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1.
Intervirology ; 62(3-4): 145-155, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31533104

RESUMO

BACKGROUND: When infected with the chikungunya virus (CHIKV), 3% to 28% of CHIKV-infected individuals remain asymptomatic, necessitating the development of improved high-throughput screening methods to overcome the limitations of molecular diagnostics or enzyme-linked immunosorbent assays (ELISAs). OBJECTIVE: In this study, two novel monoclonal antibodies (mAbs) targeting envelope 1 (E1) of CHIKV were developed and applied in a fluorescence-linked immunosorbent assay (FLISA) using coumarin-derived dendrimer as the fluorophore. METHODS: The performance of the FLISA was compared with that of ELISA. RESULTS: Using the two novel mAbs (2B5 and 2C8), FLISA could detect 1 × 105 PFU/mL of CHIKV, exhibiting a 2-fold lower limit of detection (LOD) compared to ELISA. The LOD of FICT corresponded to a comparative threshold value of 23.95 and 4 × 106 of RNA copy number/µL. In the presence of human sera and blood, virus detection by FLISA was 3-fold better than ELISA, with an LOD of 2 × 105 PFU/mL. Sera and blood interfered with the ELISA, resulting in 6 × 105 PFU/mL as the LOD. CONCLUSIONS: FLISA using two novel mAbs and coumarin-derived dendrimer is a superior diagnostic assay for detecting CHIKV in human sera and blood, compared to conventional ELISA.


Assuntos
Antígenos Virais/análise , Febre de Chikungunya/diagnóstico , Vírus Chikungunya/isolamento & purificação , Testes Diagnósticos de Rotina/métodos , Fluorometria/métodos , Técnicas Imunoenzimáticas/métodos , Proteínas do Envelope Viral/análise , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Vírus Chikungunya/imunologia , Humanos , Sensibilidade e Especificidade
2.
Vet Immunol Immunopathol ; 215: 109913, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31420069

RESUMO

The development of a rapid and efficient system to generate porcine monoclonal antibodies (mAbs) is an important step toward the discovery of critical neutralizing targets for designing rational vaccines against porcine viruses. In this study, we established a platform for producing porcine mAbs based on single cell technologies. First, we singled out an optimal donor from 507 pigs based on serum antibody neutralizing activity against porcine reproductive and respiratory syndrome virus (PRRSV). After identifying the contribution of IgG to the neutralizing activity, single CD45R+IgG+Ag+ B cells were sorted from peripheral blood mononuclear cells (PBMCs). Single B cell RT-PCR was performed using primers designed to cover the germline repertoire of the porcine VH/VL gene segments. Paired VH/VLs were cloned into a eukaryotic expression vector and transfected into 293T cells. We demonstrate that full-length porcine mAbs were produced, and antigen-specific mAbs were obtained after further validation. The approach reported in this study can be applied to generate porcine mAbs against any given antigen and may help with the screening of neutralizing antibodies against porcine pathogens.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/biossíntese , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Suínos/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Especificidade de Anticorpos , Linfócitos B/imunologia , Células HEK293 , Humanos , Região Variável de Imunoglobulina/genética , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Transfecção , Recombinação V(D)J
4.
Appl Microbiol Biotechnol ; 103(19): 8075-8086, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31463546

RESUMO

Foot-and-mouth disease virus (FMDV) has led to serious losses in the farming industry worldwide, particularly in cattle and swine. In developing countries, the control and eradication of FMD rely upon vaccination, in which the inactivated vaccine is predominant. In the preparation of inactivated vaccine, a series of purification methods were used to remove non-structural proteins (NSPs). It is necessary to develop a quantitative detection method of residual NSP and confirm a threshold value for the evaluation of the vaccine. Meanwhile, it is also important to develop a sensitive and rapid diagnostic method to distinguish infected animals from vaccinated animals (DIVA). In this study, three monoclonal antibodies (MAbs) against NSP 3ABC, designated 2G5, 9E2, and 1E10, were used. Subsequently, a series of overlapping peptides were expressed using a prokaryotic expression system to determine the minimal epitopes identified by the MAbs. Three linear B cell epitopes (BCEs), "92EYIEKA97" "23EGPYAGPLE31" and "209EPHH212", were identified by MAbs 2G5, 9E2, and 1E10, respectively. Alanine-scanning mutagenesis analysis confirmed the critical amino acid in these epitopes. The epitope "92EYIEKA97" is located in 3A, which is deleted in some natural deletion mutants that result in a change in virus tropism. MAb 9E2 that identified the epitope "23EGPYAGPLE31" reacted with 3B1 and 3B2, but did not react with 3B3. In combination with sequence alignment analysis, the epitope "23EGPYAGPLE31" is highly conserved among different FMDV isolates. Preliminary screening using the known positive and negative sera indicated the MAb 9E2 has the potential for the development of a diagnostic method for DIVA. The residual NSP in inactivated vaccines can be detected using 9E2-HRP, which indicated the MAb 9E2 is able to evaluate inactivated vaccines. The four-amino acid epitope is the first reported to date that is recognized by 1E10. These results provide valuable insight into the diagnosis of DIVA and the NSP residual evaluation in inactivated vaccines.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito B/imunologia , Vírus da Febre Aftosa/imunologia , Proteínas não Estruturais Virais/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Camundongos
5.
Rev Soc Bras Med Trop ; 52: e20180199, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31390441

RESUMO

INTRODUCTION: The Jirau hydroelectric power plant built in Rondônia state has environmental impacts that could be relevant to rabies outbreaks. METHODS: Bat populations were monitored for rabies by fluorescent antibody testing and simplified fluorescent inhibition microtesting between 2010 and 2015. RESULTS: All 1,183 bats tested negative for rabies. The prevalence of rabies antibodies was 17.5% in 1,049 bats. CONCLUSIONS: The rabies antibody dosage was not reactive in samples collected before the environmental changes, and there was a progressive increase in subsequent collections that could indicate an increase in rabies virus circulation among bats and risk of a rabies outbreak.


Assuntos
Quirópteros/virologia , Vírus da Raiva/isolamento & purificação , Raiva/veterinária , Animais , Anticorpos Antivirais/isolamento & purificação , Brasil/epidemiologia , Surtos de Doenças , Imunofluorescência/métodos , Raiva/epidemiologia , Raiva/virologia , Vírus da Raiva/imunologia , Estudos Soroepidemiológicos , Fatores de Tempo
6.
Res Vet Sci ; 125: 113-118, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31181347

RESUMO

Oral fluid (n = 564) samples collected longitudinally from twelve 14-week-old pigs vaccinated with a porcine reproductive and respiratory syndrome virus (PRRSV) modified live vaccine were used to evaluate and compare the diagnostic performance of three commercial PRRSV oral fluid (OF) ELISAs (ELISAs 1, 2, 3). Serum samples (n = 132) tested by a PRRSV serum ELISA (ELISA 'S') provided an antibody response baseline for comparison. The initial analysis comparing the rate of positivity between each OF ELISA versus ELISA 'S' and then pairwise among the three OF ELISAs determined that ELISA 2 (143 false negative results) was significantly different from ELISAs 1 and 3, and from ELISA 'S' (Cochran's Q test, p < 0.05). Receiver operating characteristic (ROC) analyses based on the manufacturers' recommended cutoff were used to estimate the diagnostic sensitivities and specificities of ELISA 1 (100%, 100%), ELISA 2 (62%, 97%), and ELISA 3 (94%, 100%). As an additional aid for interpreting results, the diagnostic sensitivities and specificities of each OF ELISA were also estimated over a range of cutoffs. Area under the curve comparisons found no significant difference between ELISAs 1 and 3, but ELISA 2 differed from both ELISA 1 and 3 (ROC Chi-square, p < 0.05). Based on these analyses, the overall diagnostic performance of the three OF ELISAs ranked ELISA 1 ≥ ELISA 3 > ELISA 2.


