RESUMEN
Bovine viral diarrhea virus (BVDV) causes ongoing economic losses to cattle industries, directly through reduced herd performance or indirectly through control program costs. ELISA assays, one of the most widely used techniques due to their ease of implementation, have been a valuable tool for mass surveillance and detection of BVDV. In this study, we developed a new indirect ELISA (rE2-ELISA) for serologic detection of BVDV. The assay considers three recombinant E2 protein subtypes as antigens, allowing serologic diagnosis of BVDV-1b (high prevalence worldwide), BVDV-1d and 1e (high prevalence in southern Chile) sub-genotypes. Recombinant E2 (rE2) proteins were successfully expressed in stably transfected CHO cells. Conditions for rE2 ELISAs were established after determining appropriate concentrations of antigen, blocking agent, secondary antibody, and serum dilutions to achieve maximum discrimination between positive and negative serum samples. The developed rE2-ELISA showed a sensitivity of 92.86% and a specificity of 98.33%. Clinical testing of 180 serum samples from herds in southern Chile showed high accuracy (kappa > 0.8) compared to the commercial BVDV Total Ab kit (IDEXX), with 95.37% positive and 87.5% negative predictive value. In addition, the rE2 ELISA has shown the capability to detect anti-BVDV antibodies from naturally infected animals with sub-genotypes 1b, 1e, or undetermined. These results indicate that the developed indirect ELISA could serve as a valid, and efficient alternative for identifying BVDV-infected animals, thus contributing to the success of disease control and eradication programs.
Asunto(s)
Diarrea Mucosa Bovina Viral , Ensayo de Inmunoadsorción Enzimática , Sensibilidad y Especificidad , Animales , Ensayo de Inmunoadsorción Enzimática/veterinaria , Ensayo de Inmunoadsorción Enzimática/métodos , Bovinos , Diarrea Mucosa Bovina Viral/diagnóstico , Diarrea Mucosa Bovina Viral/sangre , Diarrea Mucosa Bovina Viral/virología , Chile , Genotipo , Virus de la Diarrea Viral Bovina Tipo 1/inmunología , Virus de la Diarrea Viral Bovina Tipo 1/aislamiento & purificación , Virus de la Diarrea Viral Bovina/inmunología , Virus de la Diarrea Viral Bovina/aislamiento & purificación , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/genética , Antígenos Virales/inmunología , Cricetulus , Células CHO , Anticuerpos Antivirales/sangre , Proteínas Recombinantes/inmunologíaRESUMEN
Foot and mouth disease (FMD) is a highly contagious infection caused by FMD-virus (FMDV) that affects livestock worldwide with significant economic impact. The main strategy for the control is vaccination with FMDV chemically inactivated with binary ethylenimine (FMDVi). In FMDV infection and vaccination, B cell response plays a major role by providing neutralizing/protective antibodies in animal models and natural hosts. Extracellular vesicles (EVs) and small EVs (sEVs) such as exosomes are important in cellular communication. EVs secreted by antigen-presenting cells (APC) like dendritic cells (DCs) participate in the activation of B and T cells through the presentation of native antigen membrane-associated to B cells or by transferring MHC-peptide complexes to T cells and even complete antigens from DCs. In this study, we demonstrate for the first time that APC activated with the FMDVi O1 Campos vaccine-antigens secrete EVs expressing viral proteins/peptides that could stimulate FMDV-specific immune response. The secretion of EVs-FMDVi is a time-dependent process and can only be isolated within the first 24 h post-activation. These vesicles express classical EVs markers (CD9, CD81, and CD63), along with immunoregulatory molecules (MHC-II and CD86). With an average size of 155 nm, they belong to the category of EVs. Studies conducted in vitro have demonstrated that EVs-FMDVi express antigens that can stimulate a specific B cell response against FMDV, including both marginal zone B cells (MZB) and follicular B cells (FoB). These vesicles can also indirectly or directly affect T cells, indicating that they express both B and T epitopes. Additionally, lymphocyte expansion induced by EVs-FMDVi is greater in splenocytes that have previously encountered viral antigens in vivo. The present study sheds light on the role of EVs derived from APC in regulating the adaptive immunity against FMDV. This novel insight contributes to our current understanding of the immune mechanisms triggered by APC during the antiviral immune response. Furthermore, these findings may have practical implications for the development of new vaccine platforms, providing a rational basis for the design of more effective vaccines against FMDV and other viral diseases.
