RÉSUMÉ
The small (S) envelope protein of the Hepatitis B Virus (HBV), HBV-S, has the unique ability to self-assemble into highly immunogenic subviral particles (SVPs), in the absence of other viral factors, in eukaryotic cells, including those of nonhepatic origin. This feature is currently exploited for generation of SVPs exposing heterologous epitopes on their surface that can be used as vaccine candidates to target various diseases. Here, we describe a simple and robust method for production of such chimeric HBV-S protein-based SVPs in transiently transfected HEK293T cells and purification from cell supernatants by ultracentrifugation on sucrose cushion and sucrose step gradients. The SVPs obtained by this methodology have been successfully used in immunogenicity studies in animal models.
Sujet(s)
Techniques de culture cellulaire en batch , Antigènes de surface du virus de l'hépatite B/biosynthèse , Protéines de fusion recombinantes , Animaux , Techniques de culture cellulaire , Expression des gènes , Cellules HEK293 , Antigènes de surface du virus de l'hépatite B/génétique , Antigènes de surface du virus de l'hépatite B/immunologie , Antigènes de surface du virus de l'hépatite B/isolement et purification , Vaccins anti-hépatite B/biosynthèse , Vaccins anti-hépatite B/immunologie , Humains , TransfectionRÉSUMÉ
The aggregation of recombinant proteins in the different stages of purification leads to the loss of a considerable portion of target protein and reduction in the process efficiency. As the active HBsAg used in Hepatitis B vaccine production is in the form of virus-like particle (VLP), therefore the time and stages at which the VLP assembling happened through the process would be important. The aim of this study was to explore the product aggregation during different stages of large scale production of rHBsAg in Pichia pastoris at production unit of the Pasteur Institute of Iran. Dynamic light scattering (DLS) and transmission electron microscopy (TEM), and also size exclusion-high-performance liquid chromatography (SE-HPLC) were carried out on samples taken from each downstream processes steps to determine the rate of VLPs formation as the desired product and the aggregated form at each stage of the purification. Based on the results, it was found that VLPs formation started at the acid precipitation stage and reached up to 80% at the thermal treatment stage. The ultrafiltration, ion exchange chromatography and immunoaffinity chromatography stages were disclosed to have the highest contribution in the formation of VLP (virus like particle) 22â¯nm.
Sujet(s)
Antigènes de surface du virus de l'hépatite B/biosynthèse , Vaccins anti-hépatite B/biosynthèse , Agrégats de protéines , Produits biologiques/composition chimique , Chromatographie d'affinité , Chromatographie sur gel , Chromatographie en phase liquide à haute performance , Chromatographie d'échange d'ions , Disulfures/composition chimique , Fermentation , Génie génétique , Virus de l'hépatite B/immunologie , Taille de particule , Pichia/métabolisme , Protéines de fusion recombinantes/biosynthèse , Thiocyanates/composition chimique , UltrafiltrationRÉSUMÉ
BACKGROUND: Recombinant proteins expressed in host cell systems may contain host cell proteins (HCP) as impurities. While there is no clear evidence of clinical adverse events attributable to HCP, HCP levels and profiles must be documented to meet regulatory requirements and to understand the consistency of the biological product and manufacturing process. We present a general strategy for HCP characterization applied to a recombinant protein antigen, Hepatitis B surface antigen (HBsAg) used in a multivalent vaccine. METHODS: Polyclonal antisera raised against HCPs in process fractions from a mock preparation of the HBsAg yeast expression host, Hansenula polymorpha, were used to develop a quantitative sandwich ELISA to measure HCP content in batches of purified recombinant HBsAg. Batches were also subjected to SDS-PAGE and LC-MS/MS to identify detectable proteins. Batch consistency was further assessed by SDS-PAGE/densitometry purity analysis and by the ratio of specific HBsAg content (by ELISA) to total protein. RESULTS: Using the quantitative HCP ELISA, the HCP content showed no discernable trend in multiple HBsAg batches manufactured over a 5-year period. All batches were ≥95% pure by SDS-PAGE/densitometry, with consistent HBsAg/total protein ratios. In addition to the expected HBsAg antigen protein, LC-MS/MS analysis of three HBsAg batches identified several yeast proteins, none of which are known to cause adverse reactions in humans. CONCLUSIONS: Analysis of multiple HBsAg batches showed consistent HCP content and identification profiles, as well as product purity and specific antigen content, demonstrating consistent manufacturing process. Recombinant vaccines, unlike therapeutic products, are administered infrequently with only small amounts of protein injected at a time. With limited potential for adverse reactions to small quantities of HCPs in purified recombinant vaccine antigens, and considering the relevant regulatory guidelines, we conclude that once consistent manufacturing process has been demonstrated, routine HCP testing in recombinant vaccine antigens is no longer required.
