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INTRODUCTION: Patients with gastrointestinal (GI) cancers have an increased risk of serious complications and death from SARS-CoV-2 infection. The immunogenicity of vaccines in patients with GI cancers receiving anti-cancer therapies is unclear. We conducted a prospective study to evaluate the prevalence of neutralizing antibodies in a cohort of GI cancer patients receiving chemotherapy following SARS-CoV-2 vaccination. MATERIALS AND METHODS: Between September 2020 and April 2021, patients with cancer undergoing chemotherapy were enrolled. At baseline (day 0), days 28, 56, and 84, we assessed serum antibodies to SARS-CoV-2 spike (anti-S) and anti-nucleocapsid (anti-NP) and concomitantly assessed virus neutralization using a pseudovirus neutralization assay. Patients received either the Pfizer/BioNTech BNT162b2, or the Oxford/AstraZeneca ChAdOx1 vaccine. RESULTS: All 152 patients enrolled had a prior diagnosis of cancer; colorectal (n = 80, 52.6%), oesophagogastric (n = 38, 25.0%), and hepato pancreatic biliary (n = 22, 12.5%). Nearly all were receiving systemic anti-cancer therapy (99.3%). Of the 51 patients who did not receive a vaccination prior to, or during the study, 5 patients had detectable anti-NP antibodies. Ninety-nine patients received at least one dose of vaccine prior to, or during the study. Within 19 days following the first dose of vaccine, 30.0% had anti-S detected in serum which increased to 70.2% at days 20-39. In the 19 days following a second dose, anti-S positivity was 84.2% (32/38). However, pseudovirus neutralization titers (pVNT80) decreased from days 20 to 39. CONCLUSION: Despite the immunosuppressive effects of chemotherapy, 2 doses of SARS-CoV-2 vaccines are able to elicit a protective immune response in patients' ongoing treatment for gastrointestinal cancers. Decreases in pseudoviral neutralization were observed after 20-39 days, re-affirming the current recommendation for vaccine booster doses. CLINICAL TRIAL REGISTRATION NUMBER: NCT04427280.
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Vacinas contra COVID-19 , COVID-19 , Neoplasias Gastrointestinais , Imunogenicidade da Vacina , Humanos , Anticorpos , Vacina BNT162 , Neoplasias Gastrointestinais/tratamento farmacológico , Estudos Prospectivos , SARS-CoV-2RESUMO
BACKGROUND: COVID-19 vaccines have demonstrated effectiveness in reducing SARS-CoV-2 mild and severe outcomes. In vaccinated subjects with SARS-CoV-2 history, RBD-specific IgG and pseudovirus neutralization titers were rapidly recalled by a single BTN162b2 vaccine dose to higher levels than those in naïve recipients after the second dose, irrespective of waning immunity. In this study, we inspected the long-term kinetic and neutralizing responses of S-specific IgG induced by two administrations of BTN162b2 vaccine in infection-naïve subjects and in subjects previously infected with SARS-CoV-2. METHODS: Twenty-six naïve and 9 previously SARS-CoV-2 infected subjects during the second wave of the pandemic in Italy were enrolled for this study. The two groups had comparable demographic and clinical characteristics. By means of ELISA and pseudotyped-neutralization assays, we investigated the kinetics of developed IgG-RBD and their neutralizing activity against both the ancestral D614G and the SARS-CoV-2 variants of concern emerged later, respectively. The Wilcoxon matched pair signed rank test and the Kruskal-Wallis test with Dunn's correction for multiple comparison were applied when needed. RESULTS: Although after 15 weeks from vaccination IgG-RBD dropped in all participants, naïve subjects experienced a more dramatic decline than those with previous SARS-CoV-2 infection. Neutralizing antibodies remained higher in subjects with SARS-CoV-2 history and conferred broad-spectrum protection. CONCLUSIONS: These data suggest that hybrid immunity to SARS-CoV-2 has a relevant impact on the development of IgG-RBD upon vaccination. However, the rapid decay of vaccination-elicited antibodies highlights that the administration of a third dose is expected to boost the response and acquire high levels of cross-neutralizing antibodies.
