RESUMO
The study evoluated an in-house Spike Receptor Binding Domain Enzyme-Linked Immunosorbent Assay (RBD-IgG-ELISA) for detecting SARS-CoV-2 IgG antibodies in infected and vaccinated individuals. The assay demonstrated a sensitivity of 91%, specificity of 99.25%, and accuracy of 95.13%. Precision and reproducibility were highly consistent. The RBD-IgG-ELISA was able to detect 96.25% of Polymerase chain reaction (PCR) confirmed cases for SARS-CoV-2 infection, demonstrating positive and negative predictive values of 99,18% and 91,69%, respectively. In an epidemiological survey, ELISA, lateral flow immunochromatographic assay (LFIA), and electrochemiluminescence immunoassay (ECLIA) exhibited diagnostic sensitivities of 68.29%, 63.41%, and 70.73%, respectively, along with specificities of 82.93%, 80.49%, and 80.49%, respectively. Agreement between RBD-IgG-ELISA/PCR was moderate (k index 0.512). However, good agreement between different assays (RBD-IgG-ELISA/LFIA k index 0.875, RBD-IgG-ELISA/ECLIA k index 0.901). Test performance on individuals' samples were inferior due to seroconversion time and chronicity. The IgG-RBD-ELISA assay demonstrated its effectiveness in monitoring antibody levels among healthcare professionals, revealing significant differences both before and after the administration of the third vaccine dose, with heightened protection levels observed following the third dose in five Coronavirus disease (COVID-19) vaccine regimens. In conclusion, the RBD-IgG-ELISA exhibits high reproducibility, specificity, and sensitivity, making it a suitable assay validated for serosurveillance and for obtaining information about COVID-19 infections or vaccinations.
Assuntos
Anticorpos Antivirais , Vacinas contra COVID-19 , COVID-19 , Ensaio de Imunoadsorção Enzimática , Imunoglobulina G , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Humanos , Ensaio de Imunoadsorção Enzimática/métodos , COVID-19/diagnóstico , COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Reprodutibilidade dos Testes , Pessoa de Meia-Idade , Masculino , Feminino , Adulto , Teste Sorológico para COVID-19/métodos , Sensibilidade e Especificidade , Idoso , Vacinação , Adulto JovemRESUMO
COVID-19, caused by SARS-CoV-2, and meningococcal disease, caused by Neisseria meningitidis, are relevant infectious diseases, preventable through vaccination. Outer membrane vesicles (OMVs), released from Gram-negative bacteria, such as N. meningitidis, present adjuvant characteristics and may confer protection against meningococcal disease. Here, we evaluated in mice the humoral and cellular immune response to different doses of receptor binding domain (RBD) of SARS-CoV-2 adjuvanted by N. meningitidis C:2a:P1.5 OMVs and aluminum hydroxide, as a combined preparation for these pathogens. The immunization induced IgG antibodies of high avidity for RBD and OMVs, besides IgG that recognized the Omicron BA.2 variant of SARS-CoV-2 with intermediary avidity. Cellular immunity showed IFN-γ and IL-4 secretion in response to RBD and OMV stimuli, demonstrating immunologic memory and a mixed Th1/Th2 response. Offspring presented transferred IgG of similar levels and avidity as their mothers. Humoral immunity did not point to the superiority of any RBD dose, but the group immunized with a lower antigenic dose (0.5 µg) had the better cellular response. Overall, OMVs enhanced RBD immunogenicity and conferred an immune response directed to N. meningitidis too.
