RESUMO
Since the coronavirus disease 2019 (COVID-19) outbreak, laboratory diagnosis has mainly been conducted using reverse-transcription polymerase chain reaction (RT-PCR). Detecting the presence of an infectious virus in the collected sample is essential to analyze if a person can transmit infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there have been no quantitative investigations conducted for infectious SARS-CoV-2 in clinical samples. Therefore, in the present study, a rapid and simple focus-forming assay using the peroxidase-antiperoxidase technique was developed to quantify infectious SARS-CoV-2 titers in 119 samples (n = 52, nasopharyngeal swabs [NPS]; n = 67, saliva) from patients with COVID-19. Furthermore, the study findings were compared with the cycle threshold (Ct) values of real-time RT-PCR. The infectious virus titers in NPS samples and Ct values were inversely correlated, and no infectious virus could be detected when the Ct value exceeded 30. In contrast, a low correlation was observed between the infectious virus titers in saliva and Ct values (r = -0.261, p = 0.027). Furthermore, the infectious virus titers in the saliva were significantly lower than those in the NPS samples. Ten days after the onset of COVID-19 symptoms, the infectious virus was undetectable, and Ct values were more than 30 in NSP and saliva samples. The results indicate that patients whose symptoms subsided 10 days after onset, with Ct values more than 30 in NSP and saliva samples, were less likely to infect others.
Assuntos
Teste para COVID-19 , COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , Ensaio de Placa Viral , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , COVID-19/virologia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saliva/virologia , Carga Viral , Adulto JovemRESUMO
BACKGROUND: There are several types of coronaviruses that infect humans and cause disease. The latest is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is an emerging global threat with no current effective treatment. Normal intravenous immunoglobulin (N-IVIG) has been administered to coronavirus disease 2019 (COVID-19) patients to control severe inflammation and the cellular immune response. However, the neutralizing activity of N-IVIG against SARS-CoV-2 has not yet been fully evaluated. The aim of this study was to determine whether N-IVIG manufactured before the start of the COVID-19 pandemic contained IgG antibodies against the circulating human coronaviruses (HCoVs) that cross-react with the highly pathogenic coronaviruses SARS-CoV-1, Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. No cases of SARS-CoV-1 or MERS-CoV have been reported in Japan. STUDY DESIGN AND METHODS: The neutralizing and binding activities of N-IVIG against SARS-CoV-1, MERS-CoV, SARS-CoV-2, HCoV 229E, and HCoV OC43 were evaluated. Nine N-IVIG lots manufactured between 2000 and 2018, derived from donors in Japan, were tested. Binding activity was evaluated by indirect immunofluorescence assay. RESULTS: None of the N-IVIG lots tested displayed neutralizing or binding activity against SARS-CoV-1, MERS-CoV, or SARS-CoV-2. However, they displayed substantial neutralizing and binding activity against HCoV OC43 and weak neutralizing and substantial binding activity against HCoV 229E. CONCLUSION: N-IVIG derived from healthy donors in Japan before the start of the COVID-19 pandemic had no direct effect against SARS-CoV-2. Further studies are warranted to determine the effects of N-IVIG manufactured after the start of the COVID-19 pandemic against SARS-CoV-2.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Coronavirus Humano 229E/imunologia , Coronavirus Humano OC43/imunologia , Imunoglobulinas Intravenosas/imunologia , Imunoglobulinas Intravenosas/metabolismo , Humanos , Imunidade Celular/fisiologia , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Japão , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Pandemias , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
Antigen tests for infectious diseases are inexpensive and easy-to-use, but the limit of detection (LOD) is generally higher than that of PCR tests, which are considered the gold standard. In the present study, we combined a sandwich enzyme-linked immunosorbent assay (ELISA) with thionicotinamide-adenine dinucleotide (thio-NAD) cycling to improve the LOD of antigen tests for coronavirus disease 2019 (COVID-19). For recombinant nucleocapsid proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the LOD of our ELISA with thio-NAD cycling was 2.95 × 10-17 moles/assay. When UV-irradiated inactive SARS-CoV-2 was used, the minimum detectable virions corresponding to 2.6 × 104 RNA copies/assay were obtained using our ELISA with thio-NAD cycling. The assay volume for each test was 100 µL. The minimum detectable value was smaller than that of the latest antigen test using a fluorescent immunoassay for SARS-CoV-2, indicating the validity of our detection system for COVID-19 diagnosis.
