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1.
Virology ; 526: 125-137, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30388628

RESUMO

The development of a universal influenza vaccine has become a major effort to combat the high mutation rate of influenza. To explore the use of the highly conserved stem region of hemagglutinin (HA) as a universal vaccine, we produced HA-stem-based protein using yeast expression systems. The glycosylation effects on the immunogenicity and protection activities were investigated. The yield of the A/Brisbane/59/2007 HA stem produced from Pichia pastoris reached 100 mg/l. The immunogenicity of HA stem proteins in various glycoforms was further investigated and compared. All glycoforms of the HA stem protein can induce cross-reactive antibody responses, antibody-dependent cellular cytotoxicity (ADCC)-mediated protection as well as T-cell responses, with broad protection in mice. The monoglycosylated form of the A/Brisbane/59/2007 HA stem produced in yeast, together with the glycolipid C34 as the adjuvant, can elicit greater ADCC responses, better neutralizing activities against heterologous strains, and broader protection in mice.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Pichia/metabolismo , Adjuvantes Imunológicos , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Técnicas de Cultura Celular por Lotes , Reações Cruzadas , Modelos Animais de Doenças , Feminino , Glicosilação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/biossíntese , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vacinas contra Influenza/biossíntese , Vacinas contra Influenza/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Pichia/genética , Linfócitos T/metabolismo
2.
Int J Pharm ; 545(1-2): 215-228, 2018 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-29684561

RESUMO

Epidermal powder immunization (EPI) is an alternative technique to the classical immunization route using needle and syringe. In this work, we present the results of an in vivo pilot study in piglets using a dried influenza model vaccine which was applied by EPI using a novel pyrotechnically driven applicator. A liquid influenza vaccine (Pandemrix®) was first concentrated by tangential flow filtration and hemagglutinin content was determined by RP-HPLC. The liquid formulation was then transformed into a dry powder by collapse freeze-drying and subsequent cryo-milling. The vaccine powder was attached to a membrane of a novel pyrotechnical applicator using oily adjuvant components. Upon actuation of the applicator, particles were accelerated to high speed as determined by a high-speed camera setup. Piglets were immunized twice using either the novel pyrotechnical applicator or classical intramuscular injection. Blood samples of the animals were collected at various time points and analyzed by enzyme-linked immunosorbent assay. Our pilot study shows that acceleration of a dried vaccine powder to supersonic speed using the pyrotechnical applicator is possible and that the speed and impact of the particles is sufficient to breach the stratum corneum of piglet skin. Importantly, the administration of the dry vaccine powder resulted in measurable anti-H1N1 antibody titres in vivo.


Assuntos
Imunização/instrumentação , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Administração Cutânea , Animais , Animais Recém-Nascidos , Anticorpos Antivirais/sangue , Biomarcadores/sangue , Composição de Medicamentos , Epiderme , Liofilização , Imunização/métodos , Esquemas de Imunização , Imunogenicidade da Vacina , Vacinas contra Influenza/química , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/metabolismo , Injeções Intramusculares , Projetos Piloto , Pós , Sus scrofa , Tecnologia Farmacêutica/métodos , Fatores de Tempo
3.
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
4.
Drug Deliv ; 25(1): 773-779, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29542358

RESUMO

Avian influenza virus infection is a serious public health threat and preventive vaccination is the most cost-effective public health intervention strategy. Unfortunately, currently available unadjuvanted avian influenza vaccines are poorly immunogenic and alternative vaccine formulations and delivery strategies are in urgent need to reduce the high risk of avian influenza pandemics. Cationic polymers have been widely used as vectors for gene delivery in vitro and in vivo. In this study, we formulated H5N1 influenza vaccines with GenJet™ or in vivo-jetPEI®, and showed that these formulations significantly enhanced the immunogenicity of H5N1 vaccines and conferred protective immunity in a mouse model. Detailed analyses of adaptive immune responses revealed that both formulations induced mixed TH1/TH2 antigen-specific CD4 T-cell responses, antigen-specific cytotoxic CD8 T-cell and memory B-cell responses. Our findings suggest that cationic polymers merit future development as potential adjuvants for mucosal delivery of poorly immunogenic vaccines.