Sujet(s)
Expression des gènes , Antigènes de surface du virus de l'hépatite B/biosynthèse , Vaccins anti-hépatite B/biosynthèse , Virus de l'hépatite B/génétique , Pichia/métabolisme , Antigènes de surface du virus de l'hépatite B/génétique , Antigènes de surface du virus de l'hépatite B/immunologie , Vaccins anti-hépatite B/génétique , Virus de l'hépatite B/immunologie , Pichia/génétique , Protéines recombinantes/biosynthèse , Protéines recombinantes/génétique , Protéines recombinantes/immunologie , Vaccins synthétiques/biosynthèse , Vaccins synthétiques/génétique , Vaccins synthétiques/immunologieRÉSUMÉ
Hepatitis B virus (HBV) infection is a leading cause of liver damage and hepatic inflammation. Upon infection, effective antiviral responses by CD8+ T cells, CD4+ T cells, Natural killer (NK) cells, and monocytes can lead to partial or complete eradication of the viral infection. To date, many studies have shown that the production of inhibitory cytokines such as Interleukin 10 (IL-10), Transforming growth factor beta (TGF-ß), along with dysfunction of the dendritic cells (DCs), and the absence of efficient innate immune responses could lead to T cell exhaustion, development of persistent infection, and inability to eradicate the viral infection from liver. Understanding the immunopathogenesis of the virus could be useful in providing further insights toward novel strategies in the eradication of HBV infection.
Sujet(s)
Anergie clonale/effets des médicaments et des substances chimiques , Vaccins anti-hépatite B/administration et posologie , Virus de l'hépatite B/immunologie , Hépatite B chronique/immunologie , Immunité innée/effets des médicaments et des substances chimiques , Antiviraux/usage thérapeutique , Cellules dendritiques/immunologie , Cellules dendritiques/virologie , Régulation de l'expression des gènes , Vaccins anti-hépatite B/biosynthèse , Vaccins anti-hépatite B/synthèse chimique , Virus de l'hépatite B/effets des médicaments et des substances chimiques , Virus de l'hépatite B/pathogénicité , Hépatite B chronique/traitement médicamenteux , Hépatite B chronique/anatomopathologie , Hépatite B chronique/prévention et contrôle , Humains , Interleukine-10/agonistes , Interleukine-10/génétique , Interleukine-10/immunologie , Cellules tueuses naturelles/immunologie , Cellules tueuses naturelles/virologie , Foie/immunologie , Foie/virologie , Vaccination de masse/méthodes , Monocytes/immunologie , Monocytes/virologie , Lymphocytes T/immunologie , Lymphocytes T/virologie , Facteur de croissance transformant bêta/agonistes , Facteur de croissance transformant bêta/génétique , Facteur de croissance transformant bêta/immunologie , Charge virale/effets des médicaments et des substances chimiques , Charge virale/immunologieRÉSUMÉ
Vaccination is a safe and effective approach to prevent deadly diseases. To increase vaccine production, we propose that a mechanical stimulation can enhance protein production. In order to prove this hypothesis, Sf9 insect cells were used to evaluate the increase in the expression of a fusion protein from hepatitis B virus (HBV S1/S2). We discovered that the ultrasound stimulation at a frequency of 1.5 MHz, intensity of 60 mW/cm2, for a duration of 10 minutes per day increased HBV S1/S2 by 27%. We further derived a model for transport through a cell membrane under the effect of ultrasound waves, tested the key assumptions of the model through a molecular dynamics simulation package, NAMD (Nanoscale Molecular Dynamics program) and utilized CHARMM force field in a steered molecular dynamics environment. The results show that ultrasound waves can increase cell permeability, which, in turn, can enhance nutrient / waste exchange thus leading to enhanced vaccine production. This finding is very meaningful in either shortening vaccine production time, or increasing the yield of proteins for use as vaccines.
