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Background: Early studies showed the utility of pretransplant QuantiFERON-Cytomegalovirus (QF-CMV) assays for CMV-disease prediction post kidney transplant (KT). However, recent data are conflicting. Methods: This prospective cohort study enrolled adult patients undergoing KT between July 2017 and May 2019. Patients with antithymocyte globulin therapy or negative pretransplant CMV IgG were excluded. QF-CMV assays were performed on transplantation day and one month thereafter, and CMV viral loads were obtained 1, 3, and 6 months posttransplantation. The primary outcome was CMV viremia within 6 months. The QF-CMV assay-posttransplant CMV viremia association was analyzed. Results: Fifty-five patients were enrolled (male, 58.2%; mean (SD) age, 46.5 (10.2) years). Fifty-two (94.5%) received CMV-seropositive donor kidneys. Over 6 months, 29 patients developed CMV viremia (52.7%), with 14 (25.5%) having significant viremia requiring antiviral therapy. The CMV-viremia incidence of patients with nonreactive and reactive baseline QF-CMV assays did not differ significantly (55.3% and 47.1%; p = 0.573). Among patients with reactive pretransplant QF-CMV assays, there was a trend toward a lower incidence of CMV viremia for those who were persistently reactive at 1 month after KTs, although there was no statistically significant difference (50% vs 83%; p = 0.132). Conclusions: Our study could not support the use of single-timepoint pretransplant or 1-month posttransplant QF-CMV assays as a predictor for posttransplant CMV viremia in CMV seropositive KT recipients. Investigation of the association between dynamic QF-CMV-status changes and CMV-viremia incidence are needed.
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Infecções por Citomegalovirus , Transplante de Rim , Adulto , Citomegalovirus , Infecções por Citomegalovirus/diagnóstico , Infecções por Citomegalovirus/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Estudos Soroepidemiológicos , Viremia/diagnóstico , Viremia/epidemiologiaRESUMO
BACKGROUND: Prevalence and incidence of hepatitis caused by HEV infection are usually higher in developing countries. This study demonstrated the HEV seroprevalence and incidence of HEV infection in patients with clinical hepatitis in a tertiary hospital in Thailand. METHODS: A laboratory-based cross-sectional study was conducted using 1106 serum samples from patients suspected of HEV infection sent to the Serology laboratory, Siriraj Hospital, for detecting HEV antibodies during 2015-2018. Prevalence of anti-HEV IgG and IgM antibodies in general patients, including organ transplant recipients and pregnant women in a hospital setting, were determined using indirect enzyme-linked immunosorbent assay (ELISA) kits. Comparison of laboratory data between groups with different HEV serological statuses was performed. RESULTS: HEV IgG antibodies were detected in 40.82% of 904 serum samples, while HEV IgM antibodies were detected in 11.75% of 1081 serum samples. Similar IgG and IgM antibody detection rates were found in pregnant women. Interestingly, anti-HEV IgM antibodies were detected in 38.5% of patients who underwent organ transplantation. Patients who tested positive for anti-HEV IgM antibodies had higher alanine aminotransferase levels than those who had not. In contrast, patients who tested positive for anti-HEV IgG had more elevated levels of total bilirubin than those who tested negative. CONCLUSIONS: HEV seroprevalence and incidence in patients with clinical hepatitis were relatively high in the Thai population, including the pregnancy and organ transplant subgroups. The results potentially benefit the clinicians in decision-making to investigate HEV antibodies and facilitating proper management for patients.