Assuntos
Imunidade Adaptativa/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Imunidade Celular/efeitos dos fármacos , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/administração & dosagem , Influenza Aviária/imunologia , Vacinas Sintéticas/administração & dosagem , Administração Intranasal , Animais , Anticorpos Antivirais/análise , Anticorpos Antivirais/biossíntese , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Composição de Medicamentos , Feminino , Imunogenicidade da Vacina , Indicadores e Reagentes/química , Indicadores e Reagentes/metabolismo , Vacinas contra Influenza/genética , Vacinas contra Influenza/metabolismo , Vacinas contra Influenza/uso terapêutico , Influenza Aviária/metabolismo , Influenza Aviária/prevenção & controle , Influenza Aviária/virologia , Camundongos Endogâmicos BALB C , Aves Domésticas , Análise de Sobrevida , Vacinas Sintéticas/genética , Vacinas Sintéticas/metabolismo , Vacinas Sintéticas/uso terapêutico , Perda de Peso/efeitos dos fármacos
5.
BMC Biotechnol ; 17(1): 79, 2017 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-29126399

RESUMO

BACKGROUND: The lack of a universal influenza vaccine is a global health problem. Interest is now focused on structurally conserved protein domains capable of eliciting protection against a broad range of influenza virus strains. The long alpha helix (LAH) is an attractive vaccine component since it is one of the most conserved influenza hemagglutinin (HA) stalk regions. For an improved immune response, the LAH domain from H3N2 strain has been incorporated into virus-like particles (VLPs) derived from hepatitis B virus core protein (HBc) using recently developed tandem core technology. RESULTS: Fermentation conditions for recombinant HBc-LAH were established in yeast Pichia pastoris and a rapid and efficient purification method for chimeric VLPs was developed to match the requirements for industrial scale-up. Purified VLPs induced strong antibody responses against both group 1 and group 2 HA proteins in mice. CONCLUSION: Our results indicate that the tandem core technology is a useful tool for incorporation of highly hydrophobic LAH domain into HBc VLPs. Chimeric VLPs can be successfully produced in bioreactor using yeast expression system. Immunologic data indicate that HBc VLPs carrying the LAH antigen represent a promising universal influenza vaccine component.


Assuntos
Hemaglutininas Virais/isolamento & purificação , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vacinas contra Influenza/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Vírion/isolamento & purificação , Animais , Anticorpos Antivirais , Feminino , Hemaglutininas Virais/genética , Hemaglutininas Virais/imunologia , Hemaglutininas Virais/metabolismo , Vírus da Influenza A Subtipo H3N2/genética , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Pichia/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Vírion/genética , Vírion/imunologia , Vírion/metabolismo
6.
Hum Vaccin Immunother ; 13(10): 2364-2378, 2017 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-28925794

RESUMO

Spray drying is a promising method for the stabilization of vaccines, which are usually formulated as liquids. Usually, vaccine stability is improved by spray drying in the presence of a range of excipients. Unlike freeze drying, there is no freezing step involved, thus the damage related to this step is avoided. The edge of spray drying resides in its ability for particles to be engineered to desired requirements, which can be used in various vaccine delivery methods and routes. Although several spray dried vaccines have shown encouraging preclinical results, the number of vaccines that have been tested in clinical trials is limited, indicating a relatively new area of vaccine stabilization and delivery. This article reviews the current status of spray dried vaccine formulations and delivery methods. In particular it discusses the impact of process stresses on vaccine integrity, the application of excipients in spray drying of vaccines, process and formulation optimization strategies based on Design of Experiment approaches as well as opportunities for future application of spray dried vaccine powders for vaccine delivery.


Assuntos
Vacinas/administração & dosagem , Vacinas/química , Administração Oral , Animais , Química Farmacêutica/métodos , Dessecação , Composição de Medicamentos , Humanos , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/química , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/metabolismo , Camundongos , Sprays Nasais , Tamanho da Partícula , Pós , Potência de Vacina , Vacinas/imunologia , Vacinas/metabolismo
7.
Plant Biotechnol J ; 15(3): 285-296, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27483398