Assuntos
Anticorpos Antivirais/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/veterinária , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Saliva/virologia , Animais , Ensaio de Imunoadsorção Enzimática/métodos , Síndrome Respiratória e Reprodutiva Suína/diagnóstico , Curva ROC , Sensibilidade e Especificidade , Soro/virologia , Suínos
7.
J Infect Chemother ; 25(10): 786-790, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31105002

RESUMO

Japanese encephalitis (JE) is one of the most important viral encephalitis in Asia. JE is caused by the Japanese encephalitis virus (JEV), which belongs to the genus Flavivirus, family Flaviviridae. The diagnosis of JE is usually based on serological assays, and it has been reported that cross-reactivity between flaviviruses has complicated the interpretations of results from serological assays. Therefore, analysis of the cross-reactivity is an important subject for serological diagnosis of JE and other diseases caused by flaviviruses. In the present study, the cross-reactivity of the sera of patients with JE to other flaviviruses was analyzed using enzyme-linked immunosorbent assay (ELISA) and neutralization tests. Sixteen serum samples were collected from patients with JE and were tested for: i) IgM antibody against West Nile virus (WNV), dengue virus (DENV), zika virus (ZIKV), and tick-borne encephalitis virus (TBEV) using IgM-ELISA, ii) IgG antibody against DENV and TBEV using IgG-ELISA, and iii) neutralization tests with DENV 1-4, ZIKV, TBEV, and WNV. Out of the 16 samples tested using ELISA, 11 and 14 samples were positive for IgM and IgG, respectively, against at least one of the other flaviviruses. In neutralization tests, neutralizing potency against DENV, ZIKV, or TBEV was not detected in any samples. Although 13 samples showed neutralizing potency against WNV, their neutralizing antibody titers were equal to or less than one-eighth of those against JEV. These results show that neutralization tests are more specific than ELISA, indicating the importance of the neutralization tests in the diagnosis of JE.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus da Encefalite Japonesa (Espécie)/imunologia , Encefalite Japonesa/diagnóstico , Adulto , Animais , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Reações Cruzadas/imunologia , Vírus da Dengue/imunologia , Vírus da Encefalite Japonesa (Espécie)/isolamento & purificação , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Encefalite Japonesa/sangue , Encefalite Japonesa/virologia , Ensaio de Imunoadsorção Enzimática/estatística & dados numéricos , Estudos de Viabilidade , Humanos , Testes de Neutralização/métodos , Testes de Neutralização/estatística & dados numéricos , Sensibilidade e Especificidade , Células Vero , Vírus do Nilo Ocidental/imunologia , Zika virus/imunologia
8.
Emerg Microbes Infect ; 8(1): 749-759, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31130109

RESUMO

The Zika virus (ZIKV) outbreak and its link to microcephaly triggered a public health concern. To examine antibody response in a patient infected with ZIKV, we used single-cell PCR to clone 31 heavy and light chain-paired monoclonal antibodies (mAbs) that bind to ZIKV envelope (E) proteins isolated from memory B cells of a ZIKV-infected patient. Three mAbs (7B3, 1C11, and 6A6) that showed the most potent and broad neutralization activities against the African, Asian, and American strains were selected for further analysis. mAb 7B3 showed an IC50 value of 11.6 ng/mL against the circulating American strain GZ02. Epitope mapping revealed that mAbs 7B3 and 1C11 targeted residue K394 of the lateral ridge (LR) epitope of the EDIII domain, but 7B3 has a broader LR epitope footprint and recognizes residues T335, G337, E370, and N371 as well. mAb 6A6 recognized residues D67, K118, and K251 of the EDII domain. Interestingly, although the patient was seronegative for DENV infection, mAb 1C11, originating from the VH3-23 and VK1-5 germline pair, neutralized both ZIKV and DENV1. Administration of the mAbs 7B3, 1C11, and 6A6 protected neonatal SCID mice infected with a lethal dose of ZIKV. This study provides potential therapeutic antibody candidates and insights into the antibody response after ZIKV infection.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Imunização Passiva , Proteínas do Envelope Viral/imunologia , Infecção por Zika virus/prevenção & controle , Zika virus/imunologia , Adulto , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , China , Modelos Animais de Doenças , Mapeamento de Epitopos , Epitopos/imunologia , Humanos , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/isolamento & purificação , Concentração Inibidora 50 , Masculino , Camundongos , Camundongos SCID , Testes de Neutralização , Análise de Sobrevida , Resultado do Tratamento , Infecção por Zika virus/imunologia
9.
PLoS One ; 14(4): e0213978, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31009463