Assuntos
Anticorpos Antivirais , Teste para COVID-19/métodos , COVID-19/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , NAD/análogos & derivados , Proteínas do Nucleocapsídeo/imunologia , SARS-CoV-2 , Antígenos Virais , COVID-19/virologia , Humanos , Testes Imunológicos , Limite de Detecção , Nucleocapsídeo/análise , Sensibilidade e EspecificidadeRESUMO
Four serotypes of dengue virus (DENV), type 1 to 4 (DENV-1 to DENV-4), exhibit approximately 25-40% of the difference in the encoded amino acid residues of viral proteins. Reverse transcription of RNA extracted from specimens followed by PCR amplification is the current standard method of DENV serotype determination. However, since this method is time-consuming, rapid detection systems are desirable. We established several mouse monoclonal antibodies directed against DENV non-structural protein 1 and integrated them into rapid DENV detection systems. We successfully developed serotype-specific immunochromatography systems for all four DENV serotypes. Each system can detect 104 copies/mL in 15 min using laboratory and clinical isolates of DENV. No cross-reaction between DENV serotypes was observed in these DENV isolates. We also confirmed that there was no cross-reaction with chikungunya, Japanese encephalitis, Sindbis, and Zika viruses. Evaluation of these systems using serum from DENV-infected individuals indicated a serotype specificity of almost 100%. These assay systems could accelerate both DENV infection diagnosis and epidemiologic studies in DENV-endemic areas.
Assuntos
Vírus da Dengue , Dengue , Infecção por Zika virus , Zika virus , Animais , Anticorpos Monoclonais , Anticorpos Antivirais , Antígenos Virais , Cromatografia de Afinidade , Dengue/diagnóstico , Ensaio de Imunoadsorção Enzimática , Camundongos , Sensibilidade e Especificidade , Sorogrupo , Proteínas não Estruturais ViraisRESUMO
Vasorelaxation activity guided separation of the methanol extract of Calophyllum scriblitifolium bark led to the isolation of 6 chromanones (calofolic acids A-F, 1-6). Their structures were elucidated by 1D and 2D NMR spectroscopy, and their absolute configurations were investigated by a combination of CD spectroscopy and DFT calculation. All isolated chromanones showed dose-dependent vasorelaxation activity on isolated rat aorta.