RESUMO

Influenza virus-like particles (VLPs) have been shown to induce a safe and potent immune response through both humoral and cellular responses. They represent promising novel influenza vaccines. Plant-based biotechnology allows for the large-scale production of VLPs of biopharmaceutical interest using different model organisms, including Nicotiana benthamiana plants. Through this platform, influenza VLPs bud from the plasma membrane and accumulate between the membrane and the plant cell wall. To design and optimize efficient production processes, a better understanding of the plant cell wall composition of infiltrated tobacco leaves is a major interest for the plant biotechnology industry. In this study, we have investigated the alteration of the biochemical composition of the cell walls of N. benthamiana leaves subjected to abiotic and biotic stresses induced by the Agrobacterium-mediated transient transformation and the resulting high expression levels of influenza VLPs. Results show that abiotic stress due to vacuum infiltration without Agrobacterium did not induce any detectable modification of the leaf cell wall when compared to non infiltrated leaves. In contrast, various chemical changes of the leaf cell wall were observed post-Agrobacterium infiltration. Indeed, Agrobacterium infection induced deposition of callose and lignin, modified the pectin methylesterification and increased both arabinosylation of RG-I side chains and the expression of arabinogalactan proteins. Moreover, these modifications were slightly greater in plants expressing haemagglutinin-based VLP than in plants infiltrated with the Agrobacterium strain containing only the p19 suppressor of silencing.


Assuntos
Agrobacterium/metabolismo , Biotecnologia/métodos , Parede Celular/metabolismo , Hemaglutininas/metabolismo , Tabaco/metabolismo , Agrobacterium/genética , Hemaglutininas/genética , Vacinas contra Influenza/genética , Vacinas contra Influenza/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Tabaco/genética
8.
PLoS One ; 10(6): e0130375, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26091084

RESUMO

DNA vaccines can be manufactured cheaply, easily and rapidly and have performed well in pre-clinical animal studies. However, clinical trials have so far been disappointing, failing to evoke a strong immune response, possibly due to poor antigen expression. To improve antigen expression, improved technology to monitor DNA vaccine transfection efficiency is required. In the current study, we compared plasmid encoded tdTomato, mCherry, Katushka, tdKatushka2 and luciferase as reporter proteins for whole animal in vivo imaging. The intramuscular, subcutaneous and tattooing routes were compared and electroporation was used to enhance expression. We observed that overall, fluorescent proteins were not a good tool to assess expression from DNA plasmids, with a highly heterogeneous response between animals. Of the proteins used, intramuscular delivery of DNA encoding either tdTomato or luciferase gave the clearest signal, with some Katushka and tdKatushka2 signal observed. Subcutaneous delivery was weakly visible and nothing was observed following DNA tattooing. DNA encoding haemagglutinin was used to determine whether immune responses mirrored visible expression levels. A protective immune response against H1N1 influenza was induced by all routes, even after a single dose of DNA, though qualitative differences were observed, with tattooing leading to high antibody responses and subcutaneous DNA leading to high CD8 responses. We conclude that of the reporter proteins used, expression from DNA plasmids can best be assessed using tdTomato or luciferase. But, the disconnect between visible expression level and immunogenicity suggests that in vivo whole animal imaging of fluorescent proteins has limited utility for predicting DNA vaccine efficacy.


Assuntos
Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Proteínas Luminescentes/biossíntese , Vacinas de DNA/imunologia , Animais , Células CHO , Cricetinae , Cricetulus , Cães , Feminino , Expressão Gênica , Humanos , Imunização , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/genética , Vacinas contra Influenza/metabolismo , Proteínas Luminescentes/genética , Células Madin Darby de Rim Canino , Camundongos Endogâmicos BALB C , Vacinas de DNA/genética , Vacinas de DNA/metabolismo , Imagem Corporal Total
9.
BMC Biotechnol ; 15: 31, 2015 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-25981500

RESUMO

BACKGROUND: Each year, influenza is responsible for hundreds of thousand cases of illness and deaths worldwide. Due to the virus' fast mutation rate, the World Health Organization (WHO) is constantly on alert to rapidly respond to emerging pandemic strains. Although anti-viral therapies exist, the most proficient way to stop the spread of disease is through vaccination. The majority of influenza vaccines on the market are produced in embryonic hen's eggs and are composed of purified viral antigens from inactivated whole virus. This manufacturing system, however, is limited in its production capacity. Cell culture produced vaccines have been proposed for their potential to overcome the problems associated with egg-based production. Virus-like particles (VLPs) of influenza virus are promising candidate vaccines under consideration by both academic and industry researchers. METHODS: In this study, VLPs were produced in HEK293 suspension cells using the Bacmam transduction system and Sf9 cells using the baculovirus infection system. The proposed systems were assessed for their ability to produce influenza VLPs composed of Hemagglutinin (HA), Neuraminidase (NA) and Matrix Protein (M1) and compared through the lens of bioprocessing by highlighting baseline production yields and bioactivity. VLPs from both systems were characterized using available influenza quantification techniques, such as single radial immunodiffusion assay (SRID), HA assay, western blot and negative staining transmission electron microscopy (NSTEM) to quantify total particles. RESULTS: For the HEK293 production system, VLPs were found to be associated with the cell pellet in addition to those released in the supernatant. Sf9 cells produced 35 times more VLPs than HEK293 cells. Sf9-VLPs had higher total HA activity and were generally more homogeneous in morphology and size. However, Sf9 VLP samples contained 20 times more baculovirus than VLPs, whereas 293 VLPs were produced along with vesicles. CONCLUSIONS: This study highlights key production hurdles that must be overcome in both expression platforms, namely the presence of contaminants and the ensuing quantification challenges, and brings up the question of what truly constitutes an influenza VLP candidate vaccine.