RESUMO

Reticuloendotheliosis virus (REV) is the most frequent exogenous virus that contaminates attenuated vaccines. Therefore, it is extremely important to select REV-free specific-pathogen-free (SPF) chicken embryos. Generally, REV infection is assessed by detecting REV antibodies in SPF chickens. This present study seeks to evaluate REV infection by replacing serum antibody detection with yolk antibody detection. A cohort of 40 nineteen-week-old SPF chickens were artificially inoculated with REV, with 32 SPF chickens raised in another isolation environment served as a blank control. Eggs and serum from 23-week-old chickens were sampled, and yolks were diluted separately to ratios of 1:150, 1:200, 1:300 and 1:400, which were detected together with serum. We found that the yolk antibody detection findings at a dilution of 1:300 had the highest coincidence rate compared with that based on serum antibody measurements. At a dilution ratio of 1:300 for yolk antibody, 72 chickens were continuously observed for 10 weeks from 25- to 34-weeks-old. Our findings were based on serum antibody or yolk antibody detection, and the evaluation results were completely consistent. Therefore, all serum antibody-positive chickens were yolk antibody-positive, and vice versa. Accordingly, vaccine producers can estimate REV cleanliness in a poultry farm by sampling yolk antibody titers.


Assuntos
Anticorpos Antivirais/isolamento & purificação , Galinhas/virologia , Doenças das Aves Domésticas/diagnóstico , Vírus da Reticuloendoteliose/isolamento & purificação , Organismos Livres de Patógenos Específicos , Animais , Embrião de Galinha , Doenças das Aves Domésticas/virologia , Vírus da Reticuloendoteliose/imunologia , Vacinas Atenuadas , Cultura de Vírus/métodos , Cultura de Vírus/veterinária , Saco Vitelino/virologia
10.
Nat Commun ; 10(1): 1788, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996276

RESUMO

Three Ebolavirus genus viruses cause lethal disease and lack targeted therapeutics: Ebola virus, Sudan virus and Bundibugyo virus. Monoclonal antibody (mAb) cocktails against the surface glycoprotein (GP) present a potential therapeutic strategy. Here we report two crystal structures of the antibody BDBV223, alone and complexed with its GP2 stalk epitope, an interesting site for therapeutic/vaccine design due to its high sequence conservation among ebolaviruses. BDBV223, identified in a human survivor of Bundibugyo virus disease, neutralizes both Bundibugyo virus and Ebola virus, but not Sudan virus. Importantly, the structure suggests that BDBV223 binding interferes with both the trimeric bundle assembly of GP and the viral membrane by stabilizing a conformation in which the monomers are separated by GP lifting or bending. Targeted mutagenesis of BDBV223 to enhance SUDV GP recognition indicates that additional determinants of antibody binding likely lie outside the visualized interactions, and perhaps involve quaternary assembly or membrane-interacting regions.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Ebolavirus/efeitos dos fármacos , Doença pelo Vírus Ebola/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/isolamento & purificação , Anticorpos Antivirais/metabolismo , Reações Cruzadas/imunologia , Cristalografia por Raios X , Ebolavirus/imunologia , Epitopos/química , Epitopos/imunologia , Doença pelo Vírus Ebola/sangue , Doença pelo Vírus Ebola/virologia , Humanos , Hibridomas , Mutagênese , Sobreviventes , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo
11.
Clin Lab ; 65(4)2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30969068

