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The SARS-CoV-2 virus emerged in December 2019 and has caused a worldwide pandemic due to the lack of any pre-existing immunity. Accurate serology testing is urgently needed to help diagnose infection, determine past exposure of populations and assess the response to a future vaccine. The landscape of antibody responses to SARS-CoV-2 is unknown. In this study, we utilized the luciferase immunoprecipitation system to assess the antibody responses to 15 different SARS-CoV-2 antigens in patients with COVID-19. We identified new targets of the immune response to SARS-CoV-2 and show that nucleocapsid, open reading frame (ORF)8 and ORF3b elicit the strongest specific antibody responses. ORF8 and ORF3b antibodies, taken together as a cluster of points, identified 96.5% of COVID-19 samples at early and late time points of disease with 99.5% specificity. Our findings could be used to develop second-generation diagnostic tests to improve serological assays for COVID-19 and are important in understanding pathogenicity.
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Anticuerpos Antivirales/sangre , Betacoronavirus/inmunología , Técnicas de Laboratorio Clínico/métodos , Infecciones por Coronavirus/diagnóstico , Neumonía Viral/diagnóstico , Proteínas Virales/inmunología , Adulto , Anciano , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , COVID-19 , Prueba de COVID-19 , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Femenino , Hong Kong , Humanos , Masculino , Persona de Mediana Edad , Pandemias , Neumonía Viral/sangre , Neumonía Viral/inmunología , Neumonía Viral/virología , SARS-CoV-2 , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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The infectivity of severe acute respiratory syndrome coronavirus 2 in deceased persons and organisms remains unclear. We studied transgenic K18 hACE2 mice to determine the kinetics of virus infectivity after host death. Five days after death, virus infectivity in the lung declined by >96% and RNA copies declined by 48.2%.
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COVID-19 , SARS-CoV-2 , Animales , Modelos Animales de Enfermedad , Humanos , Pulmón , Ratones , Ratones TransgénicosRESUMEN
In March 2020, mild signs and symptoms of coronavirus disease developed in a healthy 33-year-old man in Hong Kong. His first infection did not produce virus neutralizing antibodies. In August, he had asymptomatic reinfection, suggesting that persons without a robust neutralizing antibody response might be at risk for reinfection.
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COVID-19/inmunología , Reinfección/diagnóstico , Formación de Anticuerpos/inmunología , Hong Kong , Humanos , Masculino , Pandemias , SARS-CoV-2 , Adulto JovenRESUMEN
BACKGROUND: Natural SARS-CoV-2 infection may elicit antibodies to a range of viral proteins including non-structural protein ORF8. RNA, adenovirus vectored and sub-unit vaccines expressing SARS-CoV-2 spike would be only expected to elicit S-antibodies and antibodies to distinct domains of nucleocapsid (N) protein may reliably differentiate infection from vaccine-elicited antibody. However, inactivated whole virus vaccines may potentially elicit antibody to wider range of viral proteins, including N protein. We hypothesized that antibody to ORF8 protein will discriminate natural infection from vaccination irrespective of vaccine type. METHODS: We optimized and validated the anti-ORF8 and anti-N C-terminal domain (NCTD) ELISA assays using sera from pre-pandemic, RT-PCR confirmed natural infection sera and BNT162b2 (BNT) or CoronaVac vaccinees. We then applied these optimized assays to a cohort of blood donor sera collected in April-July 2022 with known vaccination and self-reported infection status. RESULTS: We optimized cut-off values for the anti-ORF8 and anti-N-CTD IgG ELISA assays using receiver-operating-characteristic (ROC) curves. The sensitivity of the anti-ORF8 and anti-N-CTD ELISA for detecting past infection was 83.2% and 99.3%, respectively. Specificity of anti-ORF8 ELISA was 96.8 % vs. the pre-pandemic cohort or 93% considering the pre-pandemic and vaccine cohorts together. The anti-N-CTD ELISA specificity of 98.9% in the pre-pandemic cohort, 93% in BNT vaccinated and only 4 % in CoronaVac vaccinated cohorts. Anti-N-CTD antibody was longer-lived than anti-ORF8 antibody after natural infection. CONCLUSIONS: Anti-N-CTD antibody assays provide good discrimination between natural infection and vaccination in BNT162b2 vaccinated individuals. Anti-ORF8 antibody can help discriminate infection from vaccination in either type of vaccine and help estimate infection attack rates (IAR) in communities.