Assuntos
Antígenos Virais/química , Antígenos Virais/metabolismo , Vacinas contra Influenza/química , Vacinas contra Influenza/metabolismo , Vírion/química , Vírion/metabolismo , Animais , Antígenos Virais/genética , Antígenos Virais/isolamento & purificação , Células HEK293 , Humanos , Vacinas contra Influenza/genética , Vacinas contra Influenza/isolamento & purificação , Neuraminidase/química , Neuraminidase/genética , Neuraminidase/isolamento & purificação , Neuraminidase/metabolismo , Células Sf9 , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/isolamento & purificação , Proteínas da Matriz Viral/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Proteínas Virais/metabolismo , Vírion/genética , Vírion/isolamento & purificação
10.
Biotechnol Bioeng ; 112(11): 2267-75, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25943562

RESUMO

Dissolved carbon dioxide (dCO2 ) accumulation during cell culture has been recognized as an important parameter that needs to be controlled for successful scale-up of animal cell culture because above a certain concentration there are adverse effects on cell growth performance and protein production. We investigated the effect of accumulation of dCO2 in bioreactor cultures of expresSF+(®) insect cells infected with recombinant baculoviruses expressing recombinant influenza virus hemagglutinins (rHA). Different strategies for bioreactor cultures were used to obtain various ranges of concentrations of dCO2 (<50, 50-100, 100-200, and >200 mmHg) and to determine their effects on recombinant protein production and cell metabolic activity. We show that the accumulation of dCO2 at levels > 100 mmHg resulted in reduced metabolic activity, slowed cell growth, prolonged culture viability after infection, and decreased infection kinetics. The reduced rHA yields were not caused by the decrease in the extracellular pH that resulted from dCO2 accumulation, but were most likely due to the effect of dCO2 accumulation in cells. The results obtained here at the 2 L scale have been used for the design of large-scale processes to manufacture the rHA based recombinant vaccine Flublok™ at the 2500 L scale Biotechnol. Bioeng. 2015;112: 2267-2275. © 2015 Wiley Periodicals, Inc.


Assuntos
Dióxido de Carbono/análise , Meios de Cultura/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vacinas contra Influenza/metabolismo , Animais , Reatores Biológicos , Linhagem Celular , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Concentração de Íons de Hidrogênio , Vacinas contra Influenza/genética , Insetos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vacinas Sintéticas/genética , Vacinas Sintéticas/metabolismo
11.
Brain Behav Immun ; 47: 44-57, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25452148

RESUMO

Narcolepsy onset in children has been associated with the 2009 influenza A H1N1 pandemic and vaccination with Pandemrix. However it was not clearly observed with other adjuvanted pH1N1 vaccines such as Arepanrix or Focetria. Our aim was to characterize the differences between Pandemrix and Arepanrix that might explain the risk for narcolepsy after Pandemrix vaccination using 2D-DIGE and mass spectrometry (MS). We found that Pandemrix (2009 batch) and Arepanrix (2010 batch) showed 5 main viral proteins: hemagglutinin HA1 and HA2 subunits, neuraminidase NA, nucleoprotein NP, and matrix protein MA1 and non-viral proteins from the Gallus gallus growth matrix used in the manufacturing of the vaccines. Latticed patterns of HA1, HA2 and NA indicated charge and molecular weight heterogeneity, a phenomenon likely caused by glycosylation and sulfation. Overall, Pandemrix contained more NP and NA, while Arepanrix displayed a larger diversity of viral and chicken proteins, with the exception of five chicken proteins (PDCD6IP, TSPAN8, H-FABP, HSP and TUB proteins) that were relatively more abundant in Pandemrix. Glycosylation patterns were similar in both vaccines. A higher degree of deamidation and dioxidation was found in Pandemrix, probably reflecting differential degradation across batches. Interestingly, HA1 146N (residue 129N in the mature protein) displayed a 10-fold higher deamidation in Arepanrix versus Pandemrix. In recent vaccine strains and Focetria, 146N is mutated to D which is associated with increased production yields suggesting that 146N deamidation may have also occurred during the manufacturing of Arepanrix. The presence of 146N in large relative amounts in Pandemrix and the wild type virus and in lower relative quantities in Arepanrix or other H1N1 vaccines may have affected predisposition to narcolepsy.


