Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.717
Filtrar
1.
PLoS Pathog ; 16(8): e1008816, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32853241

RESUMO

Influenza A viruses (IAVs) cause seasonal epidemics and occasional pandemics. Most pandemics occurred upon adaptation of avian IAVs to humans. This adaptation includes a hallmark receptor-binding specificity switch of hemagglutinin (HA) from avian-type α2,3- to human-type α2,6-linked sialic acids. Complementary changes of the receptor-destroying neuraminidase (NA) are considered to restore the precarious, but poorly described, HA-NA-receptor balance required for virus fitness. In comparison to the detailed functional description of adaptive mutations in HA, little is known about the functional consequences of mutations in NA in relation to their effect on the HA-NA balance and host tropism. An understudied feature of NA is the presence of a second sialic acid-binding site (2SBS) in avian IAVs and absence of a 2SBS in human IAVs, which affects NA catalytic activity. Here we demonstrate that mutation of the 2SBS of avian IAV H5N1 disturbs the HA-NA balance. Passaging of a 2SBS-negative H5N1 virus on MDCK cells selected for progeny with a restored HA-NA balance. These viruses obtained mutations in NA that restored a functional 2SBS and/or in HA that reduced binding of avian-type receptors. Importantly, a particular HA mutation also resulted in increased binding of human-type receptors. Phylogenetic analyses of avian IAVs show that also in the field, mutations in the 2SBS precede mutations in HA that reduce binding of avian-type receptors and increase binding of human-type receptors. Thus, 2SBS mutations in NA can drive acquisition of mutations in HA that not only restore the HA-NA balance, but may also confer increased zoonotic potential.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/genética , Mutação , Neuraminidase/genética , Infecções por Orthomyxoviridae/virologia , Ácidos Siálicos/metabolismo , Replicação Viral , Substituição de Aminoácidos , Animais , Sítios de Ligação , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/isolamento & purificação , Células Madin Darby de Rim Canino , Neuraminidase/química , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/patologia , Ligação Proteica
2.
Arch Virol ; 165(11): 2503-2512, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32783078

RESUMO

Immunodominance is recognized as a key factor in the antigenic drift of seasonal influenza viruses. In the immunodominance model, each individual in a population predominantly responds to a single epitope among the five antigenic epitopes of the viral hemagglutinin (HA), driving escape mutations one at a time, and sequential mutations in multiple individuals who respond to different epitopes eventually generate a drifted strain with mutations in epitopes that are targeted by a majority of the population. A focused antibody response to the Sa epitope in people born between 1965 and 1979 was believed to contribute to a mutation at HA residue 163 and the first antigenic drift of the 2009 pandemic influenza A H1N1 virus. A serine-to-threonine mutation at HA residue 185 in the Sb epitope emerged in 2010 even before the 163 mutation. We show here that a large fraction of the population in 2010-2011 had responses to the Sb epitope, as shown by 47% of tested sera having altered titers to the S185T mutant. Responses to the Sb epitope showed an age-specific trend similar to that found for the response to Sa epitope in these subjects. Together, the focused responses to Sa and Sb epitopes may have driven the first antigenic drift of the 2009 pandemic H1N1 virus.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Variação Antigênica , Evolução Molecular , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Animais , Cães , Mapeamento de Epitopos , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , RNA Viral/genética , Seleção Genética , Análise de Sequência de DNA , Cultura de Vírus
3.
BMC Infect Dis ; 20(1): 550, 2020 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-32727378

RESUMO

BACKGROUND: Highly pathogenic influenza A (H5N8) viruses have caused several worldwide outbreaks in birds and are of potential risk to humans. Thus, a specific, rapid and sensitive method for detection is urgently needed. METHODS: In the present study, TaqMan minor groove binder probes and multiplex real-time RT-PCR primers were designed to target the H5 hemagglutinin and N8 neuraminidase genes. A total of 38 strains of avian influenza viruses and other viruses were selected to test the performance of the assay. RESULTS: The results showed that only H5 and N8 avian influenza viruses yielded a positive signal, while all other subtypes avian influenza viruses and other viruses were negative. High specificity, repeatability, and sensitivity were achieved, with a detection limit of 10 copies per reaction. CONCLUSIONS: The developed assay could be a powerful tool for rapid detection of H5N8 influenza viruses in the future.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H5N8/genética , Influenza Aviária/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Neuraminidase/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Aves/virologia , Primers do DNA/genética , Feminino , Influenza Aviária/virologia , Camundongos , Camundongos Endogâmicos BALB C , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
4.
Proc Natl Acad Sci U S A ; 117(31): 18431-18438, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690700

