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1.
NPJ Vaccines ; 4: 51, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31839997

RESUMO

Licensed influenza virus vaccines target the head domain of the hemagglutinin (HA) glycoprotein which undergoes constant antigenic drift. The highly conserved HA stalk domain is an attractive target to increase immunologic breadth required for universal influenza virus vaccines. We tested the hypothesis that immunization with a pandemic influenza virus vaccine boosts pre-existing anti-stalk antibodies. We used chimeric cH6/1, full length H2 and H18 HA antigens in an ELISA to measure anti-stalk antibodies in recipients participating in clinical trials of A/H1N1, A/H5N1 and A/H9N2 vaccines. The vaccines induced high titers of anti-H1 stalk antibodies in adults and children, with higher titers elicited by AS03-adjuvanted vaccines. We also observed cross-reactivity to H2 and H18 HAs. The A/H9N2 vaccine elicited plasmablast and memory B-cell responses. Post-vaccination serum from vaccinees protected mice against lethal challenge with cH6/1N5 and cH5/3N4 viruses. These findings support the concept of a chimeric HA stalk-based universal influenza virus vaccine. clinicaltrials.gov: NCT02415842.

2.
Lancet Infect Dis ; 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31630990

RESUMO

BACKGROUND: Influenza viruses cause substantial annual morbidity and mortality globally. Current vaccines protect against influenza only when well matched to the circulating strains. However, antigenic drift can cause considerable mismatches between vaccine and circulating strains, substantially reducing vaccine effectiveness. Moreover, current seasonal vaccines are ineffective against pandemic influenza, and production of a vaccine matched to a newly emerging virus strain takes months. Therefore, there is an unmet medical need for a broadly protective influenza virus vaccine. We aimed to test the ability of chimeric H1 haemagglutinin-based universal influenza virus vaccine candidates to induce broadly cross-reactive antibodies targeting the stalk domain of group 1 haemagglutinin-expressing influenza viruses. METHODS: We did a randomised, observer-blinded, phase 1 study in healthy adults in two centres in the USA. Participants were randomly assigned to one of three prime-boost, chimeric haemagglutinin-based vaccine regimens or one of two placebo groups. The vaccine regimens included a chimeric H8/1, intranasal, live-attenuated vaccine on day 1 followed by a non-adjuvanted, chimeric H5/1, intramuscular, inactivated vaccine on day 85; the same regimen but with the inactivated vaccine being adjuvanted with AS03; and an AS03-adjuvanted, chimeric H8/1, intramuscular, inactivated vaccine followed by an AS03-adjuvanted, chimeric H5/1, intramuscular, inactivated vaccine. In this planned interim analysis, the primary endpoints of reactogenicity and safety were assessed by blinded study group. We also assessed anti-H1 haemagglutinin stalk, anti-H2, anti-H9, and anti-H18 IgG antibody titres and plasmablast and memory B-cell responses in peripheral blood. This trial is registered with ClinicalTrials.gov, number NCT03300050. FINDINGS: Between Oct 10, 2017, and Nov 27, 2017, 65 participants were enrolled and randomly assigned. The adjuvanted inactivated vaccine, but not the live-attenuated vaccine, induced a substantial serum IgG antibody response after the prime immunisation, with a seven times increase in anti-H1 stalk antibody titres on day 29. After boost immunisation, all vaccine regimens induced detectable anti-H1 stalk antibody (2·2-5·6 times induction over baseline), cross-reactive serum IgG antibody, and peripheral blood plasmablast responses. An unsolicited adverse event was reported for 29 (48%) of 61 participants. Solicited local adverse events were reported in 12 (48%) of 25 participants following prime vaccination with intramuscular study product or placebo, in 12 (33%) of 36 after prime immunisation with intranasal study product or placebo, and in 18 (32%) of 56 following booster doses of study product or placebo. Solicited systemic adverse events were reported in 14 (56%) of 25 after prime immunisation with intramuscular study product or placebo, in 22 (61%) of 36 after immunisation with intranasal study product or placebo, and in 21 (38%) of 56 after booster doses of study product or placebo. Disaggregated safety data were not available at the time of this interim analysis. INTERPRETATION: The tested chimeric haemagglutinin-based, universal influenza virus vaccine regimens elicited cross-reactive serum IgG antibodies that targeted the conserved haemagglutinin stalk domain. This is the first proof-of-principle study to show that high anti-stalk titres can be induced by a rationally designed vaccine in humans and opens up avenues for further development of universal influenza virus vaccines. On the basis of the blinded study group, the vaccine regimens were tolerable and no safety concerns were observed. FUNDING: Bill & Melinda Gates Foundation.

