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1.
Lancet ; 396(10261): 1491-1503, 2020 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-33065035

RESUMO

BACKGROUND: Seasonal influenza remains a substantial public health threat despite the availability of egg-derived and other vaccines. Plant-based manufacturing might address some of the limitations of current vaccines. We describe two phase 3 efficacy studies of a recombinant quadrivalent virus-like particle (QVLP) influenza vaccine manufactured in plants, one in adults aged 18-64 years (the 18-64 study) and one in older people aged 65 years and older (the 65-plus study). METHODS: We did two randomised, observer-blind, multinational studies in the northern hemisphere in the 2017-18 (the 18-64 study) and 2018-19 (the 65-plus study) influenza seasons. The 18-64 study was done at 73 sites and the 65-plus study was done at 104 sites, both across Asia, Europe, and North America. In the 18-64 study, inclusion criteria were body-mass index less than 40 kg/m2; age 18-64 years at screening visit; and good health. In the 65-plus study, inclusion criteria were body-mass index of maximum 35 kg/m2; aged 65 years or older at screening visit; not living in a rehabilitation centre or care home; and no acute or evolving medical problems. Participants in the 18-64 study were randomly assigned (1:1) to receive either QVLP vaccine (30 µg haemagglutinin per strain) or placebo. Participants in the 65-plus study were randomly assigned (1:1) to receive QVLP vaccine (30 µg haemagglutinin per strain) or quadrivalent inactivated vaccine (QIV; 15 µg haemagglutinin per strain). The primary outcome in the 18-64 study was absolute vaccine efficacy to prevent laboratory-confirmed, respiratory illness caused by antigenically matched influenza strains. The primary outcome in the 65-plus study was relative vaccine efficacy to prevent laboratory-confirmed influenza-like illness caused by any influenza strain. The primary analyses were done in the per-protocol population and safety was assessed in all participants who received the assigned treatment. These studies are registered with ClinicalTrials.gov (18-64 study NCT03301051; 65-plus study NCT03739112). FINDINGS: In the 18-64 study, between Aug 30, 2017, and Jan 15, 2018, 10 160 participants were randomly assigned to receive either QVLP vaccine (5077 participants) or placebo (5083 participants). The per-protocol population consisted of 4814 participants in the QVLP group and 4812 in the placebo group. The study did not meet its primary endpoint of 70% absolute vaccine efficacy for the QVLP vaccine (35·1% [95% CI 17·9 to 48·7]) against respiratory illness caused by matched strains. 55 (1·1%) of 5064 participants in the QVLP group versus 51 (1·0%) of 5072 in the placebo group had a serious adverse event. Four (0·1%) and six [0·1%] participants had severe treatment-related treatment-emergent adverse events. In the 65-plus study, between Sept 18, 2018, and Feb 22, 2019, 12 794 participants were randomly assigned to receive either QVLP vaccine (6396 participants) or QIV (6398 participants). The per-protocol population consisted of 5996 participants in the QVLP group and 6026 in the QIV group. The study met its primary non-inferiority endpoint with a relative vaccine efficacy of the QVLP vaccine for the prevention of influenza-like illness caused by any strain of 8·8% (-16·7 to 28·7). 263 (4·1%) of 6352 participants in the QVLP group versus 266 (4·2%) of 6366 in the QIV group had serious adverse events (one [<0·1%] vs two [<0·1%] were considered treatment-related); one (<0·1%) versus three (<0·1%) participants had severe treatment-related treatment-emergent adverse events. INTERPRETATION: These efficacy studies are the first large-scale studies of any plant-derived human vaccine. Together, they show that the plant-derived QVLP vaccine can provide substantial protection against respiratory illness and influenza-like illness caused by influenza viruses in adults. QVLP vaccine was well tolerated and no major safety signal arose in participants who received QVLP vaccine across the two studies. FUNDING: Medicago.


Assuntos
Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Vacinas de Produtos Inativados/imunologia , Adolescente , Adulto , Idoso , Anticorpos Antivirais , Método Duplo-Cego , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Plantas/imunologia , Adulto Jovem
2.
PLoS One ; 15(9): e0239015, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32925936