RESUMO

Influenza hemagglutinin (HA) glycoprotein is the primary surface antigen targeted by the host immune response and a focus for development of novel vaccines, broadly neutralizing antibodies (bnAbs), and therapeutics. HA enables viral entry into host cells via receptor binding and membrane fusion and is a validated target for drug discovery. However, to date, only a very few bona fide small molecules have been reported against the HA. To identity new antiviral lead candidates against the highly conserved fusion machinery in the HA stem, we synthesized a fluorescence-polarization probe based on a recently described neutralizing cyclic peptide P7 derived from the complementarity-determining region loops of human bnAbs FI6v3 and CR9114 against the HA stem. We then designed a robust binding assay compatible with high-throughput screening to identify molecules with low micromolar to nanomolar affinity to influenza A group 1 HAs. Our simple, low-cost, and efficient in vitro assay was used to screen H1/Puerto Rico/8/1934 (H1/PR8) HA trimer against ∼72,000 compounds. The crystal structure of H1/PR8 HA in complex with our best hit compound F0045(S) confirmed that it binds to pockets in the HA stem similar to bnAbs FI6v3 and CR9114, cyclic peptide P7, and small-molecule inhibitor JNJ4796. F0045 is enantioselective against a panel of group 1 HAs and F0045(S) exhibits in vitro neutralization activity against multiple H1N1 and H5N1 strains. Our assay, compound characterization, and small-molecule candidate should further stimulate the discovery and development of new compounds with unique chemical scaffolds and enhanced influenza antiviral capabilities.


Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Polarização de Fluorescência/métodos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Influenza Humana/virologia , Bibliotecas de Moléculas Pequenas/farmacologia , Antivirais/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/metabolismo , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/metabolismo , Bibliotecas de Moléculas Pequenas/química
5.
BMC Bioinformatics ; 21(1): 316, 2020 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-32682392

RESUMO

BACKGROUND: The pandemic threat of influenza has attracted great attention worldwide. To assist public health decision-makers, new suites of tools are needed to rapidly process and combine viral information retrieved from public-domain databases for a better risk assessment. RESULTS: Using our recently developed FluConvert and IniFlu software, we automatically processed and rearranged sequence data by standard viral nomenclature, determined the group-related consensus sequences, and identified group-specific polygenic signatures. The software possesses powerful ability to integrate viral, clinical, and epidemiological data. We demonstrated that both multiple basic amino acids at the cleavage site of the HA gene and also at least 11 more evidence-based viral amino acid substitutions present in global highly pathogenic avian influenza H5N2 viruses during the years 2009-2016 that are associated with viral virulence and human infection. CONCLUSIONS: FluConvert and IniFlu are useful to monitor and assess all subtypes of influenza viruses with pandemic potential. These programs are implemented through command-line and user-friendly graphical interfaces, and identify molecular signatures with virological, epidemiological and clinical significance. FluConvert and IniFlu are available at https://apps.flutures.com or https://github.com/chinrur/FluConvert_IniFlu.


Assuntos
Vírus da Influenza A Subtipo H5N2/patogenicidade , Influenza Aviária/patologia , Interface Usuário-Computador , Sequência de Aminoácidos , Animais , Aves , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H5N2/genética , Influenza Aviária/imunologia , Influenza Aviária/virologia , Medição de Risco , Alinhamento de Sequência , Virulência
6.
Nat Med ; 26(8): 1240-1246, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32601336

RESUMO

The conserved region of influenza hemagglutinin (HA) stalk (or stem) has gained attention as a potent target for universal influenza vaccines1-5. Although the HA stalk region is relatively well conserved, the evolutionarily dynamic nature of influenza viruses6 raises concerns about the possible emergence of viruses carrying stalk escape mutation(s) under sufficient immune pressure. Here we show that immune pressure on the HA stalk can lead to expansion of escape mutant viruses in study participants challenged with a 2009 H1N1 pandemic influenza virus inoculum containing an A388V polymorphism in the HA stalk (45% wild type and 55% mutant). High level of stalk antibody titers was associated with the selection of the mutant virus both in humans and in vitro. Although the mutant virus showed slightly decreased replication in mice, it was not observed in cell culture, ferrets or human challenge participants. The A388V mutation conferred resistance to some of the potent HA stalk broadly neutralizing monoclonal antibodies (bNAbs). Co-culture of wild-type and mutant viruses in the presence of either a bNAb or human serum resulted in rapid expansion of the mutant. These data shed light on a potential obstacle for the success of HA-stalk-targeting universal influenza vaccines-viral escape from vaccine-induced stalk immunity.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/genética , Seleção Genética/genética , Animais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/farmacologia , Sequência Conservada/genética , Reações Cruzadas/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Camundongos , Seleção Genética/imunologia
7.
Science ; 368(6497): 1335-1340, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32554590