Assuntos
Anticorpos Antivirais , COVID-19 , Imunoglobulina G , Neisseria meningitidis , SARS-CoV-2 , Animais , Camundongos , Imunoglobulina G/sangue , Neisseria meningitidis/imunologia , Feminino , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , COVID-19/prevenção & controle , COVID-19/imunologia , SARS-CoV-2/imunologia , Adjuvantes Imunológicos/administração & dosagem , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , Imunidade Celular , Imunidade Humoral , Camundongos Endogâmicos BALB C , Infecções Meningocócicas/prevenção & controle , Infecções Meningocócicas/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adjuvantes de Vacinas/administração & dosagem , Hidróxido de Alumínio/administração & dosagem , Hidróxido de Alumínio/imunologia , Imunização/métodos , Afinidade de Anticorpos , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Vacinas Meningocócicas/imunologia , Vacinas Meningocócicas/administração & dosagem , Memória Imunológica , Células Th1/imunologiaRESUMO
Since their discovery in the 1990s, heavy chain antibodies have garnered significant interest in the scientific community. These antibodies, found in camelids such as llamas and alpacas, exhibit distinct characteristics from conventional antibodies due to the absence of a light chain in their structure. Furthermore, they possess a single antigen-binding domain known as VHH or Nanobody (Nb). With a small size of approximately 15 kDa, these Nbs demonstrate improved characteristics compared to conventional antibodies, including greater physicochemical stability and enhanced biodistribution, enabling them to bind inaccessible epitopes more effectively. As a result, Nbs have found numerous applications in various medical and veterinary fields, particularly in diagnostics and therapeutics. Advances in biotechnology have made the production of recombinant antibodies feasible and compatible with large-scale manufacturing. Through the construction of immune phage libraries that display VHHs and subsequent selection through biopanning, it has become possible to isolate specific Nbs targeting pharmaceutical targets of interest, such as viruses. This review describes the processes involved in nanobody production, from hyperimmunization to purification, with the aim of their application in the pharmaceutical industry. (AU)
Assuntos
Viroses , Camelídeos Americanos , Biblioteca de Peptídeos , Anticorpos Neutralizantes , Anticorpos de Domínio Único , AnticorposRESUMO
O Instituto Adolfo Lutz (IAL) foi criado em 1940 como resultado da unificação dos Institutos Bacteriológico e Bromatológico, um moderno laboratório voltado ao controle de doenças, inaugurando uma nova fase de laboratórios de saúde pública no estado de São Paulo. Os primeiros testes sorológicos oferecidos à população foram executados pelas "antigas" Seções de Sorologia e de Imunologia. Essas seções destacam-se no desenvolvimento científico do IAL pela realização de pesquisas, produção científica e inovação tecnológica, seguramente, fundamentais para a saúde pública no decorrer dos anos. O Centro de Imunologia do IAL (CIM-IAL) foi criado em 2010, com a unificação das Seções de Sorologia e Imunologia, quando ocorreu a reorganização institucional. O CIM-IAL contribuiu para importantes avanços científicos na área da saúde, reforçando sua capacidade de desenvolver pesquisas, executar e monitorar o diagnóstico e a vigilância de diferentes agravos. Este manuscrito tem como objetivo apresentar os principais acontecimentos que ressaltam o papel fundamental na busca de soluções para os problemas de saúde pública, desde a época das Seções de Sorologia e Imunologia até tornar-se o Centro de Imunologia. Na elaboração deste trabalho foram utilizadas bibliografias contendo dados históricos, científicos e relatos de profissionais da área.
A new phase of Public Health Laboratories in the state of São Paulo occurred in 1940, with the unification of Instituto Bacteriológico and Bromatológico, creating the Instituto Adolfo Lutz (IAL), a modern laboratory focused on solving problems in this area. The first diagnostic tests offered to the population were carried out by the "old" Serology and Immunology Sections. It's worth highlighting the importance of these sections in the scientific development of the IAL by carrying out research, scientific production and technological innovation, which have certainly been fundamental to public health over the years. The Immunology Center (CIM) of IAL was created in 2010, when organizational adaptation took place with the junction of the Serology and Immunology Sections. The CIM-IAL has undergone important advances in the health area, reinforcing its capacity to develop research, carry out and monitor the diagnosis and surveillance of different diseases. This manuscript aims to present the main events that highlight the fundamental role in the search for solutions to public health problems, from the time of the Serology and Immunology Sections until it became the CIM. In the preparation, bibliographies were used based on historical and scientific data and reports from professionals in the field.