Assuntos
Calophyllum/química , Cromonas/química , Vasodilatadores/química , Animais , Aorta/efeitos dos fármacos , Calophyllum/metabolismo , Cromonas/isolamento & purificação , Cromonas/farmacologia , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Conformação Molecular , Casca de Planta/química , Casca de Planta/metabolismo , Extratos Vegetais/química , Ratos , Vasodilatadores/isolamento & purificação , Vasodilatadores/farmacologiaRESUMO
In previous studies, hybridomas producing human immunoglobulin G, the antibodies 5E4 and 5A7 against influenza A and B virus were established using a novel human lymphocyte fusion partner, SPYMEG. In the present study, we succeeded in achieving the recombinant production and secretion of 5E4 and 5A7 in Chinese hamster ovary cells. Our N-glycan analysis by intact-mass detection and liquid chromatography mass spectrometry showed that recombinant 5E4 and 5A7 have one N-glycan and the typical mammalian-type N-glycan structures similar to those in hybridomas. However, the glycan distribution was slightly different among these antibodies. The amount of high-mannose-type structures was under 10% of the total N-glycans of recombinant 5E4 and 5A7, compared to 20% of the 5E4 and 5A7 produced in hybridomas. The amount of galactosylated N-glycans was increased in recombinants. Approximately 80% of the N-glycans of all antibodies was fucosylated, and no sialylated N-glycan was found. Recombinant 5E4 and 5A7 neutralized pandemic influenza A virus specifically, and influenza B virus broadly, quite similar to the 5E4 and 5A7 produced in hybridomas, respectively. Here we demonstrated that recombinants of antibodies identified from hybridomas fused with SPYMEG have normal N-glycans and that their neutralizing activities bear comparison with those of the original antibodies.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Imunoglobulina G/imunologia , Vírus da Influenza A/imunologia , Vírus da Influenza B/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/sangue , Fusão Celular/métodos , Cricetinae , Humanos , Hibridomas/imunologia , Hibridomas/metabolismo , Imunoglobulina G/biossíntese , Linfócitos/imunologia , Linfócitos/metabolismoRESUMO
Chikungunya fever is a mosquito-borne disease of key public health importance in tropical and subtropical countries. Although severe joint pain is the most distinguishing feature of chikungunya fever, diagnosis remains difficult because the symptoms of chikungunya fever are shared by many pathogens, including dengue fever. The present study aimed to develop a new immunochromatographic diagnosis test for the detection of chikungunya virus antigen in serum. Mice were immunized with isolates from patients with Thai chikungunya fever, East/Central/South African genotype, to produce mouse monoclonal antibodies against chikungunya virus. Using these monoclonal antibodies, a new diagnostic test was developed and evaluated for the detection of chikungunya virus. The newly developed diagnostic test reacted with not only the East/Central/South African genotype but also with the Asian and West African genotypes of chikungunya virus. Testing of sera from patients suspected to have chikungunya fever in Thailand (n = 50), Laos (n = 54), Indonesia (n = 2), and Senegal (n = 6) revealed sensitivity, specificity, and real-time PCR (RT-PCR) agreement values of 89.4%, 94.4%, and 91.1%, respectively. In our study using serial samples, a new diagnostic test showed high agreement with the RT-PCR within the first 5 days after onset. A rapid diagnostic test was developed using mouse monoclonal antibodies that react with chikungunya virus envelope proteins. The diagnostic accuracy of our test is clinically acceptable for chikungunya fever in the acute phase.
Assuntos
Antígenos Virais/sangue , Febre de Chikungunya/diagnóstico , Vírus Chikungunya/isolamento & purificação , Cromatografia de Afinidade/métodos , Testes Diagnósticos de Rotina/métodos , Soro/virologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Febre de Chikungunya/virologia , Humanos , Indonésia , Camundongos Endogâmicos BALB C , Senegal , Sensibilidade e Especificidade , Tailândia , Fatores de TempoRESUMO
Influenza virus has the ability to evade host immune surveillance through rapid viral genetic drift and reassortment; therefore, it remains a continuous public health threat. The development of vaccines producing broadly reactive antibodies, as well as therapeutic strategies using human neutralizing monoclonal antibodies (HuMAbs) with global reactivity, has been gathering great interest recently. Here, three hybridoma clones producing HuMAbs against influenza B virus, designated 5A7, 3A2 and 10C4, were prepared using peripheral lymphocytes from vaccinated volunteers, and were investigated for broad cross-reactive neutralizing activity. Of these HuMAbs, 3A2 and 10C4, which recognize the readily mutable 190-helix region near the receptor binding site in the hemagglutinin (HA) protein, react only with the Yamagata lineage of influenza B virus. By contrast, HuMAb 5A7 broadly neutralizes influenza B strains that were isolated from 1985 to 2006, belonging to both Yamagata and Victoria lineages. Epitope mapping revealed that 5A7 recognizes 316G, 318C and 321W near the C terminal of HA1, a highly conserved region in influenza B virus. Indeed, no mutations in the amino acid residues of the epitope region were induced, even after the virus was passaged ten times in the presence of HuMAb 5A7. Moreover, 5A7 showed significant therapeutic efficacy in mice, even when it was administered 72 hours post-infection. These results indicate that 5A7 is a promising candidate for developing therapeutics, and provide insight for the development of a universal vaccine against influenza B virus.