RESUMO
Middle East respiratory syndrome coronavirus (MERS-CoV) is a single-stranded RNA virus that causes severe respiratory disease in humans with a high fatality rate. Binding of the receptor binding domain (RBD) of the spike (S) glycoprotein to dipeptidyl peptidase 4 is the critical step in MERS-CoV infection of a host cell. No vaccines or clinically applicable treatments are currently available for MERS-CoV. Therefore, rapid diagnosis is important for improving patient outcomes through prompt treatment and protection against viral outbreaks. In this study, the aim was to establish two ELISA systems for detecting antigens and antibodies against MERS-CoV. Using a recombinant full-length S protein, an indirect ELISA was developed and found to detect MERS-CoV-specific antibodies in animal sera and sera of patient with MERS. Moreover, MAbs were induced with the recombinant S protein and RBD and used for sandwich ELISA to detect the MERS-CoV S protein. Neither ELISA system exhibited significant intra-assay or inter-assay variation, indicating good reproducibility. Moreover, the inter-day precision and sensitivity were adequate for use as a diagnostic kit. Thus, these ELISAs can be used clinically to diagnose MERS-CoV.
Assuntos
Anticorpos Antivirais/isolamento & purificação , Antígenos Virais/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/imunologia , Testes Imunológicos/métodos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Baculoviridae/genética , Infecções por Coronavirus/virologia , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/genética , Expressão Gênica , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Ligação Proteica/genética , Ligação Proteica/imunologia , Domínios e Motivos de Interação entre Proteínas , Ratos , Ratos Wistar , Receptores Virais/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Células Sf9 , Glicoproteína da Espícula de Coronavírus/genética , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/imunologiaRESUMO
BACKGROUND: As the nasal mucosa is the initial site of infection for COVID-19, intranasal vaccines are more favorable than conventional vaccines. In recent clinical studies, intranasal immunization has been shown to generate higher neutralizing antibodies; however, there is a lack of evidence on sterilizing immunity in the upper airway. Previously, we developed a recombinant measles virus encoding the spike protein of SARS-CoV-2 (rMeV-S), eliciting humoral and cellular immune responses against SARS-CoV-2. OBJECTIVES: In this study, we aim to provide an experiment on nasal vaccines focusing on a measles virus platform as well as injection routes. STUDY DESIGN: Recombinant measles viruses expressing rMeV-S were prepared, and 5 × 105 PFUs of rMeV-S were administered to Syrian golden hamsters via intramuscular or intranasal injection. Subsequently, the hamsters were challenged with inoculations of 1 × 105 PFUs of SARS-CoV-2 and euthanized 4 days post-infection. Neutralizing antibodies and RBD-specific IgG in the serum and RBD-specific IgA in the bronchoalveolar lavage fluid (BALF) were measured, and SARS-CoV-2 clearance capacity was determined via quantitative reverse-transcription PCR (qRT-PCR) analysis and viral titer measurement in the upper respiratory tract and lungs. Immunohistochemistry and histopathological examinations of lung samples from experimental hamsters were conducted. RESULTS: The intranasal immunization of rMeV-S elicits protective immune responses and alleviates virus-induced pathophysiology, such as body weight reduction and lung weight increase in hamsters. Furthermore, lung immunohistochemistry demonstrated that intranasal rMeV-S immunization induces effective SARS-CoV-2 clearance that correlates with viral RNA content, as determined by qRT-PCR, in the lung and nasal wash samples, SARS-CoV-2 viral titers in lung, nasal wash, BALF samples, serum RBD-specific IgG concentration, and RBD-specific IgA concentration in the BALF. CONCLUSION: An intranasal vaccine based on the measles virus platform is a promising strategy owing to the typical route of infection of the virus, the ease of administration of the vaccine, and the strong immune response it elicits.
Assuntos
COVID-19 , Sarampo , Orthopoxvirus , Vacinas , Animais , Cricetinae , SARS-CoV-2 , Vírus do Sarampo/genética , COVID-19/prevenção & controle , Glicoproteína da Espícula de Coronavírus , Imunização , Mucosa Nasal , Anticorpos Neutralizantes , Imunoglobulina A , Imunoglobulina G , Anticorpos Antivirais , Administração IntranasalRESUMO
Owing to the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, the development of effective and safe vaccines has become a priority. The measles virus (MeV) vaccine is an attractive vaccine platform as it has been administered to children for more than 40 years in over 100 countries. In this study, we developed a recombinant MeV expressing the full-length SARS-CoV-2 spike protein (rMeV-S) and tested its efficacy using mouse and hamster models. In hCD46Tg mice, two-dose rMeV-S vaccination induced higher Th1 secretion and humoral responses than one-dose vaccination. Interestingly, neutralizing antibodies induced by one-dose and two-dose rMeV-S immunization effectively blocked the entry of the α, ß, γ, and δ variants of SARS-CoV-2. Furthermore, two-dose rMeV-S immunization provided complete protection against SARS-CoV-2 in the hamster model. These results suggest the potential of rMeV-S as a vaccine candidate for targeting SARS-CoV-2 and its variants.