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Anticorpos Anti-Hepatite/sangue , Hepatite E , Estudos Transversais , Feminino , Hepatite E/epidemiologia , Vírus da Hepatite E/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Gravidez , Prevalência , Estudos Soroepidemiológicos , Centros de Atenção Terciária , Tailândia/epidemiologia , TransplantadosRESUMO
BACKGROUND: The epidemiology and outcomes of COVID-19 patients in Thailand are scarce. METHODS: This retrospective cohort study included adult hospitalized patients who were diagnosed with COVID-19 at Siriraj Hospital during February 2020 to April 2020. RESULTS: The prevalence of COVID-19 was 7.5% (107 COVID-19 patients) among 1409 patients who underwent RT-PCR for SARS-CoV-2 detection at our hospital during the outbreak period. Patients with COVID-19 presented with symptoms in 94.4%. Among the 104 patients who were treated with antiviral medications, 78 (75%) received 2-drug regimen (lopinavir/ritonavir or darunavir/ritonavir plus chloroquine or hydroxychloroquine), and 26 (25%) received a 3-drug regimen with favipiravir added to the 2-drug regimen. Disease progression was observed in 18 patients (16.8%). All patients with COVID-19 were discharged alive. CONCLUSIONS: The prevalence of COVID-19 was 7.5% among patients who underwent RT-PCR testing, and 10% among those having risk factors for COVID-19 acquisition. Combination antiviral therapies for COVID-19 patients were well-tolerated and produced a favorable outcome.
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COVID-19/epidemiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Amidas/uso terapêutico , Antivirais/uso terapêutico , Cloroquina/uso terapêutico , Darunavir/uso terapêutico , Progressão da Doença , Combinação de Medicamentos , Feminino , Hospitais , Hospitais Universitários , Humanos , Hidroxicloroquina/uso terapêutico , Lopinavir/uso terapêutico , Masculino , Pessoa de Meia-Idade , Pirazinas/uso terapêutico , Encaminhamento e Consulta , Estudos Retrospectivos , Ritonavir/uso terapêutico , Tailândia/epidemiologia , Resultado do Tratamento , Adulto Jovem , Tratamento Farmacológico da COVID-19RESUMO
BACKGROUND: The Coronavirus disease 2019 (COVID-19) pandemic continues to spread across the world. Hence, there is an urgent need for rapid, simple, and accurate tests to diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Performance characteristics of the rapid SARS-CoV-2 antigen detection test should be evaluated and compared with the gold standard real-time reverse transcription-polymerase chain reaction (RT-PCR) test for diagnosis of COVID-19 cases. METHODS: The rapid SARS-CoV-2 antigen detection test, Standard™ Q COVID-19 Ag kit (SD Biosensor®, Republic of Korea), was compared with the real-time RT-PCR test, Allplex™ 2019-nCoV Assay (Seegene®, Korea) for detection of SARS-CoV-2 in respiratory specimens. Four hundred fifty-four respiratory samples (mainly nasopharyngeal and throat swabs) were obtained from COVID-19 suspected cases and contact individuals, including pre-operative patients at Siriraj Hospital, Bangkok, Thailand during March-May 2020. RESULTS: Of 454 respiratory samples, 60 (13.2%) were positive, and 394 (86.8%) were negative for SARS-CoV-2 RNA by real-time RT-PCR assay. The duration from onset to laboratory test in COVID-19 suspected cases and contact individuals ranged from 0 to 14 days with a median of 3 days. The rapid SARS-CoV-2 antigen detection test's sensitivity and specificity were 98.33% (95% CI, 91.06-99.96%) and 98.73% (95% CI, 97.06-99.59%), respectively. One false negative test result was from a sample with a high real-time RT-PCR cycle threshold (Ct), while five false positive test results were from specimens of pre-operative patients. CONCLUSIONS: The rapid assay for SARS-CoV-2 antigen detection showed comparable sensitivity and specificity with the real-time RT-PCR assay. Thus, there is a potential use of this rapid and simple SARS-CoV-2 antigen detection test as a screening assay.