RESUMO

The discovery and characterization of broadly neutralizing human antibodies (bnAbs) to the highly conserved stem region of influenza hemagglutinin (HA) have contributed to considerations of a universal influenza vaccine. However, the potential for resistance to stem bnAbs also needs to be more thoroughly evaluated. Using deep mutational scanning, with a focus on epitope residues, we found that the genetic barrier to resistance to stem bnAbs is low for the H3 subtype but substantially higher for the H1 subtype owing to structural differences in the HA stem. Several strong resistance mutations in H3 can be observed in naturally circulating strains and do not reduce in vitro viral fitness and in vivo pathogenicity. This study highlights a potential challenge for development of a truly universal influenza vaccine.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Tolerância Imunológica/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Mutação
8.
Arch Virol ; 165(9): 2045-2051, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32524262

RESUMO

Data obtained from monitoring cases of severe influenza, cases of vaccinated individuals, and unique cases were used to describe influenza viruses that circulated in Russia in the 2018-2019 epidemic season. A high proportion of the mutations D222G/N in A(H1N1)pdm09 HA was detected in fatal cases. Viruses of the B/Victoria lineage with deletions in HA were detected in Russia, and a reassortant seasonal influenza A(H1N2) virus was identified. A C-terminal truncation in the NS1 protein was detected in a substantial proportion of A(H3N2) viruses.


Assuntos
Vírus da Influenza A Subtipo H1N2/isolamento & purificação , Influenza Humana/virologia , Genoma Viral , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza A Subtipo H1N2/classificação , Vírus da Influenza A Subtipo H1N2/genética , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Filogenia , Vírus Reordenados/classificação , Vírus Reordenados/genética , Vírus Reordenados/isolamento & purificação , Federação Russa , Estações do Ano
9.
PLoS One ; 15(6): e0234869, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32579578

RESUMO

The continuous variation of the seasonal influenza viruses, particularly A(H1N1)pdm09, persistently threatens human life and health around the world. In local areas of southwest china, the large time-scale genomic research on A(H1N1)pdm09 is still insufficient. Here, we sequenced 45 whole-genome sequences of influenza A(H1N1)pdm09 viruses in Lincang, China, from 2014 to 2018, by next-generation sequencing technology to characterize molecular mechanisms of their origin and evolution. Our phylogenetic analyses suggest that the A(H1N1)pdm09 strains circulating in Lincang belong to clade 6B and the subclade 6B.1A predominates in 2018. Further, the strains in 2018 possess elevated evolutionary rate as compared to strains in other years. Several newly emerged mutations for HA (hemagglutinin) in 2018 are revealed (i.e., S183P and R221K). Intriguingly, the substitution R221K falls into the RBS (receptor binding site) of HA protein, which could affect antigenic properties of influenza A(H1N1)pdm09 viruses, and another substitution S183P near to RBS with a high covering frequency (11/14 strains) in 2018 is exactly located at the epitope B. Notably, the NA (neuraminidase) protein harbors a new mutation I23T, potentially involved in N-glycosylation. Based on the background with a higher evolutionary rate in 2018 strains, we deeply evaluate the potential vaccine efficacy against Lincang strains and discover a substantive decline of the vaccine efficacy in 2018. Our analyses reaffirm that the real-time molecular surveillance and timely updated vaccine strains for prevention and control of influenza A(H1N1)pdm09 are crucial in the future.


Assuntos
Evolução Molecular , Vírus da Influenza A Subtipo H1N1/genética , Sequenciamento Completo do Genoma , Sequência de Aminoácidos , China , Análise Mutacional de DNA , Demografia , Epitopos/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vacinas contra Influenza/imunologia , Mutação/genética , Neuraminidase/genética , Filogenia , Resultado do Tratamento
10.
BMC Bioinformatics ; 21(1): 182, 2020 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-32393178