3.
Heliyon ; 5(9): e02374, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31517114

RESUMO

Ultrasonic-assisted extraction of quercetin from Dendrobium officinale was optimized by response surface methodology (RSM) using high-performance liquid chromatography as a separative method. Based on single-factor experiments and two-level factorial analysis, the ethanol concentration, solid-to-liquid ratio and ultrasonic power were selected as significant response factors. The amount of quercetin that we extracted from Dendrobium officinale was 2.506-2.594 µg/g under the extraction conditions, which showed that optimization could improve the extration rate of quercetin from Dendrobium officinale. Quercetin was extracted and detected within 12 consecutive months after the germination of Dendrobium officinale by optimizing the extraction process to analyze the accumulation of quercetin. The UV-B exposure experiments showed that the Dendrobium officinale leaves have different responses to low- and high-dose UV light. The results showed that the quercetin content in Dendrobium officinale could be changed by UV-B radiation, and the response of distinct tissue parts to varying intensities of UV-B radiation was different.

4.
Vaccines (Basel) ; 7(3)2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31540436

RESUMO

Universal influenza virus vaccine candidates that focus on the conserved hemagglutinin (HA) stalk domain and the extracellular domain of the matrix protein 2 (M2e) have been developed to increase the breadth of protection against multiple strains. In this study, we report a novel inactivated influenza virus vaccine approach that combines these two strategies. We inserted a human consensus M2e epitope into the immunodominant antigenic site (Ca2 site) of three different chimeric HAs (cHAs). Sequential immunization with inactivated viruses containing these modified cHAs substantially enhanced M2e antibody responses while simultaneously boosting stalk antibody responses. The combination of additional M2e antibodies with HA stalk antibodies resulted in superior antibody-mediated protection in mice against challenge viruses expressing homologous or heterosubtypic hemagglutinin and neuraminidase compared to vaccination strategies that targeted the HA stalk or M2e epitopes in isolation.

5.
J Virol ; 93(18)2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31375573

RESUMO

Influenza viruses express two surface glycoproteins, the hemagglutinin (HA) and the neuraminidase (NA). Anti-NA antibodies protect from lethal influenza virus challenge in the mouse model and correlate inversely with virus shedding and symptoms in humans. Consequently, the NA is a promising target for influenza virus vaccine design. Current seasonal vaccines, however, poorly induce anti-NA antibodies, partly because of the immunodominance of the HA over the NA when the two glycoproteins are closely associated. To address this issue, here we investigated whether extending the stalk domain of the NA could render it more immunogenic on virus particles. Two recombinant influenza viruses based on the H1N1 strain A/Puerto Rico/8/1934 (PR8) were rescued with NA stalk domains extended by 15 or 30 amino acids. Formalin-inactivated viruses expressing wild-type NA or the stalk-extended NA variants were used to vaccinate mice. The virus with the 30-amino-acid stalk extension induced significantly higher anti-NA IgG responses (characterized by increased in vitro antibody-dependent cellular cytotoxicity [ADCC] activity) than the wild-type PR8 virus, while anti-HA IgG levels were unaffected. Similarly, extending the stalk domain of the NA of a recent H3N2 virus enhanced the induction of anti-NA IgGs in mice. On the basis of these results, we hypothesize that the subdominance of the NA can be modulated if the protein is modified such that its height surpasses that of the HA on the viral membrane. Extending the stalk domain of NA may help to enhance its immunogenicity in influenza virus vaccines without compromising antibody responses to HA.IMPORTANCE The efficacy of influenza virus vaccines could be improved by enhancing the immunogenicity of the NA protein. One of the reasons for its poor immunogenicity is the immunodominance of the HA over the NA in many seasonal influenza virus vaccines. Here we demonstrate that, in the mouse model, extending the stalk domain of the NA protein can enhance its immunogenicity on virus particles and overcome the immunodominance of the HA without affecting antibody responses to the HA. The antibody repertoire is broadened by the extended NA and includes additional ADCC-active antibodies. Our findings may assist in the efforts toward more effective influenza virus vaccines.