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Teste de Ácido Nucleico para COVID-19 , Teste Sorológico para COVID-19 , COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , Adulto , Idoso , Antígenos Virais/análise , COVID-19/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , RNA Viral/genética , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Sensibilidade e Especificidade , Tailândia/epidemiologia , Fatores de Tempo , Adulto JovemRESUMO
Nucleic acid detection by isothermal amplification and the collateral cleavage of reporter molecules by CRISPR-associated enzymes is a promising alternative to quantitative PCR. Here, we report the clinical validation of the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) assay using the enzyme Cas13a from Leptotrichia wadei for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the virus that causes coronavirus disease 2019 (COVID-19)-in 154 nasopharyngeal and throat swab samples collected at Siriraj Hospital, Thailand. Within a detection limit of 42 RNA copies per reaction, SHERLOCK was 100% specific and 100% sensitive with a fluorescence readout, and 100% specific and 97% sensitive with a lateral-flow readout. For the full range of viral load in the clinical samples, the fluorescence readout was 100% specific and 96% sensitive. For 380 SARS-CoV-2-negative pre-operative samples from patients undergoing surgery, SHERLOCK was in 100% agreement with quantitative PCR with reverse transcription. The assay, which we show is amenable to multiplexed detection in a single lateral-flow strip incorporating an internal control for ribonuclease contamination, should facilitate SARS-CoV-2 detection in settings with limited resources.
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COVID-19/diagnóstico , Proteínas Associadas a CRISPR/genética , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/genética , SARS-CoV-2/genética , COVID-19/virologia , Humanos , Leptotrichia/enzimologia , Pandemias/prevenção & controleRESUMO
BACKGROUND: Respiratory syncytial virus (RSV) is an important virus found in adult hospitalized patients. OBJECTIVES: To study the clinical outcomes of hospitalized patients aged ≥ 15 years and diagnosed with RSV infection. STUDY DESIGN: Both retrospective and prospective cohort studies were conducted at a university hospital between May 2014 and December 2015. RESULTS: RSV was detected in 86 of 1562(5.5%) adult hospitalized patients suspected of respiratory viral infection. Sixty-nine patients were included in the study. RSV was detected by RT-PCR (82.6%), IFA (10.1%), and both RT-PCR and IFA (7.3%). Most patients (87.0%) were aged ≥ 50 years. Cardiovascular diseases, pulmonary diseases, immunocompromised hosts, and diabetes were the major comorbidities. The common manifestations were cough (92.8%), dyspnea (91.3%), sputum production (87.0%), tachypnea (75.4%), wheezing (73.9%), and fever (71.0%). Fifty- five patients (79.7%) were diagnosed with pneumonia. Hypoxemia (SpO2â¯≤â¯92%) was found in 53.6% patients. Twenty-five of 69(36.2%) patients developed respiratory failure and required ventilatory support. Cardiovascular complications were found in 24.6% of patients. Congestive heart failure, acute myocardial infarction (MI), new atrial fibrillation, and supraventricular tachycardia were found in 9(13.0%), 7(10.1%), 4(5.8%), and 3(4.3%) of 69 patients, respectively. Overall mortality was 15.9%. Pneumonia (81.8%) and acute MI (18.2%) were the major causes of death. CONCLUSIONS: Most adult hospitalized patients with RSV infection were of advanced age and had comorbidities. Cardiopulmonary complications were the major causes of death. Management and prevention of RSV infection in these vulnerable groups are necessary.
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Doenças Cardiovasculares/epidemiologia , Insuficiência Respiratória/epidemiologia , Infecções por Vírus Respiratório Sincicial/epidemiologia , Vírus Sincicial Respiratório Humano/isolamento & purificação , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Doenças Cardiovasculares/etiologia , Comorbidade , Feminino , Hospitalização , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular , Mortalidade , Estudos Prospectivos , Insuficiência Respiratória/etiologia , Infecções por Vírus Respiratório Sincicial/complicações , Vírus Sincicial Respiratório Humano/genética , Estudos Retrospectivos , Tailândia/epidemiologia , Adulto JovemRESUMO
Long-read nanopore sequencing by a MinION device offers the unique possibility to directly sequence native RNA. We combined an enzymatic poly-A tailing reaction with the native RNA sequencing to (i) sequence complex population of single-stranded (ss)RNA viruses in parallel, (ii) detect genome, subgenomic mRNA/mRNA simultaneously, (iii) detect a complex transcriptomic architecture without the need for assembly, (iv) enable real-time detection. Using this protocol, positive-ssRNA, negative-ssRNA, with/without a poly(A)-tail, segmented/non-segmented genomes were mixed and sequenced in parallel. Mapping of the generated sequences on the reference genomes showed 100% length recovery with up to 97% identity. This work provides a proof of principle and the validity of this strategy, opening up a wide range of applications to study RNA viruses.