Asunto(s)
Células Presentadoras de Antígenos , Antígenos Virales , Linfocitos B , Vesículas Extracelulares , Virus de la Fiebre Aftosa , Fiebre Aftosa , Vacunas Virales , Animales , Virus de la Fiebre Aftosa/inmunología , Vesículas Extracelulares/inmunología , Linfocitos B/inmunología , Fiebre Aftosa/inmunología , Fiebre Aftosa/prevención & control , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Antígenos Virales/inmunología , Vacunas Virales/inmunología , Proteínas Virales/inmunología , Activación de Linfocitos/inmunología , Células Dendríticas/inmunología , Presentación de Antígeno/inmunologíaRESUMEN
BACKGROUND: As new and improved antigen-detecting rapid diagnostic tests for SARS-CoV-2 infection (Ag-RDT) continue to be developed, assessing their diagnostic performance is necessary to increase test options with accurate and rapid diagnostic capacity especially in resource-constrained settings. This study aimed to assess the performance of two Ag-RDTs in a population-based study. METHODS: We conducted a diagnostic accuracy study in neighborhoods with high socioeconomic vulnerability in Salvador-Brazil, including individuals aged ≥12 years old who attended primary health services, between July and December 2022, with COVID-19 symptoms or who had been in contact with a confirmed case. Two Ag-RDTs were compared in parallel using reverse transcription polymerase chain reaction (RT-PCR) as reference standard, the PanbioTM COVID-19 Ag test (Abbott®) and Immuno-Rapid COVID-19 Ag (WAMA Diagnostic®). Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) were calculated. RESULTS: For the Abbott test the sensitivity was 52.7% (95% CI: 44.3% - 61.0%), specificity 100% (95% CI: 98.7% - 100%), PPV 100% (95% CI: 95.4% - 100%) and NPV 80.4% (95% CI: 75.9% - 84.4%). For the WAMA test, the sensitivity was 53.4% (95% CI: 45.0% - 61.6%), specificity 100% (95% CI: 98.7% - 100%), PPV 100% (95% CI: 95.4% - 100%) and NPV 80.7% (95% CI: 76.2% - 84.6%). Sensitivity for the group with Cycle Threshold (CT) <24 was 82.3% (95%CI: 72.1-90.0, n = 83) for PanbioTM COVID-19 Ag test and 87.3% (95%CI: 77.9-93.8, n = 83) for Immuno-Rapid COVID-19 Ag test. CONCLUSION: Sensitivity for both Ag-RDT was lower than reported by manufacturers. In the stratified analysis, sensitivity was higher among those with lower CT values <24. Specificity was high for both rapid antigen tests. Both Ag-RDT showed to be useful for rapid diagnostic of potential cases of COVID-19. Negative results must be assessed carefully according to clinical and epidemiological information.
Asunto(s)
Prueba Serológica para COVID-19 , COVID-19 , SARS-CoV-2 , Sensibilidad y Especificidad , Humanos , COVID-19/diagnóstico , COVID-19/inmunología , COVID-19/epidemiología , Masculino , SARS-CoV-2/inmunología , SARS-CoV-2/aislamiento & purificación , Adulto , Femenino , Persona de Mediana Edad , Brasil/epidemiología , Niño , Prueba Serológica para COVID-19/métodos , Adolescente , Antígenos Virales/inmunología , Adulto Joven , Anciano , Factores SocioeconómicosRESUMEN
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.