RESUMO
Humoral response to SARS-CoV-2 has been studied, predominantly the classical IgG and its subclasses. Although IgE antibodies are typically specifc to allergens or parasites, a few reports describe their production in response to SARS-CoV-2 and other viruses. Here, we investigated IgE specifc to receptor binding domain (RBD) of SARS-CoV-2 in a Brazilian cohort following natural infection and vaccination. Samples from 59 volunteers were assessed after infection (COVID-19), primary immunization with vectored (ChAdOx1) or inactivated (CoronaVac) vaccines, and booster immunization with mRNA (BNT162b2) vaccine. Natural COVID-19 induced IgE, but vaccination increased its levels. Subjects vaccinated with two doses of ChAdOx1 exhibited a more robust response than those immunized with two doses of CoronaVac; however, after boosting with BNT162b2, all groups presented similar IgE levels. IgE showed intermediate-to-high avidity, especially after the booster vaccine. We also found IgG4 antibodies, mainly after the booster, and they moderately correlated with IgE. ELISA results were confrmed by control assays, using IgG depletion by protein G and lack of reactivity with heterologous antigen. In our cohort, no clinical data could be associated with the IgE response. We advocate for further research on IgE and its role in viral immunity, extending beyond allergies and parasitic infections. (AU)
Assuntos
Sefarose , Imunoglobulina E , Imunoglobulina G , Vacinas contra COVID-19 , SARS-CoV-2RESUMO
Zika virus (ZIKV), a mosquito-borne pathogen, is an emerging arbovirus associated with sporadic symptomatic cases of great medical concern, particularly among pregnant women and newborns affected with neurological disorders. Serological diagnosis of ZIKV infection is still an unmet challenge due to the co-circulation of the dengue virus, which shares extensive sequence conservation of structural proteins leading to the generation of cross-reactive antibodies. In this study, we aimed to obtain tools for the development of improved serological tests for the detection of ZIKV infection. Polyclonal sera (pAb) and a monoclonal antibody (mAb 2F2) against a recombinant form of the ZIKV nonstructural protein 1 (NS1) allowed the identification of linear peptide epitopes of the NS1 protein. Based on these findings, six chemically synthesized peptides were tested both in dot blot and ELISA assays using convalescent sera collected from ZIKV-infected patients. Two of these peptides specifically detected the presence of ZIKV antibodies and proved to be candidates for the detection of ZIKV-infected subjects. The availability of these tools opens perspectives for the development of NS1-based serological tests with enhanced sensitivity regarding other flaviviruses.
Assuntos
Proteínas não Estruturais Virais , Infecção por Zika virus , Feminino , Humanos , Recém-Nascido , Gravidez , Anticorpos Monoclonais , Anticorpos Antivirais , Ensaio de Imunoadsorção Enzimática , Peptídeos , Testes Sorológicos , Proteínas não Estruturais Virais/isolamento & purificação , Zika virusRESUMO
Since their discovery in the 1990s, heavy chain antibodies have garnered significant interest in the scientific community. These antibodies, found in camelids such as llamas and alpacas, exhibit distinct characteristics from conventional antibodies due to the absence of a light chain in their structure. Furthermore, they possess a single antigen-binding domain known as VHH or Nanobody (Nb). With a small size of approximately 15 kDa, these Nbs demonstrate improved characteristics compared to conventional antibodies, including greater physicochemical stability and enhanced biodistribution, enabling them to bind inaccessible epitopes more effectively. As a result, Nbs have found numerous applications in various medical and veterinary fields, particularly in diagnostics and therapeutics. Advances in biotechnology have made the production of recombinant antibodies feasible and compatible with large-scale manufacturing. Through the construction of immune phage libraries that display VHHs and subsequent selection through biopanning, it has become possible to isolate specific Nbs targeting pharmaceutical targets of interest, such as viruses. This review describes the processes involved in nanobody production, from hyperimmunization to purification, with the aim of their application in the pharmaceutical industry.