RESUMO

BACKGROUND: In addition to causing the pandemic influenza outbreaks of 1918 and 2009, subtype H1N1 influenza A viruses (IAVs) have caused seasonal epidemics since 1977. Antigenic property of influenza viruses are determined by both protein sequence and N-linked glycosylation of influenza glycoproteins, especially hemagglutinin (HA). The currently available computational methods are only considered features in protein sequence but not N-linked glycosylation. RESULTS: A multi-task learning sparse group least absolute shrinkage and selection operator (LASSO) (MTL-SGL) regression method was developed and applied to derive two types of predominant features including protein sequence and N-linked glycosylation in hemagglutinin (HA) affecting variations in serologic data for human and swine H1N1 IAVs. Results suggested that mutations and changes in N-linked glycosylation sites are associated with the rise of antigenic variants of H1N1 IAVs. Furthermore, the implicated mutations are predominantly located at five reported antibody-binding sites, and within or close to the HA receptor binding site. All of the three N-linked glycosylation sites (i.e. sequons NCSV at HA 54, NHTV at HA 125, and NLSK at HA 160) identified by MTL-SGL to determine antigenic changes were experimentally validated in the H1N1 antigenic variants using mass spectrometry analyses. Compared with conventional sparse learning methods, MTL-SGL achieved a lower prediction error and higher accuracy, indicating that grouped features and MTL in the MTL-SGL method are not only able to handle serologic data generated from multiple reagents, supplies, and protocols, but also perform better in genetic sequence-based antigenic quantification. CONCLUSIONS: In summary, the results of this study suggest that mutations and variations in N-glycosylation in HA caused antigenic variations in H1N1 IAVs and that the sequence-based antigenicity predictive model will be useful in understanding antigenic evolution of IAVs.


Assuntos
Algoritmos , Antígenos Virais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Mutação/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Genoma Viral , Glicosilação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos , Vírus da Influenza A/imunologia , Influenza Humana/virologia , Polissacarídeos/imunologia , Reprodutibilidade dos Testes , Suínos
11.
J Med Microbiol ; 69(7): 986-998, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32459617

RESUMO

Introduction. Influenza viruses evolve rapidly and change their antigenic characteristics, necessitating biannual updates of flu vaccines.Aim. The aim of this study was to characterize influenza viruses circulating in Bulgaria during the 2018/2019 season and to identify amino acid substitutions in them that might impact vaccine effectiveness.Methodology. Typing/subtyping of influenza viruses were performed using real-time Reverse Transcription-PCR (RT-PCR) and results of phylogenetic and amino acid sequence analyses of influenza strains are presented.Results. A(H1N1)pdm09 (66 %) predominated over A(H3N2) (34 %) viruses, with undetected circulation of B viruses in the 2018/2019 season. All A(H1N1)pdm09 viruses studied fell into the recently designated 6B.1A subclade with over 50 % falling in four subgroups: 6B.1A2, 6B.1A5, 6B.1A6 and 6B.1A7. Analysed A(H3N2) viruses belonged to subclades 3C.2a1b and 3C.2a2. Amino acid sequence analysis of 36 A(H1N1)pdm09 isolates revealed the presence of six-ten substitutions in haemagglutinin (HA), compared to the A/Michigan/45/2015 vaccine virus, three of which occurred in antigenic sites Sa and Cb, together with four-nine changes at positions in neuraminidase (NA), and a number of substitutions in internal proteins. HA1 D222N substitution, associated with increased virulence, was identified in two A(H1N1)pdm09 viruses. Despite the presence of several amino acid substitutions, A(H1N1)pdm09 viruses remained antigenically similar to the vaccine virus. The 28 A(H3N2) viruses characterized carried substitutions in HA, including some in antigenic sites A, B, C and E, in NA and internal protein sequences.Conclusion. The results of this study showed the genetic diversity of circulating influenza viruses and the need for continuous antigenic and molecular surveillance.


Assuntos
Vírus da Influenza A/genética , Vacinas contra Influenza/genética , Influenza Humana/genética , Sequência de Aminoácidos/genética , Substituição de Aminoácidos/genética , Antígenos Virais/genética , Bulgária/epidemiologia , Monitoramento Epidemiológico , Evolução Molecular , Variação Genética/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Hemaglutininas , História do Século XXI , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A/imunologia , Influenza Humana/história , Influenza Humana/virologia , Neuraminidase/genética , Filogenia , RNA Viral/genética , Estações do Ano , Análise de Sequência de DNA/métodos
12.
PLoS One ; 15(5): e0233520, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32459823

RESUMO

Although vaccine delivery through the oral route remains the most convenient and safest way for mass immunization purposes, this method is limited by the requirement for large antigen doses and low vaccine efficacy. In this study, we generated recombinant baculoviruses (rBVs) expressing influenza hemagglutinin (A/PR/8/34) and orally delivered a low dose of rBVs to evaluate its vaccine efficacy in mice. Intranasal rBV vaccination was included in the whole experiment for comparison. We found that oral vaccination elicited high levels of virus-specific IgG and IgA antibody responses in both serum and mucosal samples (lung, tracheal, intestinal, fecal and vaginal). Surprisingly, complete protection from the lethal influenza challenge was observed, as indicated by reductions in the virus titer, inflammatory cytokine production, body weight change, and enhanced survival. These results suggest that oral delivery of the influenza rBV vaccine induces mucosal and systemic immunity, which protect mice from the lethal influenza virus challenge. Oral delivery of baculovirus vaccines can be developed as an effective vaccination route.