Asunto(s)
Antígenos Virales , Virus del Dengue , Dengue , Ensayo de Inmunoadsorción Enzimática , Epítopos , Dengue/diagnóstico , Dengue/inmunología , Dengue/sangre , Antígenos Virales/inmunología , Epítopos/inmunología , Humanos , Virus del Dengue/inmunología , Virus del Dengue/genética , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Reacciones Cruzadas/inmunología , Sensibilidad y EspecificidadRESUMEN
This study focuses on the development and initial assessment of an indirect IgG enzyme-linked immunosorbent assay (ELISA) specifically designed to detect of anti-SARS-CoV-2 antibodies. The unique aspect of this ELISA method lies in its utilization of a recombinant nucleocapsid (N) antigen, produced through baculovirus expression in insect cells. Our analysis involved 292 RT-qPCR confirmed positive serum samples and 54 pre-pandemic healthy controls. The process encompassed cloning, expression, and purification of the SARS-CoV-2 N gene in insect cells, with the resulted purified protein employed in our ELISA tests. Statistical analysis yielded an Area Under the Curve of 0.979, and the optimized cut-off exhibited 92 % sensitivity and 94 % specificity. These results highlight the ELISA's potential for robust and reliable serological detection of SARS-CoV-2 antibodies. Further assessments, including a larger panel size, reproducibility tests, and application in diverse populations, could enhance its utility as a valuable biotechnological solution for diseases surveillance.
Asunto(s)
Anticuerpos Antivirales , Baculoviridae , COVID-19 , Ensayo de Inmunoadsorción Enzimática , Proteínas Recombinantes , SARS-CoV-2 , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , SARS-CoV-2/inmunología , SARS-CoV-2/genética , Baculoviridae/genética , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/genética , COVID-19/diagnóstico , COVID-19/sangre , COVID-19/inmunología , Animales , Proteínas de la Nucleocápside de Coronavirus/inmunología , Proteínas de la Nucleocápside de Coronavirus/genética , Prueba Serológica para COVID-19/métodos , Células Sf9 , Antígenos Virales/inmunología , Antígenos Virales/genética , Proteínas de la Nucleocápside/inmunología , Proteínas de la Nucleocápside/genética , Sensibilidad y Especificidad , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Fosfoproteínas/inmunología , Fosfoproteínas/genéticaRESUMEN
SARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not present disulfide bonds. Intending to avoid non-specific binding of SARS-CoV-2 N to antibodies from patients who previously had different coronaviruses, a 35 kDa fragment of N was expressed without a conserved motif in E. coli as inclusion bodies (N122-419-IB). Culture media and IB washing conditions were chosen to obtain N122-419-IB with high yield (370 mg/L bacterial culture) and protein purity (90%). High pressure solubilizes protein aggregates by weakening hydrophobic and ionic interactions and alkaline pH promotes solubilization by electrostatic repulsion. The association of pH 9.0 and 2.4 kbar promoted efficient solubilization of N122-419-IB without loss of native-like tertiary structure that N presents in IB. N122-419 was refolded with a yield of 85% (326 mg/L culture) and 95% purity. The refolding process takes only 2 hours and the protein is ready for use after pH adjustment, avoiding the necessity of dialysis or purification. Antibody binding of COVID-19-positive patients sera to N122-419 was confirmed by Western blotting. ELISA using N122-419 is effective in distinguishing between sera presenting antibodies against SARS-CoV-2 from those who do not. To the best of our knowledge, the proposed condition for IB solubilization is one of the mildest described. It is possible that the refolding process can be extended to a wide range of proteins with high yields and purity, even those that are sensible to very alkaline pH.