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza B/imunologia , Influenza Humana/prevenção & controle , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Sequência de Bases , Mapeamento de Epitopos , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Hibridomas , Vírus da Influenza B/genética , Influenza Humana/tratamento farmacológico , Influenza Humana/imunologia , Injeções Intraperitoneais , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Testes de Neutralização , Alinhamento de Sequência , Análise de Sequência de DNA , Resultado do TratamentoRESUMO
Most neutralizing antibodies elicited during influenza virus infection or by vaccination have a narrow spectrum because they usually target variable epitopes in the globular head region of hemagglutinin (HA). In this study, we describe a human monoclonal antibody (HuMAb), 5D7, that was prepared from the peripheral blood lymphocytes of a vaccinated volunteer using the fusion method. The HuMAb heterosubtypically neutralizes group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H9N2, with a strong hemagglutinin inhibition activity. Selection of an escape mutant showed that the HuMAb targets a novel conformational epitope that is located in the HA head region but is distinct from the receptor binding site. Furthermore, Phe114Ile substitution in the epitope made the HA unrecognizable by the HuMAb. Amino acid residues in the predicted epitope region are also highly conserved in the HAs of H1N1 and H9N2. The HuMAb reported here may be a potential candidate for the development of therapeutic/prophylactic antibodies against H1 and H9 influenza viruses.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Proteção Cruzada , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H9N2/imunologia , Influenza Humana/prevenção & controle , Sequência de Aminoácidos , Animais , Antígenos Virais/química , Antígenos Virais/imunologia , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Hibridomas/imunologia , Vacinas contra Influenza/administração & dosagem , Influenza Humana/imunologia , Influenza Humana/virologia , Masculino , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Vacinação , Adulto JovemRESUMO
Influenza viruses are a continuous threat to human public health because of their ability to evolve rapidly through genetic drift and reassortment. Three human monoclonal antibodies (HuMAbs) were generated in this study, 1H11, 2H5 and 5G2, and they cross-neutralize a diverse range of group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H5N1 and H9N2. The three HuMAbs were prepared by fusing peripheral blood lymphocytes from an H1N1pdm-infected patient with a newly developed fusion partner cell line, SPYMEG. All the HuMAbs had little hemagglutination inhibition activity but had strong membrane-fusion inhibition activity against influenza viruses. A protease digestion assay showed the HuMAbs targeted commonly a short α-helix region in the stalk of the hemagglutinin. Furthermore, Ile45Phe and Glu47Gly double substitutions in the α-helix region made the HA unrecognizable by the HuMAbs. These two amino acid residues are highly conserved in the HAs of H1N1, H5N1 and H9N2 viruses. The HuMAbs reported here may be potential candidates for the development of therapeutic antibodies against group 1 influenza viruses.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Hemaglutininas/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Influenza Humana/virologia , Animais , Anticorpos Antivirais/imunologia , Linhagem Celular , Haplorrinos , Humanos , Rim/imunologia , Rim/virologia , Testes de NeutralizaçãoAssuntos
Colistina/farmacologia , Farmacorresistência Bacteriana/genética , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Escherichia coli/genética , Fezes/microbiologia , Feminino , Voluntários Saudáveis , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Plasmídeos , População Rural , Vietnã , Adulto JovemRESUMO