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COVID-19 , Vacunas Virales , Humanos , COVID-19/diagnóstico , COVID-19/prevención & control , Vacuna BNT162 , SARS-CoV-2 , Vacunación , Anticuerpos AntiviralesRESUMEN
Reprogramming lipid metabolic pathways is a critical feature of activating immune responses to infection. However, how these reconfigurations occur is poorly understood. Our previous screen to identify cellular deubiquitylases (DUBs) activated during influenza virus infection revealed Usp25 as a prominent hit. Here, we show that Usp25-deleted human lung epithelial A549 cells display a >10-fold increase in pathogenic influenza virus production, which was rescued upon reconstitution with the wild type but not the catalytically deficient (C178S) variant. Proteomic analysis of Usp25 interactors revealed a strong association with Erlin1/2, which we confirmed as its substrate. Newly synthesized Erlin1/2 were degraded in Usp25-/- or Usp25C178S cells, activating Srebp2, with increased cholesterol flux and attenuated TLR3-dependent responses. Our study therefore defines the function of a deubiquitylase that serves to restrict a range of viruses by reprogramming lipid biosynthetic flux to install appropriate inflammatory responses.
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Colesterol , Ubiquitina Tiolesterasa , Virosis , Humanos , Lípidos , Pulmón/metabolismo , Proteómica , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Virosis/genética , Virosis/metabolismo , Colesterol/metabolismoRESUMEN
The SARS-CoV-2 Omicron variant has demonstrated enhanced transmissibility and escape of vaccine-derived immunity. Although first-generation vaccines remain effective against severe disease and death, robust evidence on vaccine effectiveness (VE) against all Omicron infections, irrespective of symptoms, remains sparse. We used a community-wide serosurvey with 5,310 subjects to estimate how vaccination histories modulated risk of infection in infection-naive Hong Kong during a large wave of Omicron BA.2 epidemic in January-July 2022. We estimated that Omicron infected 45% (41-48%) of the local population. Three and four doses of BNT162b2 or CoronaVac were effective against Omicron infection 7 days after vaccination (VE of 48% (95% credible interval 34-64%) and 69% (46-98%) for three and four doses of BNT162b2, respectively; VE of 30% (1-66%) and 56% (6-97%) for three and four doses of CoronaVac, respectively). At 100 days after immunization, VE waned to 26% (7-41%) and 35% (10-71%) for three and four doses of BNT162b2, and to 6% (0-29%) and 11% (0-54%) for three and four doses of CoronaVac. The rapid waning of VE against infection conferred by first-generation vaccines and an increasingly complex viral evolutionary landscape highlight the necessity for rapidly deploying updated vaccines followed by vigilant monitoring of VE.
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Vacunas contra la COVID-19 , COVID-19 , Humanos , Vacuna BNT162 , Eficacia de las Vacunas , SARS-CoV-2RESUMEN
The antibody response magnitude and kinetics may impact clinical severity, serological diagnosis and long-term protection of COVID-19, which may play a role in why children experience lower morbidity. We therefore tested samples from 122 children in Hong Kong with symptomatic (n = 78) and asymptomatic (n = 44) SARS-CoV-2 infections up to 200 days post infection, relative to 71 infected adults (symptomatic n = 61, and asymptomatic n = 10), and negative controls (n = 48). We assessed serum IgG antibodies to a 14-wide antigen panel of structural and accessory proteins by Luciferase Immuno-Precipitation System (LIPS) assay and circulating cytokines. Infected children have lower levels of Spike, Membrane, ORF3a, ORF7a, ORF7b antibodies, comparable ORF8 and elevated E-specific antibodies than adults. Combination of two unique antibody targets, ORF3d and ORF8, can accurately discriminate SARS-CoV-2 infection in children. Principal component analysis reveals distinct pediatric serological signatures, and the highest contribution to variance from adults are antibody responses to non-structural proteins ORF3d, NSP1, ORF3a and ORF8. From a diverse panel of cytokines that can modulate immune priming and relative inflammation, IL-8, MCP-1 and IL-6 correlate with the magnitude of pediatric antibody specificity and severity. Antibodies to SARS-CoV-2 internal proteins may become an important sero surveillance tool of infection with the roll-out of vaccines in the pediatric population.