Sujet(s)
Vaccins anti-hépatite B/biosynthèse , Ondes ultrasonores , 4-Chloro-7-nitro-2,1,3-benzoxadiazole/analogues et dérivés , 4-Chloro-7-nitro-2,1,3-benzoxadiazole/métabolisme , Animaux , Technique de Western , Perméabilité des membranes cellulaires , Désoxyglucose/analogues et dérivés , Désoxyglucose/métabolisme , Vaccins anti-hépatite B/immunologie , Simulation de dynamique moléculaire , Phosphatidylcholines/composition chimique , Protéines/métabolisme , Cellules Sf9 , Sonication , ThermodynamiqueRÉSUMÉ
Nowadays, gold is used in (nano-)medicine, usually in the form of nanoparticles, due to the solid proofs given of its therapeutic effects on several diseases. Gold also plays an important role in the vaccine field as an adjuvant and a carrier, reducing toxicity, enhancing immunogenic activity, and providing stability in storage. An even brighter golden future is expected for gold applications in this area.
Sujet(s)
Vaccins contre le SIDA/administration et posologie , Vaccins anticancéreux/administration et posologie , Or/immunologie , Vaccins anti-hépatite B/administration et posologie , Nanoparticules métalliques/administration et posologie , Nanomédecine/tendances , Vaccins contre le SIDA/biosynthèse , Syndrome d'immunodéficience acquise/immunologie , Syndrome d'immunodéficience acquise/anatomopathologie , Syndrome d'immunodéficience acquise/prévention et contrôle , Adjuvants immunologiques/administration et posologie , Vaccins anticancéreux/biosynthèse , Or/administration et posologie , Hépatite B/immunologie , Hépatite B/anatomopathologie , Hépatite B/prévention et contrôle , Vaccins anti-hépatite B/biosynthèse , Humains , Nanoparticules métalliques/composition chimique , Nanomédecine/méthodes , Tumeurs/immunologie , Tumeurs/anatomopathologie , Tumeurs/prévention et contrôleRÉSUMÉ
Chronic hepatitis B is a serious liver disease and puts people at high risk of death from cirrhosis and liver cancer. Although DNA vaccination has been emerged as a potential immunotherapeutic strategy for the treatment of chronic hepatitis B, the efficiencies were not adequate in clinical trials. Here we describe the design, synthesis, and evaluation of mannosylated phenylalanine grafted chitosan (Man-CS-Phe) as a DNA delivery vector for direct transfection of antigen presenting cells to improve cellular and humoral immunity to plasmid-coded antigen. The cationic Man-CS-Phe micelles condense plasmid DNA into nanoscale polyplexes and provide efficient protection of complexed DNA from nuclease degradation. The mannose receptor-mediated enhanced cell uptake and high in vitro transfection efficiency of the polyplexes were demonstrated in RAW 264.7 and DC 2.4 cells using GFP-expressing plasmid DNA. Furthermore, intradermal immunization of BALB/c mice indicated that hepatitis B DNA vaccine/Man-CS-Phe polyplexes not only induced multi-fold higher serum antibody titer in comparison to all other formulations including FuGENE HD, but also significantly stimulated T-cell proliferation and skewed T helper toward Th1 polarization. These results illustrate that the Man-CS-Phe can serve as a promising DNA delivery vector to harness both cellular and humoral arms of immune system.
Sujet(s)
Cellules présentatrices d'antigène/immunologie , Chitosane/composition chimique , Vecteurs de médicaments , Antigènes de surface du virus de l'hépatite B/administration et posologie , Vaccins anti-hépatite B/administration et posologie , Mannose/composition chimique , Phénylalanine/composition chimique , Administration par voie cutanée , Animaux , Cellules présentatrices d'antigène/virologie , Marqueurs biologiques/sang , Prolifération cellulaire , Chimie pharmaceutique , Chitosane/analogues et dérivés , Chitosane/toxicité , Femelle , Anticorps de l'hépatite B/sang , Antigènes de surface du virus de l'hépatite B/biosynthèse , Antigènes de surface du virus de l'hépatite B/génétique , Antigènes de surface du virus de l'hépatite B/immunologie , Vaccins anti-hépatite B/biosynthèse , Vaccins anti-hépatite B/génétique , Vaccins anti-hépatite B/immunologie , Immunité cellulaire , Immunité humorale , Immunisation , Activation des lymphocytes , Mannose/analogues et dérivés , Mannose/toxicité , Souris , Souris de lignée BALB C , Micelles , Phénylalanine/analogues et dérivés , Phénylalanine/toxicité , Cellules RAW 264.7 , Lymphocytes T auxiliaires/immunologie , Transfection , Vaccins à ADN/administration et posologie , Vaccins à ADN/immunologieRÉSUMÉ
The concept of occult infection caused by hepatitis B virus (HBV) is determined as the presence of HBV DNA in blood sera or liver with the absence of detectable HBsAg. The actuality of this problem is associated with the fact, that occult hepatitis B (OHB) can be transmitted during hemotransfusions, cause reactivation of chronic hepatitis B in immune compromised individuals, facilitate development of liver cirrhosis and hepatocellular carcinoma. Several different hypotheses of OHB immunopathogenesis have been proposed, including a low number of copies of HBV DNA, altered immune response of the macroorganism, genetic variability of the S gene, integration of viral DNA into host genome, infection of mononuclear cells of peripheral blood, presence of immune complexes that hide HBsAg, and interference by other viruses such as HCV and HIV. Molecular mechanisms of HBV virus in HBsAg-negative individuals are not fully understood, however, viral mutations seem a very significant factor. Approaches of OHB prophylaxis including use of a polyvalent vaccine, that allows vaccination against wild and mutant HBV viruses, are examined.