RESUMO

Understanding the extent and limitation of viral genome evolution can provide insight about potential drug and vaccine targets. Influenza B Viruses (IBVs) infect humans in a seasonal manner and causes significant morbidity and mortality. IBVs are negative-sense single-stranded RNA viruses with a segmented genome and can be divided into two antigenically distinct lineages. The two lineages have been circulating and further evolving for almost four decades. The immune response to IBV infection can lead to antibodies that target the strain causing the infection. Some antibodies are cross-reactive and are able to bind strains from both lineages but, because of antigenic drift and immunodominance, both lineages continue to evolve and challenge human health. Here we investigate changes in the genomes of an IBVs from each lineage after passage in tissue culture in the presence of human sera containing polyclonal antibodies directed toward antigenically and temporally distinct viruses. Our previous analysis of the fourth segment, which encodes the major surface protein HA, revealed a pattern of change in which signature sequences from one lineage mutated to the signature sequences of the other lineage. Here we analyze genes from the other genomic segments and observe that most of the quasispecies' heterogeneity occurs at the same loci in each lineage. The nature of the variants at these loci are investigated and possible reasons for this pattern are discussed. This work expands our understanding of the extent and limitations of genomic change in IBV.


Assuntos
Variação Antigênica/genética , Epitopos/genética , Vírus da Influenza B/genética , Animais , Anticorpos Antivirais/sangue , Cães , Genoma Viral/genética , Genômica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vírus da Influenza B/crescimento & desenvolvimento , Influenza Humana/virologia , Células Madin Darby de Rim Canino
3.
PLoS Pathog ; 16(9): e1008583, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32970783

RESUMO

The prospect of universal influenza vaccines is generating much interest and research at the intersection of immunology, epidemiology, and viral evolution. While the current focus is on developing a vaccine that elicits a broadly cross-reactive immune response in clinical trials, there are important downstream questions about global deployment of a universal influenza vaccine that should be explored to minimize unintended consequences and maximize benefits. Here, we review and synthesize the questions most relevant to predicting the population benefits of universal influenza vaccines and discuss how existing information could be mined to begin to address these questions. We review three research topics where computational modeling could bring valuable evidence: immune imprinting, viral evolution, and transmission. We address the positive and negative consequences of imprinting, in which early childhood exposure to influenza shapes and limits immune responses to future infections via memory of conserved influenza antigens. However, the mechanisms at play, their effectiveness, breadth of protection, and the ability to "reprogram" already imprinted individuals, remains heavily debated. We describe instances of rapid influenza evolution that illustrate the plasticity of the influenza virus in the face of drug pressure and discuss how novel vaccines could introduce new selective pressures on the evolution of the virus. We examine the possible unintended consequences of broadly protective (but infection-permissive) vaccines on the dynamics of epidemic and pandemic influenza, compared to conventional vaccines that have been shown to provide herd immunity benefits. In conclusion, computational modeling offers a valuable tool to anticipate the benefits of ambitious universal influenza vaccine programs, while balancing the risks from endemic influenza strains and unpredictable pandemic viruses. Moving forward, it will be important to mine the vast amount of data generated in clinical studies of universal influenza vaccines to ensure that the benefits and consequences of these vaccine programs have been carefully modeled and explored.


Assuntos
Anticorpos Antivirais/imunologia , Pesquisa Biomédica/tendências , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Ensaios Clínicos como Assunto , Humanos , Influenza Humana/epidemiologia , Influenza Humana/imunologia , Influenza Humana/virologia
4.
Nat Commun ; 11(1): 4566, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917903

RESUMO

Influenza virus exposures in childhood can establish long-lived memory B cell responses that can be recalled later in life. Here, we complete a large serological survey to elucidate the specificity of antibodies against contemporary H3N2 viruses in differently aged individuals who were likely primed with different H3N2 strains in childhood. We find that most humans who were first infected in childhood with H3N2 viral strains from the 1960s and 1970s possess non-neutralizing antibodies against contemporary 3c2.A H3N2 viruses. We find that 3c2.A H3N2 virus infections boost non-neutralizing H3N2 antibodies in middle-aged individuals, potentially leaving many of them in a perpetual state of 3c2.A H3N2 viral susceptibility.


Assuntos
Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/imunologia , Antígenos Virais/imunologia , Criança , Pré-Escolar , Suscetibilidade a Doenças , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Philadelphia , Proteínas Recombinantes , Estações do Ano , Adulto Jovem
5.
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
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.
Proc Natl Acad Sci U S A ; 117(29): 17221-17227, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32631992

RESUMO

Immunity to influenza viruses can be long-lived, but reinfections with antigenically distinct viral strains and subtypes are common. Reinfections can boost antibody responses against viral strains first encountered in childhood through a process termed "original antigenic sin." It is unknown how initial childhood exposures affect the induction of antibodies against the hemagglutinin (HA) stalk domain of influenza viruses. This is an important consideration since broadly reactive HA stalk antibodies can protect against infection, and universal vaccine platforms are being developed to induce these antibodies. Here we show that experimentally infected ferrets and naturally infected humans establish strong "immunological imprints" against HA stalk antigens first encountered during primary influenza virus infections. We found that HA stalk antibodies are surprisingly boosted upon subsequent infections with antigenically distinct influenza A virus subtypes. Paradoxically, these heterosubtypic-boosted HA stalk antibodies do not bind efficiently to the boosting influenza virus strain. Our results demonstrate that an individual's HA stalk antibody response is dependent on the specific subtype of influenza virus that they first encounter early in life. We propose that humans are susceptible to heterosubtypic influenza virus infections later in life since these viruses boost HA stalk antibodies that do not bind efficiently to the boosting antigen.


