Construction Of Recombinant Adenovirus Of HPV-11 E7 And Analysis Of Transfection Dendritic Cells

Condyloma acuminata (CA) is one of the most common sexually transmitted disease(STD) caused by Human Papillomavirus (HPV) in China. During the period of 1991-2001, the average annual growth of incidence was 19.48% in 31 provinces of China. In 2000, the incidence of CA is 17.55/100 thousands and the constituent ratio is up to 25.47 % in all STD. In recent years, the incidence of CA is still very high. CA has become a serious public health problem.HPV is a small, double-stranded DNA virus predominantly infecting the cutaneous and mucosal epithelium of the genital tract, and more than 100 different subsets of HPV have been identified. HPV-6/11 is the genotype most commonly found in CA. CA may be caused by different HPV subsets together and no cross immunization among HPV subsets. Persons infected with HPV may be clinical, subclinical, or latent. In most persons, HPV infections appear to be transient and result in no sequelae. Some untreated CA lesions may regress spontaneously. In a small proportion, HPV infections may persist and progress to cancer. The exact reason for this is unknown. Some studies suggest that CA may be attributed to the poor local immune response to HPV infection at the site of the lesion, but there are different opinions for the role of Th1 and Th2 cytokine in the process of HPV infection.Dendritic cells(DC) are the most potent and the only antigen-presenting cells(APC),which are capable of activating naive T cells and induce the specific cytotoxic T cells(CTL) response. Recently, more and more evidences demonstrate that DC with HPV genetic modification could elicit HPV specific CTL in vivo and in vitro, efficiently boost protective anti-HPV immune response. Studies show the injection DC pulsed with HPV-16 antigen at the site of the lesion have got the satisfactory therapeutic efficacy to recurrent and stubborn CA. But mostresearches focus on high-risk types HPV-16/18 that associated with cervical carcinomas, and few to low-risk types HPV-6/11.The effective immune response against HPV infection involves three phases, including virus antigen presenting, activating immune cells and eliminating the virally infected cells by effector cells. Any one of the three phases disturbance, the immune response can be affected. HPV replicative cycle is tired to the keratinocyte differentiation programme. Viral early protein E7 is produced in insufficient quantities and localized mainly in the nucleus of undifferentiated keratinocyte in basal layers of stratified epithelium. Later proteins are produced in more distal layers where capsids are formed and shed in sloughed-off epithelial, probably which days of information. Langerhans cells(LC) are a skin-specific member of DC. LC density was found to be decreased and morphology altered at the site of HPV infection. Several factors minimize or prevent exposure of HPV virus antigens to the immune system. There is no opportunity for APC to engulf HPV virus and present virion-derived antigens to immune system. DC is the pivotal APC for regulating immune response. HPV E7 protein could be an ideal candidate as potential specific targets for immunotherapy. Obviously, vaccine targeted at presetting antigen through DC may be a very efficient way to induce immune response and eliminate the infection.Currently, there is no definitive therapy for CA and recurrence rates relatively high, ranging from 60% to 70%, a vaccine would be effective treatment. In this study, We firstly globally analyze genes involved in the immune modulation of CA lesion by the powerful DNA microarray technique, then construct recombinant adenovirus encoding the HPV-11 E7 gene, analyze the change of the mature DC surface markers and function after transfected with the vector. It might develop a sound approach to the treatment of CA with HPV-11 E7 transfection DC.The main results are as following:1. Analysis of local immunity in CA lesions with DNA microarray technique(1) The extraction and purification of total RNA in CA lesionsTo check HPV infection of clinical samples, DNA extractions of clinical samples were used as PCR templates and PCR were carried out.The general primer was designed according to HPV gene sequences from GenBank. The amplification products were analyzed by electrophoresis in 1% agarose gel. The total RNA of CA and normal foreskin samples were extracted by a TRIZOL method. The integrity of the purified RNA was determined with 1.2% denaturing agarose gel electrophoresis. The results show the 150bp HPV segments from all CA patient samples have been amplified successfully. No amplification segment from 1 health adult. Three bands of ribosomal RNA 28S,18S,5S could be found with denaturing agarose gel electrophoresis. The extraction RNA exhibit OD260/OD 280 ratio about 2.0 .These results indicate that RNA of CA and normal foreskin samples have been extracted successfully.