Assuntos
Baculoviridae , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza , Infecções por Orthomyxoviridae , Administração Oral , Animais , Anticorpos Antivirais/imunologia , Baculoviridae/genética , Baculoviridae/imunologia , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Vírus da Influenza A Subtipo H1N1/genética , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Células Sf9 , Spodoptera
13.
Biotechnol Bioeng ; 117(7): 1990-2007, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32297972

RESUMO

High-quality antibody (Ab) production depends on the availability of immunologically relevant antigens. We present a potentially universal platform for generating soluble antigens from bacterial hosts, tailored to immunized animals for Ab production. A novel RNA-dependent chaperone, in which the target antigen is genetically fused with an RNA-interacting domain (RID) docking tag derived from the immunized host, promotes the solubility and robust folding of the target antigen. We selected the N-terminal tRNA-binding domain of lysyl-tRNA synthetase (LysRS) as the RID for fusion with viral proteins and demonstrated the expression of the RID fusion proteins in their soluble and native conformations; immunization predominantly elicited Ab responses to the target antigen, whereas the "self" RID tag remained nonimmunogenic. Differential immunogenicity of the fusion proteins greatly enriched and simplified the screening of hybridoma clones of monoclonal antibodies (mAbs), enabling specific and sensitive serodiagnosis of MERS-CoV infection. Moreover, mAbs against the consensus influenza hemagglutinin stalk domain enabled a novel assay for trivalent seasonal influenza vaccines. The Fc-mediated effector function was demonstrated, which could be harnessed for the design of next-generation "universal" influenza vaccines. The nonimmunogenic built-in antigen folding module tailored to a repertoire of immunized animal hosts will drive immunochemical diagnostics, therapeutics, and designer vaccines.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/biossíntese , Antígenos Virais/química , Infecções por Coronavirus/diagnóstico , Hibridomas/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Chaperonas Moleculares , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Antígenos Virais/genética , Antígenos Virais/imunologia , Infecções por Coronavirus/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Imunização , Vacinas contra Influenza , Lisina-tRNA Ligase/química , Lisina-tRNA Ligase/genética , Camundongos , Camundongos Endogâmicos BALB C , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Testes Sorológicos , Solubilidade
14.
Int J Infect Dis ; 95: 413-420, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32276045

RESUMO

BACKGROUND: Influenza viruses evolve rapidly and cause regular seasonal epidemics in humans challenging effective vaccination. The virus surface HA glycoprotein is the primary target for the host immune response. Here, we investigated the vaccine efficacy and evolution patterns of human influenza A/H3N2 viruses that circulated in Kenyan in the period before and after the 2009 A/H1N1 pandemic, targeting the HA1 domain. MATERIALS AND METHODS: A hundred and fifteen HA sequences of Kenyan virus viruses were analyzed relative to the corresponding WHO vaccine reference strains using bioinformatics approaches. RESULTS: Our analyses revealed varied amino acid substitutions at all the five antigenic sites (A-E) of the HA1 domain, with a majority the changes occurring at sites A and B. The Kenyan A/H3N2 viruses isolated during 2007/2008 seasons belonged to A/Brisbane/10/2007-like viruses lineage, while those circulating in 2009-2012 belonged to the lineage of A/Victoria/361/2011-like viruses. The 2013 viruses clustered in clade 3C.3 of the A/Samara/73/2013-like viruses. The mean evolutionary rate of the A/H3N2 viruses analyzed in the study was at 4.17×10-3 (95% HPD=3.09×10-3-5.31×10-3) nucleotide substitutions per site per year, whereas the TMRCA was estimated at 11.18 (95% HPD=9.00-14.12) years ago from 2013. The prediction of vaccine efficacy revealed modest vaccine efficaciousness during 2008, and 2010 influenza seasons, whilst sub-optimal effectiveness was registered in 2007, 2009, 2012 and 2013. Further, the overall selective pressure acting on the HA1 domain was estimated at 0.56 (ω<1), suggesting that a majority of codon sites in the HA1 epitopes were evolving under purifying selection. CONCLUSIONS: Generally, our results highlight the genetic plasticity of A/H3N2 viruses and reveal considerable disparity in vaccine efficaciousness against the A/H3N2 viruses that circulated in Kenya, specifically during 2007, 2009, 2012, and 2013 influenza seasons. Our findings underscore the importance and need for consistent surveillance and molecular characterization of influenza viruses, to inform decision making and enhance early of detection of strains with epidemic/pandemic potential as well as benefit in guiding decisions regarding the appropriate annual influenza vaccine formulations.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/virologia , Substituição de Aminoácidos , Epitopos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Imunogenicidade da Vacina , Vírus da Influenza A Subtipo H3N2/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Quênia , Filogenia , Domínios Proteicos/imunologia , Estações do Ano
15.
J Virol ; 94(12)2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32269119