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Formação de Anticorpos , Vacina BNT162/imunologia , COVID-19 , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Humanos , SARS-CoV-2 , VacinaçãoRESUMO
The virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the global coronavirus disease-2019 (COVID-19) pandemic, spread rapidly around the world causing high morbidity and mortality. However, there are four known, endemic seasonal coronaviruses in humans (HCoVs), and whether antibodies for these HCoVs play a role in severity of COVID-19 disease has generated a lot of interest. Of these seasonal viruses NL63 is of particular interest as it uses the same cell entry receptor as SARS-CoV-2. We use functional, neutralizing assays to investigate cross-reactive antibodies and their relationship with COVID-19 severity. We analyzed the neutralization of SARS-CoV-2, NL63, HKU1, and 229E in 38 COVID-19 patients and 62 healthcare workers, and a further 182 samples to specifically study the relationship between SARS-CoV-2 and NL63. We found that although HCoV neutralization was very common there was little evidence that these antibodies neutralized SARS-CoV-2. Despite no evidence in cross-neutralization, levels of NL63 neutralizing antibodies become elevated after exposure to SARS-CoV-2 through infection or following vaccination.
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COVID-19 , Coronavirus Humano NL63 , Anticorpos Antivirais , Reações Cruzadas , Humanos , Pandemias , SARS-CoV-2 , Estações do Ano , Glicoproteína da Espícula de CoronavírusRESUMO
BACKGROUND: A notable feature of coronavirus disease 2019 (COVID-19) is that children are less susceptible to severe disease. Children are known to experience more infections with endemic human coronaviruses (HCoVs) compared to adults. Little is known whether HCoV infections lead to cross-reactive anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies. METHODS: We investigated the presence of cross-reactive anti-SARS-CoV-2 IgG antibodies to spike 1 (S1), S1-receptor-binding domain (S1-RBD), and nucleocapsid protein (NP) by enzyme-linked immunosorbent assays, and neutralizing activity by a SARS-CoV-2 pseudotyped virus neutralization assay, in prepandemic sera collected from children (n = 50) and adults (n = 45), and compared with serum samples from convalescent COVID-19 patients (n = 16). RESULTS: A significant proportion of children (up to 40%) had detectable cross-reactive antibodies to SARS-CoV-2 S1, S1-RBD, and NP antigens, and the anti-S1 and anti-S1-RBD antibody levels correlated with anti-HCoV-HKU1 and anti-HCoV-OC43 S1 antibody titers in prepandemic samples (P < .001). There were marked increases of anti-HCoV-HKU1 and - OC43 S1 (but not anti-NL63 and -229E S1-RBD) antibody titers in serum samples from convalescent COVID-19 patients (P < .001), indicating an activation of cross-reactive immunological memory to ß-coronavirus spike. CONCLUSIONS: We demonstrated cross-reactive anti-SARS-CoV-2 antibodies in prepandemic serum samples from children and young adults. Promoting this cross-reactive immunity and memory response derived from common HCoV may be an effective strategy against SARS-COV-2 and future novel coronaviruses.
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Anticorpos Antivirais/sangue , COVID-19/imunologia , Imunoglobulina G/sangue , SARS-CoV-2/imunologia , Adolescente , Adulto , Anticorpos Antivirais/imunologia , COVID-19/sangue , COVID-19/virologia , Criança , Pré-Escolar , Convalescença , Coronavirus Humano 229E/imunologia , Proteínas do Envelope de Coronavírus/imunologia , Coronavirus Humano OC43/imunologia , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Feminino , Células HEK293 , Humanos , Imunoglobulina G/imunologia , Memória Imunológica , Masculino , Pessoa de Meia-Idade , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto JovemRESUMO
SARS-CoV-2 continues to circulate in the human population necessitating regular booster immunization for its long-term control. Ideally, vaccines should ideally not only protect against symptomatic disease, but also prevent transmission via asymptomatic shedding and cover existing and future variants of the virus. This may ultimately only be possible through induction of potent and long-lasting immune responses in the nasopharyngeal tract, the initial entry site of SARS-CoV-2. To this end, we have designed a vaccine based on recombinantly expressed receptor binding domain (RBD) of SARS-CoV-2, fused to the C-terminus of C. perfringens enterotoxin, which is known to target Claudin-4, a matrix molecule highly expressed on mucosal microfold (M) cells of the nasal and bronchial-associated lymphoid tissues. To further enhance immune responses, the vaccine was adjuvanted with a novel toll-like receptor 3/RIG-I agonist (Riboxxim™), consisting of synthetic short double stranded RNA. Intranasal prime-boost immunization of mice induced robust mucosal and systemic anti-SARS-CoV-2 neutralizing antibody responses against SARS-CoV-2 strains Wuhan-Hu-1, and several variants (B.1.351/beta, B.1.1.7/alpha, B.1.617.2/delta), as well as systemic T-cell responses. A combination vaccine with M-cell targeted recombinant HA1 from an H1N1 G4 influenza strain also induced mucosal and systemic antibodies against influenza. Taken together, the data show that development of an intranasal SARS-CoV-2 vaccine based on recombinant RBD adjuvanted with a TLR3 agonist is feasible, also as a combination vaccine against influenza.