RESUMO

BACKGROUND: The aim of this study was to prove the difference between 37°C water incubation method and the BEP III automated immunoassay analyzer incubation method in anti-EBNA1-IgA antibody detection and to find the best way to improve the consistency of the two incubation methods. METHODS: The 37°C water incubation method and BEP III analyzer incubation method were used with the same panel of samples (n = 39) in anti-EBNA1-IgA antibody detection. Except for incubation, the rest of the steps were performed by the BEP III analyzer in both groups. All the data were analyzed by SPSS 17.0 software. Line charts and bar charts were used to compare the difference between the two incubation methods in anti-EBNA1-IgA antibody detection. We planned to find the best incubation scheme for BEP III analyzer, consistent with the water incubation method, using three groups of prolonged incubation time experiments. RESULTS: A sample panel of 39 outpatients were analyzed by two incubation methods. The results showed by line charts that the water incubation method had higher S/CO values than the BEP III analyzer incubation method. Meanwhile the water incubation group had more positive results (61.5%) and less borderline positive results (35.9%) than that of the BEP III analyzer incubation group which were 43.5% and 51.2%, respectively, in the stacked bar charts. We found that by prolonging the incubation time in the BEP III analyzer for 6 minutes in the first and second incubation steps the S/CO values we increased and achieved statistically coincident results with water incubation group. CONCLUSIONS: There were biases between the 37°C water incubation method and the BEP III analyzer incubation method in anti-EBNA1-IgA antibody detection. The water incubation method had higher S/CO values than the BEP III analyzer incubation method in paired groups and led partly to a difference in test results. By prolonging the BEP III analyzer incubation time properly, it can reduce the difference to some extent resulting in statistically similar results with the water incubation method.


Assuntos
Anticorpos Antivirais/isolamento & purificação , Infecções por Vírus Epstein-Barr/imunologia , Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Imunoensaio/métodos , Imunoglobulina A/isolamento & purificação , Antígenos Virais/imunologia , Automação , Proteínas do Capsídeo/imunologia , Ensaio de Imunoadsorção Enzimática , Herpesvirus Humano 4/imunologia , Humanos , Pacientes Ambulatoriais , Controle de Qualidade , Reprodutibilidade dos Testes , Manejo de Espécimes
13.
Mol Immunol ; 109: 12-19, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30849663

RESUMO

The persistent infection of high-risk human papillomavirus (HPV) is one of the most common causes of cervical cancer. It is well documented that expression of two oncogenes (E6/E7) plays a key role in tumor progression. HPV16E7 -targeting via nanobody (Nb) therefore could be beneficial for HPV16-associated cancer diagnosis and therapy. In this work, phage-display approach was employed to select the high affinity HPV16E7-specific Nb. Firstly; a high-quality immune library was constructed. After three round of biopanning, high-affinity HPV16 E7-specific nanobodies were retrieved. By phage ELISA and sequencing, four different sequences of anti- HPV16E7 nanobodies were selected. Then recombinant nanobody Nb2 was cloned and expressed in E. coli, and the specificity and thermal stability of purified Nb2 was evaluated. To examine the potential of Nb2 as an inhibitor of E7 function, Nb2 was expressed within HPV16 positive cells. Proliferation assay showed that the intracellular expressed Nb2 as an intrabody can decrease the growth of HPV16-positive cells. The results indicate that Nb2 as an intracellular antibody directed towards HPV oncoprotein E7 has great promise in applications for the therapy of HPV16-associated disease.


Assuntos
Anticorpos Antivirais , Carcinoma de Células Escamosas/imunologia , Papillomavirus Humano 16/imunologia , Proteínas E7 de Papillomavirus/antagonistas & inibidores , Proteínas E7 de Papillomavirus/imunologia , Anticorpos de Domínio Único , Neoplasias do Colo do Útero/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Anticorpos Antivirais/farmacologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/virologia , Linhagem Celular Tumoral , Clonagem Molecular , Escherichia coli , Feminino , Expressão Gênica , Papillomavirus Humano 16/genética , Humanos , Proteínas E7 de Papillomavirus/genética , Biblioteca de Peptídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/isolamento & purificação , Anticorpos de Domínio Único/farmacologia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia
14.
Bioengineered ; 10(1): 33-42, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30913952

RESUMO

The diagnosis of influenza A virus is essential since it can be confused with influenza A like illness and lead to inaccurate drug prescription. In this study, the M2e peptide, a strategic antigen that is conserved in all virus subtypes, was used as a diagnostic marker of influenza A. For the first time, M2e-specific IgY antibody was covalently conjugated to alkaline phosphatase (ALP) enzyme in the presence of glutaraldehyde. The antibody-enzyme bioconjugate was characterized by fluorescence and Fourier-transform infrared spectroscopy. Subsequently, the diagnostic value of this bioconjugate was evaluated by direct sandwich ELISA using nasopharyngeal swab samples positive/negative for H1N1 and H3N2, which were previously analyzed by rRT-PCR for influenza. In conclusion, the M2e-specific IgY-ALP bioconjugate demonstrated positive results for Influenza A in samples that were diagnosed as Influenza A via the RT-PCR method.