Sujet(s)
Carcinome hépatocellulaire/prévention et contrôle , ADN viral/sang , Vaccins anti-hépatite B/biosynthèse , Hépatite B chronique/prévention et contrôle , Cirrhose du foie/prévention et contrôle , Tumeurs du foie/prévention et contrôle , Complexe antigène-anticorps/sang , Complexe antigène-anticorps/composition chimique , Carcinome hépatocellulaire/étiologie , Carcinome hépatocellulaire/immunologie , Carcinome hépatocellulaire/anatomopathologie , Variations de nombre de copies de segment d'ADN , ADN viral/immunologie , Antigènes de surface du virus de l'hépatite B/immunologie , Vaccins anti-hépatite B/administration et posologie , Vaccins anti-hépatite B/immunologie , Virus de l'hépatite B/génétique , Virus de l'hépatite B/immunologie , Hépatite B chronique/complications , Hépatite B chronique/immunologie , Hépatite B chronique/anatomopathologie , Humains , Échappement immunitaire , Foie/immunologie , Foie/virologie , Cirrhose du foie/étiologie , Cirrhose du foie/immunologie , Cirrhose du foie/anatomopathologie , Tumeurs du foie/étiologie , Tumeurs du foie/immunologie , Tumeurs du foie/anatomopathologie , Mutation , VaccinationRÉSUMÉ
Monoclonal antibodies, modern vaccines and gene therapy have become a major field in modern biotechnology, especially in the area of human health and fascinating developments achieved in the past decades are impressive examples of an interdisciplinary interplay between medicine, biology and engineering. Among the classical products from cells one can find viral vaccines, monoclonal antibodies, and interferons, as well as recombinant therapeutic proteins. Gene therapy opens up challenging new areas. In this review, a definitions of these processes are given and fields of application and products, as well as the future prospects, are discussed.
Sujet(s)
Anticorps monoclonaux/biosynthèse , Biotechnologie/méthodes , Génie génétique , Thérapie génétique , Protéines recombinantes/biosynthèse , Vaccins synthétiques/biosynthèse , Alemtuzumab , Animaux , Anticorps monoclonaux/génétique , Anticorps monoclonaux/usage thérapeutique , Anticorps monoclonaux humanisés/biosynthèse , Anticorps monoclonaux humanisés/usage thérapeutique , Antinéoplasiques/métabolisme , Antinéoplasiques/usage thérapeutique , Clonage moléculaire , Vaccin diphtérie-tétanos-coqueluche/biosynthèse , Vaccin diphtérie-tétanos-coqueluche/usage thérapeutique , Techniques de transfert de gènes , Vecteurs génétiques , Vaccins anti-hépatite B/biosynthèse , Vaccins anti-hépatite B/usage thérapeutique , Humains , Hyperlipoprotéinémie de type II/génétique , Hyperlipoprotéinémie de type II/thérapie , Vaccins antigrippaux/biosynthèse , Vaccins antigrippaux/usage thérapeutique , Vaccin antipoliomyélitique inactivé/biosynthèse , Vaccin antipoliomyélitique inactivé/usage thérapeutique , Interférence par ARN , Protéines recombinantes/génétique , Protéines recombinantes/usage thérapeutique , Immunodéficience combinée grave/génétique , Immunodéficience combinée grave/thérapie , Vaccins synthétiques/génétique , Vaccins synthétiques/usage thérapeutiqueRÉSUMÉ
Hepatitis B surface antigen (HBsAg) carrying preS sequences could be an ideal candidate for a new hepatitis B virus (HBV) vaccine with higher efficacy. Here we report the success in achieving efficient and stable expression of hepatitis B virus S antigen and preS1 epitope fusion protein (S/preS1) in CHO cells. The HMRCHEF53u/Neo-S/preS1 expression vector carrying S/preS1 gene was constructed and transfected into CHO-S cells. A stable and high-expression CHO cell line, named 10G6, was selected by ELISA and limiting dilution analysis. Western blotting analysis showed S/preS1 expressed from 10G6 cells possessed both S and preS1 antigenicity. 10G6 cells displayed characters of favorable growth and stable S/preS1 expression in repeated batch cultures as evaluated by viable cell density, viability and S/preS1 concentration. And cultivation of 10G6 cells in fed-batch mode resulted in S/preS1 production at 17-20 mg/L with viable cell density at 7 x 10(6)-10 x 10(6) cells/mL.