Assuntos
Vacinas contra Influenza/metabolismo , Narcolepsia/etiologia , Vacinação/efeitos adversos , Proteínas Virais/metabolismo , Humanos , Vacinas contra Influenza/efeitos adversos , Influenza Humana/prevenção & controle , Espectrometria de Massas
12.
Microb Cell Fact ; 13: 162, 2014 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-25421093

RESUMO

BACKGROUND: The Autotransporter pathway, ubiquitous in Gram-negative bacteria, allows the efficient secretion of large passenger proteins via a relatively simple mechanism. Capitalizing on its crystal structure, we have engineered the Escherichia coli autotransporter Hemoglobin protease (Hbp) into a versatile platform for secretion and surface display of multiple heterologous proteins in one carrier molecule. RESULTS: As proof-of-concept, we demonstrate efficient secretion and high-density display of the sizeable Mycobacterium tuberculosis antigens ESAT6, Ag85B and Rv2660c in E. coli simultaneously. Furthermore, we show stable multivalent display of these antigens in an attenuated Salmonella Typhimurium strain upon chromosomal integration. To emphasize the versatility of the Hbp platform, we also demonstrate efficient expression of multiple sizeable antigenic fragments from Chlamydia trachomatis and the influenza A virus at the Salmonella cell surface. CONCLUSIONS: The successful efficient cell surface display of multiple antigens from various pathogenic organisms highlights the potential of Hbp as a universal platform for the development of multivalent recombinant bacterial vector vaccines.


Assuntos
Antígenos de Bactérias , Antígenos Virais , Sistemas de Secreção Bacterianos , Vacinas Bacterianas , Endopeptidases , Escherichia coli , Vacinas contra Influenza , Salmonella typhimurium , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Antígenos Virais/genética , Antígenos Virais/metabolismo , Vacinas Bacterianas/genética , Vacinas Bacterianas/metabolismo , Chlamydia trachomatis/genética , Endopeptidases/genética , Endopeptidases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Vírus da Influenza A/genética , Vacinas contra Influenza/genética , Vacinas contra Influenza/metabolismo , Mycobacterium tuberculosis/genética , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo
13.
PLoS Pathog ; 10(5): e1004103, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24788925

RESUMO

Recent studies have shown high usage of the IGHV1-69 germline immunoglobulin gene for influenza hemagglutinin stem-directed broadly-neutralizing antibodies (HV1-69-sBnAbs). Here we show that a major structural solution for these HV1-69-sBnAbs is achieved through a critical triad comprising two CDR-H2 loop anchor residues (a hydrophobic residue at position 53 (Ile or Met) and Phe54), and CDR-H3-Tyr at positions 98±1; together with distinctive V-segment CDR amino acid substitutions that occur in positions sparse in AID/polymerase-η recognition motifs. A semi-synthetic IGHV1-69 phage-display library screen designed to investigate AID/polη restrictions resulted in the isolation of HV1-69-sBnAbs that featured a distinctive Ile52Ser mutation in the CDR-H2 loop, a universal CDR-H3 Tyr at position 98 or 99, and required as little as two additional substitutions for heterosubtypic neutralizing activity. The functional importance of the Ile52Ser mutation was confirmed by mutagenesis and by BCR studies. Structural modeling suggests that substitution of a small amino acid at position 52 (or 52a) facilitates the insertion of CDR-H2 Phe54 and CDR-H3-Tyr into adjacent pockets on the stem. These results support the concept that activation and expansion of a defined subset of IGHV1-69-encoded B cells to produce potent HV1-69-sBnAbs does not necessarily require a heavily diversified V-segment acquired through recycling/reentry into the germinal center; rather, the incorporation of distinctive amino acid substitutions by Phase 2 long-patch error-prone repair of AID-induced mutations or by random non-AID SHM events may be sufficient. We propose that these routes of B cell maturation should be further investigated and exploited as a pathway for HV1-69-sBnAb elicitation by vaccination.


