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Particulate Recombinant Hepatitis E Virus Capsid Protein and Its Antigenicity and Immunogenicity
颗粒化重组戊型肝炎病毒衣壳蛋白及其抗原性与免疫原性

HE Zhi_Qiang,ZHANG Jun,LI Shao_Wei,LIN Jian,LIU Ru_ShiCHEN Yi_Xin,WANG Ying_Bin,XIA Ning_Shao,
何志强
,张军,李少伟,林鉴,刘如石,陈毅歆,王颖彬,夏宁邵

生物工程学报 , 2004,
Abstract: An E. coli expressed recombinant antigen NE2 was reported to aggregate into homo-oligomer, and can induce protective antibodies on rhesus monkey, but its immunogenicty was much weak after being purified. In this study, three N-terminal extension mutant of NE2 were expressed in E. coli, one of which named HEV 239 was found to aggregate into particle. HEV 239 antigen had good reactivity with sera of hepatitis E patients. The reactivity of HEV 239 against neutralization monoclonal antibody 8C11 was similar as NE2 antigen, while the reactivity of it against another neutralization monoclonal antibody 8H3 is much better than NE2 antigen, which indicated better antigenicity of HEV 239 than NE2. The diameter of purified HEV 239 particulate antigen was between 15 nm to 30 nm. The ED50 of immunization of HEV 239 particle adsorbed by aluminum adjuvant to BALB/c mice was between 0.08 microg to 0.25 microg. In contrast, the seraconversion rate of mice immunized by NE2 antigen adsorbed by aluminium adjuvant was only 25% on 60 microg vaccination. These results suggested that HEV 239 antigen particle has better immunogenicity as well as antigenicity than those of NE2 antigen, so it is a better vaccine candidate against HEV.
Comparison of immunogenicity of Aluminum salts as adjuvant for recombinant Hepatitis-B vaccine
Fazeli MR,Abbaspour M,Ghahremani MH,Alimian M
Tehran University Medical Journal , 2007,
Abstract: Background: Aluminum salts are common adjuvants in human and animal vaccine preparations. The two adjuvants aluminum phosphate and aluminum hydroxide show acceptable immunoadjuvant properties with many antigens. These two salts have different physicochemical characteristics that make each one suitable for certain antigens. The surface antigen of Hepatitis B (HBsAg) has several antigenic epitopes that bind to aluminum adjuvants by a ligand exchange mechanism. Although HBV vaccines using an aluminum hydroxide adjuvant are available, higher antigenicity is needed for the subgroup of people who do not respond sufficiently to the currently available vaccines. Methods: A solution of recombinant HBsAg for making different formulations of vaccines with aluminum phosphate (Adju-Phos ) and aluminum hydroxide (Alhydrogel ) adjuvants was obtained from Darupakhsh Pharmaceutical Company. The total protein content, antigenicity, and purity of HBsAg solution were determined using BCA, ELISA, and SDS-PAGE methods, respectively. The different formulations were prepared in the lab and administered i.p. to two test groups of Balb/C mice and a third test group received the Engerix vaccine, which is currently available on the market and uses an aluminum hydroxide adjuvant. The control group of animals received the solution without antigen. After 28 days, heart blood samples were collected and serum was separated to determine the antibody titer against HBsAg using an ELISA kit. Results: This study shows that the vaccine formulated with aluminum phosphate exerted more immunogenicity than both the aluminum hydroxide laboratory formulation and the Engerix vaccines. Conclusion: Although the results of our study indicate higher immunogenic properties of the vaccine formulated with the aluminum phosphate adjuvant, complementary experiments are needed to further evaluate the biological properties with respect to effectiveness, adverse effects, product stability and finally possibility for manufacturing and distribution of this new formulation as a Hepatitis B vaccine.