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Vacinas contra COVID-19 , COVID-19 , Influenza Humana , Animais , Humanos , Camundongos , Adjuvantes Imunológicos , Adjuvantes Farmacêuticos , Anticorpos Neutralizantes , Anticorpos Antivirais , Clostridium perfringens , COVID-19/prevenção & controle , Vacinas contra COVID-19/imunologia , Mucosa Gástrica , Vírus da Influenza A Subtipo H1N1 , Vacinas contra Influenza , Células M , SARS-CoV-2 , Receptor 3 Toll-LikeRESUMO
Background: The virus neutralization assay is a principal method to assess the efficacy of antibodies in blocking viral entry. Due to biosafety handling requirements of viruses classified as hazard group 3 or 4, pseudotyped viruses can be used as a safer alternative. However, it is often queried how well the results derived from pseudotyped viruses correlate with authentic virus. This systematic review and meta-analysis was designed to comprehensively evaluate the correlation between the two assays. Methods: Using PubMed and Google Scholar, reports that incorporated neutralisation assays with both pseudotyped virus, authentic virus, and the application of a mathematical formula to assess the relationship between the results, were selected for review. Our searches identified 67 reports, of which 22 underwent a three-level meta-analysis. Results: The three-level meta-analysis revealed a high level of correlation between pseudotyped viruses and authentic viruses when used in an neutralisation assay. Reports that were not included in the meta-analysis also showed a high degree of correlation, with the exception of lentiviral-based pseudotyped Ebola viruses. Conclusion: Pseudotyped viruses identified in this report can be used as a surrogate for authentic virus, though care must be taken in considering which pseudotype core to use when generating new uncharacterised pseudotyped viruses.
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Ebolavirus , Pseudotipagem ViralRESUMO
Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in addition to their better-known use in gene therapy for the delivery of a gene of interest. PVs are replication defective because the viral genome is divided into different plasmids that are not incorporated into the PVs. This safe and versatile system allows the use of PVs in biosafety level 2 laboratories. Here, we present a general methodology to produce lentiviral PVs based on three plasmids as mentioned here: (1) the backbone plasmid carrying the reporter gene needed to monitor the infection; (2) the packaging plasmid carrying the genes for all the structural proteins needed to generate the PVs; (3) the envelope surface glycoprotein expression plasmid that determines virus tropism and mediates viral entry into the host cell. In this work, SARS-CoV-2 Spike is the envelope glycoprotein used for the production of non-replicative SARS-CoV-2 pseudotyped lentiviruses. Briefly, packaging cells (HEK293T) were co-transfected with the three different plasmids using standard methods. After 48 h, the supernatant containing the PVs was harvested, filtered, and stored at -80 °C. The infectivity of SARS-CoV-2 PVs was tested by studying the expression of the reporter gene (luciferase) in a target cell line 48 h after infection. The higher the value for relative luminescence units (RLUs), the higher the infection/transduction rate. Furthermore, the infectious PVs were added to the serially diluted serum samples to study the neutralization process of pseudoviruses' entry into target cells, measured as the reduction in RLU intensity: lower values corresponding to high neutralizing activity.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Imunidade Humoral , Pseudotipagem Viral , Células HEK293 , Lentivirus/genética , Testes de Neutralização , Anticorpos AntiviraisRESUMO