Sujet(s)
Épitopes/biosynthèse , Antigènes de surface du virus de l'hépatite B/biosynthèse , Vaccins anti-hépatite B/biosynthèse , Précurseurs de protéines/biosynthèse , Animaux , Cellules CHO , Cricetulus , Épitopes/génétique , Antigènes de surface du virus de l'hépatite B/génétique , Antigènes de surface du virus de l'hépatite B/immunologie , Vaccins anti-hépatite B/génétique , Virus de l'hépatite B , Précurseurs de protéines/génétique , Précurseurs de protéines/immunologie , Protéines de fusion recombinantes/biosynthèse , Protéines de fusion recombinantes/génétique , TransfectionRÉSUMÉ
OBJECTIVE: To evaluate the long-term efficacy and duration of yeast-derived recombinant hepatitis B vaccine in hepatitis B virus (HBV)-endemic areas. METHOD: A cross-sectional investigation was carried out in five HBV-endemic areas. Children who were born between 1997 and 2008 and vaccinated with yeast-derived recombinant hepatitis B vaccine were selected. Serum samples were taken to test HBV infection markers by microparticle enzyme immunoassay, and the results were compared to those before vaccination. RESULTS: 7066 subjects were enrolled. The average adjusted hepatitis B surface antigen (HBsAg) prevalence was 1.02%. HBV core antibody (anti-HBc) prevalence was 3.54%. The overall percentage of HBsAg(-)&Anti-HBc(-)&Anti-HBs(+) was 61.34%. With time after immunization, the percentage annually decreases from 86.11% in 2008 to 49.80% in 1997. Geometric mean concentration (GMC) of anti-HBs decreased significantly annually. The portion of GMC=100-999.9 mIU/ml was 48.0% in 2008, and decreased to 16.7% in 1997. CONCLUSION: HBsAg prevalence decreased dramatically. This shows that the yeast-derived recombinant hepatitis B vaccine is effective and stable after being used for 12 years in HBV-endemic areas. It is not suggested to carry out booster immunization.
Sujet(s)
Vaccins anti-hépatite B/usage thérapeutique , Hépatite B/prévention et contrôle , Chine/épidémiologie , Études transversales , Femelle , Hépatite B/épidémiologie , Anticorps de l'hépatite B/sang , Antigènes de surface du virus de l'hépatite B/sang , Vaccins anti-hépatite B/biosynthèse , Vaccins anti-hépatite B/immunologie , Humains , Nourrisson , Mâle , Prévalence , Vaccins synthétiques/biosynthèse , Vaccins synthétiques/immunologie , Vaccins synthétiques/usage thérapeutiqueRÉSUMÉ
Plant-based oral vaccines are a promising emergent technology that could help alleviate disease burden worldwide by providing a low-cost, heat-stable, oral alternative to parenterally administered commercial vaccines. Here, we describe high-level accumulation of the hepatitis B surface antigen (HBsAg) at a mean concentration of 0.51%TSP in maize T1 seeds using an improved version of the globulin1 promoter. This concentration is more than fourfold higher than any previously reported lines. HBsAg expressed in maize seeds was extremely heat stable, tolerating temperatures up to 55 °C for 1 month without degradation. Optimal heat stability was achieved after oil extraction of ground maize material, either by supercritical fluid extraction or hexane treatment. The contributions of this material towards the development of a practical oral vaccine delivery system are discussed.