Extinction of Hepatitis C Virus by Ribavirin in Hepatoma Cells Involves Lethal Mutagenesis  [PDF]
Ana M. Ortega-Prieto, Julie Sheldon, Ana Grande-Pérez, Héctor Tejero, Josep Gregori, Josep Quer, Juan I. Esteban, Esteban Domingo, Celia Perales
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0071039
Abstract: Lethal mutagenesis, or virus extinction produced by enhanced mutation rates, is under investigation as an antiviral strategy that aims at counteracting the adaptive capacity of viral quasispecies, and avoiding selection of antiviral-escape mutants. To explore lethal mutagenesis of hepatitis C virus (HCV), it is important to establish whether ribavirin, the purine nucleoside analogue used in anti-HCV therapy, acts as a mutagenic agent during virus replication in cell culture. Here we report the effect of ribavirin during serial passages of HCV in human hepatoma Huh-7.5 cells, regarding viral progeny production and complexity of mutant spectra. Ribavirin produced an increase of mutant spectrum complexity and of the transition types associated with ribavirin mutagenesis, resulting in HCV extinction. Ribavirin-mediated depletion of intracellular GTP was not the major contributory factor to mutagenesis since mycophenolic acid evoked a similar decrease in GTP without an increase in mutant spectrum complexity. The intracellular concentration of the other nucleoside-triphosphates was elevated as a result of ribavirin treatment. Mycophenolic acid extinguished HCV without an intervening mutagenic activity. Ribavirin-mediated, but not mycophenolic acid-mediated, extinction of HCV occurred via a decrease of specific infectivity, a feature typical of lethal mutagenesis. We discuss some possibilities to explain disparate results on ribavirin mutagenesis of HCV.
Toolbox for Non-Intrusive Structural and Functional Analysis of Recombinant VLP Based Vaccines: A Case Study with Hepatitis B Vaccine  [PDF]
Anke M. Mulder, Bridget Carragher, Victoria Towne, Yuan Meng, Yang Wang, Lance Dieter, Clinton S. Potter, Michael W. Washabaugh, Robert D. Sitrin, Qinjian Zhao
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0033235
Abstract: Background Fundamental to vaccine development, manufacturing consistency, and product stability is an understanding of the vaccine structure-activity relationship. With the virus-like particle (VLP) approach for recombinant vaccines gaining popularity, there is growing demand for tools that define their key characteristics. We assessed a suite of non-intrusive VLP epitope structure and function characterization tools by application to the Hepatitis B surface antigen (rHBsAg) VLP-based vaccine. Methodology The epitope-specific immune reactivity of rHBsAg epitopes to a given monoclonal antibody was monitored by surface plasmon resonance (SPR) and quantitatively analyzed on rHBsAg VLPs in-solution or bound to adjuvant with a competitive enzyme-linked immunosorbent assay (ELISA). The structure of recombinant rHBsAg particles was examined by cryo transmission electron microscopy (cryoTEM) and in-solution atomic force microscopy (AFM). Principal Findings SPR and competitive ELISA determined relative antigenicity in solution, in real time, with rapid turn-around, and without the need of dissolving the particulate aluminum based adjuvant. These methods demonstrated the nature of the clinically relevant epitopes of HBsAg as being responsive to heat and/or redox treatment. In-solution AFM and cryoTEM determined vaccine particle size distribution, shape, and morphology. Redox-treated rHBsAg enabled 3D reconstruction from CryoTEM images – confirming the previously proposed octahedral structure and the established lipid-to-protein ratio of HBsAg particles. Results from these non-intrusive biophysical and immunochemical analyses coalesced into a comprehensive understanding of rHBsAg vaccine epitope structure and function that was important for assuring the desired epitope formation, determinants for vaccine potency, and particle stability during vaccine design, development, and manufacturing. Significance Together, the methods presented here comprise a novel suite of non-intrusive VLP structural and functional characterization tools for recombinant vaccines. Key VLP structural features were defined and epitope-specific antigenicity was quantified while preserving epitope integrity and particle morphology. These tools should facilitate the development of other VLP-based vaccines.
Comparison of Two Recombinant Hepatitis B Vaccines
Hasan Nikui Nejad,Gholamali Ghorbani,Reza Razaghi,Hossain Akbari
Hepatitis Monthly , 2009,
Abstract: Background and Aims: Hepatitis B virus (HBV) infection is an important public health problem. Hepatitis B vaccine induces protective response in the majority of vaccinated persons. In our country, we do not have any evaluation for the efficacy of each type of vaccines used in adult and our objective was therefore to evaluate the efficacy of vaccines..Methods: In a randomized double-blind clinical trial 347 military personnel and their family in Kashan city, central Iran, were studied during August 2007 to April 2008. Participates who did not have history of HBV vaccination were included in this study. Five-mL blood samples were taken from each person and tested for hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (HBsAb) and total hepatitis B core antibody (HBcAb). If the test results were negative, they were then divided into two groups and vaccinated with either recombinant Cuban or Korean vaccine. One month after the latest vaccine dose, we assessed the antibody to hepatitis B surface antigen (anti-HBs Ab) titer..Results: 347 subjects (207 men, 140 women) were studied. All participants were more than 15 years old. The mean±SD age of participants was 32.3±7.2 years. The mean±SD titer of anti-HBs Ab was 253.6±95.4 MIU/mL in Cuban group and 315.7±163.5 in Korean vaccine group (P<0.001). The Korean vaccine induced a higher titer of antibody in ages less than 40. Four (2.3%) subjects in the Cuban and 2 (1.1%) in the Korean vaccine group did not develop protection..Conclusions: The Korean vaccine induces more protection and higher titer in subjects aged less than 40 years than the Cuban vaccine. Therefore, we believe that the Korean hepatitis B vaccine is a better choice in comparison to the Cuban vaccine for prevention of hepatitis B virus infection and mass vaccination in our country.
