RESUMO
Pharmacodynamic assessment of T-cell-based cancer immunotherapies often focus on detecting rare circulating T-cell populations. The therapy-induced immune cells in blood-derived clinical samples are often present in very low frequencies and with the currently available T-cell analytical assays, amplification of the cells of interest prior to analysis is often required. Current approaches aiming to enrich antigen-specific T cells from human Peripheral Blood Mononuclear Cells (PBMCs) depend on in vitro culturing in presence of their cognate peptides and cytokines. In the present work, we improved a standard, publicly available protocol for T-cell immune analyses based on the in vitro expansion of T cells. We used PBMCs from healthy subjects and well-described viral antigens as a model system for optimizing the experimental procedures and conditions. Using the standard protocol, we first demonstrated significant enrichment of antigen-specific T cells, even when their starting frequency ex vivo was low. Importantly, this amplification occurred with high specificity, with no or neglectable enrichment of irrelevant T-cell clones being observed in the cultures. Testing of modified culturing timelines suggested that the protocol can be adjusted accordingly to allow for greater cell yield with strong preservation of the functionality of antigen-specific T cells. Overall, our work has led to the refinement of a standard protocol for in vitro stimulation of antigen-specific T cells and highlighted its reliability and reproducibility. We envision that the optimized protocol could be applied for longitudinal monitoring of rare blood-circulating T cells in scenarios with limited sample material.
Assuntos
Linfócitos T , Humanos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Antígenos Virais/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Células Cultivadas , Vacinas Anticâncer/imunologiaRESUMO
Immunodiagnostic tests for detecting dengue virus infections encounter challenges related to cross-reactivity with other related flaviviruses. Our research focuses on the development of a synthetic multiepitope antigen tailored for dengue immunodiagnostics. Selected dengue epitopes involved structural linearity and dissimilarity from the proteomes of Zika and Yellow fever viruses which served for computationally modeling the three-dimensional protein structure, resulting in the design of two proteins: rDME-C and rDME-BR. Both proteins consist of seven epitopes, separated by the GPGPG linker, and a carboxy-terminal 6 × -histidine tag. The molecular weights of the final proteins rDME-C and rDME-BR are 16.83 kDa and 16.80 kDa, respectively, both with an isoelectric point of 6.35. The distinguishing factor between the two proteins lies in the origin of their epitope sequences, where rDME-C is based on the reference dengue proteome, while rDME-BR utilizes sequences from prevalent Dengue genotypes in Brazil from 2008 to 2019. PyMol analysis revealed exposure of epitopes in the secondary structure. Successful expression of the antigens was achieved in soluble form and fluorescence experiments indicated a disordered structure. In subsequent testing, rDME-BR and rDME-C antigens were assessed using an indirect Elisa protocol against Dengue infected serum, previously examined with a commercial diagnostic test. Optimal concentrations for antigens were determined at 10 µg/mL for rDME-BR and 30 µg/mL for rDME-C, with serum dilutions ranging from 1:50 to 1:100. Both antigens effectively detected IgM and IgG antibodies in Dengue fever patients, with rDME-BR exhibiting higher sensitivity. Our in-house test showed a sensitivity of 77.3 % and 82.6 % and a specificity of 89.4 % and 71.4 % for rDME-C and rDEM-BR antigens. No cross-reactivity was observed with serum from Zika-infected mice but with COVID-19 serum samples. Our findings underscore the utility of synthetic biology in crafting Dengue-specific multiepitope proteins and hold promise for precise clinical diagnosis and monitoring responses to emerging Dengue vaccines.