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BACKGOUND: Intravenous drug users (IVDUs) are among the high-risk groups who are most vulnerable to HIV infection. Several illicit drugs alter host immune function with increased incidence of infections including that of HIV. Many studies of the immune response of NK cells in HIV-1 seronegative IVDUs and HIV-1 seropositive IVDUs have been published from the Western countries and yet no data is available from Thailand. OBJECTIVE: To determine natural killer cell cytotoxicity and lymphocyte subsets in Thai HIV-1 infected intravenous drug users. METHODS: The NK cell cytotoxic function was determined using our well-established EGFP-K562 flow cytometric assay in 30 IVDUs with HIV-1 infection (IVH) comparing with those from the same number of non-infected IVDUs (IVX), HIV-1 seropositive individuals (HIV-1+ve) and healthy controls. The percentage and the absolute number of NK cells, helper CD4+ T cells and cytotoxic CD8+ T cells were also investigated. RESULTS: Among the study groups, IVH showed not only the lowest percentage of lytic activity by NK cells, but also a decline in the percentage and absolute count of NK cells. A decline in helper CD4+ T cells and an increase of cytotoxic CD8+ T cells of IVH group when compared to those of other 3 groups were also demonstrated. CONCLUSIONS: The failure of innate immune NK cell function and their number in IVH may support the involvement of additional components of the immune system in the control of HIV-1 disease.
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Citotoxicidade Imunológica , Usuários de Drogas/estatística & dados numéricos , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos/imunologia , Adulto , Contagem de Linfócito CD4 , Relação CD4-CD8 , Linhagem Celular , Feminino , Infecções por HIV/epidemiologia , Infecções por HIV/transmissão , Humanos , Células Matadoras Naturais/metabolismo , Subpopulações de Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Vigilância da População , Estudos Soroepidemiológicos , Tailândia/epidemiologia , Adulto JovemRESUMO
BACKGROUND: There have been a few studies aimed at identifying epitopes of ADCC-inducing antibodies when compared to those of neutralizing antibodies and cytotoxic T lymphocytes against a variety of HIV-1 clades. OBJECTIVE: To map the common ADCC epitopes of HIV-1 CRF01_AE. METHODS: We screened 65 sera of confirmed early HIV-1 CRF01_AE infected individuals for ADCC antibody against gp120 utilizing an EGFP-CEM-NKr flow cytometric assay. Sera with high ADCC antibody were then examined against ADCC epitopes using the complete HIV-1 CRF01_AE gp160- and subtype A Gag-overlapping peptide sets which were divided into 7 pools:E1-E7 and 5 pools:G1-G5, respectively. Each positive peptide pool was further investigated for fine ADCC epitope mapping using matrix formats. RESULTS: Twenty, 25 and 20 sera demonstrated the high-, medium- and low-ADCC antibody activities against gp120, respectively. Interestingly, 11 Env- and 6 Gag-peptides of pools E3, E4, E7 and pools G1, G2, G4 with high ADCC responses were also responded by at least 20%, 12% and 5%, 10% of medium- and low-ADCC antibody sera, respectively. These eleven common Env ADCC epitopes were localized at C2-V3-C3-V4 regions of gp120 and cytoplasmic tail of gp41 while six common Gag ADCC epitopes were localized at p17-p24-p2 regions. CONCLUSIONS: Although the degree of ADCC antibody responses to the gp120 protein varied from high to low, there were certain consensus Env and Gag peptides that could induce the ADCC antibody responses of 21.54-58.46% and 23.08-41.54%, respectively of the early infected individuals. This epitope information should be useful as the new antibody-based vaccine immunogens.