Assuntos
Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Infecções por Orthomyxoviridae/imunologia , Animais , Furões , Hemaglutininas , Humanos , Imunização Secundária , Imunoglobulina G/sangue , Proteínas Recombinantes
8.
Proc Natl Acad Sci U S A ; 117(30): 17757-17763, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32669430

RESUMO

Vaccination has been used to control the spread of seasonal flu; however, the virus continues to evolve and escape from host immune response through mutation and increasing glycosylation. Efforts have been directed toward development of a universal vaccine with broadly protective activity against multiple influenza strains and subtypes. Here we report the design and evaluation of various chimeric vaccines based on the most common avian influenza H5 and human influenza H1 sequences. Of these constructs, the chimeric HA (cHA) vaccine with consensus H5 as globular head and consensus H1 as stem was shown to elicit broadly protective CD4+ and CD8+ T cell responses. Interestingly, the monoglycosylated cHA (cHAmg) vaccine with GlcNAc on each glycosite induced more stem-specific antibodies, with higher antibody-dependent cellular cytotoxicity (ADCC), and better neutralizing and stronger cross-protection activities against H1, H3, H5, and H7 strains and subtypes. Moreover, the cHAmg vaccine combined with a glycolipid adjuvant designed for class switch further enhanced the vaccine efficacy with more IFN-γ, IL-4, and CD8+ memory T cells produced.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteção Cruzada/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Orthomyxoviridae/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Modelos Animais de Doenças , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos , Influenza Humana/virologia , Camundongos , Modelos Moleculares , Testes de Neutralização , Orthomyxoviridae/classificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Relação Estrutura-Atividade , Vacinação
9.
BMC Bioinformatics ; 21(1): 256, 2020 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-32560624

RESUMO

BACKGROUND: In 2009, a novel influenza vaccine was distributed worldwide to combat the H1N1 influenza "swine flu" pandemic. However, antibodies induced by the vaccine display differences in their specificity and cross-reactivity dependent on pre-existing immunity. Here, we present a computational model that can capture the effect of pre-existing immunity on influenza vaccine responses. The model predicts the region of the virus hemagglutinin (HA) protein targeted by antibodies after vaccination as well as the level of cross-reactivity induced by the vaccine. We tested our model by simulating a scenario similar to the 2009 pandemic vaccine and compared the results to antibody binding data obtained from human subjects vaccinated with the monovalent 2009 H1N1 influenza vaccine. RESULTS: We found that both specificity and cross-reactivity of the antibodies induced by the 2009 H1N1 influenza HA protein were affected by the viral strain the individual was originally exposed. Specifically, the level of antigenic relatedness between the original exposure HA antigen and the 2009 HA protein affected antigenic-site immunodominance. Moreover, antibody cross-reactivity was increased when the individual's pre-existing immunity was specific to an HA protein antigenically distinct from the 2009 pandemic strain. Comparison of simulation data with antibody binding data from human serum samples demonstrated qualitative and quantitative similarities between the model and real-life immune responses to the 2009 vaccine. CONCLUSION: We provide a novel method to evaluate expected outcomes in antibody specificity and cross-reactivity after influenza vaccination in individuals with different influenza HA antigen exposure histories. The model produced similar outcomes as what has been previously reported in humans after receiving the 2009 influenza pandemic vaccine. Our results suggest that differences in cross-reactivity after influenza vaccination should be expected in individuals with different exposure histories.