Assuntos
Antígenos Virais , Vírus da Dengue , Dengue , Ensaio de Imunoadsorção Enzimática , Epitopos , Dengue/diagnóstico , Dengue/imunologia , Dengue/sangue , Antígenos Virais/imunologia , Epitopos/imunologia , Humanos , Vírus da Dengue/imunologia , Vírus da Dengue/genética , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Reações Cruzadas/imunologia , Sensibilidade e EspecificidadeRESUMO
Lateral flow assays (LFAs) are currently the most popular point-of-care diagnostics, rapidly transforming disease diagnosis from expensive doctor checkups and laboratory-based tests to potential on-the-shelf commodities. Yet, their sensitive element, a monoclonal antibody, is expensive to formulate, and their long-term storage depends on refrigeration technology that cannot be met in resource-limited areas. In this work, LCB1 affibodies (antibody mimetic miniproteins) were conjugated to bovine serum albumin (BSA) to afford a high-avidity synthetic capture (LCB1-BSA) capable of detecting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and virus like particles (VLPs). Substituting the monoclonal antibody 2B04 for LCB1-BSA (stable up to 60 °C) significantly improved the thermal stability, shelf life, and affordability of plasmonic-fluor-based LFAs (p-LFAs). Furthermore, this substitution significantly improved the sensitivity of p-LFAs toward the spike protein and VLPs with precise quantitative ability over 2 and 3 orders of magnitude, respectively. LCB1-BSA sensors could detect VLPs at 100-fold lower concentrations, and this improvement, combined with their robust nature, enabled us to develop an aerosol sampling technology to detect aerosolized viral particles. Synthetic captures like LCB1-BSA can increase the ultrasensitivity, availability, sustainability, and long-term accuracy of LFAs while also decreasing their manufacturing costs.
Assuntos
Aerossóis , Antígenos Virais , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação , Aerossóis/química , Glicoproteína da Espícula de Coronavírus/imunologia , Antígenos Virais/análise , Antígenos Virais/imunologia , Soroalbumina Bovina/química , COVID-19/diagnóstico , COVID-19/virologia , Humanos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/química , Imunoensaio/métodos , Temperatura , Limite de DetecçãoRESUMO
The recent emergence of a SARS-CoV-2 saltation variant, BA.2.87.1, which features 65 spike mutations relative to BA.2, has attracted worldwide attention. In this study, we elucidate the antigenic characteristics and immune evasion capability of BA.2.87.1. Our findings reveal that BA.2.87.1 is more susceptible to XBB-induced humoral immunity compared to JN.1. Notably, BA.2.87.1 lacks critical escaping mutations in the receptor binding domain (RBD) thus allowing various classes of neutralizing antibodies (NAbs) that were escaped by XBB or BA.2.86 subvariants to neutralize BA.2.87.1, although the deletions in the N-terminal domain (NTD), specifically 15-23del and 136-146del, compensate for the resistance to humoral immunity. Interestingly, several neutralizing antibody drugs have been found to restore their efficacy against BA.2.87.1, including SA58, REGN-10933 and COV2-2196. Hence, our results suggest that BA.2.87.1 may not become widespread until it acquires multiple RBD mutations to achieve sufficient immune evasion comparable to that of JN.1.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19 , Evasão da Resposta Imune , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Anticorpos Neutralizantes/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , COVID-19/imunologia , COVID-19/virologia , Anticorpos Antivirais/imunologia , Humanos , Mutação , Animais , Antígenos Virais/imunologia , Antígenos Virais/genética , Imunidade HumoralRESUMO
Porcine circovirus type 2 (PCV2) is a globally prevalent infectious pathogen affecting swine, with its capsid protein (Cap) being the sole structural protein critical for vaccine development. Prior research has demonstrated that PCV2 Cap proteins produced in Escherichia coli (E. coli) can form virus-like particles (VLPs) in vitro, and nuclear localization signal peptides (NLS) play a pivotal role in stabilizing PCV2 VLPs. Recently, PCV2d has emerged as an important strain within the PCV2 epidemic. In this study, we systematically optimized the PCV2d Cap protein and successfully produced intact PCV2d VLPs containing NLS using E. coli. The recombinant PCV2d Cap protein was purified through affinity chromatography, yielding 7.5 mg of recombinant protein per 100 ml of bacterial culture. We augmented the conventional buffer system with various substances such as arginine, ß-mercaptoethanol, glycerol, polyethylene glycol, and glutathione to promote VLP assembly. The recombinant PCV2d Cap self-assembled into VLPs approximately 20 nm in diameter, featuring uniform distribution and exceptional stability in the optimized buffer. We developed the vaccine and immunized pigs and mice, evaluating the immunogenicity of the PCV2d VLPs vaccine by measuring PCV2-IgG, IL-4, TNF-α, and IFN-γ levels, comparing them to commercial vaccines utilizing truncated PCV2 Cap antigens. The HE staining and immunohistochemical tests confirmed that the PCV2 VLPs vaccine offered robust protection. The results revealed that animals vaccinated with the PCV2d VLPs vaccine exhibited high levels of PCV2 antibodies, with TNF-α and IFN-γ levels rapidly increasing at 14 days post-immunization, which were higher than those observed in commercially available vaccines, particularly in the mouse trial. This could be due to the fact that full-length Cap proteins can assemble into more stable PCV2d VLPs in the assembling buffer. In conclusion, our produced PCV2d VLPs vaccine elicited stronger immune responses in pigs and mice compared to commercial vaccines. The PCV2d VLPs from this study serve as an excellent candidate vaccine antigen, providing insights for PCV2d vaccine research.