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Citotoxicidade Celular Dependente de Anticorpos/imunologia , Epitopos/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/imunologia , Epitopos/química , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/genética , Humanos , Soros Imunes/imunologia , Peptídeos/química , Peptídeos/imunologiaRESUMO
We present here the complete genome sequences of Zika virus strains isolated from aborted fetal tissue (brain and placenta) and amniotic fluid of a microcephaly patient in Thailand in 2017. The virus genomes that were sequenced have an average length of 10,807 nucleotides.
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We sequenced the virus genomes from 3 pregnant women in Thailand with Zika virus diagnoses. All had infections with the Asian lineage. The woman infected at gestational week 9, and not those infected at weeks 20 and 24, had a fetus with microcephaly. Asian lineage Zika viruses can cause microcephaly.
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Microcefalia/diagnóstico , Complicações Infecciosas na Gravidez , Infecção por Zika virus , Zika virus/isolamento & purificação , Feminino , Humanos , Recém-Nascido , Microcefalia/etiologia , Gravidez , Primeiro Trimestre da Gravidez , Tailândia , Zika virus/genéticaRESUMO
BACKGROUND: Although smallpox was completely eliminated by 1980, it remains possible that variola virus could be intentionally released in an act of bioterrorism. Thus, several studies have been performed to detect antibody levels after smallpox vaccination of the current population in various countries to indicate the duration of maintenance of immunological memory. Our study endeavored to investigate the level of neutralizing (Nt) antibody responses of Thai individuals who had been immunized with smallpox vaccine during childhood. METHODS: The plaque reduction neutralization test (PRNT) was used to study vaccinia Nt antibody responses in sera of individuals ranging in age from 35-4, 45-54, 55-64, 65-74, 75-84 and > 84 years old, referred to as groups 1-6, respectively. Each group included 200 sera: 100 male sera and 100 female sera. RESULTS: An incubation time of 15 hours for sera and vaccinia virus was confirmed to be the optimal incubation period for PRNT. Positive Nt antibody titers (≥32) were detected in 135 (11.25%) of 1,200 sera: 81 (6.75%) male sera and 54 (4.5%) female sera. There were 4 (2%), 11 (5.5%), 19 (9.5%), 16 (8%), 33 (16.5%), and 52 (26%) positive sera in groups 1-6, respectively. Interestingly, the oldest individual with positive Nt antibody was a 98-year-old female. Two males aged 96 and 91 years old had the highest Nt antibody titers. CONCLUSIONS: Our data suggests that the vaccinia-specific Nt antibody response in the current Thai population could be maintained for more than 90 years after vaccination. However, the majority of the Thai population aged ≥35-74 years old is still highly susceptible to infection.
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Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vacina Antivariólica/imunologia , Varíola/imunologia , Vírus da Varíola/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Bioterrorismo , Feminino , Humanos , Memória Imunológica , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Tailândia , VacinaçãoRESUMO
This article was migrated. The article was marked as recommended. Background: A thorough understanding of infectious diseases is needed by medical professionals; therefore effective microbiological teaching is critical. Although faculty lectures are a convenient means of educating large groups of students, they may fail to engage students and convey an understanding of the subject. Therefore, we developed peer teaching methods based on game-based learning using a reality musical talent show format. Methods: A group of student representatives were trained to lecture to a class of 300 third-year medical students via a game show format over a 3-year period (2013-2015). Results: The students reported a higher level of understanding (3.6-4.2 vs 3.6-3.9 out of 5; p Conclusions: Peer teaching did improve the students' attitude towards learning and conferred teaching skills, but the learning activity needs adjustment to reduce the out-of-class preparation time.