Sujet(s)
Antigènes de surface du virus de l'hépatite B/biosynthèse , Vaccins anti-hépatite B/biosynthèse , Végétaux génétiquement modifiés/métabolisme , Graines/composition chimique , Zea mays/immunologie , Antigènes de surface du virus de l'hépatite B/analyse , Extraits de plantes/analyse , Extraits de plantes/immunologie , Huiles végétales/analyseRÉSUMÉ
CB.Hep-1 monoclonal antibody (mAb) is used for a recombinant Hepatitis B vaccine manufacturing, which is included in a worldwide vaccination program against Hepatitis B disease. The use of this mAb as immunoligand has been addressed into one of the most efficient steps of active pharmaceutical ingredient purification process. Regarding this, Quality Risk Management (QRM) provides an excellent framework for the risk management use in pharmaceutical manufacturing and quality decision-making applications. Consequently, this study sought applying a prospective risk analysis methodology Failure Mode Effects Analysis (FMEA) as QRM tool for analyzing different CB.Hep-1 mAb manufacturing technologies. As main conclusions FMEA was successfully used to assess risks associated with potential problems in CB.Hep-1 mAb manufacturing processes. The severity and occurrence of risks analysis evidenced that the percentage of very high severe risks ranged 31.0-38.7% of all risks and the huge majority of risks have a very low occurrence level (61.9-83.3%) in all assessed technologies. Finally, additive Risk Priority Number, was descending ordered as follow: transgenic plants (2636), ascites (2577), transgenic animals (2046) and hollow fiber bioreactors (1654), which also corroborated that in vitro technology, should be the technology of choice for CB.Hep-1 mAb manufacturing in terms of risks and mAb molecule quality.
Sujet(s)
Anticorps monoclonaux/biosynthèse , Vaccins anti-hépatite B/biosynthèse , Animaux , Animal génétiquement modifié , Anticorps monoclonaux/immunologie , Anticorps monoclonaux d'origine murine/biosynthèse , Anticorps monoclonaux d'origine murine/immunologie , Biotechnologie/méthodes , Chromatographie d'affinité , Anticorps de l'hépatite B/biosynthèse , Anticorps de l'hépatite B/immunologie , Vaccins anti-hépatite B/immunologie , Vaccins anti-hépatite B/isolement et purification , Vaccins anti-hépatite B/normes , Humains , Souris , Souris de lignée BALB C , Végétaux génétiquement modifiés , Gestion du risqueSujet(s)
Antigènes de surface du virus de l'hépatite B/immunologie , Vaccins anti-hépatite B/immunologie , Tubercules/immunologie , Végétaux génétiquement modifiés/immunologie , Solanum tuberosum/immunologie , Animaux , Anticorps/immunologie , Antigènes de surface du virus de l'hépatite B/biosynthèse , Vaccins anti-hépatite B/biosynthèse , Souris , Lignées consanguines de souris , Tubercules/génétique , Végétaux génétiquement modifiés/génétique , Solanum tuberosum/génétique , Vaccins comestibles/biosynthèse , Vaccins comestibles/immunologieRÉSUMÉ
In the last few years, plants have become an increasingly attractive platform for recombinant protein production. This builds on two decades of research, starting with transgenic approaches to develop oral vaccines in which antigens or therapeutics can be delivered in processed plant biomass, and progressing to transient expression approaches whereby high yields of purified targets are administered parenterally. The advantages of plant-based expression systems include high scalability, low upstream costs, biocontainment, lack of human or animal pathogens, and ability to produce target proteins with desired structures and biological functions. Using transgenic and transient expression in whole plants or plant cell culture, a variety of recombinant subunit vaccine candidates, therapeutic proteins, including monoclonal antibodies, and dietary proteins have been produced. Some of these products have been tested in early phase clinical trials, and show safety and efficacy. Among those are mucosal vaccines for diarrheal diseases, hepatitis B and rabies; injectable vaccines for non-Hodgkin's lymphoma, H1N1 and H5N1 strains of influenza A virus, and Newcastle disease in poultry; and topical antibodies for the treatment of dental caries and HIV. As lead plant-based products have entered clinical trials, there has been increased emphasis on manufacturing under current Good Manufacturing Practice (cGMP) guidelines, and the preparation and presentation to the relevant government agencies of regulatory packages.