Introduction: The Eidolon helvum fruit bat is one of the most widely distributed fruit bats in Africa and known to be a reservoir for several pathogenic viruses that can cause disease in animals and humans. To assess the risk of zoonotic spillover, we conducted a serological survey of 304 serum samples from E. helvum bats that were captured for human consumption in Makurdi, Nigeria. Methods: Using pseudotyped viruses, we screened 304 serum samples for neutralizing antibodies against viruses from the Coronaviridae, Filoviridae, Orthomyxoviridae and Paramyxoviridae families. Results: We report the presence of neutralizing antibodies against henipavirus lineage GH-M74a virus (odds ratio 6.23; p < 0.001), Nipah virus (odds ratio 4.04; p = 0.00031), bat influenza H17N10 virus (odds ratio 7.25; p < 0.001) and no significant association with Ebola virus (odds ratio 0.56; p = 0.375) in this bat cohort. Conclusion: The data suggest a potential risk of zoonotic spillover including the possible circulation of highly pathogenic viruses in E. helvum populations. These findings highlight the importance of maintaining sero-surveillance of E. helvum, and the necessity for further, more comprehensive investigations to monitor changes in virus prevalence, distribution over time, and across different geographic locations.
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Quirópteros , Viroses , Animais , Humanos , Nigéria/epidemiologia , Zoonoses/epidemiologia , Anticorpos NeutralizantesRESUMO
Coronaviruses infections, culminating in the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic beginning in 2019, have highlighted the importance of effective vaccines to induce an antibody response with cross-neutralizing activity. COVID-19 vaccines have been rapidly developed to reduce the burden of SARS-CoV-2 infections and disease severity. Cross-protection from seasonal human coronaviruses (hCoVs) infections has been hypothesized but is still controversial. Here, we investigated the neutralizing activity against ancestral SARS-CoV-2 and the variants of concern (VOCs) in individuals vaccinated with two doses of either BNT162b2, mRNA-1273, or AZD1222, with or without a history of SARS-CoV-2 infection. Antibody neutralizing activity to SARS-CoV-2 and the VOCs was higher in BNT162b2-vaccinated subjects who were previously infected with SARS-CoV-2 and conferred broad-spectrum protection. The Omicron BA.1 variant was the most resistant among the VOCs. COVID-19 vaccination did not confer protection against hCoV-HKU1. Conversely, antibodies induced by mRNA-1273 vaccination displayed a boosting in their neutralizing activity against hCoV-NL63, whereas AZD1222 vaccination increased antibody neutralization against hCoV-229E, suggesting potential differences in antigenicity and immunogenicity of the different spike constructs used between various vaccination platforms. These data would suggest that there may be shared epitopes between the HCoVs and SARS-CoV-2 spike proteins.
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BACKGROUND: Neutralizing antibodies are important for protection against the pandemic SARS-CoV-2 virus, and long-term memory responses determine the risk of re-infection or boosting after vaccination. T-cellular responses are considered important for partial protection against novel variants of concern. METHODS: A prospective cohort of hospitalized (n = 14) and community (n = 38) patients with rt-PCR confirmed SARS-CoV-2 infection were recruited. Blood samples and clinical data were collected when diagnosed and at 6 months. Serum samples were analyzed for SARS-CoV-2-spike specific antibodies using ELISA (IgG, IgA, IgM), pseudotype neutralization and microneutralization assays. Peripheral blood mononuclear cells were investigated for virus-specific T-cell responses in the interferon-γ and interleukin-2 fluorescent-linked immunosorbent spot (FluroSpot) assay. RESULTS: We found durable SARS-CoV-2 spike- and internal protein specific T-cellular responses in patients with persistent antibodies at 6 months. Significantly higher IL-2 and IFN-γ secreting T-cell responses as well as SARS-CoV-2 specific IgG and neutralizing antibodies were detected in hospitalized compared to community patients. The immune response was impacted by age, gender, comorbidity and severity of illness, reflecting clinical observations. CONCLUSIONS: SARS-CoV-2 specific T-cellular and antibody responses persisted for 6 months post confirmed infection. In previously infected patients, re-exposure or vaccination will boost long-term immunity, possibly providing protection against re-infection with variant viruses.