Asunto(s)
Anticuerpos Antivirales/sangre , Antígenos Virales/química , COVID-19/sangre , COVID-19/diagnóstico , Proteínas de la Nucleocápside de Coronavirus/química , Inmunoglobulina G/sangre , Cuerpos de Inclusión/química , Replegamiento Proteico , SARS-CoV-2/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , COVID-19/virología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Presión Hidrostática , Inmunoglobulina G/inmunología , Fosfoproteínas/química , Fosfoproteínas/inmunología , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , SolubilidadRESUMEN
Macrophages can be polarized toward a proinflammatory phenotype (M1) (CD68+) or to an anti-inflammatory one (M2) (CD163+). Polarization can be triggered by cytokines such as IFN-γ for M1, or IL-10 and TGF-ß, for M2. In the context of pediatric Epstein Barr virus (EBV) infection, little is known about macrophage polarization in EBV primary or persistent infection. When studying tonsils of patients undergoing primary infection (PI), healthy carrier (HC), reactivation (R), and not infected (NI), M1 profile prevailed in all infection status. However, an increase in M2 cells was observed in those patients with broader expression of latency antigens, in particular EBNA2. Tonsils from primary infected patients showed an increased IL-10 expression, whereas, unexpectedly, TGF-ß expression correlated with M1 marker. Furthermore, an inverse correlation was demonstrated between CD68 and IFN-γ. Therefore, in the context of asymptomatic infection in children, M1 macrophage polarization prevails, even in the presence of IL-10 and TGF-ê´ immunomodulatory cytokines, and it might be independent from lymphomagenesis process. Our finding indicates that macrophages may have a significant plasticity in response to different types of extrinsic stimuli, and further studies are required to investigate M1 polarization under anti-inflammatory stimuli. IMPORTANCE Most studies on Epstein Barr virus (EBV) primary infection have been performed in adolescents and young adult populations with Infectious Mononucleosis (IM) in developed countries. Furthermore, studies related to macrophage polarization were assessed in EBV-associated lymphomas, but little is known about macrophage polarization in the context of primary infection at the site of viral entry and replication, the tonsils. Therefore, the aim of this study was to characterize macrophage response in children undergoing EBV primary or persistent infection, in order to enlighten the role of macrophages in viral pathogenesis, in a population with a high incidence of EBV-associated lymphomas in children younger than 10 years old. This study may contribute to explain, at least in part, the asymptomatic viral infection in children from an underdeveloped region, given that M1 polarization pattern prevails, but in a regulatory environment.
Asunto(s)
Microambiente Celular/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/fisiología , Inmunomodulación , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Adolescente , Antígenos Virales/inmunología , Biomarcadores , Niño , Preescolar , Citocinas/metabolismo , Infecciones por Virus de Epstein-Barr/diagnóstico , Femenino , Interacciones Huésped-Patógeno/inmunología , Humanos , Lactante , Macrófagos/metabolismo , Masculino , Pruebas Serológicas , Carga Viral , Proteínas Virales/inmunologíaAsunto(s)
Vacunas contra la COVID-19/administración & dosificación , COVID-19/virología , SARS-CoV-2/aislamiento & purificación , Antígenos Virales/genética , Antígenos Virales/inmunología , COVID-19/inmunología , COVID-19/prevención & control , COVID-19/transmisión , Vacunas contra la COVID-19/inmunología , Humanos , Mutación , SARS-CoV-2/genética , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Vacunas de ARNmRESUMEN
Serological testing is a powerful tool in epidemiological studies for understanding viral circulation and assessing the effectiveness of virus control measures, as is the case of SARS-CoV-2, the pathogenic agent of COVID-19. Immunoassays can quantitatively reveal the concentration of antiviral antibodies. The assessment of antiviral antibody titers may provide information on virus exposure, and changes in IgG levels are also indicative of a reduction in viral circulation. In this work, we describe a serological study for the evaluation of antiviral IgG and IgM antibodies and their correlation with antiviral activity. The serological assay for IgG detection used two SARS-CoV-2 proteins as antigens, the nucleocapsid N protein and the 3CL protease. Cross-reactivity tests in animals have shown high selectivity for detection of antiviral antibodies, using both the N and 3CL antigens. Using samples of human serum from individuals previously diagnosed by PCR for COVID-19, we observed high sensitivity of the ELISA assay. Serological results with human samples also suggest that the combination of higher titers of antiviral IgG antibodies to different antigen targets may be associated with greater neutralization activity, which can be enhanced in the presence of antiviral IgM antibodies.