6.
NPJ Vaccines ; 4: 31, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31341648

RESUMO

Current seasonal influenza virus vaccines only provide limited, short-lived protection, and antigenic drift in the hemagglutinin surface glycoprotein necessitates their annual re-formulation and re-administration. To overcome these limitations, universal vaccine strategies that aim at eliciting broadly protective antibodies to conserved epitopes of the hemagglutinin show promise for protecting against diverse and drifted influenza viruses. Here a vaccination strategy that focuses antibody responses to conserved epitopes of the H3 hemagglutinin is described. The approach is based on antigenic silencing of the immunodominant major antigenic sites of an H3 protein from 2014 by replacing them with corresponding sequences of exotic avian hemagglutinins, yielding "mosaic" hemagglutinins. In mice, vaccination with inactivated viruses expressing mosaic hemagglutinins induced highly cross-reactive antibodies against the H3 stalk domain that elicited Fc-mediated effector functions in vitro. In addition, the mosaic viruses elicited head-specific antibodies with neutralizing and hemagglutination-inhibiting activity against recent H3N2 viruses in vitro. Immune sera protected mice from heterologous challenge with viruses carrying H3 proteins from 1968 and 1982, whereas immune sera generated with a seasonal vaccine did not protect. Consequently, the mosaic vaccination approach provides a promising avenue toward a universal influenza virus vaccine.

7.
J Virol ; 93(12)2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30944178

RESUMO

Influenza B viruses cause seasonal epidemics and are a considerable burden to public health. However, protection by current seasonal vaccines is suboptimal due to the antigenic changes of the circulating strains. In this study, we report a novel universal influenza B virus vaccination strategy based on "mosaic" hemagglutinins. We generated mosaic B hemagglutinins by replacing the major antigenic sites of the type B hemagglutinin with corresponding sequences from exotic influenza A hemagglutinins and expressed them as soluble trimeric proteins. Sequential vaccination with recombinant mosaic B hemagglutinin proteins conferred cross-protection against both homologous and heterologous influenza B virus strains in the mouse model. Of note, we rescued recombinant influenza B viruses expressing mosaic B hemagglutinins, which could serve as the basis for a universal influenza B virus vaccine.IMPORTANCE This work reports a universal influenza B virus vaccination strategy based on focusing antibody responses to conserved head and stalk epitopes of the hemagglutinin. Recombinant mosaic influenza B hemagglutinin proteins and recombinant viruses have been generated as novel vaccine candidates. This vaccine strategy provided broad cross-protection in the mouse model. Our findings will inform and drive development toward a more effective influenza B virus vaccine.

8.
J Virol ; 93(2)2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30381487

RESUMO

The influenza B virus hemagglutinin contains four major antigenic sites (the 120 loop, the 150 loop, the 160 loop, and the 190 helix) within the head domain. These immunodominant antigenic sites are the main targets of neutralizing antibodies and are subject to antigenic drift. Yet little is known about the specific antibody responses toward each site in terms of antibody prevalence and hemagglutination inhibition activity. In this study, we used modified hemagglutinins of influenza B virus which display only one or none of the major antigenic sites to measure antibody responses toward the classical as well as the noncanonical epitopes in mice, ferrets, and humans. With our novel reagents, we found that both hemagglutination inhibition antibodies and total IgGs were mostly induced by the major antigenic sites. However, in human adults, we observed high hemagglutination inhibition antibody responses toward the noncanonical epitopes. By stratifying the human samples into age groups, we found that the noncanonical antibody responses appeared to increase with age.IMPORTANCE This study dissected the specific antibody responses toward the major antigenic sites and the noncanonical epitopes of influenza B virus hemagglutinin in animals and humans using novel reagents. These findings will guide the design of the next generation of influenza virus vaccines.