Assuntos
Fosfatase Alcalina/química , Anticorpos Antivirais/química , Antígenos Virais/imunologia , Imunoglobulinas/química , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Influenza Humana/diagnóstico , Fosfatase Alcalina/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/isolamento & purificação , Antígenos Virais/administração & dosagem , Antígenos Virais/química , Galinhas , Reagentes para Ligações Cruzadas/química , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/química , Feminino , Glutaral/química , Humanos , Imunização , Imunoconjugados/química , Imunoglobulinas/biossíntese , Imunoglobulinas/isolamento & purificação , Vírus da Influenza A Subtipo H1N1/química , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/química , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/imunologia , Influenza Humana/virologia , Nasofaringe/virologia , Peptídeos/administração & dosagem , Peptídeos/química , Peptídeos/imunologia , Espectroscopia de Infravermelho com Transformada de Fourier
15.
PLoS One ; 14(3): e0212811, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30897117

RESUMO

Adeno-associated virus (AAV) vectors represent promising candidates for gene therapy; however, pre-existing neutralizing antibodies (NAb) may reduce AAV vector delivery efficiency. In this study, the presence of AAV NAb was investigated in cats, which serve as a larger and outbred animal model for the prediction of gene therapy outcomes in humans but also in cats.Serum/plasma samples from 230 client-owned Swiss cats and 20 specified pathogen-free cats were investigated for NAb to AAV1, AAV2, AAV5, AAV6, AAV7, AAV8 and AAV9 using in vitro transduction inhibition and a beta-galactosidase assay. NAb to all tested AAV serotypes were found. Of the client-owned cats, 53% had NAb to one or more of the AAV serotypes. NAb (≥1:10) were found at frequencies of 5% (AAV6) to 28% (AAV7). The highest titers were found against AAV7 (≥1:160). The NAb prevalence to AAV2, AAV7 and AAV9 differed geographically. Regarding titers ≥1:10 against single AAV serotypes, age, breed and sex of the cats were not associated with the NAb prevalence. Cats with titers ≥1:20 against AAV2 and titers ≥1:40 against AAV7 were significantly younger than cats with low/no titers, and purebred cats were significantly more likely than non-purebred cats to have NAb to AAV2 (≥1:40). Additionally, regarding NAb to all AAV combined, female cats were significantly more likely than male cats to have NAb titers ≥1:40. Preliminary data using AAV-DJ indicated that less pre-existing NAb to the hybrid AAV-DJ can be expected compared to the wild-type AAV serotypes. AAV NAb will need to be taken into account for future in vivo gene therapy studies in cats.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Dependovirus/imunologia , Terapia Genética/efeitos adversos , Vetores Genéticos/imunologia , Fatores Etários , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Doenças do Gato/genética , Doenças do Gato/terapia , Gatos , Linhagem Celular Tumoral , Dependovirus/genética , Feminino , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/terapia , Terapia Genética/métodos , Vetores Genéticos/genética , Células HEK293 , Humanos , Masculino , Modelos Animais , Sorogrupo , Fatores Sexuais
17.
Res Vet Sci ; 124: 178-185, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30904721