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Anticuerpos Antivirales/inmunología , Prueba Serológica para COVID-19/métodos , COVID-19/diagnóstico , COVID-19/prevención & control , Vigilancia Inmunológica , SARS-CoV-2/inmunología , Animales , Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , COVID-19/epidemiología , COVID-19/inmunología , Prueba Serológica para COVID-19/normas , Reacciones Cruzadas , Virus del Dengue/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/normas , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Ratones , Ratones Endogámicos BALB C , Sensibilidad y Especificidad , Virus Zika/inmunologíaRESUMEN
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.
Asunto(s)
Anticuerpos Antivirales/sangre , Prueba Serológica para COVID-19/métodos , COVID-19/diagnóstico , Inmunoglobulina G/sangre , SARS-CoV-2/aislamiento & purificación , Anticuerpos Antivirales/inmunología , Antígenos Virales/sangre , Antígenos Virales/inmunología , COVID-19/sangre , COVID-19/inmunología , Prueba Serológica para COVID-19/economía , Ensayo de Inmunoadsorción Enzimática/economía , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Inmunoglobulina G/inmunología , Imanes/química , Níquel/química , SARS-CoV-2/inmunología , Sensibilidad y Especificidad , Seroconversión , Factores de TiempoRESUMEN
Dendritic cells (DCs) are the most potent antigen-presenting cells, unique to initiate and coordinate the adaptive immune response. In pigs, conventional DCs (cDCs), plasmacytoid DCs (pDCs), and monocyte-derived DCs (moDCs) have been described in blood and tissues. Different pathogens, such as viruses, could infect these cells, and in some cases, compromise their response. The understanding of the interaction between DCs and viruses is critical to comprehend viral immunopathological responses. Porcine reproductive and respiratory syndrome virus (PRRSV) is the most important respiratory pathogen in the global pig population. Different reports support the notion that PRRSV modulates pig immune response in addition to their genetic and antigenic variability. The interaction of PRRSV with DCs is a mostly unexplored area with conflicting results and lots of uncertainties. Among the scarce certainties, cDCs and pDCs are refractory to PRRSV infection in contrast to moDCs. Additionally, response of DCs to PRRSV can be different depending on the type of DCs and maybe is related to the virulence of the viral isolate. The precise impact of this virus-DC interaction upon the development of the specific immune response is not fully elucidated. The present review briefly summarizes and discusses the previous studies on the interaction of in vitro derived bone marrow (bm)- and moDCs, and in vivo isolated cDCs, pDCs, and moDCs with PRRSV1 and 2.
Asunto(s)
Células Dendríticas/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Animales , Presentación de Antígeno , Antígenos Virales/inmunología , Médula Ósea , Células Dendríticas/clasificación , Predicción , Monocitos , Virus del Síndrome Respiratorio y Reproductivo Porcino/patogenicidad , Porcinos , Linfocitos T Reguladores/inmunología , Vacunas Virales , VirulenciaRESUMEN
Zika virus (ZIKV) is a flavivirus that has emerged as a global health threat after the 2015 outbreak in the Americas, where devastating congenital defects were documented. There are currently no vaccines to prevent ZIKV infections nor commercially available clinical diagnostic tests demonstrated to identify ZIKV without cross-reactive interference of related flaviviruses. Early diagnosis is critical when treating symptomatic patients and in preventing ZIKV transmission. In this context, the development of sensitive and accurate diagnostic methods are urgently needed for the detection of ZIKV acute infection. The aim of this study consisted of obtaining monoclonal antibodies (mAbs) against denatured monomeric ZIKV Nonstructural protein 1 (ZNS1), a useful diagnostic marker for flavivirus early detection, in order to develop a highly specific and sensitive ZNS1 indirect competitive ELISA (icELISA). The production of hybridomas secreting ZNS1 mAbs was carried out through immunizations with denatured monomeric ZNS1. We selected 1F5 and 6E2 hybridoma clones, which recognized the heat-denatured ZNS1 hexameric form by indirect ELISA. Cross-reaction studies indicated that these mAbs specifically bind to a ZNS1 linear epitope, and that they do not cross-react with the NS1 protein from other related flaviviruses. The 1F5 mAb enabled the development of a sensitive and reproducible icELISA to detect and quantify small amounts of ZNS1 disease marker in heat-denatured human sera. Here, we establish a reliable 1F5 based-icELISA that constitutes a promising diagnostic tool for control strategies and the prevention of ZIKV propagation.