Assuntos
Anticorpos Antivirais , Proteínas do Capsídeo , Circovirus , Escherichia coli , Proteínas Recombinantes , Vacinas de Partículas Semelhantes a Vírus , Animais , Circovirus/imunologia , Circovirus/genética , Suínos , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas de Partículas Semelhantes a Vírus/genética , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Camundongos , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/imunologia , Doenças dos Suínos/prevenção & controle , Vacinas Virais/imunologia , Vacinas Virais/genética , Desenvolvimento de Vacinas , Antígenos Virais/imunologia , Antígenos Virais/genética , Imunoglobulina G/sangue , Análise Custo-Benefício , Feminino , Interferon gama/metabolismo , Imunogenicidade da VacinaAssuntos
Antígenos Virais , COVID-19 , Medições Luminescentes , SARS-CoV-2 , Humanos , COVID-19/diagnóstico , COVID-19/imunologia , SARS-CoV-2/imunologia , Imunoensaio/métodos , Antígenos Virais/imunologia , Antígenos Virais/sangue , Antígenos Virais/análise , Teste Sorológico para COVID-19/métodos , Sensibilidade e EspecificidadeRESUMO
Coronavirus disease 2019, caused by SARS-CoV-2, remains an on-going pandemic, partly due to the emergence of variant viruses that can "break-through" the protection of the current vaccines and neutralizing antibodies (nAbs), highlighting the needs for broadly nAbs and next-generation vaccines. We report an antibody that exhibits breadth and potency in binding the receptor-binding domain (RBD) of the virus spike glycoprotein across SARS coronaviruses. Initially, a lead antibody was computationally discovered and crystallographically validated that binds to a highly conserved surface of the RBD of wild-type SARS-CoV-2. Subsequently, through experimental affinity enhancement and computational affinity maturation, it was further developed to bind the RBD of all concerning SARS-CoV-2 variants, SARS-CoV-1 and pangolin coronavirus with pico-molar binding affinities, consistently exhibited strong neutralization activity against wild-type SARS-CoV-2 and the Alpha and Delta variants. These results identify a vulnerable target site on coronaviruses for development of pan-sarbecovirus nAbs and vaccines.
Assuntos
Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Antivirais/genética , Anticorpos Antivirais/metabolismo , Afinidade de Anticorpos , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Antígenos Virais/química , Antígenos Virais/genética , Anticorpos Amplamente Neutralizantes/genética , Anticorpos Amplamente Neutralizantes/metabolismo , Cristalografia por Raios X , Epitopos/química , Epitopos/imunologia , Humanos , Fragmentos de Imunoglobulinas/imunologia , Simulação de Acoplamento Molecular , Método de Monte Carlo , Testes de Neutralização , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Domínios Proteicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
We report on sample IS/17575 since it generated highly divergent results in the Belgian SARS-CoV-2 serology external quality assessment scheme. Sample IS/17575 was serum originating from a 30 years old male patient. 124 diagnostic laboratories analysed this sample. A total of 168 results was returned (including 5 doubles). Overall, 38 were positive. All tests against S1 were positive except the Euroimmun IgG ELISA and the Ortho clinical Diagnostics VITROS IgG CLIA. All tests against S1/S2 (Liaison, Diasorin) resulted in a signal above cutoff. Assays against RBD, mostly generate a negative result. An exception are the Wantai SARS-CoV-2 ELISA's. All tests targeting N protein were negative. The survey shows, when >6 months post-infection, assays targeting at least S1, and preferably S1 combined with S2, are the most sensitive. This finding accentuates the necessity of external quality assessment schedules and importance of antigenic composition of serologic SARS-CoV-2 assays.