Assuntos
Anticorpos Neutralizantes/metabolismo , Mapeamento de Epitopos , Hemaglutinação por Vírus/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/imunologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Epitopos/química , Epitopos/genética , Epitopos/metabolismo , Hemaglutinação por Vírus/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos , Vacinas contra Influenza/química , Vacinas contra Influenza/genética , Vacinas contra Influenza/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Terapia de Alvo Molecular , Engenharia de Proteínas/métodos , Estrutura Quaternária de Proteína , Homologia de Sequência de Aminoácidos
14.
Biotechnol J ; 9(9): 1206-14, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24753388

RESUMO

The baculovirus/insect cell system has proven to be a very powerful tool for the expression of several therapeutics. Nevertheless, these products sometimes suffer from reduced biological activity and unwanted side effects. Several studies have demonstrated that glycosylation can greatly influence the structure, function, half-life, antigenicity and immunogenicity of various glycoproteins. Yet, the glycosylation pattern of insect cell-derived products is not favorable for many applications. Especially, the presence of core α1,3-linked fucose bears the risk of causing immediate hypersensitivity reactions in patients with allergy. In this study, we evaluated the impact of fucose residues on the allergenic potential of an insect cell-expressed vaccine candidate. In order to block the GDP-L-fucose de novo synthesis pathway, we integrated the Pseudomonas aeruginosa GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD) gene into a baculovirus backbone. This virus was then used for the expression of soluble influenza A virus hemagglutinin (HA). Expression studies showed that the co-expression of RMD did not influence the overall level of recombinant protein secretion. We confirmed the result of our strategy by analyzing PNGase A-released N-glycans using MALDI-TOF-MS. In order to evaluate the biological impact of defucosylation of influenza HA we tested the binding activity of IgE derived from the sera of patients with allergy to the purified antigen. The non-fucosylated HA showed a 10-fold decrease in IgE binding levels as compared to wildtype variants.


Assuntos
Anticorpos/imunologia , Fucose/metabolismo , Glicoproteínas/imunologia , Hipersensibilidade/imunologia , Soros Imunes/imunologia , Imunoglobulina E/imunologia , Insetos/metabolismo , Animais , Anticorpos/metabolismo , Antígenos/imunologia , Antígenos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Baculoviridae/genética , Baculoviridae/metabolismo , Fucose/imunologia , Glicoproteínas/metabolismo , Glicosilação , Hemaglutininas/genética , Hemaglutininas/metabolismo , Humanos , Hipersensibilidade/metabolismo , Imunoglobulina E/metabolismo , Vírus da Influenza A/metabolismo , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/metabolismo , Oxirredutases Atuantes sobre Doadores de Grupos Aldeído ou Oxo/genética , Oxirredutases Atuantes sobre Doadores de Grupos Aldeído ou Oxo/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Células Sf9
15.
PLoS Pathog ; 10(1): e1003831, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24391498

RESUMO

The 2009 H1N1 pandemic (H1N1pdm) viruses have evolved to contain an E47K substitution in the HA2 subunit of the stalk region of the hemagglutinin (HA) protein. The biological significance of this single amino acid change was investigated by comparing A/California/7/2009 (HA2-E47) with a later strain, A/Brisbane/10/2010 (HA2-K47). The E47K change was found to reduce the threshold pH for membrane fusion from 5.4 to 5.0. An inter-monomer salt bridge between K47 in HA2 and E21 in HA1, a neighboring highly conserved residue, which stabilized the trimer structure, was found to be responsible for the reduced threshold pH for fusion. The higher structural and acid stability of the HA trimer caused by the E47K change also conferred higher viral thermal stability and infectivity in ferrets, suggesting a fitness advantage for the E47K evolutionary change in humans. Our study indicated that the pH of HA fusion activation is an important factor for influenza virus replication and host adaptation. The identification of this genetic signature in the HA stalk region that influences vaccine virus thermal stability also has significant implications for influenza vaccine production.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Infecções por Orthomyxoviridae/metabolismo , Internalização do Vírus , Replicação Viral/fisiologia , Substituição de Aminoácidos , Animais , Membrana Celular , Embrião de Galinha , Cães , Furões , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Vacinas contra Influenza/genética , Vacinas contra Influenza/metabolismo , Células Madin Darby de Rim Canino , Mutação de Sentido Incorreto , Infecções por Orthomyxoviridae/genética , Estrutura Terciária de Proteína
16.
J Pharm Sci ; 103(3): 821-7, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24425059