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Antivirales/química , Antígenos Virales/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas no Estructurales Virales/genética , Infección por el Virus Zika/diagnóstico , Virus Zika/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/aislamiento & purificación , Antígenos Virales/administración & dosificación , Antígenos Virales/inmunología , Unión Competitiva , Clonación Molecular , Diagnóstico Precoz , Ensayo de Inmunoadsorción Enzimática/normas , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Células HEK293 , Humanos , Hibridomas/química , Hibridomas/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Multimerización de Proteína , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Proteínas no Estructurales Virales/administración & dosificación , Proteínas no Estructurales Virales/inmunología , Virus Zika/genética , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/virologíaRESUMEN
Bats are hosts of a range of viruses, and their great diversity and unique characteristics that distinguish them from all other mammals have been related to the maintenance, evolution, and dissemination of these pathogens. Recently, very divergent hantaviruses have been discovered in distinct species of bats worldwide, but their association with human disease remains unclear. Considering the low success rates of detecting hantavirus RNA in bat tissues and that to date no hantaviruses have been isolated from bat samples, immunodiagnostic tools could be very helpful to understand pathogenesis, epidemiology, and geographic range of bat-borne hantaviruses. In this sense, we aimed to identify in silico immunogenic B-cell epitopes present on bat-borne hantaviruses nucleoprotein (NP) and verify if they are conserved among them and other selected members of Mammantavirinae, using a combination of (the three most used) different prediction algorithms, ELLIPRO, Discotope 2.0, and PEPITO server. To support our data, we in silico modeled 3D structures of NPs from representative members of bat-borne hantaviruses, using comparative and ab initio methods due to the absence of crystallographic structures of studied proteins or similar models in the Protein Data Bank. Our analysis demonstrated the antigenic complexity of the bat-borne hantaviruses group, showing a low sequence conservation of epitopes among members of its own group and a minor conservation degree in comparison to Orthohantavirus, with a recognized importance to public health. Our data suggest that the use of recombinant rodent-borne hantavirus NPs to cross-detect antibodies against bat- or shrew-borne viruses could underestimate the real impact of this virus in nature.
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Antígenos Virales/inmunología , Quirópteros/virología , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/inmunología , Orthohantavirus/inmunología , Algoritmos , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Antígenos Virales/química , Secuencia Conservada , Orthohantavirus/química , Orthohantavirus/aislamiento & purificación , Orthohantavirus/fisiología , Especificidad del Huésped , Modelos Moleculares , Filogenia , Conformación Proteica , Estructura Secundaria de Proteína , Musarañas/virologíaRESUMEN
BACKGROUND: Infectious bursal disease (IBD), also known as Gumboro disease, is a viral infection that causes mortality and immunosuppression in chickens (Gallus gallus). VP2 and VP3 are the major structural viral capsid components and are the most immunogenic proteins of IBD virus (IBDV). Reliable diagnostic tests using VP2 and VP3 produced in heterologous systems are important tools to control this infection. One advantage of an IBD diagnostic based on VP3, over those that use VP2, is that VP3 has linear epitopes, enabling its production in bacteria. RESULTS: We tested the suitability of recombinant VP3 (rVP3) as a diagnostic reagent in an enzyme-linked immunosorbent assay (ELISA). Compared with a commercial test, rVP3 ELISA showed high sensitivity and specificity as a diagnostic tool for vaccinated animals. In addition, rVP3, but not the commercial ELISA, was able to detect antibodies in nonvaccinated chickens, probably developed against circulating IBDV strains. It was possible the assessment of VP3 regions antigenicity using chicken antisera. CONCLUSIONS: The full-length recombinant VP3 can be used to assess post vaccination immunological status of chickens and its production is feasible and inexpensive. The evaluation of VP3 regions as candidates for general use in the diagnosis of IBD in chickens should be conducted with caution. Our work was the first to identify several regions of VP3 recognized by chicken antibodies.