Assuntos
Antígenos Virais/imunologia , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Anticorpos Antivirais/imunologia , Bélgica , Testes Diagnósticos de Rotina , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Masculino , Fosfoproteínas/imunologia , Sensibilidade e EspecificidadeRESUMO
To monitor the levels of protecting antibodies raised in the population in response to infection and/or to immunization with SARS-CoV-2, we need a technique that allows high throughput and low-cost quantitative analysis of human IgG antibodies reactive against viral antigens. Here we describe an ultra-fast, high throughput and inexpensive assay to detect SARS-CoV-2 seroconversion in humans. The assay is based on Ni2+ magnetic particles coated with His tagged SARS-CoV-2 antigens. A simple and inexpensive 96 well plate magnetic extraction/homogenization process is described which allows the simultaneous analysis of 96 samples and delivers results in 7 min with high accuracy.
Assuntos
Anticorpos Antivirais/sangue , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Imunoglobulina G/sangue , SARS-CoV-2/isolamento & purificação , Anticorpos Antivirais/imunologia , Antígenos Virais/sangue , Antígenos Virais/imunologia , COVID-19/sangue , COVID-19/imunologia , Teste Sorológico para COVID-19/economia , Ensaio de Imunoadsorção Enzimática/economia , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Imunoglobulina G/imunologia , Imãs/química , Níquel/química , SARS-CoV-2/imunologia , Sensibilidade e Especificidade , Soroconversão , Fatores de TempoRESUMO
A versatile portfolio of diagnostic tests is essential for the containment of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic. Besides nucleic acid-based test systems and point-of-care (POCT) antigen (Ag) tests, quantitative, laboratory-based nucleocapsid Ag tests for SARS-CoV-2 have recently been launched. Here, we evaluated four commercial Ag tests on automated platforms and one POCT to detect SARS-CoV-2. We evaluated PCR-positive (n = 107) and PCR-negative (n = 303) respiratory swabs from asymptomatic and symptomatic patients at the end of the second pandemic wave in Germany (February-March 2021) as well as clinical isolates EU1 (B.1.117), variant of concern (VOC) Alpha (B.1.1.7) or Beta (B.1.351), which had been expanded in a biosafety level 3 laboratory. The specificities of automated SARS-CoV-2 Ag tests ranged between 97.0 and 99.7% (Lumipulse G SARS-CoV-2 Ag (Fujirebio): 97.03%, Elecsys SARS-CoV-2 Ag (Roche Diagnostics): 97.69%; LIAISON® SARS-CoV-2 Ag (Diasorin) and SARS-CoV-2 Ag ELISA (Euroimmun): 99.67%). In this study cohort of hospitalized patients, the clinical sensitivities of tests were low, ranging from 17.76 to 52.34%, and analytical sensitivities ranged from 420,000 to 25,000,000 Geq/ml. In comparison, the detection limit of the Roche Rapid Ag Test (RAT) was 9,300,000 Geq/ml, detecting 23.58% of respiratory samples. Receiver-operating-characteristics (ROCs) and Youden's index analyses were performed to further characterize the assays' overall performance and determine optimal assay cutoffs for sensitivity and specificity. VOCs carrying up to four amino acid mutations in nucleocapsid were detected by all five assays with characteristics comparable to non-VOCs. In summary, automated, quantitative SARS-CoV-2 Ag tests show variable performance and are not necessarily superior to a standard POCT. The efficacy of any alternative testing strategies to complement nucleic acid-based assays must be carefully evaluated by independent laboratories prior to widespread implementation.