A Single Amino Acid Substitution Changes Antigenicity of ORF2-Encoded Proteins of Hepatitis E Virus  [PDF]
Jiu-Hong Liang,Xing Dai,Chen Dong,Ji-Hong Meng
International Journal of Molecular Sciences , 2010, DOI: 10.3390/ijms11082962
Abstract: Extensive genomic diversity has been observed among hepatitis E virus (HEV) strains. However, the implication of the genetic heterogeneity on HEV antigenic properties is uncertain. In this study, monoclonal antibodies (Mabs) against truncated ORF2-encoded proteins (aa452?617, designated p166 proteins) derived from HEV strains of Burma (genotype 1a, p166Bur), Pakistan (1b, p166Pak) and Morocco (1c, p166Mor) were raised and used for identification of HEV antigenic diversity. Six Mabs reacted to these 3 p166 proteins as well as p166 proteins constructed from strains derived from Mexico (genotype?2), US?(genotype?3) and China (genotype 4), indicating the existence of pan?genotypic epitopes. Two Mabs, 1B5 and 6C7, reacted with p166Bur and p166Mor, but not p166Pak or p166s derived from genotypes 2, 3, and 4, indicating that these 2 Mabs recognized strain-specific HEV epitopes. Both the common and specific epitopes could not be mapped by 23 synthetic peptides spanning the p166Bur sequence, suggesting that they are confirmation?dependent. Comparative sequence analysis showed that p166Bur and p166Mor shared an identical aa sequence along their entire lengths, whereas for p166Pak the aas occupying positions 606 and 614 are different from aas at corresponding positions of p166Bur and p166Mor. Reactivity between 1B5 and p166Bur was abrogated with mutation of p166Bur/A606V, whereas p166Pak acquired the reactivity to 1B5 with mutation of p166Pak/V606A. However, mutations of p166Bur/L614M and P166Pak/M614L did not affect the immunoreactivity. Therefore, the aa occupying position 606 plays a critical role in maintaining the antigenicity of the HEV p166 proteins.
Immunogenicity and antigenicity of the recombinant EMA-1 protein of Theileria equi expressed in the yeast Pichia pastoris
Nizoli, Leandro Q.;Concei??o, Fabrício R.;Silva, Sérgio S.;Dummer, Luana A.;Santos Jr., Alceu G.;Leite, Fábio P. L.;
Revista Brasileira de Parasitologia Veterinária , 2009, DOI: 10.4322/rbpv.01802001
Abstract: the equine piroplasmosis caused by theileria equi is one of the most important parasitic diseases of the equine, causing damage to animal health and economic losses. in t. equi, 2 merozoite surface proteins, equi merozoite antigen ema-1 and ema-2, have been identified as the most immunodominant antigens. this suggests that these antigens might be used as immunobiological tools. the ema-1 of theileria equi was cloned and expressed in the yeast pichia pastoris. the transformed yeast was grown at high cell density, expressing up to 389 mg.l-1 of recombinant protein. the protein was concentrated and detected in dot blot. the recombinant product was antigenically similar to the native protein as determined using monoclonal antibodies, and polyclonal antibodies obtained from equines naturally infected with t. equi. the immunogenicity of rema-1 protein was demonstrated by ifat using sera from recombinant-protein-immunized mice using aluminum hydroxide as adjuvant. all animals vaccinated with rema-1 developed a high specific antibody response. this results suggest that rema-1expressed in p. pastoris might be a strong candidate to be used as an antigen for immune diagnostics as well as a vaccine antigen.