RESUMO

IgA antibodies on mucosal surfaces are known to play an important role in protection from influenza A virus (IAV) infection and are believed to be more potent than IgG for cross-protective immunity against IAVs of multiple hemagglutinin (HA) subtypes. However, in general, neutralizing antibodies specific to HA are principally HA subtype specific. Here, we focus on nonneutralizing but broadly cross-reactive HA-specific IgA antibodies. Recombinant IgG, monomeric IgA (mIgA), and polymeric secretory IgA (pSIgA) antibodies were generated based on the sequence of a mouse anti-HA monoclonal antibody (MAb) 5A5 that had no neutralizing activity but showed broad binding capacity to multiple HA subtypes. While confirming that there was no neutralizing activity of the recombinant MAbs against IAV strains A/Puerto Rico/8/1934 (H1N1), A/Adachi/2/1957 (H2N2), A/Hong Kong/483/1997 (H5N1), A/shearwater/South Australia/1/1972 (H6N5), A/duck/England/1/1956 (H11N6), and A/duck/Alberta/60/1976 (H12N5), we found that pSIgA, but not mIgA and IgG, significantly reduced budding and release of most of the viruses from infected cells. Electron microscopy demonstrated that pSIgA deposited newly produced virus particles on the surfaces of infected cells, most likely due to tethering of virus particles. Furthermore, we found that pSIgA showed significantly higher activity to reduce plaque sizes of the viruses than IgG and mIgA. These results suggest that nonneutralizing pSIgA reactive to multiple HA subtypes may play a role in intersubtype cross-protective immunity against IAVs.IMPORTANCE Mucosal immunity represented by pSIgA plays important roles in protection from IAV infection. Furthermore, IAV HA-specific pSIgA antibodies are thought to contribute to cross-protective immunity against multiple IAV subtypes. However, the mechanisms by which pSIgA exerts such versatile antiviral activity are not fully understood. In this study, we generated broadly cross-reactive recombinant IgG and pSIgA having the same antigen-recognition site and compared their antiviral activities in vitro These recombinant antibodies did not show "classical" neutralizing activity, whereas pSIgA, but not IgG, significantly inhibited the production of progeny virus particles from infected cells. Plaque formation was also significantly reduced by pSIgA, but not IgG. These effects were seen in infection with IAVs of several different HA subtypes. Based on our findings, we propose an antibody-mediated host defense mechanism by which mucosal immunity may contribute to broad cross-protection from IAVs of multiple HA subtypes, including viruses with pandemic potential.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunoglobulina A/imunologia , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/genética , Proteção Cruzada , Reações Cruzadas , Cães , Feminino , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/classificação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Imunidade nas Mucosas , Imunoglobulina A/genética , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H2N2/genética , Vírus da Influenza A Subtipo H2N2/imunologia , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/imunologia , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Liberação de Vírus
16.
PLoS One ; 15(3): e0227962, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32155152

