and IgGs to HKU1 N recognised SARS-CoV-2 N. Crossreactivity with SARS-CoV-2 was stronger for alpha-rather than beta-HCoV despite having less sequence identity, revealing the importance of conformational recognition. Higher preexisting IgG to OC43 N correlated with lower IgG to SARS-CoV-2 in rRT-PCR negative individuals, reflecting less exposure and indicating a potential protective association. Antibodies to SARS-CoV-2 N were higher in patients with more severe and longer symptoms and in females. IgGs remained stable for at least 3 months, while IgAs and IgMs declined faster. In conclusion, N is a primary target of SARS-CoV-2-specific and HCoV crossreactive antibodies, both of which may affect the acquisition of immunity to COVID-19.

SummaryThe production of SARS-CoV-2-specific neutralizing antibodies is widely considered as a key mechanism for COVID-19 resolution and protection. However, beyond their protective function, antibodies to SARS-CoV-2 may also participate in disease pathogenesis. To explore the potential relationship between virus-specific humoral responses and COVID-19 immunopathology, we measured serum antibody classes and subclasses to the receptor-binding domain of the SARS-CoV-2 spike protein and the nucleoprotein in a cohort of hospitalized COVID-19 patients with moderate to severe disease. We found that RBD-specific IgG1 and IgG3 dominated the humoral response to SARS-CoV-2, were more abundant in severe patients, and positively correlated with several clinical parameters of inflammation. In contrast, a virus-specific IgA2 response skewed toward RBD rather than NP associated with a more favorable clinical course. Interestingly, RBD-dominant IgA2 responses were mostly detected in patients with gastrointestinal symptoms, suggesting the possible involvement of intrinsically tolerogenic gut immune pathways in the attenuation of virus-induced inflammation and disease resolution.

Summary Background Two doses of mRNA vaccination have shown >94% efficacy at preventing COVID-19 mostly in naïve adults, but it is not clear if the second dose is needed to maximize effectiveness in those previously exposed to SARS-CoV-2 and what other factors affect responsiveness. Methods We measured IgA, IgG and IgM levels against SARS-CoV-2 spike (S) and nucleocapsid (N) antigens from the wild-type and S from the Alpha, Beta and Gamma variants of concern, after BNT162b2 (Pfizer/BioNTech) or mRNA-1273 (Moderna) vaccination in a cohort of health care workers (N=578). Neutralizing capacity and antibody avidity were evaluated. Data were analyzed in relation to COVID-19 history, comorbidities, vaccine doses, brand and adverse events. Findings Vaccination induced robust IgA and IgG levels against all S antigens. Neutralization capacity and S IgA and IgG levels were higher in mRNA-1273 vaccinees, previously SARS-CoV-2 exposed, particularly if symptomatic, and in those experiencing systemic adverse effects (p

The blood-brain barrier (BBB) is a formidable obstacle for delivery of biologic therapeutics to central nervous system (CNS) targets. Whilst the BBB prevents passage of the vast majority of molecules, it also selectively transports a wide variety of molecules required to maintain brain homeostasis. Receptor-mediated transcytosis is one example of a macromolecule transport system that is employed by cells of the BBB to supply essential proteins to the brain and which can be utilized to deliver biologic payloads, such as antibodies, across the BBB. In this study, we performed phage display selections on the mouse brain endothelial cell line, bEND.3, to enrich for antibody single-chain variable fragments (scFvs) that could compete for binding with a known BBB-crossing antibody fragment, FC5. A number of these scFvs were converted to IgGs and characterized for their ability to bind to mouse, rat and human brain endothelial cells, and subsequent ability to transport across the BBB. We demonstrated that these newly identified BBB-targeting IgGs had increased brain exposure when delivered peripherally in mice and were also able to transport a biologically active molecule, interleukin-1 receptor antagonist (IL-1RA), into the CNS. The antagonism of the interleukin-1 system within the CNS can result in the relief of neuropathic pain. We demonstrated that the BBB-targeting IgGs were able to elicit an analgesic response in a mouse model of nerve ligation-induced hypersensitivity when fused to IL-1RA.

The blood-brain barrier (BBB) is a formidable obstacle for brain delivery of therapeutic antibodies. However, antibodies against the transferrin receptor (TfR), enriched in brain endothelial cells, have been developed as delivery carriers of therapeutic cargos into the brain via a receptor-mediated transcytosis pathway. In vitro and in vivo studies demonstrated that either a low-affinity or mono-valent binding of these antibodies to the TfR improves their release on the abluminal side of the BBB and target engagement in brain parenchyma. However, these studies have been performed with mouse-selective TfR antibodies that recognize different TfR epitopes and have varied binding characteristics. In this study, we evaluated serum pharmacokinetics, brain and CSF exposure of the rat TfR-binding antibody OX26 affinity variants, having Kds of 5 nM, 76 nM, 108 nM and 174 nM; all binding the same epitope in bi-valent format. Pharmacodynamic responses were tested in Hargreaves chronic pain model after conjugation of OX26 affinity variants with the analgesic and anti-epileptic peptide, galanin. OX26 variants with affinities of 76 nM and 108 nM showed enhanced brain and CSF exposure and higher potency in Hargreaves model, compared to a 5 nM affinity variant; lowering affinity to 174 nM resulted in prolonged serum pharmacokinetics, but reduced brain and CSF exposure. The study demonstrates that binding affinity optimization of TfR-binding antibodies could improve their brain and CSF exposure even in the absence of mono-valent TfR engagement.