Asunto(s)
Antígenos Virales/inmunología , Infecciones por Birnaviridae/veterinaria , Pollos , Virus de la Enfermedad Infecciosa de la Bolsa/genética , Enfermedades de las Aves de Corral/virología , Proteínas Estructurales Virales/inmunología , Animales , Infecciones por Birnaviridae/epidemiología , Infecciones por Birnaviridae/virología , Brasil/epidemiología , Regulación Viral de la Expresión Génica , Enfermedades de las Aves de Corral/epidemiologíaRESUMEN
Besides antigen-specific responses to viral antigens, humoral immune response in virus infection can generate polyreactive and autoreactive antibodies. Dengue and Zika virus infections have been linked to antibody-mediated autoimmune disorders, including Guillain-Barré syndrome. A unique feature of flaviviruses is the secretion of nonstructural protein 1 (NS1) by infected cells. NS1 is highly immunogenic, and antibodies targeting NS1 can have both protective and pathogenic roles. In the present study, we investigated the humoral immune response to Zika virus NS1 and found NS1 to be an immunodominant viral antigen associated with the presence of autoreactive antibodies. Through single B cell cultures, we coupled binding assays and BCR sequencing, confirming the immunodominance of NS1. We demonstrate the presence of self-reactive clones in germinal centers after both infection and immunization, some of which present cross-reactivity with NS1. Sequence analysis of anti-NS1 B cell clones showed sequence features associated with pathogenic autoreactive antibodies. Our findings demonstrate NS1 immunodominance at the cellular level as well as a potential role for NS1 in ZIKV-associated autoimmune manifestations.
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Reacciones Cruzadas/inmunología , Proteínas no Estructurales Virales/inmunología , Infección por el Virus Zika/inmunología , Animales , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Linfocitos B/virología , Femenino , Centro Germinal/patología , Centro Germinal/virología , Inmunización , Inmunoglobulina M/sangre , Ratones Endogámicos BALB C , Proteínas no Estructurales Virales/sangre , Infección por el Virus Zika/virologíaAsunto(s)
Humanos , Vacunas contra la COVID-19/administración & dosificación , SARS-CoV-2/aislamiento & purificación , COVID-19/virología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Vacunas contra la COVID-19/inmunología , SARS-CoV-2/genética , COVID-19/inmunología , COVID-19/prevención & control , COVID-19/transmisión , Mutación , Antígenos Virales/genética , Antígenos Virales/inmunologíaRESUMEN
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.
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Subtipo H7N9 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/biosíntesis , Gripe Humana/prevención & control , Pandemias/prevención & control , Animales , Antígenos Virales/biosíntesis , Antígenos Virales/inmunología , Composición de Medicamentos/métodos , Composición de Medicamentos/estadística & datos numéricos , Industria Farmacéutica/normas , Femenino , Humanos , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/aislamiento & purificación , Gripe Humana/inmunología , Gripe Humana/virología , Ratones , Ratones Endogámicos BALB C , Vacunas de Productos Inactivados/biosíntesis , Vacunas de Productos Inactivados/inmunología , Vacunas de Productos Inactivados/aislamiento & purificaciónRESUMEN
BACKGROUND: The heat-labile nature of Dengue virus (DENV) in serum samples must be considered when applying routine diagnostic tests to avoid issues that could impact the accuracy of test results with direct implications for case management and disease reporting. OBJECTIVES: To check if pre-analytical variables, such as storage time and temperature, have an impact on the accuracy of the main routine diagnostic tests for dengue. METHODS: Virus isolation, reverse transcription real-time polymerase chain reaction (RT-PCR) and NS1 enzyme-linked immunosorbent assay (ELISA) were evaluated using 84 samples submitted to different pre-analytical conditions. FINDINGS: Sensitivity and negative predictive value were directly affected by sample storage conditions. RT-PCR and virus isolation showed greater dependence on well-conserved samples for an accurate diagnosis. Interestingly, even storage at -30ºC for a relatively short time (15 days) was not adequate for accurate results using virus isolation and RT-PCR tests. On the other hand, NS1 ELISA showed no significant reduction in positivity for aliquots tested under the same conditions as in the previous tests. MAIN CONCLUSIONS: Our results support the stability of the NS1 marker in ELISA diagnosis and indicate that the accuracy of routine tests such as virus isolation and RT-PCR is significantly affected by inadequate transport and storage conditions of serum samples.