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BACKGROUND: There have been very few reports of HIV-1 subtypes and drug resistance mutations (DRMs) from Nepal which is geographically located between two high-prevalence HIV-1 infection countries, China and India. OBJECTIVE: The aim of this study was to determine prevalence of acquired and transmitted DRMs and HIV-1 subtypes in Nepal. METHODS: Thirty-five HIV-1 seropositive samples from central region of Nepal were collected in 2011. The subjects were divided into two groups, antiretroviral (ARV) drug naïve group (n=15) and antiretroviral treatment (ART) group (n=20), 90% (18/20) of them received zidovudine, lamivudine and nevirapine (AZT/3TC/NVP) regimen. HIV pol (protease and reverse transcriptase regions) nucleotide sequences were analyzed by Viroseq HIV-1 Genotyping System. Nearly full-length genomic (NFLG) sequences of 10 samples were performed. RESULTS: NFLG genotyping revealed that 80% of samples were infected with subtype C and 20% with recombinants (C/D/H and C/A). Phylogenetic analysis of 35 pol sequences from Nepal were subtype C. The prevalence of acquired DRMs to NNRTIs and NRTIs was 15% (3/20). DRMs to NVP, K103N and V179D, and to NRTIs were observed at 11.1% (2/18) and 5% (1/20), respectively. The prevalence of DRMs to rilpivirine for E138A/G was 5.7%. The minor protease inhibitors (PI) associated mutations (A71T/V and T74S) were observed in 5/35 (14.3%) subjects. CONCLUSION: This is the first report of NFLG HIV-1 genomic sequences and DRMs from Nepal. National surveillance of HIV DRMs to ARVs and molecular epidemiology study should be done annually for better prevention and treatment of HIV infection in Nepal.
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Farmacorresistência Viral , Genótipo , Infecções por HIV/virologia , HIV-1/classificação , HIV-1/efeitos dos fármacos , Mutação , Adulto , Antirretrovirais/uso terapêutico , Criança , Feminino , Infecções por HIV/tratamento farmacológico , HIV-1/genética , HIV-1/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Nepal , Prevalência , Análise de Sequência de DNA , Adulto Jovem , Produtos do Gene pol do Vírus da Imunodeficiência Humana/genéticaRESUMO
Influenza B virus, which causes acute respiratory infections, has increased in prevalence in recent years. Based on the nucleotide sequence of the hemagglutinin (HA) gene, influenza B virus can be divided into two lineages, Victoria and Yamagata, that co-circulate during the influenza season. However, analysis of the potential association between the clinical and virological characteristic and the lineage of influenza B viruses isolated in Thailand was lacking. To investigate influenza B virus genetically and determine its neuraminidase (NA) inhibitor susceptibility phenotype, a total of 6920 nasopharyngeal-wash samples were collected from patients with influenza-like illness between the years 2011 and 2014 and were screened for influenza B virus by real-time PCR. Of these samples, 3.1% (216/6920) were confirmed to contain influenza B viruses, and 110 of these influenza viruses were randomly selected for nucleotide sequence analysis of the HA and NA genes. Phylogenetic analysis of the HA sequences showed clustering into various clades: Yamagata clade 3 (11/110, 10%), Yamagata clade 2 (71/110, 64.5%), and Victoria clade 1 (28/110, 25.5%). The analysis of clinical characteristic demonstrated that the Victoria lineage was significantly associated with the duration of hospitalization, number of deceased cases, pneumonia, secondary bacterial infection and underlying disease. When combined with phylogenetic analysis of the NA sequences, four samples showed viruses with reassortant sequences between the Victoria and Yamagata lineages. Statistical analysis of the clinical outcomes and demographic data for the reassortant strains did not differ from those of the other strains in circulation. Oseltamivir-resistant influenza B viruses were not detected. Our findings indicated the co-circulation of the Victoria and Yamagata lineages over the past four cold seasons in Bangkok. We also demonstrated differences in the clinical symptoms between these lineages.