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Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Imunidade Celular , SARS-CoV-2/imunologia , Linfócitos T/imunologia , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , COVID-19/mortalidade , COVID-19/terapia , Feminino , Seguimentos , Hospitalização , Humanos , Interferon gama/imunologia , Interleucina-2/imunologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores de RiscoRESUMO
RaTG13 is a close relative of SARS-CoV-2, the virus responsible for the COVID-19 pandemic, sharing 96% sequence similarity at the genome-wide level. The spike receptor binding domain (RBD) of RaTG13 contains a number of amino acid substitutions when compared to SARS-CoV-2, likely impacting affinity for the ACE2 receptor. Antigenic differences between the viruses are less well understood, especially whether RaTG13 spike can be efficiently neutralised by antibodies generated from infection with, or vaccination against, SARS-CoV-2. Using RaTG13 and SARS-CoV-2 pseudotypes we compared neutralisation using convalescent sera from previously infected patients or vaccinated healthcare workers. Surprisingly, our results revealed that RaTG13 was more efficiently neutralised than SARS-CoV-2. In addition, neutralisation assays using spike mutants harbouring single and combinatorial amino acid substitutions within the RBD demonstrated that both spike proteins can tolerate multiple changes without dramatically reducing neutralisation. Moreover, introducing the 484 K mutation into RaTG13 resulted in increased neutralisation, in contrast to the same mutation in SARS-CoV-2 (E484K). This is despite E484K having a well-documented role in immune evasion in variants of concern (VOC) such as B.1.351 (Beta). These results indicate that the future spill-over of RaTG13 and/or related sarbecoviruses could be mitigated using current SARS-CoV-2-based vaccination strategies.
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COVID-19 , Quirópteros , Animais , COVID-19/terapia , Quirópteros/metabolismo , Humanos , Imunização Passiva , Glicoproteínas de Membrana/metabolismo , Pandemias , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Proteínas do Envelope Viral/genética , Soroterapia para COVID-19RESUMO
The rise of SARS-CoV-2 variants has made the pursuit to define correlates of protection more troublesome, despite the availability of the World Health Organisation (WHO) International Standard for anti-SARS-CoV-2 Immunoglobulin sera, a key reagent used to standardise laboratory findings into an international unitage. Using pseudotyped virus, we examine the capacity of convalescent sera, from a well-defined cohort of healthcare workers (HCW) and Patients infected during the first wave from a national critical care centre in the UK to neutralise B.1.1.298, variants of interest (VOI) B.1.617.1 (Kappa), and four VOCs, B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta), including the B.1.617.2 K417N, informally known as Delta Plus. We utilised the WHO International Standard for anti-SARS-CoV-2 Immunoglobulin to report neutralisation antibody levels in International Units per mL. Our data demonstrate a significant reduction in the ability of first wave convalescent sera to neutralise the VOCs. Patients and HCWs with more severe COVID-19 were found to have higher antibody titres and to neutralise the VOCs more effectively than individuals with milder symptoms. Using an estimated threshold for 50% protection, 54 IU/mL, we found most asymptomatic and mild cases did not produce titres above this threshold.
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COVID-19 , SARS-CoV-2 , Anticorpos Antivirais , COVID-19/terapia , Humanos , Imunização Passiva , SARS-CoV-2/genética , Índice de Gravidade de Doença , Soroterapia para COVID-19RESUMO
Some filoviruses can be transmitted to humans by zoonotic spillover events from their natural host and filovirus outbreaks have occured with increasing frequency in the last years. The filovirus Lloviu virus (LLOV), was identified in 2002 in Schreiber's bats (Miniopterus schreibersii) in Spain and was subsequently detected in bats in Hungary. Here we isolate infectious LLOV from the blood of a live sampled Schreiber's bat in Hungary. The isolate is subsequently sequenced and cultured in the Miniopterus sp. kidney cell line SuBK12-08. It is furthermore able to infect monkey and human cells, suggesting that LLOV might have spillover potential. A multi-year surveillance of LLOV in bats in Hungary detects LLOV RNA in both deceased and live animals as well as in coupled ectoparasites from the families Nycteribiidae and Ixodidae. This correlates with LLOV seropositivity in sampled Schreiber's bats. Our data support the role of bats, specifically Miniopterus schreibersii as hosts for LLOV in Europe. We suggest that bat-associated parasites might play a role in the natural ecology of filoviruses in temperate climate regions compared to filoviruses in the tropics.