RESUMO

The recombinant hemagglutinin (rHA)-based influenza vaccine Flublok® has recently been approved in the United States as an alternative to the traditional egg-derived flu vaccines. Flublok is a purified vaccine with a hemagglutinin content that is threefold higher than standard inactivated influenza vaccines. When rHA derived from an H3N2 influenza virus was expressed, purified, and stored for 1 month, a rapid loss of in vitro potency (∼50%) was observed as measured by the single radial immunodiffusion (SRID) assay. A comprehensive characterization of the rHA protein antigen was pursued to identify the potential causes and mechanisms of this potency loss. In addition, the biophysical and chemical stability of the rHA in different formulations and storage conditions was evaluated over time. Results demonstrate that the potency loss over time did not correlate with trends in changes to the higher order structure or hydrodynamic size of the rHA. The most likely mechanism for the early loss of potency was disulfide-mediated cross-linking of rHA, as the formation of non-native disulfide-linked multimers over time correlated well with the observed potency loss. Furthermore, a loss of free thiol content, particularly in specific cysteine residues in the antigen's C-terminus, was correlated with potency loss measured by SRID.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Vírus da Influenza A Subtipo H3N2/metabolismo , Vacinas contra Influenza/química , Fenômenos Químicos , Cisteína/análise , Cisteína/química , Cistina/análise , Cistina/química , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Excipientes/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/farmacologia , Hidrodinâmica , Imunodifusão , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H3N2/imunologia , Vacinas contra Influenza/genética , Vacinas contra Influenza/metabolismo , Vacinas contra Influenza/farmacologia , Octoxinol/química , Oxirredução , Mapeamento de Peptídeos , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura Ambiente , Tioglicolatos/química
17.
PLoS One ; 8(9): e75460, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24086536

RESUMO

Influenza vaccines that target the highly variable surface glycoproteins hemagglutinin and neuraminidase cause inconvenience of having vaccination every year. For this reason, development of universal vaccines targeting conserved viral components is needed. In this study, we generated recombinant adenovirus (rAd) vaccine encoding nucleoprotein (NP) of A/PR/8/34 influenza virus, designated rAd/NP. BALB/c mice were immunized intranasally or sublingually with rAd/NP vaccine and subsequently challenged with lethal doses of heterologous as well as homologous influenza viruses. We found that intranasal immunization of rAd/NP elicited strong mucosal IgA responses as well as stronger CD8 T-cell responses toward immunodominant K(d)-restricted NP147-155 epitope than sublingual immunization. Importantly, only single intranasal but not sublingual immunization of rAd/NP provides potent protection against both homologous and heterologous influenza virus challenges. These results suggest that recombinant rAd/NP could be a universal vaccine candidate for mucosal administration against influenza virus.


Assuntos
Adenoviridae/metabolismo , Vacinas contra Influenza/uso terapêutico , Influenza Humana/prevenção & controle , Nucleoproteínas/metabolismo , Vacinas Sintéticas/uso terapêutico , Adenoviridae/genética , Administração através da Mucosa , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Linfócitos T CD8-Positivos/imunologia , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Humanos , Imunoglobulina A/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/metabolismo
18.
Analyst ; 138(20): 6073-80, 2013 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-23961535

RESUMO

Influenza is a viral pandemic that affects millions of people worldwide. Seasonal variations due to genetic shuffling and antigenic drifts in the influenza viruses have necessitated continual updating of therapeutics. The growing resistance to current influenza drugs has increased demand for new antivirals. The highly conserved nature of NP, a multi-functional viral protein that is serotypically distinct and abundantly expressed during infection, has led to its use in developing universal biotherapeutics and vaccines that could be effective against the virus, irrespective of its strain variations. Compounds causing aggregation of NP have recently been shown to be potent antivirals but require the development of new high-throughput assays capable of screening compounds with similar modes of action. Here, we describe the development of a new bioassay for the Influenza A nucleoprotein (NP). The assay was developed to quantify ligand-induced aggregation of a GFP-tagged NP and was validated with aggregation-inducing compounds such as nucleozin and a NP-specific antibody. The new NP-GFP aggregation assay can be performed with partially purified or mixtures of proteins and is amenable to a high-throughput format. Using this assay, we demonstrate the potential of a new anti-NP polyclonal antibody that we have obtained from chicken. This cost-effective high-yield source of anti-NP IgY has potential for large-scale production and development of therapeutic antibodies. The simplicity, speed and flexibility of this assay make it an invaluable tool for timely development of effective antivirals that can help to control future epidemics.