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COVID-19 , SARS-CoV-2 , Adulto , Especificidad de Anticuerpos , Niño , Citocinas , Humanos , Inmunoglobulina GRESUMEN
We present an interim analysis of a registered clinical study (NCT04800133) to establish immunobridging with various antibody and cellular immunity markers and to compare the immunogenicity and reactogenicity of 2-dose BNT162b2 and CoronaVac in healthy adolescents as primary objectives. One-dose BNT162b2, recommended in some localities for risk reduction of myocarditis, is also assessed. Antibodies and T cell immune responses are non-inferior or similar in adolescents receiving 2 doses of BNT162b2 (BB, N = 116) and CoronaVac (CC, N = 123) versus adults after 2 doses of the same vaccine (BB, N = 147; CC, N = 141) but not in adolescents after 1-dose BNT162b2 (B, N = 116). CC induces SARS-CoV-2 N and N C-terminal domain seropositivity in a higher proportion of adolescents than adults. Adverse reactions are mostly mild for both vaccines and more frequent for BNT162b2. We find higher S, neutralising, avidity and Fc receptor-binding antibody responses in adolescents receiving BB than CC, and a similar induction of strong S-specific T cells by the 2 vaccines, in addition to N- and M-specific T cells induced by CoronaVac but not BNT162b2, possibly implying differential durability and cross-variant protection by BNT162b2 and CoronaVac, the 2 most used SARS-CoV-2 vaccines worldwide. Our results support the use of both vaccines in adolescents.
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COVID-19 , Vacunas Virales , Adolescente , Adulto , Vacuna BNT162 , COVID-19/prevención & control , Vacunas contra la COVID-19/efectos adversos , Humanos , SARS-CoV-2RESUMEN
SARS-CoV-2 virions are composed of structural proteins, but during virus infection, an additional 30 proteins could be expressed according to putative open reading frames (ORFs) of the viral genome. Some of these additional proteins modulate cellular processes through direct interactions, their truncations can affect disease pathogenesis and they can also serve as antigenic targets for more specific serology. In addition to structural proteins, the ORF1a/b polyprotein and accessory proteins can stimulate antibody responses during infection. Antibodies that target non-structural proteins can impact viral infection, through Fc mediated effector functions, through interactions during virus entry, fusion, replication and egress within infected cells. Characterization of the serological responses to additional proteins, provides a snapshot of the 'antibody landscape', which includes the antibody magnitude, antigenic specificity and informs the biological relevance of SARS-CoV-2 proteins.
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Anticuerpos Antivirales/sangre , Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , SARS-CoV-2/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/diagnóstico , COVID-19/prevención & control , HumanosRESUMEN
OBJECTIVES: Current inactivated influenza vaccines provide suboptimal protection against antigenic drift, and repeated annual vaccinations shape antibody specificity but the effect on protection from infection is not well understood. METHODS: We studied the effects of cumulative and staggered vaccinations in mice to determine the effect of influenza vaccination on protection from infection and immune quality. RESULTS: We found that the timing of vaccination and antigenic change impacted the quality of immune responses. When mice received two different H3N2 strains (A/Hong Kong/4801/2014 and A/Singapore/INFIMH-16-0019/2016) by staggered timing of vaccination, there were higher H3HA antibody and B-cell memory responses than four cumulative vaccinations or when two vaccinations were successive. Interestingly, after challenge with a lethal-drifted H3N2 virus (A/Hong Kong/1/1968), mice with staggered vaccination were unable to produce high titres of antibodies specific to the challenge strain compared to other vaccination regimens because of high levels of vaccine-specific cross-reactive antibodies. All vaccination regimens resulted in protection, in terms of viral loads and survival, from lethal challenge, while lung IL-6 and inflammation were lowest in staggered or cumulative vaccination groups, indicating further advantage. CONCLUSION: Our findings help justify influenza vaccination policies that currently recommend repeat vaccination in infants and annual seasonal vaccination, with no evidence for impaired immunity by repeated seasonal vaccination.
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T cell responses are a key cornerstone to viral immunity to drive high-quality antibody responses, establishing memory for recall and for viral clearance. Inefficient recruitment of T cell responses plays a role in the development of severe COVID-19 and is also represented by reduced cellular responses in men, children, and diversity compared with other epitope-specific subsets and available T cell receptor diversity. SARS-CoV-2-specific T cell responses are elicited by multiple vaccine formats and augmented by prior infection for hybrid immunity. Epitope conservation is relatively well-maintained leading to T cell crossreactivity for variants of concern that have diminished serological responses.