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Quirópteros , Dípteros , Filoviridae , Animais , Humanos , Hungria/epidemiologia , ZoonosesRESUMO
This protocol details a rapid and reliable method for the production and titration of high-titre viral pseudotype particles with the SARS-CoV-2 spike protein (and D614G or other variants of concern, VOC) on a lentiviral vector core, and use for neutralisation assays in target cells expressing angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). It additionally provides detailed instructions on substituting in new spike variants via gene cloning, lyophilisation and storage/shipping considerations for wide deployment potential. Results obtained with this protocol show that SARS-CoV-2 pseudotypes can be produced at equivalent titres to SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) pseudotypes, neutralised by human convalescent plasma and monoclonal antibodies, and stored at a range of laboratory temperatures and lyophilised for distribution and subsequent application.
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Precision monitoring of antibody responses during the COVID-19 pandemic is increasingly important during large scale vaccine rollout and rise in prevalence of Severe Acute Respiratory Syndrome-related Coronavirus-2 (SARS-CoV-2) variants of concern (VOC). Equally important is defining Correlates of Protection (CoP) for SARS-CoV-2 infection and COVID-19 disease. Data from epidemiological studies and vaccine trials identified virus neutralising antibodies (Nab) and SARS-CoV-2 antigen-specific (notably RBD and S) binding antibodies as candidate CoP. In this study, we used the World Health Organisation (WHO) international standard to benchmark neutralising antibody responses and a large panel of binding antibody assays to compare convalescent sera obtained from: a) COVID-19 patients; b) SARS-CoV-2 seropositive healthcare workers (HCW) and c) seronegative HCW. The ultimate aim of this study is to identify biomarkers of humoral immunity that could be used to differentiate severe from mild or asymptomatic SARS-CoV-2 infections. Some of these biomarkers could be used to define CoP in further serological studies using samples from vaccination breakthrough and/or re-infection cases. Whenever suitable, the antibody levels of the samples studied were expressed in International Units (IU) for virus neutralisation assays or in Binding Antibody Units (BAU) for ELISA tests. In this work we used commercial and non-commercial antibody binding assays; a lateral flow test for detection of SARS-CoV-2-specific IgG/IgM; a high throughput multiplexed particle flow cytometry assay for SARS-CoV-2 Spike (S), Nucleocapsid (N) and Receptor Binding Domain (RBD) proteins); a multiplex antigen semi-automated immuno-blotting assay measuring IgM, IgA and IgG; a pseudotyped microneutralisation test (pMN) and an electroporation-dependent neutralisation assay (EDNA). Our results indicate that overall, severe COVID-19 patients showed statistically significantly higher levels of SARS-CoV-2-specific neutralising antibodies (average 1029 IU/ml) than those observed in seropositive HCW with mild or asymptomatic infections (379 IU/ml) and that clinical severity scoring, based on WHO guidelines was tightly correlated with neutralisation and RBD/S antibodies. In addition, there was a positive correlation between severity, N-antibody assays and intracellular virus neutralisation.