Assuntos
Anticorpos Antivirais/análise , Descoberta de Drogas/métodos , Vírus da Influenza A Subtipo H1N1/química , Vacinas contra Influenza/síntese química , Nucleoproteínas/química , Animais , Galinhas , Feminino , Humanos , Imunoglobulinas/química , Imunoglobulinas/metabolismo , Vírus da Influenza A Subtipo H1N1/metabolismo , Vacinas contra Influenza/metabolismo , Influenza Humana/prevenção & controle , Nucleoproteínas/metabolismo
19.
PLoS One ; 8(6): e66719, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23799128

RESUMO

Highly pathogenic avian influenza H5N1 viruses can result in poultry and occasionally in human mortality. A safe and effective H5N1 vaccine is urgently needed to reduce the pandemic potential. Hemagglutinin (HA), a major envelope protein accounting for approximately 80% of spikes in influenza virus, is often used as a major antigen for subunit vaccine development. In this study, we conducted a systematic study of the immune response against influenza virus infection following immunization with recombinant HA proteins expressed in insect (Sf9) cells, insect cells that contain exogenous genes for elaborating N-linked glycans (Mimic) and mammalian cells (CHO). While the antibody titers are higher with the insect cell derived HA proteins, the neutralization and HA inhibition titers are much higher with the mammalian cell produced HA proteins. Recombinant HA proteins containing tri- or tetra-antennary complex, terminally sialylated and asialyated-galactose type N-glycans induced better protective immunity in mice to lethal challenge. The results are highly relevant to issues that should be considered in the production of fragment vaccines.


Assuntos
Hemaglutininas/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Processamento de Proteína Pós-Traducional , Proteínas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Células Produtoras de Anticorpos/metabolismo , Células Apresentadoras de Antígenos/imunologia , Células CHO , Configuração de Carboidratos , Sequência de Carboidratos , Cricetinae , Cricetulus , Feminino , Glicosilação , Hemaglutininas/química , Hemaglutininas/metabolismo , Humanos , Isotipos de Imunoglobulinas/sangue , Vacinas contra Influenza/química , Vacinas contra Influenza/metabolismo , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Mananas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Células Sf9 , Spodoptera , Linfócitos T/imunologia , Vacinação , Proteínas Virais/química , Proteínas Virais/metabolismo
20.
BMC Biotechnol ; 13: 50, 2013 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-23767961

RESUMO

BACKGROUND: The production process for the current influenza vaccine takes about 6 months and its antigenic composition must be modified annually. In the attempt towards developing influenza vaccine production that would be faster, safer and cheaper we engineered an influenza vaccine in which multiple copies of hemagglutinin (HA) would be delivered by a vector, adenovirus dodecahedron (Ad Dd). Dd is a virus-like particle, formed by assembly of twelve copies of pentameric penton base (Pb) proteins responsible for virus penetration. In order to attach HA to the vector, an adaptor containing WW domains was used. The WW domain is a linear peptide fragment identified as a partner of proline-proline-x-tyrosine (PPxY) motif present at the N-terminal extremity of the Pb protein, which is a building block of Dd. That tandem of three WW domains in fusion with the protein of interest enables interaction with Dd and efficient translocation to the cytoplasm of cells in culture. RESULTS: Since HA is an oligomeric protein with complicated processing, we prepared six different constructs of HA (A/swan/Poland/467/2006(H5N1)) in fusion with the WW adaptor. Herein we report baculovirus expression and functional analysis of six HA-WW variants. The best behaving variant was successfully delivered into human cells in vitro. CONCLUSIONS: Engineering of a soluble complex of HA with Dd, a virus-like particle that serves as a vector, an adjuvant and as a multivalent presentation platform, is an important step toward a novel influenza vaccine.


Assuntos
Adenoviridae/genética , Hemaglutininas/genética , Vacinas contra Influenza/genética , Engenharia de Proteínas/métodos , Proteínas Virais/metabolismo , Células Cultivadas , Clonagem Molecular , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Testes de Inibição da Hemadsorção , Hemaglutininas/metabolismo , Vacinas contra Influenza/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Complexos Ubiquitina-Proteína Ligase/genética , Proteínas Virais/genética
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