The number of dengue cases has increased dramatically in recent years. In Latin America, the number of cases and deaths in 2023 was the highest ever recorded. We report on a patient who had been infected with dengue virus during his stay in Costa Rica in September 2023, and developed the disease after returning to Japan. Plasma obtained from the patient was used for diagnosis and dengue virus serotyping by real-time PCR. The nucleotide sequence of the envelope region of dengue virus was then determined by the direct sequencing method, and this sequence was used for phylogenetic analyses. The patient was found to be infected with dengue virus type 3 genotype III. The sequence from the present case was more homologous with sequences registered in Florida, USA, associated with travel to Cuba in 2022 than with sequences registered in Costa Rica 10 years ago. The Pan American Health Organization reported that only dengue virus type 1 and 2 cases were reported in Costa Rica in 2019-2021, whereas dengue virus type 3 and 4 cases started being reported in 2022. In 2023, the reported numbers of cases with dengue virus types 3 and 4 exceeded those of dengue virus types 1 and 2. In addition, regional differences in endemic strains have been observed in Costa Rica. Our findings suggest that the dengue virus type 3 that infected the patient was more likely an influx of a strain that had been circulating in Caribbean countries such as Cuba in recent years, rather than a re-emergence of an indigenous virus in Costa Rica. The serotypes of dengue virus prevalent in Costa Rica have been changing since 2022. All four serotypes were prevalent in 2023, with a particularly sharp increase in the number of cases of dengue virus types 3 and 4. Future monitoring and surveillance are essential because changes in endemic serotypes can cause antibody-dependent enhancement, which can lead to severe dengue disease presentations.
RESUMO
Dengue is a mosquito-borne disease that has spread to over 100 countries. Its symptoms vary from the relatively mild acute febrile illness called dengue fever to the much more severe dengue shock syndrome. Dengue is caused by dengue virus (DENV), which belongs to the Flavivirus genus of the family Flaviviridae. There are four serotypes of DENV, i.e., DENV1 to DENV4, and each serotype is divided into distinct genotypes. Thailand is an endemic area where all four serotypes of DENV co-circulate. Genome sequencing of the DENV2 that was isolated in Thailand in 2016 and 2017 revealed the emergence of the Cosmopolitan genotype and its co-circulation with the Asian-I genotype. However, it was unclear whether different genotypes have different levels of viral replication and pathogenicity. Focus-forming assay (FFA) results showed that clinical isolates of these genotypes differed in focus size and proliferative capacity. Using circular polymerase extension reaction, we generated parental and chimeric viruses with swapped genes between these two DENV2 genotypes, and compared their focus sizes and infectivity titers using FFA. The results showed that the focus size was larger when the structural proteins and/or non-structural NS1-NS2B proteins were derived from the Cosmopolitan virus. The infectious titers were consistent with the focus sizes. Single-round infectious particle assay results confirmed that chimeric viruses with Cosmopolitan type structural proteins, particularly prM/E, had significantly increased luciferase activity. Replicon assay results showed that Cosmopolitan NS1-NS2B proteins had increased reporter gene expression levels. Furthermore, in interferon-receptor knock-out mice, viruses with Cosmopolitan structural and NS1-NS2B proteins had higher titers in the blood, and caused critical disease courses. These results suggested that differences in the sequences within the structural and NS1-NS2B proteins may be responsible for the differences in replication, pathogenicity, and infectivity between the Asian-I and Cosmopolitan viruses.
Assuntos
Vírus da Dengue , Dengue , Animais , Camundongos , Dengue/epidemiologia , Virulência , Sorogrupo , Genótipo , Replicação ViralRESUMO
Thailand-Japan Research Collaboration Center on Emerging and Re-emerging Infections (RCC-ERI) was established in 2005 as a research collaboration center cooperated by Research Institute for Microbial Diseases (RIMD), Osaka University and Department of Medical Sciences, Ministry of Public Health of Thailand. In addition, Mahidol-Osaka Center for Infectious Diseases (MOCID) was established in 2010, also as another research collaboration center cooperated by RIMD, Osaka University and Faculty of Tropical Medicine, Mahidol University. Epidemiological and basic studies on human pathogenic viruses have been conducted in RCC-ERI and MOCID. In this report, brief overviews of the research activities of both centers, as well as the phenotypic studies on the envelope glycoproteins of HIV-1 Thai strains that have been performed at RCC-ERI, are presented. SATREPS is a Japanese government program by the Japan Science and Technology Agency (JST) and the Japan International Cooperation Agency (JICA) that promotes international joint research targeting global issues. Our research group at RIMD have collaborated with the Ministry of Public Health - National Institute of Health (NIH) and Mahidol University in Thailand from 2008 as a four-years project. Our aim on this collaboration is to generate human monoclonal antibodies neutralizing infectious viral agents, hopefully to apply them to clinical field as "therapeutic antibodies". Here we introduce our trials in this project, especially on the generation of human monoclonal antibodies against dengue virus as well as against influenza virus that have been performed as collaboration between Thai and Japanese research groups.
Assuntos
Doenças Transmissíveis/virologia , Comportamento Cooperativo , Infectologia , Pesquisa , Anticorpos Monoclonais , Anticorpos Antivirais , Doenças Transmissíveis/epidemiologia , Vírus da Dengue/imunologia , HIV-1 , Humanos , Infectologia/organização & administração , Japão , Orthomyxoviridae/imunologia , Pesquisa/organização & administração , Tailândia , Medicina Tropical/organização & administração , Vírus/genéticaRESUMO
Immunoglobulin A (IgA) has been showing potential as a new therapeutic antibody. However, recombinant IgA suffers from low yield. Supplementation of the medium is an effective approach to improving the production and quality of recombinant proteins. In this study, we adapted IgA1-producing CHO-K1 suspension cells to a high concentration (150 mM) of different disaccharides, namely sucrose, maltose, lactose, and trehalose, to improve the production and quality of recombinant IgA1. The disaccharide-adapted cell lines had slower cell growth rates, but their cell viability was extended compared to the nonadapted IgA1-producing cell line. Glucose consumption was exhausted in all cell lines except for the maltose-adapted one, which still contained glucose even after the 9th day of culturing. Lactate production was higher among the disaccharide-adapted cell lines. The specific productivity of the maltose-adapted IgA1-producing line was 4.5-fold that of the nonadapted line. In addition, this specific productivity was higher than in previous productions of recombinant IgA1 with a lambda chain. Lastly, secreted IgA1 aggregated in all cell lines, which may have been caused by self-aggregation. This aggregation was also found to begin inside the cells for maltose-adapted cell line. These results suggest that a high concentration of disaccharide-supplemented induced hyperosmolarity in the IgA1-producing CHO-K1 cell lines. In addition, the maltose-adapted CHO-K1 cell line benefited from having an additional source of carbohydrate. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-023-00571-5.
RESUMO
A 25-year-old patient with a primary immunodeficiency lacking immunoglobulin production experienced a relapse after a 239-day period of persistent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Viral genetic sequencing demonstrated that SARS-CoV-2 had evolved during the infection period, with at least five mutations associated with host cellular immune recognition. Among them, the T32I mutation in ORF3a was found to evade recognition by CD4+ T cells. The virus found after relapse showed an increased proliferative capacity in vitro. SARS-CoV-2 may have evolved to evade recognition by CD4+ T cells and increased in its proliferative capacity during the persistent infection, likely leading to relapse. These mutations may further affect viral clearance in hosts with similar types of human leukocyte antigens. The early elimination of SARS-CoV-2 in immunocompromised patients is therefore important not only to improve the condition of patients but also to prevent the emergence of mutants that threaten public health.
RESUMO
The Omicron variant continuously evolves under the humoral immune pressure exerted by vaccination and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and the resulting Omicron subvariants display further immune evasion and antibody escape. An engineered angiotensin-converting enzyme 2 (ACE2) decoy composed of high-affinity ACE2 and an IgG1 Fc domain could offer an alternative modality to neutralize SARS-CoV-2. We previously reported its broad spectrum and therapeutic potential in rodent models. Here, we demonstrate that the engineered ACE2 decoy retains neutralization activity against Omicron subvariants, including the currently emerging XBB and BQ.1 strains, which completely evade antibodies currently in clinical use. SARS-CoV-2, under the suboptimal concentration of neutralizing drugs, generated SARS-CoV-2 mutants escaping wild-type ACE2 decoy and monoclonal antibodies, whereas no escape mutant emerged against the engineered ACE2 decoy. Furthermore, inhalation of aerosolized decoys improved the outcomes of rodents infected with SARS-CoV-2 at a 20-fold lower dose than that of intravenous administration. Last, the engineered ACE2 decoy exhibited therapeutic efficacy for cynomolgus macaques infected with SARS-CoV-2. These results indicate that this engineered ACE2 decoy represents a promising therapeutic strategy to overcome immune-evading SARS-CoV-2 variants and that liquid aerosol inhalation could be considered as a noninvasive approach to enhance the efficacy of COVID-19 treatments.
Assuntos
COVID-19 , Animais , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2 , Anticorpos Monoclonais , Macaca fascicularisRESUMO
The global spread of the four dengue virus serotypes (DENV-1 to -4) has made this virus a major and growing public health concern. Generally, pre-existing neutralizing antibodies derived from primary infection play a significant role in protecting against subsequent infection with the same serotype. By contrast, these pre-existing antibodies are believed to mediate a non-protective response to subsequent heterotypic DENV infections, leading to the onset of dengue illness. In this study, we prepared hybridomas producing human monoclonal antibodies (HuMAbs) against DENV using peripheral blood mononuclear cells (PBMCs) from patients in the acute phase (around 1 week after the onset of illness) or the convalescent phase (around 2weeks after the onset of illness) of secondary infection. Interestingly, a larger number of hybridoma clones was obtained from patients in the acute phase than from those in the convalescent phase. Most HuMAbs from acute-phase infections were cross-reactive with all four DENV serotypes and showed significant neutralization activity to all four DENV serotypes. Thus, secondary DENV infection plays a significant role in stimulating memory cells to transiently increase the number of antibody-secreting plasma cells in patients in the early phase after the secondary infection. These HuMAbs will enable us to better understand the protective and pathogenic effects of DENV infection, which could vary greatly among secondarily-infected individuals.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Neutralizantes/biossíntese , Vírus da Dengue/imunologia , Dengue/imunologia , Linfócitos/imunologia , Proteínas Virais/imunologia , Adulto , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Chlorocebus aethiops , Coinfecção , Feminino , Imunofluorescência , Humanos , Hibridomas , Masculino , Testes de Neutralização , Sorotipagem , Células Vero , Adulto JovemRESUMO
Influenza virus RNA was amplified by a continuous-flow polydimethylsiloxane microfluidic RT-PCR chip within 15-20 min. The amplified influenza virus RNA was observed with the naked eye, as the red color at the test line, using a lateral flow immunoassay within 1 min.
Assuntos
DNA Viral/análise , Imunoensaio/métodos , Técnicas Analíticas Microfluídicas/métodos , Orthomyxoviridae/genética , Orthomyxoviridae/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , DNA Viral/genética , Orthomyxoviridae/imunologiaRESUMO
The recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major burden for health care systems worldwide, and is a threat to our daily lives. Various effective ingredients against SARS-CoV-2 were already reported, however, since products contain various ingredients, it is also important to evaluate the effectiveness of commercially available disinfectants per se. In this study, the virucidal efficacy of forty-eight commercially available products were evaluated according to the standardized suspension method EN 14476 and the following results were obtained: Alcohol-based disinfectants, hand soaps, wet wipes, alkaline cleaners, quaternary ammonium compound sanitizers and oxygen bleach had great virucidal efficacy against SARS-CoV-2. Enveloped viruses such as SARS-CoV-2 are among the most susceptible of pathogens to formulated microbicidal actives and detergents, but as the results of this study showed, it is also necessary to pay attention to the concentration at the time of use and the required contact time.