Assuntos
Anticorpos Antivirais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Modelos Imunológicos , Sequência de Aminoácidos , Anticorpos Antivirais/sangue , Antígenos Virais/química , Antígenos Virais/imunologia , Simulação por Computador , Reações Cruzadas , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos
10.
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
11.
DNA Cell Biol ; 39(9): 1730-1740, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32580635

RESUMO

Polyinosinic-polycytidylic acid (PIC) is a potent double-stranded RNA (dsRNA) adjuvant useful in intranasal influenza vaccination. In mice, the intensity and duration of immune responses to PIC correlated with the double-stranded chain length. A rational method to avoid PIC chain extension in PIC production is to use multiple short poly(I) molecules and one long poly(C) molecule for PIC assembly. In this study, we elucidate that a newly developed uPIC100-400 molecule comprising multiple 0.1 kb poly(I) molecules and one 0.4 kb poly(C) molecule effectively enhanced the immune responses in mice, by preventing the challenged viral propagation and inducing hemagglutinin-specific IgA, after intranasal A(H1N1)pdm09 influenza vaccination. Reduced intraperitoneal toxicity of PIC prepared with multiple short poly(I) molecules in mice indicates the widened effective range of uPIC100-400 as an adjuvant. In contrast to uPIC100-400, the PIC molecule comprising multiple 0.05 kb poly(I) molecules failed to elicit mouse mucosal immunity. These results were consistent with TLR3 response but not retinoic acid inducible gene I (RIG-I)-like receptor response in the cell assays, which suggests that the adjuvant effect of PIC in mouse intranasal immunization depends on TLR3 signaling. In conclusion, the double-stranded PIC with reduced toxicity developed in this study would contribute to the development of PIC-adjuvanted vaccines.


Assuntos
Adjuvantes Imunológicos/uso terapêutico , Indutores de Interferon/uso terapêutico , Infecções por Orthomyxoviridae/imunologia , Poli I-C/uso terapêutico , Receptor 3 Toll-Like/metabolismo , Vacinação/métodos , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/efeitos adversos , Animais , Células Cultivadas , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunoglobulina A/imunologia , Vacinas contra Influenza/imunologia , Indutores de Interferon/administração & dosagem , Indutores de Interferon/efeitos adversos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/prevenção & controle , Poli I-C/administração & dosagem , Poli I-C/efeitos adversos , Transdução de Sinais
12.
Nat Commun ; 11(1): 2445, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415074

RESUMO

The 2019-2020 Northern Hemisphere influenza vaccine includes antigens from 3c3.A H3N2 viruses; however, over half of circulating H3N2 viruses belong to subclade 3c2.A1b. Here, we analyze antibody responses elicited by the egg-adapted 3c3.A H3N2 vaccine strain in ferrets and humans. We find that this vaccine strain elicits antibodies that have reduced reactivity to a wild-type 3c3.A strain and very limited reactivity to 3c2.A strains, including the currently circulating 3c2.A1b strain.


Assuntos
Antígenos Virais/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Vacinas contra Influenza/imunologia , Adulto , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/química , Furões/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Óvulo/imunologia
13.
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
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.
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
16.
Vet Microbiol ; 243: 108640, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32273019

RESUMO

In the present study, we have generated several H5N2 HA recombinant baculoviruses for production of a HA subunit vaccine against the lethal H5N2 avian influenza virus (AIV). The effective display of functional HA on the cell membrane and baculoviral envelope was examined. Our results reveal that chickens immunized with the chimeric AIV HA protein fused with the baculovirus gp64 cytoplasmic domain (CTD) induced higher HI titer. To further increase the expression level of the H5N2 AIV HA protein, the HA gene of H5N2 AIV was amplified and cloned into three novel baculovirus surface display vectors BacDual DisplayEGFP-2HA, BacDual DisplayEGFP-3HA, BacDual DisplayEGFP-4HA which contains multiple expression cassettes for higher level display of HA proteins on the cell membrane and baculovirus envelope. To determine the optimum conditions for producing HA protein, various MOI, infection times, and shaker times for virus transfection were tested. Our results reveal that the conditions of an MOI of 5, 3 day post infection, and 15 min of shaker time have higher efficiency for HA protein production. Our results reveal that the baculovirus surface display vector pBacDual DisplayEGFP-4HA increases significantly the expression level of the H5N2 AIV HA protein. Chickens that received two doses of BacDual DisplayEGFP-4HA cell lysates formulated with Montanide ISA70 adjuvant elicited efficient immunogenicity and had an average HI titer of 7 log2 at 2 weeks post-vaccination. Challenge studies revealed that vaccinated chickens with HI titers 5 log2 were completely protected against the lethal H5N1 AIV challenge. Furthermore, HI titers could be maintained at 5 log2 for 20 weeks for laying hens. This study suggests that the HA protein expression from the baculovirus surface display system could be a safe and efficacious subunit vaccine for chickens.