Sujet(s)
Vaccins synthétiques/immunologie , Vaccins/immunologie , Anticorps/immunologie , Anticorps monoclonaux/immunologie , Vaccins anticancéreux/biosynthèse , Essais cliniques comme sujet , Escherichia coli entérotoxigène/immunologie , Vaccins anti-Escherichia coli/biosynthèse , Maladie de Gaucher/traitement médicamenteux , Glucosylceramidase/immunologie , Glucosylceramidase/usage thérapeutique , Vaccins anti-hépatite B/biosynthèse , Humains , Nourrisson , Sous-type H1N1 du virus de la grippe A/immunologie , Vaccins antigrippaux/biosynthèse , Lymphome malin non hodgkinien/immunologie , Virus de la maladie de Newcastle/immunologie , Virus de Norwalk/immunologie , Plantes/immunologie , Végétaux génétiquement modifiés/immunologie , Vaccins antirabiques/biosynthèse , Vaccins antirabiques/immunologie , Vaccins antiviraux/biosynthèseRÉSUMÉ
Mucosal vaccination is a perspective for the control of infectious diseases, since it is capable of inducing humoral and cell-mediated responses. In addition, the delivery of vaccines to mucosal surfaces makes immunization practice safe and acceptable, and eliminates needle-associated risks. Transgenic plants can be used as bioreactors for the production of mucosally delivered protective antigens. This technology shows great promise to simplify and decrease the cost of vaccine delivery. Herein, we review the development of mucosally administered vaccines expressed in transgenic plants. In particular, we evaluate the advantages and disadvantages of using plants for the production of mucosal vaccines against widespread infectious diseases such as HIV, hepatitis B and TB.
Sujet(s)
Biotechnologie/méthodes , Maladies transmissibles/immunologie , Immunité muqueuse , Végétaux génétiquement modifiés/métabolisme , Technologie pharmaceutique/méthodes , Vaccination/méthodes , Vaccins synthétiques/biosynthèse , Vaccins contre le SIDA/biosynthèse , Vaccins contre le SIDA/génétique , Vaccins anti-hépatite B/biosynthèse , Vaccins anti-hépatite B/génétique , Humains , Solanum lycopersicum/génétique , Solanum lycopersicum/métabolisme , Végétaux génétiquement modifiés/génétique , Nicotiana/génétique , Nicotiana/métabolisme , Vaccins antituberculeux/biosynthèse , Vaccins antituberculeux/génétique , Vaccins synthétiques/génétiqueRÉSUMÉ
Human hepatitis B virus (HBV) causes a communicable disease that spreads worldwide and has brought about considerable economic losses due to human mortality and morbidity. HBV fails to reproduce in both cell cultures and laboratory animals; however, it is known that excess virion surface protein named hepatitis B surface antigen (HBsAg) is produced during viral replication and circulates in the blood of carriers as noninfectious particles of 22-nm diameter. It had been shown that purified HBsAg particles induce an efficient systemic immune response after injection. Consequently, subunit HBV vaccines based on HBsAg synthesized in yeasts or mammalian cell culture are currently used. Taking into account that hepatitis B is a sexually transmitted disease, development of a mucosal HBV vaccine would be beneficial. In this article, we analyze the data on development of plant-based HBV vaccines.
Sujet(s)
Biotechnologie/méthodes , Vaccins anti-hépatite B/biosynthèse , Immunité muqueuse , Végétaux génétiquement modifiés/métabolisme , Technologie pharmaceutique/méthodes , Vaccins synthétiques/biosynthèse , Antigènes de surface du virus de l'hépatite B/biosynthèse , Antigènes de surface du virus de l'hépatite B/génétique , Vaccins anti-hépatite B/génétique , Humains , Végétaux génétiquement modifiés/génétique , Vaccins synthétiques/génétiqueRÉSUMÉ
BACKGROUND: The hepatitis B virus (HBV) DNA vaccine can generate both HBsAg-specific humoral and cellular immune responses. The immune response can be improved by inclusion of an adjuvant, such as the cytokine GM-CSF which is known to be a very good adjuvant. METHODS: To investigate the ability of GM-CSF to enhance HBV-DNA vaccines, we constructed the plasmids by fusion of GM-CSF gene to the HBV-S gene. Normal and HBV-transgenic mice were then immunized with these plasmids. RESULTS: Our results show that pCDNA3.1-GM-CSF-S induced the most powerful HBsAg-specific humoral and cellular immune response, and that it was able to overcome the non-response to HBsAg in HBV-transgenic mice. In contrast, pCDNA3.1-S-GM-CSF was able to induce only a very poor immune response. CONCLUSIONS: When the HBV-S gene is fused to the GM-CSF gene, the immune effects of the HBV DNA vaccine both in normal and HBV-transgenic mice can be strengthened and HBV-DNA plasmids fused with GM-CSF may be useful for both preventative and therapeutic purposes.