Assuntos
Anticorpos Neutralizantes/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Vírus da Influenza B/imunologia , Influenza Humana/imunologia , Adulto , Fatores Etários , Idoso , Animais , Anticorpos Antivirais/metabolismo , Pré-Escolar , Cães , Furões , Deriva Genética , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Epitopos Imunodominantes/imunologia , Lactente , Vírus da Influenza B/genética , Vacinas contra Influenza/imunologia , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Camundongos , Pessoa de Meia-Idade , Especificidade da Espécie
9.
J Clin Invest ; 128(11): 4992-4996, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30188868

RESUMO

Hemagglutination inhibition (HI) titers are a major correlate of protection for influenza-related illness. The influenza virus hemagglutinin possesses antigenic sites that are the targets of HI active antibodies. Here, a panel of mutant viruses each lacking a classically defined antigenic site was created to compare the species-specific immunodominance of the antigenic sites in a clinically relevant hemagglutinin. HI active antibodies of antisera from influenza virus-infected mice targeted sites Sb and Ca2. HI active antibodies of guinea pigs were not directed against any specific antigenic site, although trends were observed toward Sb, Ca2, and Sa. HI titers of antisera from infected ferrets were significantly affected by site Sa. HI active antibodies of adult humans followed yet another immunodominance pattern, in which sites Sb and Sa were immunodominant. When comparing the HI profiles among different species by antigenic cartography, animals and humans grouped separately. This study provides characterizations of the antibody-mediated immune responses against the head domain of a recent H1 hemagglutinin in animals and humans.


Assuntos
Anticorpos Antivirais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/imunologia , Animais , Antígenos Virais , Feminino , Furões , Cobaias , Humanos , Camundongos
10.
J Virol ; 92(20)2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30045991

RESUMO

The hemagglutinin protein of H3N2 influenza viruses is the major target of neutralizing antibodies induced by infection and vaccination. However, the virus frequently escapes antibody-mediated neutralization due to mutations in the globular head domain. Five topologically distinct antigenic sites in the head domain of H3 hemagglutinin, A to E, have been previously described by mapping the binding sites of monoclonal antibodies, yet little is known about the contribution of each site to the immunogenicity of modern H3 hemagglutinins, as measured by hemagglutination inhibition activity, which is known to correlate with protection. To investigate the hierarchy of antibody immunodominance, five Δ1 recombinant influenza viruses expressing hemagglutinin of the A/Hong Kong/4801/2014 (H3N2) strain with mutations in single antigenic sites were generated. Next, the Δ1 viruses were used to determine the hierarchy of immunodominance by measuring the hemagglutination inhibition reactivity of mouse antisera and plasma from 18 human subjects before and after seasonal influenza vaccination in 2017-2018. In both mice and humans, mutations in antigenic site B caused the most significant decrease in hemagglutination inhibition titers compared to wild-type hemagglutinin. This study revealed that antigenic site B is immunodominant in the H3N2 influenza virus strain included in the current vaccine preparations.IMPORTANCE Influenza viruses rapidly evade humoral immunity through antigenic drift, making current vaccines poorly effective and antibody-mediated protection short-lived. The majority of neutralizing antibodies target five antigenic sites in the head domain of the hemagglutinin protein that are also the most sequence-variable regions. A better understanding of the contribution of each antigenic site to the overall antibody response to hemagglutinin may help in the design of improved influenza virus vaccines.