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COVID-19/imunologia , Convalescença , Imunidade Humoral , SARS-CoV-2/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Biomarcadores/sangue , COVID-19/sangue , COVID-19/diagnóstico , Teste Sorológico para COVID-19/normas , Calibragem , Humanos , Isotipos de Imunoglobulinas/sangue , Isotipos de Imunoglobulinas/imunologia , Padrões de Referência , Índice de Gravidade de DoençaRESUMO
The novel coronavirus SARS-CoV-2 is the seventh identified human coronavirus. Understanding the extent of pre-existing immunity induced by seropositivity to endemic seasonal coronaviruses and the impact of cross-reactivity on COVID-19 disease progression remains a key research question in immunity to SARS-CoV-2 and the immunopathology of COVID-2019 disease. This paper describes a panel of lentiviral pseudotypes bearing the spike (S) proteins for each of the seven human coronaviruses (HCoVs), generated under similar conditions optimized for high titre production allowing a high-throughput investigation of antibody neutralization breadth. Optimal production conditions and most readily available permissive target cell lines were determined for spike-mediated entry by each HCoV pseudotype: SARS-CoV-1, SARS-CoV-2 and HCoV-NL63 best transduced HEK293T/17 cells transfected with ACE2 and TMPRSS2, HCoV-229E and MERS-CoV preferentially entered HUH7 cells, and CHO cells were most permissive for the seasonal betacoronavirus HCoV-HKU1. Entry of ACE2 using pseudotypes was enhanced by ACE2 and TMPRSS2 expression in target cells, whilst TMPRSS2 transfection rendered HEK293T/17 cells permissive for HCoV-HKU1 and HCoV-OC43 entry. Additionally, pseudotype viruses were produced bearing additional coronavirus surface proteins, including the SARS-CoV-2 Envelope (E) and Membrane (M) proteins and HCoV-OC43/HCoV-HKU1 Haemagglutinin-Esterase (HE) proteins. This panel of lentiviral pseudotypes provides a safe, rapidly quantifiable and high-throughput tool for serological comparison of pan-coronavirus neutralizing responses; this can be used to elucidate antibody dynamics against individual coronaviruses and the effects of antibody cross-reactivity on clinical outcome following natural infection or vaccination.
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Anticorpos Antivirais/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , COVID-19/imunologia , Coronavirus/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Amplamente Neutralizantes/sangue , Linhagem Celular , Coronavirus Humano 229E/imunologia , Coronavirus Humano 229E/fisiologia , Coronavirus Humano NL63/imunologia , Coronavirus Humano NL63/fisiologia , Coronavirus Humano OC43/imunologia , Coronavirus Humano OC43/fisiologia , Reações Cruzadas , Humanos , Lentivirus/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Testes de Neutralização , Plasmídeos , SARS-CoV-2/fisiologia , Transfecção , Internalização do VírusRESUMO
Peste des petits ruminants (PPR) is a viral disease of goats and sheep that occurs in Africa, the Middle East and Asia with a severe impact on livelihoods and livestock trade. Many wild artiodactyls are susceptible to PPR virus (PPRV) infection, and some outbreaks have threatened endangered wild populations. The role of wild species in PPRV epidemiology is unclear, which is a knowledge gap for the Global Strategy for the Control and Eradication of PPR. These studies aimed to investigate PPRV infection in wild artiodactyls in the Greater Serengeti and Amboseli ecosystems of Kenya and Tanzania. Out of 132 animals purposively sampled in 2015-2016, 19.7% were PPRV seropositive by ID Screen PPR competition enzyme-linked immunosorbent assay (cELISA; IDvet, France) from the following species: African buffalo, wildebeest, topi, kongoni, Grant's gazelle, impala, Thomson's gazelle, warthog and gerenuk, while waterbuck and lesser kudu were seronegative. In 2018-2019, a cross-sectional survey of randomly selected African buffalo and Grant's gazelle herds was conducted. The weighted estimate of PPRV seroprevalence was 12.0% out of 191 African buffalo and 1.1% out of 139 Grant's gazelles. All ocular and nasal swabs and faeces were negative by PPRV real-time reverse transcription-polymerase chain reaction (RT-qPCR). Investigations of a PPR-like disease in sheep and goats confirmed PPRV circulation in the area by rapid detection test and/or RT-qPCR. These results demonstrated serological evidence of PPRV infection in wild artiodactyl species at the wildlife-livestock interface in this ecosystem where PPRV is endemic in domestic small ruminants. Exposure to PPRV could be via spillover from infected small ruminants or from transmission between wild animals, while the relatively low seroprevalence suggests that sustained transmission is unlikely. Further studies of other major wild artiodactyls in this ecosystem are required, such as impala, Thomson's gazelle and wildebeest.