Expression and characterization of hepatitis C virus core protein fused to hepatitis B virus core antigen
Li Yang,Chunlin Wang,Yuan Wang,Guangdi Li
Science China Life Sciences , 1999, DOI: 10.1007/BF03183605
Abstract: Recombinant plasmids were constructed by fusing the gene fragments encoding the full-length (1-191aa) and the truncated (1-40aa and l-69aa) HCV core proteins (HCc) respectively to the core gene of HBV at the position of amino acid 144 and expressed inE. coli. The products were analyzed by ELISA, Western blotting as well as the immunization of the mice. The results showed that those fusion proteins (B144C191, B144C69, B144C40) possessed the dual antigenicity and immunogenicity of both hepatitis B virus core antigen (HBcAg) and hepatitis C virus core protein (HCc). Analysis by electron microscopy and CsCI density gradient ultra-centrifugation revealed that similar to the HBcAg itself, all fusion proteins were able to form particles. Comparison of the antigenicity and immunogenicity of those fusion proteins showed that the length of HCc gene fused to HBcAg had no much effect on the antigenicity and immunogenicity of HBcAg, however, B144C69 and B144C40 induced higher titres antibodies against HCc than B144C191. Using those fusion proteins, ELISA for screening of antibodies against both HBV and HCV in human sera was also established.
Expression and characterization of hepatitis C virus core protein fused to hepatitis B virus core antigen
YANG Li,WANG Chunlin,WANG Yuan,LI Guangdi,
杨莉
,王春林,汪垣,李光地

中国科学C辑(英文版) , 1999,
Abstract: Recombinant plasmids were constructed by fusing the gene fragments encoding the full-length (l-191aa) and the truncated (l-40aa and l-69aa) HCV core proteins (HCc) respectively to the core gene of HBV at the position of amino acid 144 and expressed in E. coli. The products were analyzed by ELISA, Western blotting as well as the immunization of the mice. The results showed that those fusion proteins (B144C191, B144C69, B144C40) possessed the dual antigenicity and immunogenicity of both hepatitis B virus core antigen (HBcAg) and hepatitis C virus core protein (HCc). Analysis by electron microscopy and CsCl density gradient ultra-centrifugation revealed that similar to the HBcAg itself, all fusion proteins were able to form particles. Comparison of the antigenicity and immunogenicity of those fusion proteins showed that the length of HCc gene fused to HBcAg had no much effect on the antigenicity and immunogenicity of HBcAg, however, B144C69 and B144C40 induced higher titres antibodies against HCc than B144C191. Using those fusion proteins, ELISA for screening of antibodies against both HBV and HCV in human sera was also established.
Comparison of two recombinant hepatitis B vaccines and their interchangeability in Argentine infants
Tregnaghi,Miguel; Ussher,José; Baudagna,Ana María; Calvari,Miriam; Gra?a,Gabriela;
Revista Panamericana de Salud Pública , 2004, DOI: 10.1590/S1020-49892004000100006
Abstract: objective: to compare two pediatric recombinant hepatitis b vaccines-the engerix-b reference vaccine and the euvax-b vaccine-in terms of immunogenicity and reactogenicity, and also to investigate their interchangeability, that is, whether a three-dose hepatitis b vaccination schedule begun with a first dose of engerix-b could be completed with two doses of euvax-b. methods: this study was conducted in the city of córdoba, argentina, from march 1999 through february 2000. three groups of argentine newborns (100 per group) were vaccinated at 0, 1, and 6 months of age with hepatitis b vaccine: group a, three doses of euvax-b; group b, three doses of engerix-b; and group c, one dose of engerix-b followed by two doses of euvax-b. reactogenicity was evaluated based on parental reporting of any solicited local or systemic event occurring during the 7-day period following vaccination. whether euvax-b and engerix-b were clinically identical was assessed in terms of the seroprotection rates (antibodies to hepatitis b surface antigen (anti-hbsag) >10 milli-international units per ml (miu/ml) 2 months after the third vaccination). results: reactogenicity was low in all three groups. five months after the second dose (that is, immediately prior to the third vaccination), seroprotection rates were 95.9%, 94.7%, and 90.2% for groups a, b, and c, respectively. two months after the third dose all subjects were seroprotected, with geometric mean concentrations of anti-hbsag of 2 468.1, 1 714.8, and 2 075.3 miu/ml for groups a, b, and c, respectively. conclusions: both of the recombinant hepatitis b vaccines that we studied were well tolerated and highly immunogenic. euvax-b was clinically identical (not inferior) to the engerix-b reference vaccine, and either vaccine could be used to achieve the world health organization goal of immunizing all infants against hepatitis b. further, euvax-b can be safely used in infants given an initial dose of either euvax-b or engerix-b.
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