Assuntos
Antígenos Virais/análise , Teste Sorológico para COVID-19/métodos , COVID-19/virologia , SARS-CoV-2/isolamento & purificação , Antígenos Virais/imunologia , Automação/economia , Automação/métodos , COVID-19/diagnóstico , Teste Sorológico para COVID-19/economia , Estudos de Coortes , Reações Falso-Negativas , Alemanha , Humanos , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Sensibilidade e EspecificidadeRESUMO
The COVID-19 pandemic has caused significant morbidity and mortality. There is an urgent need for serological tests to detect antibodies against SARS-CoV-2, which could be used to assess past infection, evaluate responses to vaccines in development, and determine individuals who may be protected from future infection. Current serological tests developed for SARS-CoV-2 rely on traditional technologies such as enzyme-linked immunosorbent assays (ELISA) and lateral flow assays, which have not scaled to meet the demand of hundreds of millions of antibody tests so far. Herein, we present an alternative method of antibody testing that depends on one protein reagent being added to patient serum/plasma or whole blood with direct, visual readout. Two novel fusion proteins, RBD-2E8 and B6-CH1-RBD, were designed to bind red blood cells (RBCs) via a single-chain variable fragment (scFv), thereby displaying the receptor-binding domain (RBD) of SARS-CoV-2 spike protein on the surface of RBCs. Mixing mammalian-derived RBD-2E8 and B6-CH1-RBD with convalescent COVID-19 patient serum and RBCs led to visible hemagglutination, indicating the presence of antibodies against SARS-CoV-2 RBD. B6-CH1-RBD made in bacteria was not as effective in inducing agglutination, indicating better recognition of RBD epitopes from mammalian cells. Given that our hemagglutination test uses methods routinely used in hospital clinical labs across the world for blood typing, we anticipate the test can be rapidly deployed at minimal cost. We anticipate our hemagglutination assay may find extensive use in low-resource settings for detecting SARS-CoV-2 antibodies.
Assuntos
Anticorpos Antivirais/análise , Anticorpos Antivirais/imunologia , Teste Sorológico para COVID-19/métodos , COVID-19/sangue , COVID-19/imunologia , Testes de Hemaglutinação/métodos , Sistemas Automatizados de Assistência Junto ao Leito , SARS-CoV-2/imunologia , Antígenos Virais/imunologia , COVID-19/diagnóstico , COVID-19/virologia , Teste Sorológico para COVID-19/economia , Eritrócitos/imunologia , Testes de Hemaglutinação/economia , Humanos , Sistemas Automatizados de Assistência Junto ao Leito/economia , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Fatores de TempoRESUMO
OBJECTIVES: Novel point-of-care antigen assays present a promising opportunity for rapid screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. The purpose of this study was the clinical assessment of the new Roche SARS-CoV-2 Rapid Antigen Test. METHODS: The clinical performance of Roche SARS-CoV-2 Rapid Antigen Test was evaluated vs. a reverse transcription polymerase chain reaction (RT-PCR) laboratory-based assay (Seegene AllplexTM2019-nCoV) in nasopharyngeal swabs collected from a series of consecutive patients referred for SARS-CoV-2 diagnostics to the Pederzoli Hospital (Peschiera del Garda, Verona, Italy) over a 2-week period. RESULTS: The final study population consisted of 321 consecutive patients (mean age, 46 years and IQR, 32-56 years; 181 women, 56.4%), with 149/321 (46.4%) positive for SARS-CoV-2 RNA via the Seegene AllplexTM2019-nCoV Assay, and 109/321 (34.0%) positive with Roche SARS-CoV-2 Rapid Antigen Test, respectively. The overall accuracy of Roche SARS-CoV-2 Rapid Antigen Test compared to molecular testing was 86.9%, with 72.5% sensitivity and 99.4% specificity. Progressive decline in performance was observed as cycle threshold (Ct) values of different SARS-CoV-2 gene targets increased. The sensitivity was found to range between 97-100% in clinical samples with Ct values <25, between 50-81% in those with Ct values between 25 and <30, but low as 12-18% in samples with Ct values between 30 and <37. CONCLUSIONS: The clinical performance of Roche SARS-CoV-2 Rapid Antigen Test is excellent in nasopharyngeal swabs with Ct values <25, which makes it a reliable screening test in patients with high viral load. However, mass community screening would require the use of more sensitive techniques.