Sujet(s)
Facteur de stimulation des colonies de granulocytes et de macrophages/immunologie , Antigènes de surface du virus de l'hépatite B/immunologie , Vaccins anti-hépatite B/immunologie , Hépatite B/prévention et contrôle , Vaccins à ADN/immunologie , Animaux , Cytokines/analyse , Femelle , Facteur de stimulation des colonies de granulocytes et de macrophages/génétique , Anticorps de l'hépatite/sang , Hépatite B/immunologie , Antigènes de surface du virus de l'hépatite B/génétique , Vaccins anti-hépatite B/biosynthèse , Vaccins anti-hépatite B/génétique , Immunité cellulaire , Immunité humorale , Mâle , Souris , Souris de lignée BALB C , Souris transgéniques , Plasmides/immunologie , Vaccins à ADN/biosynthèse , Vaccins à ADN/génétiqueRÉSUMÉ
We previous reported the development of novel hepatitis B virus(HBV) vaccine containing the surface antigen(S) plus PreS1 fusion derived from Chinese hamster ovary (CHO) cells system. In this study, we analyzed the impact of different adjuvants on immunogenicity of the HBV particle vaccine in Balb/C mice, including alum alone, CpG oligodeoxynucleotides (CpG-ODN) alone and CpG-ODN in combination with alum adjuvant. We first detected the antigen specific humoral response in mice, including total IgG antibody and IgG subtyping. Then, we characterized the specific cell-mediated immune (CMI) response by detection of gamma-interferon secreting splenocytes after stimulating with S or PreS1 peptide pool. Our results showed that: CpG-ODN adjuvanted vaccine could rapidly induce higher level of anti-PreS 1 and anti-S antibodies, and a higher ratio of IgG2a/IgG1 antibody than that of alum adjuvanted vaccine. At the same time, CpG-ODN adjuvanted vaccine induced robust antigen-specific cellular immune responses in mice, which was superior to that of alum adjuvanted vaccine and CpG-ODN in combination with alum adjuvanted vaccine; however, the vaccine candidate with CpG-ODN in combination with alum adjuvant induced highest anti-S antibody and mixed IgG subclasses in mice after twice immunization. There exists dominant HBV CMI epitopes in the N-terminal of S antigen. These results provided important evidence that CpG-ODN adjuvanted HBSS1 particles vaccine may serve as a novel candidate in the development of new preventive and therapeutic agents against hepatitis B infection.
Sujet(s)
Adjuvants immunologiques/pharmacologie , Antigènes de surface du virus de l'hépatite B/immunologie , Vaccins anti-hépatite B/immunologie , Oligodésoxyribonucléotides/immunologie , Précurseurs de protéines/immunologie , Animaux , Cellules CHO , Cricetinae , Cricetulus , Femelle , Antigènes de surface du virus de l'hépatite B/biosynthèse , Antigènes de surface du virus de l'hépatite B/génétique , Vaccins anti-hépatite B/biosynthèse , Immunisation , Souris , Souris de lignée BALB C , Précurseurs de protéines/biosynthèse , Précurseurs de protéines/génétique , Protéines de fusion recombinantes/biosynthèse , Protéines de fusion recombinantes/génétique , Protéines de fusion recombinantes/immunologie , Vaccins synthétiques/biosynthèse , Vaccins synthétiques/immunologieRÉSUMÉ
Since 1983, several recombinant antibodies have been expressed in important agronomic plant species. However, to date no evaluation has been published about prolonged antibody stability within plant tissues under cryo-preservation conditions. This current report presents an approach to the KDEL-plantibody HB-01 (PHB-01) stability in frozen tobacco leaves by presenting scientific evidence about the stability of a plantibody to a prolonged low temperature exposure in this biological source. Results clearly show that the PHB-01 amount is maintained during the storage of tobacco leaves at -20 degrees C for 90 days. The PHB-01 recovery was not affected by any irreversible physical and/or chemical change produced in tobacco leaves after this cryo-preservation time. The amount of total soluble proteins in the clarified extract decreased in proportion with the storage time and the PHB-01 molecules isolated from frozen leaf extracts were highly pure, >95%, according to an SDS-PAGE assessment under reducing conditions. Low temperature exposure of tobacco leaves did not reveal visible changes in frozen leaves, which is essential for the further extractability of proteins. The PHB-01 is stable in tobacco leaves at -20 degrees C during 90 days, which offers the possibility to overcome problems associated with detrimental climate conditions and optimize purification capabilities.