Assuntos
Anticorpos Antivirais/sangue , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Epitopos Imunodominantes/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Animais , Voluntários Saudáveis , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Camundongos , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia
11.
J Virol ; 92(16)2018 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-29899093

RESUMO

Influenza A and B viruses can continuously evade humoral immune responses by developing mutations in the globular head of the hemagglutinin (HA) that prevent antibody binding. However, the influenza B virus HA over time displays less antigenic variation despite being functionally and structurally similar to the influenza A virus HA. To determine if the influenza B virus HA is under constraints that limit its antigenic variation, we performed a transposon screen to compare the mutational tolerance of the currently circulating influenza A virus HAs (H1 and H3 subtypes) and influenza B virus HAs (B/Victoria87 and B/Yamagata88 antigenic lineages). A library of insertional mutants for each HA was generated and deep sequenced after passaging to determine where insertions were tolerated in replicating viruses. The head domains of both viruses tolerated transposon mutagenesis, but the influenza A virus head was more tolerant to insertions than the influenza B virus head domain. Furthermore, all five of the known antigenic sites of the influenza A virus HA were tolerant of 15 nucleotide insertions, while insertions were detected in only two of the four antigenic sites in the influenza B virus head domain. Our analysis demonstrated that the influenza B virus HA is inherently less tolerant of transposon-mediated insertions than the influenza A virus HA. The reduced insertional tolerance of the influenza B virus HA may reveal genetic restrictions resulting in a lower capacity for antigenic evolution.IMPORTANCE Influenza viruses cause seasonal epidemics and result in significant human morbidity and mortality. Influenza viruses persist in the human population through generating mutations in the hemagglutinin head domain that prevent antibody recognition. Despite the similar selective pressures on influenza A and B viruses, influenza A virus displays a higher rate and breadth of antigenic variability than influenza B virus. A transposon mutagenesis screen was used to examine if the reduced antigenic variability of influenza B virus was due to inherent differences in mutational tolerance. This study demonstrates that the influenza A virus head domain and the individual antigenic sites targeted by humoral responses are more tolerant to insertions than those of influenza B virus. This finding sheds light on the genetic factors controlling the antigenic evolution of influenza viruses.


Assuntos
Variação Antigênica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/fisiologia , Vírus da Influenza B/fisiologia , Mutagênese Insercional , Mutagênese , Replicação Viral , Análise Mutacional de DNA , Elementos de DNA Transponíveis , Variação Genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Vírus da Influenza A/genética , Vírus da Influenza B/genética , Análise de Sequência de DNA
12.
PLoS One ; 13(4): e0194830, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29617394

RESUMO

The standard method to quantify the hemagglutinin content of influenza virus vaccines is the single radial immunodiffusion assay. This assay primarily relies on polyclonal antibodies against the head domain of the influenza virus hemagglutinin, which is the main target antigen of influenza virus vaccines. Novel influenza virus vaccine candidates that redirect the immune response towards the evolutionary more conserved hemagglutinin stalk, including chimeric hemagglutinin and headless hemagglutinin constructs, are highly dependent on the structural integrity of the protein to present conformational epitopes for neutralizing antibodies. In this study, we describe a novel enzyme-linked immunosorbent assay that allows quantifying the amount of hemagglutinin with correctly folded stalk domains and which could be further developed into a potency assay for stalk-based influenza virus vaccines.


Assuntos
Ensaio de Imunoadsorção Enzimática , Glicoproteínas de Hemaglutininação de Vírus da Influenza/análise , Vacinas contra Influenza/análise , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Vírus da Influenza A/metabolismo , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/metabolismo , Domínios Proteicos , Dobramento de Proteína , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química
13.
J Virol ; 91(14)2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28468881