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Background: Children are less clinically affected by SARS-CoV-2 infection than adults with the majority of cases being mild or asymptomatic and the differences in infection outcomes are poorly understood. The kinetics, magnitude and landscape of the antibody response may impact the clinical severity and serological diagnosis of COVID-19. Thus, a comprehensive investigation of the antibody landscape in children and adults is needed. Methods: We tested 254 plasma from 122 children with symptomatic and asymptomatic SARS-CoV-2 infections in Hong Kong up to 206 days post symptom onset, including 146 longitudinal samples from 58 children. Adult COVID-19 patients and pre-pandemic controls were included for comparison. We assessed antibodies to a 14-wide panel of SARS-CoV-2 structural and accessory proteins by Luciferase Immunoprecipitation System (LIPS). Findings: Children have lower levels of Spike and Nucleocapsid antibodies than adults, and their cumulative humoral response is more expanded to accessory proteins (NSP1 and Open Reading Frames (ORFs)). Sensitive serology using the three N, ORF3b, ORF8 antibodies can discriminate COVID-19 in children. Principal component analysis revealed distinct serological signatures in children and the highest contribution to variance were responses to non-structural proteins ORF3b, NSP1, ORF7a and ORF8. Longitudinal sampling revealed maintenance or increase of antibodies for at least 6 months, except for ORF7b antibodies which showed decline. It was interesting to note that children have higher antibody responses towards known IFN antagonists: ORF3b, ORF6 and ORF7a. The diversified SARS-CoV-2 antibody response in children may be an important factor in driving control of SARS-CoV-2 infection.
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Nucleocapsid proteins are essential for SARS-CoV-2 life cycle. Here, we describe protocols to gather domain-specific insights about essential properties of nucleocapsids. These assays include dynamic light scattering to characterize oligomerization, fluorescence polarization to quantify RNA binding, hydrogen-deuterium exchange mass spectrometry to map RNA binding regions, negative-stain electron microscopy to visualize oligomeric species, interferon reporter assay to evaluate interferon signaling modulation, and a serology assay to reveal insights for improved sensitivity and specificity. These assays are broadly applicable to RNA-encapsidated nucleocapsids. For complete details on the use and execution of this protocol, please refer to Wu et al. (2021).
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COVID-19/sangre , Proteínas de la Nucleocápside de Coronavirus/sangre , Interferones/metabolismo , Nucleocápside/metabolismo , ARN Viral/metabolismo , SARS-CoV-2/aislamiento & purificación , Antivirales/metabolismo , COVID-19/virología , Proteínas de la Nucleocápside de Coronavirus/genética , Humanos , Nucleocápside/genética , Fosfoproteínas/sangre , Fosfoproteínas/genética , Unión Proteica , ARN Viral/genéticaRESUMEN
Nucleocapsid (N) encoded by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays key roles in the replication cycle and is a critical serological marker. Here, we characterize essential biochemical properties of N and describe the utility of these insights in serological studies. We define N domains important for oligomerization and RNA binding and show that N oligomerization provides a high-affinity RNA-binding platform. We also map the RNA-binding interface, showing protection in the N-terminal domain and linker region. In addition, phosphorylation causes reduction of RNA binding and redistribution of N from liquid droplets to loose coils, showing how N-RNA accessibility and assembly may be regulated by phosphorylation. Finally, we find that the C-terminal domain of N is the most immunogenic, based on antibody binding to patient samples. Together, we provide a biochemical description of SARS-CoV-2 N and highlight the value of using N domains as highly specific and sensitive diagnostic markers.
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SARS-CoV-2 infection of children leads to a mild illness and the immunological differences with adults remains unclear. We quantified the SARS-CoV-2 specific T cell responses in adults and children (<13 years of age) with RT-PCR confirmed asymptomatic and symptomatic infection for long-term memory, phenotype and polyfunctional cytokines. Acute and memory CD4+ T cell responses to structural SARS-CoV-2 proteins significantly increased with age, whilst CD8+ T cell responses increased with time post infection. Infected children had significantly lower CD4+ and CD8+ T cell responses to SARS-CoV-2 structural and ORF1ab proteins compared to infected adults. SARS-CoV-2-specific CD8+ T cell responses were comparable in magnitude to uninfected negative adult controls. In infected adults CD4+ T cell specificity was skewed towards structural peptides, whilst children had increased contribution of ORF1ab responses. This may reflect differing T cell compartmentalisation for antigen processing during antigen exposure or lower recruitment of memory populations. T cell polyfunctional cytokine production was comparable between children and adults, but children had a lower proportion of SARS-CoV-2 CD4+ T cell effector memory. Compared to adults, children had significantly lower levels of antibodies to ß-coronaviruses, indicating differing baseline immunity. Total T follicular helper responses was increased in children during acute infection indicating rapid co-ordination of the T and B cell responses. However total monocyte responses were reduced in children which may be reflective of differing levels of inflammation between children and adults. Therefore, reduced prior ß-coronavirus immunity and reduced activation and recruitment of de novo responses in children may drive milder COVID-19 pathogenesis.