RESUMO

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating viral disease of cloven-hoofed animals. Vaccination is a key element in the control of FMD among countries where the disease is enzootic. Differentiating infected from vaccinated animals in herds after immunization is an important component of effective eradication strategies. Non-structural protein (NSP) 3A of FMDV is as part of a larger detected antigen that is used for this differential diagnosis. Here, we generated a specific monoclonal antibody (MAb) against FMDV non-structural protein called 3A10, and further defined the linear epitopes recognized by the MAb 3A10 using a series of peptides that expressed GST-fused protein. Using Western blot, it was showed that the 5-aa peptide 126ERTLP130 of 3A was the minimal epitope reactive to MAb 3A10. Alanine-scanning mutagenesis analysis revealed that Arg127 and Leu129 were crucial for MAb 3A10 binding to 126ERTLP130. Furthermore, sequence alignment analysis, indicated that the epitope 126ERTLP130 recognized by 3A10 was shown to be conserved among seven serotypes of FMDV strains. The synthetic peptide Elisa demonstrated that this epitope peptide could be recognized by sera from FMDV-infected pigs and cattle, but negative reactivity to unvaccinated and vaccinated healthy animal sera. Thus, the MAb reagents and the linear epitopes defined herein provide theoretical and technical support for the development of diagnostic tools for infection differentiating FMDV infected from vaccinated animals.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Epitopos/imunologia , Vírus da Febre Aftosa/imunologia , Vacinação/veterinária , Proteínas não Estruturais Virais/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Western Blotting , Linhagem Celular , Sequência Conservada , Mapeamento de Epitopos , Epitopos/genética , Feminino , Vírus da Febre Aftosa/genética , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , Ligação Proteica , Proteínas não Estruturais Virais/genética
18.
Biosens Bioelectron ; 131: 46-52, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30822687

RESUMO

Detection of viral infection is commonly performed using serological techniques like the enzyme-linked immunosorbent assay (ELISA) to detect antibody responses. Such assays may also be used to determine the infection phase based on isotype prevalence. However, ELISAs demonstrate limited sensitivity and are difficult to perform at the point of care. Here, we present a novel technique for label-free, rapid detection of ultra-low concentrations of virus specific antibodies. We have developed a simple, robust capacitive biosensor using microwires coated with Zika or Chikungunya virus envelope antigen. With little discernable nonspecific binding, the sensor can detect as few as 10 antibody molecules in a small volume (10 molecules/30 µL) within minutes. It can also be used to rapidly, specifically, and accurately determine the isotype of antigen-specific antibodies. Finally, we demonstrate that anti-Zika virus antibody can be sensitively and specifically detected in dilute mouse serum and can be isotyped using the sensor. Overall, our findings suggest that our microwire sensor platform has the potential to be used as a reliable, sensitive, and inexpensive diagnostic tool to detect immune responses at the point of care.


Assuntos
Anticorpos Antivirais/isolamento & purificação , Técnicas Biossensoriais , Infecção por Zika virus/diagnóstico , Zika virus/isolamento & purificação , Anticorpos Antivirais/sangue , Formação de Anticorpos/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Proteínas do Envelope Viral/sangue , Proteínas do Envelope Viral/isolamento & purificação , Zika virus/patogenicidade , Infecção por Zika virus/virologia
19.
J Nanobiotechnology ; 17(1): 35, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30823927

RESUMO

BACKGROUND: Sensitive and specific antibodies can be used as essential probes to develop competitive enzyme-linked immunosorbent assay (cELISA). However, traditional antibodies are difficult to produce, only available in limited quantities, and ineffective as enzymatic labels. Nanobodies, which are single-domain antibodies (sdAbs), offer an alternative, more promising tool to circumvent these limitations. In the present work, a cELISA using nanobody-horseradish peroxidase (HRP) fusion protein firstly designed as a probe was developed for detecting anti-Newcastle disease virus (NDV) antibodies in chicken sera. RESULTS: In the study, a platform for the rapid and simple production of nanobody-HRP fusion protein was constructed. First, a total of 9 anti-NDV-NP protein nanobodies were screened from a immunised Bactrian camel. Then, the Nb5-HRP fusions were produced with the platform and used for the first time as sensitive reagents for developing cELISA to detect anti-NDV antibodies. The cut-off value of the cELISA was 18%, and the sensitivity and specificity were respectively 100% and 98.6%. The HI test and commercial ELISA kit (IDEXX) separately agreed 97.83% and 98.1% with cELISA when testing clinical chicken sera and both agreed 100% when testing egg yolks. However, for detecting anti-NDV antibodies in the sequential sera from the challenged chickens, cELISA demonstrated to be more sensitive than the HI test and commercial ELISA kit. Moreover, a close correlation (R2 = 0.914) was found between the percent competitive inhibition values of cELISA and HI titers. CONCLUSIONS: A platform was successfully designed to easily and rapidly produce the nanobody-HRP fusion protein, which was the first time to be used as reagents for establishing cELISA. Results suggest that the platform supports the development of a cELISA with high sensitivity, simplicity, and rapid detection of anti-NDV antibodies. Overall, we believe that the platform based on nanobody-HRP fusions can be widely used for future investigations and treatment other diseases and viruses.