Assuntos
Antígenos Virais/análise , Teste Sorológico para COVID-19/normas , COVID-19/diagnóstico , COVID-19/virologia , Técnicas de Diagnóstico Molecular/normas , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação , Adulto , Antígenos Virais/imunologia , COVID-19/imunologia , Teste de Ácido Nucleico para COVID-19/normas , Feminino , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Nasofaringe/imunologia , Nasofaringe/virologia , Sistemas Automatizados de Assistência Junto ao Leito , Sensibilidade e Especificidade , Carga ViralAssuntos
Teste para COVID-19 , COVID-19/diagnóstico , Acessibilidade aos Serviços de Saúde , Pandemias , SARS-CoV-2/isolamento & purificação , Antígenos Virais/análise , Antígenos Virais/imunologia , COVID-19/epidemiologia , Teste de Ácido Nucleico para COVID-19 , Teste para COVID-19/economia , Teste para COVID-19/métodos , Teste para COVID-19/estatística & dados numéricos , Proteínas do Nucleocapsídeo de Coronavírus/análise , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Análise Custo-Benefício , Estudos de Avaliação como Assunto , Financiamento Governamental , Humanos , Invenções , National Institutes of Health (U.S.) , Testes Imediatos , Parcerias Público-Privadas/organização & administração , RNA Viral/análise , Kit de Reagentes para Diagnóstico/provisão & distribuição , SARS-CoV-2/imunologia , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/análise , Glicoproteína da Espícula de Coronavírus/imunologia , Estados Unidos/epidemiologiaRESUMO
In March 2013 it was reported by the World Health Organization (WHO) the first cases of human infections with avian influenza virus A (H7N9). From 2013 to December 2019, 1568 cases have been reported with 616 deaths. H7N9 infection has been associated with high morbidity and mortality rates, and vaccination is currently the most effective way to prevent infections and consequently flu-related severe illness. Developing and producing vaccines against pandemic influenza viruses is the main strategy for a response to a possible pandemic. This study aims to present the production of three industrial lots under current Good Manufacturing Practices (cGMP) of the active antigen used to produce the pandemic influenza vaccine candidate against A(H7N9). These batches were characterized and evaluated for quality standards and tested for immunogenicity in mice. The average yield was 173.50 ± 7.88 µg/mL of hemagglutinin and all the preparations met all the required specifications. The formulated H7N9 vaccine is poorly immunogenic and needs to be adjuvanted with an oil in water emulsion adjuvant (IB160) to achieve a best immune response, in a prime and in a boost scheme. These data are important for initial production planning and preparedness in the case of a H7N9 pandemic.
Assuntos
Subtipo H7N9 do Vírus da Influenza A/imunologia , Vacinas contra Influenza/biossíntese , Influenza Humana/prevenção & controle , Pandemias/prevenção & controle , Animais , Antígenos Virais/biossíntese , Antígenos Virais/imunologia , Composição de Medicamentos/métodos , Composição de Medicamentos/estatística & dados numéricos , Indústria Farmacêutica/normas , Feminino , Humanos , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/isolamento & purificação , Influenza Humana/imunologia , Influenza Humana/virologia , Camundongos , Camundongos Endogâmicos BALB C , Vacinas de Produtos Inativados/biossíntese , Vacinas de Produtos Inativados/imunologia , Vacinas de Produtos Inativados/isolamento & purificaçãoRESUMO
BACKGROUND: The ongoing coronavirus disease 19 (COVID-19) is posing a threat to the public health globally. Serological test for SARS-CoV-2 antibody can improve early diagnosis of COVID-19 and serves as a valuable supplement to RNA detection. METHOD: A SARS-CoV-2 IgG/IgM combined antibody test strip based on colloidal gold immunochromatography assay was developed, with both spike protein and nucleocapsid protein of SARS-CoV-2 antigen used for antibody detection. From 3 medical institutions across China, serum or plasma of 170 patients with confirmed COVID-19 diagnosis and 300 normal controls were collected and tested with the strip. Sensitivity, specificity, kappa coefficient, receiver operating characteristic (ROC) curve, and area under the curve (AUC) were analyzed. Positive rates in different medical centers, age group, gender, and different disease course were compared. RESULTS: 158 out 170 samples from confirmed COVID-19 patients had positive results from the test, and 296 out of 300 samples from normal controls had negative results. The kit was 92.9% sensitive and 98.7% specific. The positive rate was 77.3% during the first week after disease onset, but reached 100% since day 9. AUC and kappa coefficient were 0.958 and 0.926, respectively, which showed the consistency of the test results with the standard diagnosis. Age or gender caused little variations in the kit sensitivity. CONCLUSION: The rapid, easy-to-use SARS-CoV-2 IgG/IgM combined antibody test kit has a superior performance, which can help with accurate diagnosis and thus timely treatment and isolation of COVID-19 patients, that contributes to the better control of the global pandemic.
