《【病毒外文文獻】2006 A recombinant baculovirus-expressed S glycoprotein vaccine elicits high titers of SARS-associated coronavirus (SARS》由會員分享，可在線閱讀，更多相關(guān)《【病毒外文文獻】2006 A recombinant baculovirus-expressed S glycoprotein vaccine elicits high titers of SARS-associated coronavirus (SARS（8頁珍藏版）》請在裝配圖網(wǎng)上搜索。

Abstract truncated a without strong reacti viral indicate neutralizing K 1 disease ness tion recommendations SARS SARS CoV as by 0264 410X doi 10 1016 j v Vaccine 24 2006 3624 3631 A recombinant baculovirus expressed S glycoprotein vaccine elicits high titers of SARS associated coronavirus SARS CoV neutralizing antibodies in mice Zhimin Zhou a Penny Post a Rick Chubet a Katherine Holtz a Clifton McPherson a Martin Petric b Manon Cox a a Protein Sciences Corporation 1000 Research Parkway Meriden CT 06540 USA b BC Centre for Disease Control 655W 12th Ave Vancouver BC Canada V5Z 4R4 Received 22 November 2005 received in revised form 17 January 2006 accepted 24 January 2006 Available online 9 February 2006 A recombinant SARS CoV spike S glycoprotein vaccine produced in insect cells in a pre clinical development stage is described A version of S glycoprotein containing only the ecto domain as well as a His tagged full length version were cloned and expressed in serum free insect cell line ExpresSF The proteins purified to apparent homogeneity by liquid column chromatography were formulated adjuvant at 3 9 27 and 50H9262g per dose in phosphate saline and used to immunize mice Both antigens in each formulation elicited a immune response after two or three vaccinations with the antigen Neutralizing antibody titers correlated closely with standard ELISA vity against the S glycoprotein The truncated S protein was also formulated with an adjuvant aluminum hydroxide at 1H9262g per dose adjuvant and 5H9262g per dose adjuvant Significantly enhanced immune responses manifested by higher titers of serum ELISA and neutralizing antibodies were achieved in adjuvanted groups with fewer doses and lower concentration of S glycoprotein These findings that the ecto domain of SARS CoV S glycoprotein vaccine with or without adjuvant is immunogenic and induces high titers of virus antibodies to levels similar to those achieved with the full S glycoprotein 2006 Elsevier Ltd All rights reserved eywords SARS CoV Recombinant protein vaccine Virus neutralization Adjuv Introduction Severe acute respiratory syndrome SARS is a respiratory whose main symptoms include fever cough short of breath and pneumonia The World Health Organiza reported 8114 cases with 775 fatalities WHO Updated October 2004 The etiologic agent of was shown to be a coronavirus now designated as 1 5 The disease has the potential to re appear a new naturally acquired outbreak by accidental 6 or intentional release Corresponding author Tel 1 203 686 0800 fax 1 203 686 0268 E mail address zzhou Z Zhou priority care lation v e virus There opment the candidates combination inacti DN proteins see front matter 2006 Elsevier Ltd All rights reserved accine 2006 01 059 vaccine ant Aluminum hydroxide The development of SARS CoV vaccines has become a for preparedness for future outbreaks to protect health workers managing new cases as well as the general popu A Phase I clinical trial using killed whole SARS CoV accine has been conducted by Sinovac Biotech Ltd How ver concerns have been raised for the use of inactivated vaccines based on observations with feline coronavirus are also several subunit vaccines in pre clinical devel targeting SARS CoV structural proteins including spike S and nucleocapsid N proteins Other vaccine include vector based DNA vaccines 7 11 a of whole killed virus and DNA vaccines 12 vated whole virus vaccines 13 14 a combination of A and S peptide vaccines 15 and other recombinant and their fragments 24 2006 3624 3631 3625 protein this mediate cell 16 19 coprotein comple at also 20 34 cations using sisting of e protein body of 2 2 1 a T Dr cloned 5 w tein ulo w The inserts presence then assemble flanking mic PCR assist w Both sequences CoV A gr DN S Z Zhou et al Vaccine One of the most promising candidates for SARS CoV vaccine is the S glycoprotein protein Oligomers of glycoprotein form large spikes in the viral envelope and the binding of SARS CoV to host cell through host receptor the angiotensin converting enzyme II ACE2 The receptor binding domain RBD of the S gly has been mapped to residues 318 510 20 22 A x structure of RBD and soluble ACE2 was determined 2 9 A 23 The S protein and its fragments were shown to induce neutralizing antibodies 16 24 33 The S glycoprotein has been biochemically characterized and its features were explored for therapeutic appli 20 35 This report describes two pre clinical studies in mice purified subunit vaccines of SARS S glycoprotein con of the ecto domain transmembrane domain deleted this protein and a His tagged full length S glycoprotein xpressed in an insect cell system Immunogenicity of the vaccines in an ELISA and in viral neutralizing anti assays was determined together with the adjuvant effect aluminum hydroxide Materials and methods Recombinant protein vaccine The gene encoding the S glycoprotein was cloned from lysate of culture SARS CoV 3200300841 Passage 3 in rizol LS Reagent Sigma which was kindly provided by Dean Erdman CDC Atlanta Briefly the cDNA was in two steps due to the size of the gene 3 5 kb The prime one third of the ORF Front containing the N terminus as cloned into a baculovirus transfer vector pPSC12 Pro Sciences Corporation PSC downstream from the bac virus very late promoter of the polyhedrin gene The insert as also in frame with the chitinase secretion signal sequence remainder of the gene was cloned into pUC18 Both were sequenced MWG Biotech in full to confirm of entire gene Two fragments of the ORF were joined in pPSC12 using convenient restriction sites to the complete SARS CoV ORF and the insert and regions of the vector were sequenced The regions encoding the transmembrane and cytoplas domains of SARS CoV were deleted using site directed This construct was named Delta1TMS Fig 1A To purification of the full length S protein a His 6 tag as constructed to the C terminus of the protein Fig 1A constructs were sequenced in full and their nucleotide were identical to the published sequence of SARS S gene for the protein region Gen Bank Accession No Y278741 To generate a recombinant baculovirus linearized Auto apha California nuclear polyhedrosis virus AcNPV A and the recombination plasmid DNA containing the gene were mixed co precipitated with calcium phosphate Fig His membrane of and identified gle further cells binant of ity infected this e infected w were matography identity and body His tagged determined 1 Two recombinant SARS spike proteins used in the study C terminal 6 tagged full length S His 6 tag FL S and the ecto domain of S trans domain deleted Delta1TM S proteins A schematic representations the S proteins B purified His 6 tag FL S C purified Delta1TM S Sf9 cells were transfected Recombinant viruses were by their distinctive plaque morphology A sin recombinant virus plaque was isolated The virus was amplified using Sf9 cells for P1 and ExpresSF in the absence of serum for P2 and P3 The recom virus was further scaled up before added to 45 liters ExpresSF cell culture in a bioreactor at a multiplic of infection of 1 0 plaque forming unit pfu cell The culture was incubated at 27 C for 60 64 h During period recombinant SARS CoV Delta1TM S protein was xpressed in and secreted from the recombinant baculovirus cells while the His tagged full length S glycoprotein as not secreted Both Delta1TM S and His tagged full length S glycoproteins purified to apparent homogeneity by column chro Fig 1B and C before formulation Protein was determined by N terminal peptide sequencing Western blotting with anti His 6 tag monoclonal anti QIAGEN and or anti SARS S sera PSC Purity of full length S 90 and Delta1TM S 95 was by densitometry Quantity One Densitometry 3626 24 T Mouse Group sampling Day 15 1 8888 2 3 8888 4 5 8888 6 4 4 For example in immunized Softw Systems A monoclonal reagent cal of e teins 2 2 formulated dose full length PBS Alhydrogel tion 2 5 aluminum ml lated 50 glycoprotein 2 5 adjuv adjuv 2 3 T Mouse Group 1 2 3 4 5 6 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