RESUMO

Hendra virus (HeV) is a zoonotic paramyxovirus that causes deadly illness in horses and humans. An intriguing feature of HeV is the utilization of endosomal protease for activation of the viral fusion protein (F). Here we investigated how endosomal F trafficking affects HeV assembly. We found that the HeV matrix (M) and F proteins each induced particle release when they were expressed alone but that their coexpression led to coordinated assembly of virus-like particles (VLPs) that were morphologically and physically distinct from M-only or F-only VLPs. Mutations to the F protein transmembrane domain or cytoplasmic tail that disrupted endocytic trafficking led to failure of F to function with M for VLP assembly. Wild-type F functioned normally for VLP assembly even when its cleavage was prevented with a cathepsin inhibitor, indicating that it is endocytic F trafficking that is important for VLP assembly, not proteolytic F cleavage. Under specific conditions of reduced M expression, we found that M could no longer induce significant VLP release but retained the ability to be incorporated as a passenger into F-driven VLPs, provided that the F protein was competent for endocytic trafficking. The F and M proteins were both found to traffic through Rab11-positive recycling endosomes (REs), suggesting a model in which F and M trafficking pathways converge at REs, enabling these proteins to preassemble before arriving at plasma membrane budding sites.IMPORTANCE Hendra virus and Nipah virus are zoonotic paramyxoviruses that cause lethal infections in humans. Unlike that for most paramyxoviruses, activation of the henipavirus fusion protein occurs in recycling endosomal compartments. In this study, we demonstrate that the unique endocytic trafficking pathway of Hendra virus F protein is required for proper viral assembly and particle release. These results advance our basic understanding of the henipavirus assembly process and provide a novel model for the interplay between glycoprotein trafficking and paramyxovirus assembly.


Assuntos
Vírus Hendra/genética , Multimerização Proteica , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Virossomos/metabolismo , Linhagem Celular , Endossomos/metabolismo , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Domínios Proteicos , Transporte Proteico , Proteínas da Matriz Viral/metabolismo , Virossomos/genética
14.
J Virol ; 91(12)2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28356526

RESUMO

Seasonal influenza virus epidemics represent a significant public health burden. Approximately 25% of all influenza virus infections are caused by type B viruses, and these infections can be severe, especially in children. Current influenza virus vaccines are an effective prophylaxis against infection but are impacted by rapid antigenic drift, which can lead to mismatches between vaccine strains and circulating strains. Here, we describe a broadly protective vaccine candidate based on chimeric hemagglutinins, consisting of globular head domains from exotic influenza A viruses and stalk domains from influenza B viruses. Sequential vaccination with these constructs in mice leads to the induction of broadly reactive antibodies that bind to the conserved stalk domain of influenza B virus hemagglutinin. Vaccinated mice are protected from lethal challenge with diverse influenza B viruses. Results from serum transfer experiments and antibody-dependent cell-mediated cytotoxicity (ADCC) assays indicate that this protection is antibody mediated and based on Fc effector functions. The present data suggest that chimeric hemagglutinin-based vaccination is a viable strategy to broadly protect against influenza B virus infection.IMPORTANCE While current influenza virus vaccines are effective, they are affected by mismatches between vaccine strains and circulating strains. Furthermore, the antiviral drug oseltamivir is less effective for treating influenza B virus infections than for treating influenza A virus infections. A vaccine that induces broad and long-lasting protection against influenza B viruses is therefore urgently needed.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza B/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Modelos Animais de Doenças , Glicoproteínas de Hemaglutininação de Vírus da Influenza/administração & dosagem , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Vírus da Influenza A/química , Vírus da Influenza A/genética , Vírus da Influenza A/imunologia , Vírus da Influenza B/química , Camundongos , Infecções por Orthomyxoviridae/virologia , Receptores Fc/imunologia , Vacinação
15.
J Virol ; 88(22): 13099-110, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25210190

RESUMO

UNLABELLED: Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections. IMPORTANCE: Henipaviruses cause deadly infections in humans, with a mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998, killing 109 people, and smaller outbreaks have since occurred in Bangladesh and India. In this study, we have defined, for the first time, host factors that interact with henipavirus M proteins and contribute to viral particle assembly. We have also defined a new host protein-viral protein binding interface that can potentially be targeted for the inhibition of paramyxovirus infections.


Assuntos
Complexo 3 de Proteínas Adaptadoras/metabolismo , Subunidades beta do Complexo de Proteínas Adaptadoras/metabolismo , Vírus Hendra/fisiologia , Interações Hospedeiro-Patógeno , Vírus Nipah/fisiologia , Mapeamento de Interação de Proteínas , Proteínas da Matriz Viral/metabolismo , Liberação de Vírus , Humanos , Imunoprecipitação , Espectrometria de Massas
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