Assuntos
Baculoviridae/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Virus da Influenza A Subtipo H5N1/patogenicidade , Vacinas contra Influenza/imunologia , Influenza Aviária/prevenção & controle , Animais , Anticorpos Antivirais/sangue , Galinhas/imunologia , Galinhas/virologia , Feminino , Vírus da Influenza A Subtipo H5N2/genética , Vacinas contra Influenza/genética , Influenza Aviária/imunologia , Vacinas de Subunidades/genética , Vacinas de Subunidades/imunologia
17.
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
18.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(1): 26-32, 2020 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-32314721

RESUMO

Objective To study B cell epitopes of the influenza virus hemagglutinin (HA) by computer simulation and ELISA blocking experiment, and to establish a new method for pathogen microbial epitope detection. Methods The 2009 H1N1 influenza virus inactivated vaccine was used as the immunogen, and the monoclonal antibodies (mAbs) were prepared by conventional hybridoma fusion and screening techniques. The characteristics of mAbs were identified by ELISA, hemagglutination inhibition (HI) and Western blot analysis. The obtained mAbs were used as a tool to predict the epitope of H1N1 influenza virus by ELISA blocking experiment combined with computer simulation method. Results Four mAbs against the HA antigen on H1N1 influenza virus were obtained, and HA epitopes were classified into two types by ELISA blocking experiment. The computer simulation revealed that the four antibodies could bind to two epitopes on HA. Conclusion The results of computer simulation and ELISA blocking experiment are consistent, and a new method for predicting the epitopes of other pathogenic microorganisms had been established.


Assuntos
Simulação por Computador , Epitopos de Linfócito B/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Ensaio de Imunoadsorção Enzimática
19.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(1): 69-74, 2020 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-32314726

RESUMO

Objective To prepare the monoclonal antibodies (mAb) against hemagglutinin of H4 subtype avian influenza virus (AIV), and develop a sandwich ELISA for the detection of H4 subtype AIV. Methods The BALB/c mice were immunized with inactive H4 subtype AIV. A mAb against H4 subtype AIV, designated as 6G4, was obtained by cell fusion, hemagglutination inhibition (HI) screening and subcloing. Immuofluorescence cytochemistry and Western blotting were used to detect the reactivity of 6G4 with H4 subtype AIV, and the specificity, broad spectrum and stability of 6G4 were characterized by HI assay. Subclass of 6G4 was determined by kit. With chicken polyclonal antibody against H4 subtype AIV as coated antibody, 6G4 mAb as capture antibody and HRP-labeled goat anti-mouse IgG as the enzyme-labeled antibody, a sandwich ELISA for the detection of H4 subtype AIV was established by optimization of the reaction conditions and serial verification. Results 6G4 belonged to IgG1 subclass, and the light chain belonged to κ. It could secrete antibody stably and had good reactivity, specificity, broad spectrum and stability. ELISA based on 6G4 was specific, sensitive, accurate and suitable for the detection of a large number of samples. Conclusion We successfully achieved the anti-H4 subtype AIV mAb, and developed the sandwich ELISA for the detection of H4 subtype AIV.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/biossíntese , Ensaio de Imunoadsorção Enzimática , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Testes de Inibição da Hemaglutinação , Vírus da Influenza A , Camundongos , Camundongos Endogâmicos BALB C
20.
J Gen Virol ; 101(5): 473-483, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32209169

RESUMO

Newcastle disease virus (NDV) vectors expressing avian influenza virus (AIV) haemagglutinin (HA) of subtype H5 simultaneously protect chickens from Newcastle disease (ND) as well as avian influenza (AI). The expressed, membrane-bound surface protein HA is incorporated into virions while soluble HA has been described as a potent antigen. We tested whether co-expression of both HA variants from the same NDV vector increased the overall level of HA, which could be important for optimal immunogenicity. Recombinant NDVsolH5_H5 co-expressed membrane-bound H5 of highly pathogenic (HP) AIV H5N1, detectable in infected cells, and soluble H5, which was secreted into the supernatant. This virus was compared to recombinant NDV that express either membrane-bound (rNDVH5) or soluble H5 (rNDVsolH5). Replication in embryonated chicken eggs (ECEs) and in cell culture, as well as pathogenicity in ECEs, was not influenced by the second heterologous transcriptional unit. However, the co-expression of soluble H5 with membrane-bound H5 increased total protein level about 5.25-fold as detected by MS quantification. Hence, this virus is very interesting as a vaccine virus in chickens against HPAIV infections in situations in which previous H5-expressing NDVs have reached their limit, such as in the face of pre-existing AIV maternal immunity.


Assuntos
Antígenos Virais/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Influenza Aviária/imunologia , Vírus da Doença de Newcastle/imunologia , Animais , Anticorpos Antivirais/imunologia , Linhagem Celular , Galinhas , Cricetinae , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Doença de Newcastle/imunologia , Eliminação de Partículas Virais/imunologia
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