Assuntos
Anticorpos de Domínio Único , Viroses , Humanos , Distribuição Tecidual , Anticorpos/metabolismo , Epitopos/metabolismoRESUMO
Zika virus (ZIKV), a mosquito-borne pathogen, is an emerging arbovirus associated with sporadic symptomatic cases of great medical concern, particularly among pregnant women and newborns affected with neurological disorders. Serological diagnosis of ZIKV infection is still an unmet challenge due to the co-circulation of the dengue virus, which shares extensive sequence conservation of structural proteins leading to the generation of cross-reactive antibodies. In this study, we aimed to obtain tools for the development of improved serological tests for the detection of ZIKV infection. Polyclonal sera (pAb) and a monoclonal antibody (mAb 2F2) against a recombinant form of the ZIKV nonstructural protein 1 (NS1) allowed the identification of linear peptide epitopes of the NS1 protein. Based on these findings, six chemically synthesized peptides were tested both in dot blot and ELISA assays using convalescent sera collected from ZIKV-infected patients. Two of these peptides specifically detected the presence of ZIKV antibodies and proved to be candidates for the detection of ZIKV-infected subjects. The availability of these tools opens perspectives for the development of NS1-based serological tests with enhanced sensitivity regarding other flaviviruses.
RESUMO
According to the World Health Organization (WHO), in the second half of 2022, there are about 606 million confirmed cases of COVID-19 and almost 6,500,000 deaths around the world. A pandemic was declared by the WHO in March 2020 when the new coronavirus spread around the world. The short time between the first cases in Wuhan and the declaration of a pandemic initiated the search for ways to stop the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or to attempt to cure the disease COVID-19. More than ever, research groups are developing vaccines, drugs, and immunobiological compounds, and they are even trying to repurpose drugs in an increasing number of clinical trials. There are great expectations regarding the vaccine's effectiveness for the prevention of COVID-19. However, producing sufficient doses of vaccines for the entire population and SARS-CoV-2 variants are challenges for pharmaceutical industries. On the contrary, efforts have been made to create different vaccines with different approaches so that they can be used by the entire population. Here, we summarize about 8162 clinical trials, showing a greater number of drug clinical trials in Europe and the United States and less clinical trials in low-income countries. Promising results about the use of new drugs and drug repositioning, monoclonal antibodies, convalescent plasma, and mesenchymal stem cells to control viral infection/replication or the hyper-inflammatory response to the new coronavirus bring hope to treat the disease.
RESUMO
Spike (S) protein has been recognized as a promising molecular target for diagnostic, vaccines and antiviral drugs development for COVID-19. In this study, we analyzed the most predominant mutations in the S protein of Brazilian isolates and predicted the effect of these amino acid alterations to protein conformation. A total of 25,924 sequences were obtained from GISAID for five regions of Brazilian territory (Midwest, North, Northeast, South, and Southeast), according to exclusion criteria. Most of the SARS-CoV-2 isolates belongs to the G clade and showed a large occurrence of D614G, N501Y and L18F substitutions. Prediction effects of these amino acid substitutions on the structure dynamics of the spike protein indicated a positive ΔΔG values and negative ΔΔSVib in most cases which is associated to structural stabilization and flexibility reduction of the S protein. Mutations E484K, N501Y and K417N belong to several SARS-CoV-2 variants of concern such as Alpha, Beta, Gamma and Delta, and showed high incidence among Brazilian isolates. These mutations have been described to increase RBD affinity to ACE-2 host and abolishment of RBD affinity to potent neutralizing ant-RBD. The increase in rates of infection and reinfection requires continuous genomic surveillance studies in order to characterize emerging mutations and monitor vaccine efficacy, and thus consideration structural data and dynamics in the observed phenotypes.Communicated by Ramaswamy H. Sarma.