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Antígenos Virales/sangre , Virus del Dengue/aislamiento & purificación , Dengue/diagnóstico , Ensayo de Inmunoadsorción Enzimática/métodos , Pruebas Inmunológicas/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Proteínas no Estructurales Virales/inmunología , Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , Dengue/sangre , Dengue/virología , Virus del Dengue/genética , Virus del Dengue/inmunología , Humanos , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad , Proteínas no Estructurales Virales/genéticaRESUMEN
Management of SARS-CoV-2 requires safe decision-making to minimize contamination. Healthcare workers and professionals in confined areas are affected by the risk of the activity and the environment. Isolation of contaminated workers and healthcare professionals requires clinical and diagnostic criteria. On the other hand, interrupting the isolation of healthcare employees and professionals is critical because diagnostic tests do not support clinical decisions. In addition to defining the best test in view of its accuracy, it is necessary to consider aspects such as the stage of the disease or cure, the viral load and the individual's own immunity. Uncertainty about natural and herd immunity to the disease leads to the development of appropriate antivirals, diagnostic tests and vaccines.
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Prueba de Ácido Nucleico para COVID-19 , Prueba Serológica para COVID-19 , COVID-19/transmisión , Aislamiento de Pacientes/normas , Inmunidad Adaptativa/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/virología , COVID-19/diagnóstico , COVID-19/inmunología , COVID-19/prevención & control , Prueba de COVID-19 , Toma de Decisiones Clínicas , Heces/química , Heces/virología , Personal de Salud , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Nasofaringe/química , Nasofaringe/virología , Aislamiento de Pacientes/métodos , ARN Viral/análisis , SARS-CoV-2 , Esputo/química , Esputo/virología , Carga ViralRESUMEN
The impact of influenza vaccination is largely measured by estimating vaccine effectiveness (VE), which vary in different seasons. Strain mutations and waning immunity present two key mechanisms affecting VE. We sought to quantify the relative effect of these mechanisms by projecting VE and the reduction of illness due to vaccination. We developed a stochastic age-structured agent-based simulation model of influenza transmission dynamics to encapsulate intraseason waning of immunity post-vaccination, and mutation-induced antigenic distance between circulating strains and vaccine strains. Parameterizing the model with published estimates, we projected the temporal and overall VE during an epidemic season, and estimated the reduction of infection for high (70%) and low (30%) vaccine efficacies to reflect the levels of vaccine-induced protection in randomized control trials. Both temporal and overall VE decreased as the attack rate increased, with lower median values for epidemics starting with strains that were antigenically more distant from vaccine strains. We observed a higher rate of temporal decline with considerably lower median values of the overall VE in the presence of intraseason waning of immunity compared with only the antigenic distance effect. The highest benefit of vaccination in preventing influenza infection was achieved at moderate attack rates in the range of 6%-15%. The results show that even when VE is relatively low in the population and almost negligible for older age groups (i.e., 50+ years), vaccination can still prevent significant illness in high-risk individuals; thereby reducing healthcare resource utilization and economic burden. Our study indicates that early vaccination remains an important strategy for alleviating the burden of seasonal influenza. Policy discussions on optimal timing of vaccination to reduce the effect of intraseason waning of immunity should be considered in the context of strain mutations within the epidemic course.