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SARS-CoV-2 infection of children leads to a mild illness and the immunological differences with adults are unclear. Here, we report SARS-CoV-2 specific T cell responses in infected adults and children and find that the acute and memory CD4+ T cell responses to structural SARS-CoV-2 proteins increase with age, whereas CD8+ T cell responses increase with time post-infection. Infected children have lower CD4+ and CD8+ T cell responses to SARS-CoV-2 structural and ORF1ab proteins when compared with infected adults, comparable T cell polyfunctionality and reduced CD4+ T cell effector memory. Compared with adults, children have lower levels of antibodies to ß-coronaviruses, indicating differing baseline immunity. Total T follicular helper responses are increased, whilst monocyte numbers are reduced, indicating rapid adaptive co-ordination of the T and B cell responses and differing levels of inflammation. Therefore, reduced prior ß-coronavirus immunity and reduced T cell activation in children might drive milder COVID-19 pathogenesis.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , COVID-19/inmunología , SARS-CoV-2/inmunología , Adolescente , Adulto , Factores de Edad , Anciano , Anticuerpos Antivirales/inmunología , COVID-19/metabolismo , COVID-19/patología , COVID-19/virología , Niño , Preescolar , Femenino , Humanos , Lactante , Inflamación/inmunología , Estudios Longitudinales , Masculino , Persona de Mediana Edad , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/metabolismo , Adulto JovenRESUMEN
Influenza-specific antibody dependent cellular cytotoxicity (ADCC) antibodies have a broad cross reactivity and potential as an immune correlate for universal vaccines. Peptide-mapping for ADCC reactivity of H1-HA and H7-HA proteins from human serum samples identified high ADCC-inducing peptides in both the HA1 and HA2 regions. Vaccination of mice with single ADCC-peptides induced ADCC activity leading to partial protection from lethal influenza challenge, with increased survival, reduced viral loads, and reduced activation of NK cells in the lungs. Targeted vaccination strategies to elicit ADCC responses may provide an approach for universal vaccines.
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Subtipo H1N1 del Virus de la Influenza A , Vacunas contra la Influenza , Gripe Humana , Animales , Anticuerpos Antivirales , Citotoxicidad Celular Dependiente de Anticuerpos , Epítopos , Glicoproteínas Hemaglutininas del Virus de la Influenza , Gripe Humana/prevención & control , Ratones , Péptidos , VacunaciónRESUMEN
OBJECTIVES: Enhanced inactivated influenza vaccines (eIIV) aim to increase immunogenicity and protection compared with the widely used standard IIV (S-IIV). METHODS: We tested four vaccines in parallel, FluZone high dose, FluBlok and FluAd versus S-IIV in a randomised controlled trial of older adults and in a mouse infection model to assess immunogenicity, protection from lethal challenge and mechanisms of action. RESULTS: In older adults, FluAd vaccination stimulated a superior antibody profile, including H3-HA antibodies that were elevated for up to 1 year after vaccination, higher avidity H3HA IgG and larger HA stem IgG responses. In a mouse model, FluAd also elicited an earlier and larger induction of HA stem antibodies with increased germinal centre responses and upregulation and long-term expression of B-cell switch transcription factors. Long-term cross-reactive memory responses were sustained by FluAd following lethal heterosubtypic influenza challenge, with reduced lung damage and viral loads, coinciding with increased T- and B-cell recall. Advantages were also noted for the high-dose FluZone vaccine in both humans and mice. CONCLUSION: The early, broadly reactive and long-lived antibody response of FluAd indicates a potential advantage of this vaccine, particularly in years when there is a mismatch between the vaccine strain and the circulating strain of influenza viruses.
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Nucleocapsid protein (N) is the most abundant viral protein encoded by SARS-CoV-2, the causative agent of COVID-19. N plays key roles at different steps in the replication cycle and is used as a serological marker of infection. Here we characterize the biochemical properties of SARS-CoV-2 N. We define the N domains important for oligomerization and RNA binding that are associated with spherical droplet formation and suggest that N accessibility and assembly may be regulated by phosphorylation. We also map the RNA binding interface using hydrogen-deuterium exchange mass spectrometry. Finally, we find that the N protein C-terminal domain is the most immunogenic by sensitivity, based upon antibody binding to COVID-19 patient samples from the US and Hong Kong. Together, these findings uncover domain-specific insights into the significance of SARS-CoV-2 N and highlight the diagnostic value of using N domains as highly specific and sensitive markers of COVID-19.