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Vírus da Influenza B , Influenza Humana/epidemiologia , Influenza Humana/virologia , Centros de Atenção Terciária , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Análise por Conglomerados , Análise Mutacional de DNA , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Neuraminidase/antagonistas & inibidores , Neuraminidase/genética , Oseltamivir/uso terapêutico , Fenótipo , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Tailândia , Adulto JovemRESUMO
The antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism involves both innate and adaptive immune systems. While a number of epitope mapping studies of neutralizing (Nt) antibodies and cytotoxic T lymphocyte (CTL) against a variety of HIV-1 clades have been reported, there has been a paucity of similar studies aimed at identifying epitopes of ADCC-inducing antibodies. Herein we screened 35 sera from HIV-1 CRF01_AE-infected blood donors for ADCC antibody activity against gp120 utilizing an EGFP-CEM-NK(r) flow cytometric assay. Eighteen sera with high ADCC antibody activity were then comprehensively examined for ADCC antibody epitopes using the HIV-1 subtype CRF01_AE TH023 gp120 peptide set consisting of 126 peptides of 15 amino acids in length, overlapping by 11 amino acids. This peptide set was divided into five pools (E1-E5). Each positive peptide pool was further investigated for fine epitope mapping of ADCC antibody activity using a 5 by 5 peptide matrix format. Interestingly, six and three peptides from peptide pools E1 and E2, respectively, responded to at least 33.33% of the tested sera. These nine common ADCC epitopes were localized to the C1 and V2 region of gp120. Furthermore, 5/9 epitopes were also shown to serve as full or partial Nt antibody targets for HIV-1 subtypes B and CRF01_AE. We submit these data on epitope mapping of ADCC or dual ADCC-Nt antibodies against HIV-1 gp120 that should be considered in the formulation of vaccines against HIV-1.
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Citotoxicidade Celular Dependente de Anticorpos/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Soropositividade para HIV/imunologia , HIV-1/imunologia , Anticorpos Neutralizantes/imunologia , Citotoxicidade Celular Dependente de Anticorpos/genética , Epitopos/genética , Feminino , Citometria de Fluxo , Anticorpos Anti-HIV/genética , Proteína gp120 do Envelope de HIV/genética , Soropositividade para HIV/genética , HIV-1/genética , HIV-1/fisiologia , Humanos , Masculino , Testes de Neutralização , Vacinas ViraisRESUMO
BACKGROUND: Conserved neutralizing epitopes are considered to be a key role for eliciting broadly neutralizing antibody (NAb). Previously, two conserved neutralizing epitopes of HIV-1 CRF01_AE envelope were identified at amino acid 93-112 of the C1 (C1E) and at 218-239 of the C2 (C2E) regions. To access the potency of antibody directed against conserved epitopes, a monoclonal antibody (MAb) specific to the C2E region was developed and characterized. METHODS: The immunogenicity of two epitopes was examined by immunizing BALB/c mice with the matching synthetic peptides. One MAb, C2EB5, directed against peptide C2E, was generated by conventional methods, while C1E1 and C1E2 peptides induced slight antibody response in mice. The neutralizing activity of MAb C2EB5 was examined using a peripheral blood mononuclear cell (PBMC) based method and various HIV-1 subtypes including A, B, C, D, and CRF01_AE; C2EB5 was compared with other known neutralizing MAbs (4E10, 447-52D) and with sCD4. The exposure of the C2 epitope on native virus was investigated using virus capture by these MAbs. RESULTS: The MAb C2EB5 demonstrated cross-neutralization against various HIV-1 subtypes. The overall potency of MAb C2EB5 against 5 subtypes was ranked in the following order: subtype C> CRF01_AE> subtype D> subtype A> subtype B. The epitope exposure for MAb C2EB5 was also correlated with the neutralization properties of each subtype. CONCLUSION: This study demonstrates the cross-clade neutralizing activity of a MAb directed against an epitope located in the C2 region of the HIV-1 env and highlights differences in the exposure of antigenic epitopes on the surface of various HIV-1 subtypes. The epitope for this newly identified neutralizing MAb made against a subtype CRF01_AE peptide is particularly exposed in subtype C viral isolates.