RESUMO

OBJECTIVE: Since the 2009 influenza pandemic, Latin American (LA) countries have strengthened their influenza surveillance systems. We analyzed influenza genetic sequence data from the 2017 through 2018 Southern Hemisphere (SH) influenza season from selected LA countries, to map the availability of influenza genetic sequence data from, and to describe, the 2017 through 2018 SH influenza seasons in LA. METHODS: We analyzed influenza A/H1pdm09, A/H3, B/Victoria and B/Yamagata hemagglutinin sequences from clinical samples from 12 National Influenza Centers (NICs) in ten countries (Argentina, Brazil, Chile, Colombia, Costa Rica, Ecuador, Mexico, Paraguay, Peru and Uruguay) with a collection date from epidemiologic week (EW) 18, 2017 through EW 43, 2018. These sequences were generated by the NIC or the WHO Collaborating Center (CC) at the U.S Centers for Disease Control and Prevention, uploaded to the Global Initiative on Sharing All Influenza Data (GISAID) platform, and used for phylogenetic reconstruction. FINDINGS: Influenza hemagglutinin sequences from the participating countries (A/H1pdm09 n = 326, A/H3 n = 636, B n = 433) were highly concordant with the genetic groups of the influenza vaccine-recommended viruses for influenza A/H1pdm09 and influenza B. For influenza A/H3, the concordance was variable. CONCLUSIONS: Considering the constant evolution of influenza viruses, high-quality surveillance data-specifically genetic sequence data, are important to allow public health decision makers to make informed decisions about prevention and control strategies, such as influenza vaccine composition. Countries that conduct influenza genetic sequencing for surveillance in LA should continue to work with the WHO CCs to produce high-quality genetic sequence data and upload those sequences to open-access databases.


Assuntos
Evolução Molecular , Vacinas contra Influenza/administração & dosagem , Influenza Humana/prevenção & controle , Orthomyxoviridae/genética , Pandemias/prevenção & controle , Conjuntos de Dados como Assunto , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vacinas contra Influenza/imunologia , Influenza Humana/epidemiologia , Influenza Humana/microbiologia , América Latina/epidemiologia , Orthomyxoviridae/imunologia , Orthomyxoviridae/isolamento & purificação , Filogenia
17.
PLoS One ; 15(3): e0229601, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32130243

RESUMO

A community outbreak of human influenza A(H1N1)pdm09 virus strains was observed in Myanmar in 2017. We investigated the circulation patterns, antigenicity, and drug resistance of 2017 influenza A(H1N1)pdm09 viruses from Myanmar and characterized the full genome of influenza virus strains in Myanmar from in-patients and out-patients to assess the pathogenicity of the viruses. Nasopharyngeal swabs were collected from out-patients and in-patients with acute respiratory tract infections in Yangon and Pyinmana City in Myanmar during January-December 2017. A total of 215 out-patients and 18 in-patients infected with A(H1N1)pdm09 were detected by virus isolation and real-time RT-PCR. Among the positive patients, 90.6% were less than 14 years old. Hemagglutination inhibition (HI) antibody titers against A(H1N1)pdm09 viruses in Myanmar were similar to the recommended Japanese influenza vaccine strain for 2017-2018 seasons (A/Singapore/GP1908/2015) and WHO recommended 2017 southern hemisphere vaccine component (A/Michigan/45/2015). Phylogenetic analysis of the hemagglutinin sequence showed that the Myanmar strains belonged to the genetic subclade 6B.1, possessing mutations of S162N and S164T at potential antigenic sites. However, the amino acid mutation at position 222, which may enhance the severity of disease and mortality, was not found. One case with no prior history of oseltamivir treatment possessed H275Y mutated virus in neuraminidase (NA), which confers resistance to oseltamivir and peramivir with elevated IC50 values. The full genome sequence of Myanmar strains showed no difference between samples from in-patients and out-patients, suggesting no additional viral mutations associated with patient severity. Several amino acid changes were observed in PB2, PB1, and M2 of Myanmar strains when compared to the vaccine strain and other Asian strains. However, no mutations associated with pathogenicity were found in the Myanmar strains, suggesting that viral factors cannot explain the underlying reasons of the massive outbreak in Myanmar. This study reported the first detection of an oseltamivir-resistant influenza virus in Myanmar, highlighting the importance of continuous antiviral monitoring and genetic characterization of the influenza virus in Myanmar.


Assuntos
Epidemias , Vírus da Influenza A Subtipo H1N1 , Influenza Humana/epidemiologia , Adolescente , Adulto , Substituição de Aminoácidos , Antígenos Virais , Antivirais/farmacologia , Criança , Pré-Escolar , Farmacorresistência Viral/genética , Feminino , Genoma Viral , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Lactente , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Mianmar/epidemiologia , Oseltamivir/farmacologia , Filogenia , Adulto Jovem
18.
Nat Commun ; 11(1): 1233, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32144244

RESUMO

Antigenic drift of influenza virus hemagglutinin (HA) is enabled by facile evolvability. However, HA antigenic site B, which has become immunodominant in recent human H3N2 influenza viruses, is also evolutionarily constrained by its involvement in receptor binding. Here, we employ deep mutational scanning to probe the local fitness landscape of HA antigenic site B in six different human H3N2 strains spanning from 1968 to 2016. We observe that the fitness landscape of HA antigenic site B can be very different between strains. Sequence variants that exhibit high fitness in one strain can be deleterious in another, indicating that the evolutionary constraints of antigenic site B have changed over time. Structural analysis suggests that the local fitness landscape of antigenic site B can be reshaped by natural mutations via modulation of the receptor-binding mode. Overall, these findings elucidate how influenza virus continues to explore new antigenic space despite strong functional constraints.