Assuntos
Teste para COVID-19/métodos , Imunoensaio/métodos , Adulto , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Teste para COVID-19/instrumentação , Estudos de Casos e Controles , China , Feminino , Coloide de Ouro , Humanos , Imunoensaio/instrumentação , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Nucleocapsídeo/imunologia , Fitas Reagentes , SARS-CoV-2/imunologia , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
Estimates of seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies have been hampered by inadequate assay sensitivity and specificity. Using an enzyme-linked immunosorbent assay-based approach that combines data about immunoglobulin G responses to both the nucleocapsid and spike receptor binding domain antigens, we show that excellent sensitivity and specificity can be achieved. We used this assay to assess the frequency of virus-specific antibodies in a cohort of elective surgery patients in Australia and estimated seroprevalence in Australia to be 0.28% (95% Confidence Interval, 0-1.15%). These data confirm the low level of transmission of SARS-CoV-2 in Australia before July 2020 and validate the specificity of our assay.
Assuntos
Anticorpos Antivirais/análise , COVID-19/diagnóstico , Ensaio de Imunoadsorção Enzimática , Estudos Soroepidemiológicos , Antígenos Virais/imunologia , Austrália , COVID-19/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Humanos , Imunoglobulina G/análise , Fosfoproteínas/imunologia , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
The potency of all modern tissue culture human rabies vaccines is measured based on the National Institute of Health (NIH) potency test that is laborious, time-consuming, involves large test variations and requires sacrifice of large number of animals. To circumvent these limitations, several researchers and WHO expert working groups have discussed development of alternative in vitro methods to replace the NIH potency test. Although several immunochemical methods have been proposed to quantify rabies glycoprotein (G-protein) using multiple murine monoclonal antibodies, we report an In vitro competitive inhibition ELISA (CIA) method based on the use of a neutralizing rabies glycoprotein site III directed novel therapeutic human rabies monoclonal antibody (RAB1) that shows equivalence to the mice NIH potency test in recognition of neutralization site of the glycoprotein. In vitro potency testing of WHO 7th International Standard for rabies vaccine (IS) by CIA using RAB1 and In-house reference standard (IHRS) as a standard to assess its suitability for the assessment of validation parameters showed accurate and precise values with <15% coefficient variance. The method was validated using 5PL standard curve with linearity r2 > 0.98 and LLOQ of 0.125 IU/mL indicating sensitivity of the method. The method was found to be precise, robust and accurate to quantitate intact rabies glycoprotein in final vaccine and showed a strong correlation (Pearson's r = 0.81) with the NIH potency values of licensed Vero cell rabies vaccine. The CIA test using RAB1 was able to accurately quantitate degradation of rabies vaccine and assess loss in antigenicity of lyophilized and reconstituted liquid rabies vaccine under thermal stress conditions. The method was able to differentiate between potent and reduced potency vaccine samples. The new in vitro competitive inhibition ELISA method using RAB1 thus can be a valid alternative to the NIH test.
Assuntos
Antígenos Virais/imunologia , Vacina Antirrábica/imunologia , Vírus da Raiva/imunologia , Raiva/prevenção & controle , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos Virais/administração & dosagem , Chlorocebus aethiops , Ensaio de Imunoadsorção Enzimática/métodos , Mapeamento de Epitopos/métodos , Humanos , Profilaxia Pós-Exposição/métodos , Raiva/imunologia , Raiva/virologia , Vacina Antirrábica/administração & dosagem , Potência de Vacina , Células Vero , Proteínas do Envelope Viral/administração & dosagemRESUMO
Human papillomavirus (HPV) is the causative agent of cervical and other epithelial cancers. Naturally occurring variants of HPV have been classified into lineages and sublineages based on their whole-genome sequences, but little is known about the impact of this diversity on the structure and function of viral gene products. The HPV capsid is an icosahedral lattice comprising 72 pentamers of the major capsid protein (L1) and the associated minor capsid protein (L2). We investigated the potential impact of this genome variation on the capsid antigenicity of lineage and sublineage variants of seven vaccine-relevant, oncogenic HPV genotypes by using a large panel of monoclonal antibodies (MAbs) raised against the L1 proteins of lineage A antigens. Each genotype had at least one variant that displayed a ≥4-fold reduced neutralizing antibody sensitivity against at least one MAb, demonstrating that naturally occurring variation can affect one or more functional antigenic determinants on the HPV capsid. For HPV16, HPV18, HPV31, and HPV45, the overall impact was of a low magnitude. For HPV33 (sublineages A2 and A3 and lineages B and C), HPV52 (lineage D), and HPV58 (lineage C), however, variant residues in the indicated lineages and sublineages reduced their sensitivity to neutralization by all MAbs by up to 1,000-fold, suggesting the presence of key antigenic determinants on the surface of these capsids. These determinants were resolved further by site-directed mutagenesis. These data improve our understanding of the impact of naturally occurring variation on the antigenicity of the HPV capsid of vaccine-relevant oncogenic HPV genotypes.IMPORTANCE Human papillomavirus (HPV) is the causative agent of cervical and some other epithelial cancers. HPV vaccines generate functional (neutralizing) antibodies that target the virus particles (or capsids) of the most common HPV cancer-causing genotypes. Each genotype comprises variant forms that have arisen over millennia and which include changes within the capsid proteins. In this study, we explored the potential for these naturally occurring variant capsids to impact recognition by neutralizing monoclonal antibodies. All genotypes included at least one variant form that exhibited reduced recognition by at least one antibody, with some genotypes affected more than others. These data highlight the impact of naturally occurring variation on the structure of the HPV capsid proteins of vaccine-relevant oncogenic HPV genotypes.