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HIV-1 tat gene function and immunogenicity of HIV-1 Tat protein from 3 low (PS01, PS40, PS58) and 3 high (PS19, PS65, LP22) viral load infected, untreated and asymptomatic individuals from Thailand were compared. Levels of Tat-dependent chloramphenicol acetyltransferase (CAT) induced in HL3T1 cells with tat1 gene from HIV-1 isolates of high viral load group was significantly higher than those from low viral load group. HIV-1 subtype determination using env (C2-V4) gene demonstrated that 2/3 (PS01 and PS40) and 1/3 (PS58) from low viral load group were CRF01_AE and subtype B, while all 3 HIV-1 isolates from high viral load group were CRF01_AE. However, all 3 HIV-1 tat nucleotide sequences from low viral load group, which contained env CRF01_AE sequence, belonged to subtype B whereas all those from high viral load group contained CRF01_AE sequence. HIV Tat recombinant proteins from these groups were tested for immunogenicity in mice. All recombinant Tat proteins (except from PS58) were immunogenic in a dose-dependent manner, but with significantly differences of the immunogenicity levels between high and low viral load groups. These results indicated that HIV-1 subtype B tat gene activities might be associated with reduced disease progression of HIV-1 CRF01_AE infected individuals.
Assuntos
Genes tat/fisiologia , Infecções por HIV/virologia , HIV-1/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/fisiologia , Adulto , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Progressão da Doença , Epitopos/genética , Feminino , HIV-1/imunologia , Humanos , Masculino , Camundongos , Proteínas Recombinantes , Carga Viral , Adulto JovemRESUMO
The recombinant envelope protein (gp120) of the human immunodeficiency virus type 1 (HIV-1) CRF01_AE env gene isolated from the corresponding blood (rgp120-F36PC) and genital fluid (rgp120-F36VC) specimens obtained from HIV infected individuals was successfully produced in both prokaryote and eukaryote cells. The yields of HIV-1 recombinant envelope proteins rgp120-F36PC and rgp120-F36VC produced in E. coli and in mammalian cells were 1.0 and 1.2, and 0.3 and 0.5 mg/ml, respectively. Antibody responses in mice immunized with rgp120-F36VC protein were not significantly higher than those with rgp120-F36PC protein. The level of antibody response in mice immunized with V3 deleted recombinant gp120 proteins from rgp120-F36VC and rgp120-F36PC was not significantly different from wild type rgp120 proteins. beta-strands at the tip of the V3 loop of the HIV-1 envelope protein were predicted for the wild type genital fluid isolate but not for the wild type blood isolate. The replication capacity of both F36PC and F36VC was quite efficient. The infectivity assay of the epithelial cell line for pNL4-3/gp120F36VC was better than for pNL4-3/gp120F36PC. The extra beta-strands in the V3 loop may be involved in cell tropism.
Assuntos
Líquidos Corporais/virologia , Anticorpos Anti-HIV , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , Animais , Feminino , Proteína gp120 do Envelope de HIV/sangue , Infecções por HIV/imunologia , Infecções por HIV/virologia , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/imunologia , Vagina/virologiaRESUMO
NC37 cells containing the Epstein-Barr virus (EBV) genome do not express the viral glycoprotein-350 (gp350) on the cell surface. Despite being a cancer cell line, NC37 cells show resistance to natural killer (NK) cell cytotoxicity by the standard chromium ((51)Cr) release assay (CRA). EBV-gp350 has been identified as a ligand for antibody dependent cell-mediated cytotoxicity (ADCC). The stable expression of gp350 on the NC37 cell surface membrane could make this cell line a suitable target for measuring ADCC antibody. The pcDNA3.1-gp350 was transfected into the stably expressing enhanced green fluorescent protein (EGFP)-NC37 cell line. The transfected cells were then selected for expression of gp350 on the cell surface using immunomagnetic bead-based sorting. The gp350-EGFP-NC37 cell line was then re-examined for resistance to NK cytotoxicity, and compared with the standard K562 and EGFP-K562 cell lines using the CRA and a flow cytometric method, respectively. Surprisingly, the gp350-EGFP-NC37 cells, like the parental NC37 cell line, showed comparable resistance to NK cell-mediated cytotoxic activity by the CRA, while demonstrating susceptibility to NK cell cytotoxicity comparable to EGFP expressing K562 cells by the flow cytometric method. The susceptibility of gp350-EGFP-NC37 cells to NK cell cytotoxic activity is dependent on the type of assay.