Assuntos
Antígenos Virais/genética , Evolução Molecular , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H3N2/genética , Receptores de Superfície Celular/metabolismo , Animais , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Sítios de Ligação/genética , Cristalografia por Raios X , Análise Mutacional de DNA , Cães , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza A Subtipo H3N2/metabolismo , Células Madin Darby de Rim Canino , Mutação , Domínios Proteicos/genética , Domínios Proteicos/imunologia , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA
19.
Microb Cell Fact ; 19(1): 53, 2020 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-32122351

RESUMO

BACKGROUND: Existing methods for preparing influenza vaccines pose the greatest challenge against highly pandemic avian influenza H7N9 outbreak in the poultry and humans. Exploring a new strategy for manufacturing and delivering a safe and effective H7N9 vaccine is needed urgently. RESULTS: An alternative approach is to develop an influenza H7N9 oral vaccine based on yeast display technology in a timely manner. Hemagglutinin (HA) of A/Anhui/1/2013 (AH-H7N9) is used as a model antigen and characterized its expression on the surface of Saccharomyces cerevisiae (S.cerevisiae) EBY 100. Mice administrated orally with S.cerevisiae EBY100/pYD5-HA produced significant titers of IgG antibody as well as significant amounts of cytokines IFN-γ and IL-4. Importantly, S.cerevisiae EBY100/pYD5-HA could provide effective immune protection against homologous A/Anhui/1/2013 (AH-H7N9) virus challenge. CONCLUSIONS: Our findings suggest that platform based on yeast surface technology provides an alternative approach to prepare a promising influenza H7N9 oral vaccine candidate that can significantly shorten the preparedness period and result in effective protection against influenza A pandemic.


Assuntos
Anticorpos Antivirais/sangue , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/administração & dosagem , Infecções por Orthomyxoviridae/prevenção & controle , Administração Oral , Animais , Técnicas de Visualização da Superfície Celular , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Imunoglobulina G/sangue , Subtipo H7N9 do Vírus da Influenza A , Interferon gama/imunologia , Interleucina-4/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Saccharomyces cerevisiae
20.
Arch Virol ; 165(5): 1141-1150, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32222822

RESUMO

Pigs are capable of harbouring influenza A viruses of human and avian origin in their respiratory tracts and thus act as an important intermediary host to generate novel influenza viruses with pandemic potential by genetic reassortment between the two viruses. Here, we show that two distinct H1N2 swine influenza viruses contain avian-like or classical swine-like hemagglutinins with polymerase acidic (PA) and nucleoprotein (NP) genes from 2009 pandemic H1N1 influenza viruses that were found to be circulating in Korean pigs in 2018. Swine H1N2 influenza virus containing an avian-like hemagglutinin gene had enhanced pathogenicity, causing severe interstitial pneumonia in infected pigs and mice. The mortality rate of mice infected with swine H1N2 influenza virus containing an avian-like hemagglutinin gene was higher by 100% when compared to that of mice infected with swine H1N2 influenza virus harbouring classical swine-like hemagglutinin. Further, chemokines attracting inflammatory cells were strongly induced in lung tissues of pigs and mice infected by swine H1N2 influenza virus containing an avian-like hemagglutinin gene. In conclusion, it is necessary for the well-being of humans and pigs to closely monitor swine influenza viruses containing avian-like hemagglutinin with PA and NP genes from 2009 pandemic H1N1 influenza viruses.


Assuntos
Vírus da Influenza A Subtipo H1N2/crescimento & desenvolvimento , Infecções por Orthomyxoviridae/veterinária , Doenças dos Suínos/virologia , Fatores de Virulência/genética , Animais , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N2/genética , Vírus da Influenza A Subtipo H1N2/isolamento & purificação , Vírus da Influenza A Subtipo H1N2/patogenicidade , Camundongos , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Proteínas de Ligação a RNA/genética , Análise de Sobrevida , Suínos , Doenças dos Suínos/patologia , Proteínas do Core Viral/genética , Virulência
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA