| Background and ObjectiveAdenovirus is a virion which consists of a 70-90 nm diameter nonenveloped icosahedral capsid that packages a single double-stranded linear DNA genome of approximately 35 kb in length.The capsid is composed of 252 capsomeres,of which 240 are homotrimeric hexons and 12 are pentameric pentons located at the vertices of the icosahedron and from which project a single.variable-length homotrimeric fiber.Type-specific antigenic determinants that give rise to neutralizing antibodies are located primarily on the surface of the hexon capsomere,the fiber,and,to a lesser extent.the penton protein.HAdVs,located within the genus Mastadenovirus.are divided into seven species.A-G.Species B can be further divided into two subspecies.B1(3,7,16,21,50)and B2(11,14,34,35).There are 70 recognized HAdV genotypes defined classically by genome data analysis.It is strong infective and can cause acute pharyngitis,lethal pneumonia in infants,epidemic conjunctivitis,cystitis,enteritis and other multi-system organ infections,in which the most common is respiratory infections.HAdV infections occur throughout the year but are more commonly found in late winter,spring,and early summer in temperate regions.Infants and young children have the highest attack rates of endemic HAdV infections.Most children have been exposed to several types of endemic HAdVs by the time they enter school.Infections with the less common species E HAdV-4 and species B serotypes can occur later in life.The species E HAdV-4 and species B HAdV-7 occur mostly in epidemics;species B HAdV-3 is sometimes endemic and sometimes epidemic.In particular,HAdV-7 has been observed to be one of the most pathogenic serotypes,causing a number of fatal lower respiratory tract infections.The majority of HAdV infections are self-limited and do not require specific therapy.There are no agents licensed specifically for treatment of HAdV disease.Adenovirus vectors are widely used in gene therapy fields.A large number of studies have investigated replication defective,E1-deleted adenovirus vectors for gene therapy.Species C HAdV-5 has been the primary platform for the construction of vectors.Adenovirus vectors can infect a variety of cell types,including nondividing as well as dividing cells,and can be prepared readily in large quantities in tissue culture.These vectors can accommodate large inserts(up to 8 kb)and express higher levels of recombinant proteins than most other viral vectors.In the early time,we had successfully constructed HAdV-3 hexon chimeric replication-defective adenoviral vector,which expressing type 3 hexon protein.In this topic,we try to clone three kinds of hexon genes/peptides from HAdV-3.4.7 types into the Ad5 replication-defective adenovirus vector and construct a new chimeric vector expressing the three interested proteins.After that,we tranfected the chimeric vector into AD293 for virus assembly,and rescuing replication-defective recombinant adenovirus.In mice immunization,neutralization test will be done to compare the level of cellular and humoral immunity genenated by intranasal and intramuscular delivery,so as to make an evaluation to the immunogenic ability utilizing adenovirus-mediated gene immunization.MethodsDNA sequencing and classification of human adenoviruseA total of 195 children with ARI requiring hospitalization from 2012 to 2013 were studied.Throat swab specimens were collected from each patient.DNA was extracted and 1.6-kb HVR fragment was amplified by PCR.The seven from HAdVs-positive samples collected were sequenced for classification.Generation of adenoviral recombinantsA shuttle vector from AdEasyTM adenovirus vector system,pShuttle-IRES-hrGFP-1,was digested with the restriction endonucleases NotⅠ and XhoⅠ,and a 2.8-kb hexon fragment amplified by PCR was inserted into the NotⅠ/XhoⅠ sites of shuttle vector to construct pSAd7H shuttle.The resulting shuttle vector was used for homologous recombination with pAdEasy-1 in E.coli strain BJ5183 to generate the rAd genome prAd-Ad7H.The reconbinant genome vector was verified by means of PCR.DNA sequencing.restricted enzyme analysis(REA).Prior to transfection into AD293 cells,prAd-Ad7H vector was digested with Pacl to isolate the recombinant genome from the vector backbone.Transfected cells were incubated at 37℃ with 5%CO2.Green fluorescein protein(GFP)and cytopathic effect(CPE)were observed under fluorescein microscope everyday.Recombinant adenovirus was harvested when 95 to 100%cytopathic effect was observed.To verify the infectivity of P0 production,One-tenth of the crude virus solution was used for the next round of infection.Meanwhile,RT-PCR and Western Blot would be performed to detect the hexon gene expression.Six to eight-week-old female BALB/c mice were prepared for mice immunization experiments and divided into 5 groups.Each group contained 4 mice.With the prime-double boost regimen,mice were administered intranasally and intramusally 1 × 109 v.p.particles of recombinant adenovirus at week 1,3and 4.The blood of BALB/c mice was collected.harvested the supernatants at week 5.6.The antibody activ ity of these sera was determined by neutralization test.Rescue of trivalent gene/peptides-chimeric recombinant virusConstruction of a hexon-chimeric vector prAd3H.It was hypervariable region 1(HVR1)modification that the specific mutation from Ad3-hexon was introduced into the Ad5-hexon by splice-overlap extension PCR to generate Ad3HVR1-LR.For DNA amplification,the 5.3-kb hybrid fragment containing mutation was subcloned into SphⅠ and EcoRI sites of pUC19 vector.Prior to homologous recombination,the backbone vector pAdEasy-1 was linearized by digestion with AsiSI,and then cotransfered with Ad3HVR1-LR to E.coli strain BJ5183.The mutation that native HVR1 was replaced with Ad3-HVR1 was confirmed by PCR and DNA sequencing.Construction of E3 region chimeric vector prAd4H3H.Ad4L(1.5 kb),Ad4H(2.8 kb).and Ad4R(2.1kb),were amplified by PCR expectively,and then ligated into pUC19 vector one after another to generate a hybrid fragment Ad4-LHR.This resulting DNA fragment was isolated from pUC19 using Bsu36Ⅰ and KpnⅠ digestion,and inserted the interesting gene Ad4 hexon into SpeI-digested vecter prAd3H(described aboved)by homologous recombination in E.coli strain BJ5183,constructing prAd4H3H.PCR,REA and DNA sequencing were performed to verify the 2.8-kb insertion in E3 chimeric vector prAd4H3H.The shuttle vector pSAd7H covered Ad7 hexon was digested and linearized with PmeⅠ.This PmeⅠ-digest fragment was cotranformed with prAd4H3H in BJ51 83 for construction of prAd7H4H3H.The insertions or replacement in recombinant vector confirmed by PCR,REA and DNA fragment sequencing.Purified rAd genome was digested with PacⅠ and used for transfection of AD293 cells to produce trivalent recombinant adenovirus.RT-PCR was used to confirmed the three fragment transcription events,and Western Blot was performed to detect protein expression.ResultClassification of human adenovirus samples in hospitalized children in Guangzhou,2012-2013The molecular types of these samples were determined by the Blastn of the sequences in GenBank.In 2012.71.4%were identified as type 3 and 28.6%were type 7.In 2013,three types of HAdVs were identified:HAdV-3,-7 and-55.HAdV-3 account for 69.8%(30/43),followed by HAdV-7(12/43,27.9%).One case of HAdV-55 in 2013 was identified.Rescue of Ad7 hexon-chimeric recombinant virusThe recombinant vector prAd-Ad7H was constructed successfully.REA and DNA sequencing showed there was no mutation in hexon gene.To produce viruses,prAd-Ad7H was digested with PacI to liberate linear adenoviral genomes,then transfected into 293 cells.It was 4 days post-transfection that packaged viral particles could be observed by GFP expression.For Western blotting of Ad7 hexon,as expected,the intensity of bands in Western blotting correlated with the copy number of the capsid proteins where the gene was inserted(106 kDa).Neutralization test was performed to assess antibody activity.The results showed as follows.The sera from intranasal group were at a ratio of 1:179.1:357 respectively;while sera from intramuscular group were at a ratio of 1:357.1:370.However,anti-virus activity of sera of recombinant immunization was much lower than that of wildtype strain Ad7.Further study will be done to improve the efficiency of immunization experiment.Construction of trivalent hexon-chimeric Ad5-based vectorGenome of recombinat vector prAd7H4H3H contained three genes/peptides:Ad3HVR1(39 bp),Ad4 hexon(2.8 kb)and Ad7 hexon(2.8 kb).These exogenous fragments were confirmed by DNA sequencing and no mutation was found.Restricted enzyme analysis showed that bands in agarose gel were consistent with that performed by vectorNTI software,suggesting three interesting fragments located at the right region we designed prev ious.To recombinant vector.prAd7H4H3H was digested with Pacl to liberate linear adenoviral genome,then transfected into AD293 cells.However,it was 10 days post-transfection that packaged viral particles could be observed by GFP expression.It was about 2-fold slower than prAd-Ad7H as described before.For virus DNA detection,positive bands could be found in both P0 and P1 recombinant adenovirus.Results of RT-PCT indicated that transcription of three exogenous genes/peptides.Western Blotting and animal immunization experiments will be done in the next step.ConclusionSeveral hexon-chimeric rAd5 vectors we generated showed high efficiency and growth stability of homologous recombination in E.coli cells.Ad5-based replication defective recombinant adenovirus was rescued in AD293 cells,and hexon protein expression could be verify by Western blotting.In mice immunization,level of antibody titer was not high,more study should be done to improve the efficiency of immunization experiment.To recombinant vector,prAd7H4H3H was rescued afterl0 days transfection and it was also verified by RT-PCR,suggesting Ad5-based vector is reasonable and feasible to serve as trivalent vaccine.However,more work,such as Western Blotting and animals immunization experiments.will be further done to confirm and develop this trivalent vaccine candidate. |
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