Assuntos
Alphapapillomavirus/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Proteínas do Capsídeo/imunologia , Genótipo , Vacinas contra Papillomavirus/imunologia , Alphapapillomavirus/genética , Anticorpos Monoclonais/genética , Antígenos Virais/genética , Proteínas do Capsídeo/genética , Epitopos , Genes Virais/genética , Variação Genética , Papillomavirus Humano 16/genética , Papillomavirus Humano 31/genética , Humanos , Testes de Neutralização , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/imunologia , Oncogenes , Papillomaviridae , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/genéticaRESUMO
Convalescent serum with a high abundance of neutralization IgG is a promising therapeutic agent for rescuing COVID-19 patients in the critical stage. Knowing the concentration of SARS-CoV-2 S1-specific IgG is crucial in selecting appropriate convalescent serum donors. Here, we present a portable microfluidic ELISA technology for rapid (15 min), quantitative, and sensitive detection of anti-SARS-CoV-2 S1 IgG in human serum with only 8 µL sample volume. We first identified a humanized monoclonal IgG that has a high binding affinity and a relatively high specificity towards SARS-CoV-2 S1 protein, which can subsequently serve as the calibration standard of anti-SARS-CoV-2 S1 IgG in serological analyses. We then measured the abundance of anti-SARS-CoV-2 S1 IgG in 16 convalescent COVID-19 patients. Due to the availability of the calibration standard and the large dynamic range of our assay, we were able to identify "qualified donors" for convalescent serum therapy with only one fixed dilution factor (200 ×). Finally, we demonstrated that our technology can sensitively detect SARS-CoV-2 antigens (S1 and N proteins) with pg/mL level sensitivities in 40 min. Overall, our technology can greatly facilitate rapid, sensitive, and quantitative analysis of COVID-19 related markers for therapeutic, diagnostic, epidemiologic, and prognostic purposes.
Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Infecções por Coronavirus/virologia , Ensaio de Imunoadsorção Enzimática/instrumentação , Imunoglobulina G/sangue , Técnicas Analíticas Microfluídicas/instrumentação , Pneumonia Viral/virologia , Adolescente , Adulto , Anticorpos Antivirais/imunologia , Antígenos Virais/sangue , Antígenos Virais/imunologia , Técnicas Biossensoriais/economia , Técnicas Biossensoriais/instrumentação , COVID-19 , Infecções por Coronavirus/terapia , Ensaio de Imunoadsorção Enzimática/economia , Desenho de Equipamento , Humanos , Imunização Passiva , Imunoglobulina G/imunologia , Limite de Detecção , Medições Luminescentes/economia , Medições Luminescentes/instrumentação , Técnicas Analíticas Microfluídicas/economia , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/terapia , SARS-CoV-2 , Fatores de Tempo , Adulto Jovem , Soroterapia para COVID-19RESUMO
Several protein vaccine candidates are among the COVID-19 vaccines in development. The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) has prepared a standardized template to describe the key considerations for the benefit-risk assessment of protein vaccines. This will help key stakeholders to assess potential safety issues and understand the benefit-risk of such a vaccine platform. The structured and standardized assessment provided by the template would also help contribute to improved public acceptance and communication of licensed protein vaccines.