Assuntos
Anticorpos Antivirais/sangue , Ensaio de Imunoadsorção Enzimática , Vírus da Doença de Newcastle/imunologia , Anticorpos de Domínio Único/imunologia , Animais , Anticorpos Antivirais/isolamento & purificação , Camelus , Galinhas , Escherichia coli , Biblioteca Gênica , Células HEK293 , Peroxidase do Rábano Silvestre/química , Humanos , Proteínas Recombinantes/química , Sensibilidade e Especificidade
20.
mBio ; 10(1)2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30782664

RESUMO

Paramyxoviruses, specifically, the childhood pathogen human parainfluenza virus type 3, are internalized into host cells following fusion between the viral and target cell membranes. The receptor binding protein, hemagglutinin (HA)-neuraminidase (HN), and the fusion protein (F) facilitate viral fusion and entry into the cell through a coordinated process involving HN activation by receptor binding, which triggers conformational changes in the F protein to activate it to reach its fusion-competent state. Interfering with this process through premature activation of the F protein has been shown to be an effective antiviral strategy in vitro. Conformational changes in the F protein leading to adoption of the postfusion form of the protein-prior to receptor engagement of HN at the host cell membrane-render the virus noninfectious. We previously identified a small compound (CSC11) that implements this antiviral strategy through an interaction with HN, causing HN to activate F in an untimely process. To assess the functionality of such compounds, it is necessary to verify that the postfusion state of F has been achieved. As demonstrated by Melero and colleagues, soluble forms of the recombinant postfusion pneumovirus F proteins and of their six helix bundle (6HB) motifs can be used to generate postfusion-specific antibodies. We produced novel anti-HPIV3 F conformation-specific antibodies that can be used to assess the functionality of compounds designed to induce F activation. In this study, using systematic chemical modifications of CSC11, we synthesized a more potent derivative of this compound, CM9. Much like CSC11, CM9 causes premature triggering of the F protein through an interaction with HN prior to receptor engagement, thereby preventing fusion and subsequent infection. In addition to validating the potency of CM9 using plaque reduction, fusion inhibition, and binding avidity assays, we confirmed the transition to a postfusion conformation of F in the presence of CM9 using our novel anti-HPIV3 conformation-specific antibodies. We present both CM9 and these newly characterized postfusion antibodies as novel tools to explore and develop antiviral approaches. In turn, these advances in both our molecular toolset and our understanding of HN-F interaction will support development of more-effective antivirals. Combining the findings described here with our recently described physiologically relevant ex vivo system, we have the potential to inform the development of therapeutics to block viral infection.IMPORTANCE Paramyxoviruses, including human parainfluenza virus type 3, are internalized into host cells by fusion between viral and target cell membranes. The receptor binding protein, hemagglutinin-neuraminidase (HN), and the fusion protein (F) facilitate viral fusion and entry into cells through a process involving HN activation by receptor binding, which triggers conformational changes in F to activate it to reach its fusion-competent state. Interfering with this process through premature activation of the F protein may be an effective antiviral strategy in vitro We identified and optimized small compounds that implement this antiviral strategy through an interaction with HN, causing HN to activate F in an untimely fashion. To address that mechanism, we produced novel anti-HPIV3 F conformation-specific antibodies that can be used to assess the functionality of compounds designed to induce F activation. Both the novel antiviral compounds that we present and these newly characterized postfusion antibodies are novel tools for the exploration and development of antiviral approaches.


Assuntos
Antivirais/farmacologia , Proteína HN/metabolismo , Vírus da Parainfluenza 3 Humana/efeitos dos fármacos , Vírus da Parainfluenza 3 Humana/fisiologia , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus/efeitos dos fármacos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Antivirais/síntese química , Linhagem Celular , Humanos , Ligação Proteica , Conformação Proteica , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/imunologia , Ensaio de Placa Viral
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