(2) Analysis of local immunity in CA lesions with DNA microarry techniqueTotal RNA extracted from CA samples was reverse-transcribed into cDNA, and then transcribed into biotin-labeled cRNA and fragmented. The cRNA fragments was hybridized with the GeneChip Human U133A Plus2.0 array slides. The hybridization slides were scanned and analyzed with the Microarray Suite Software version 5.0. Some different gene was verified by Real-time Q-PCR . Our study showed that 3676(29.5%) out of total 12473 probe sets is present, 8517(68.3%)absent, 280(2.2%) marginal. The average present signal is 69.7. The probe sets number of signal increase^ decrease and no change is 1472(11.8%), 1784( 14.3% ),9217(73.9 %), respectively. Compared with the normal control ,the intensities of Thl (IFN- y ,IL-12,TNF- a )andTh2(IL-4,IL-10)type cytokines is lower and higher Respectively ,and no change IL-2JL-5, IL-13. The transcript level of slgAJgA, IgG, IgM is increased. The data of Real-time Q-PCR were consistent with the microarrarys. Our study verified that imbalance of Thl/Th2 response in CA lesions, representing immune suppression of Thl response and over activation of Th2 response.2. Generation of Recombinant Adenovirus Vectors of HPV-11 E7 and Expression in Eukarytoic Cells(1) Molecular cloning and sequence analysis of HPV-11 E7HPV-11 E7 gene was amplified from E7/pUCm-T vector by PCR, thendirectional cloned into entry vector pENTR-TOPO? vector to form TOPO-E7 plasmid and transformed into the competent E.coil DH5 a . The recombinants were screened by plate with kanamycin. The recombinant plasmid TOPO-E7 was identified and confirmed with enzyme digestion and sequencing. The results show that 300bp bands of enzyme digestion segments could be visualized with 1% agarose gel electrophoresis. The sequences of nucleotides was 100% homologous to HPV-11 known sequence in GenBank (ID:333026). It suggests that HPV-11 E7 has been cloned successfully, which was the foundation of the further study of the immunological activity and epidemiology.(2) Generation of Recombinant Adenovirus Vectors of HPV-11 E7 andExpression in Eukarytoic CellsWith the LR recombination reaction between pAD/CMV/V5-DEST? gateway vectors and TOPO-E7 plasmid, the E7 gene was transferred into the pAD/CMV/V5-DEST? gateway vector. The recombinant was digested by Pac I enzyme and transfected into 293A cell by Lipofectamine method for recombinant adenovirus vectors pAD-E7. The expression of E7 on HaCaT cells infected with pAD-E7 vectors was analyzed by confocal microscope and SDS-PAGE methods. The results show the recombinant plasmid TOPO-E7 was identified and confirmed with enzyme digestion and sequencing. The recombinant adenovirus vectors pAD-E7 was generated efficiently with titer of 5x108 pfu/ml on transfection 293A cells. The E7 protein could be seen on HaCaT cells with confocaljnicroscope 48h post infected with the recombinant adenovirus vector. The cell protein infected with the vector presented an additional thick band of 15kDa molecular weight compared with the control cells on SDS-PAGE. These suggested the HPV11 E7 gene could be expressed efficiently in eukarytoic cells by recombinant adenovirus mediated transfer, and it was the foundation of further researching in its function and developing the therapeutic vaccine against CA.3. Effects of transfection with HPV-11 E7 recombinant adenovirus on DC(1) Induction and identification of DC from human peripheral bloodmonocytesThe moncytes were isolated from human peripheral blood mononuclear cells (PBMC) and cultured with the cytokins(GM-CSF,IL-4,TNF- a ).The morphological changes were observed by microscope. The surface antigens were analyzed by flow cyometry (FCM). The results show that cultured cells appear floating and typical dendritical branches after 9-10 days .The DC relatively specific surface molecules of CD83 ( 89.6% X CDla (87.3%) and co-stimulating molecules CD40 (74.4%), CD80 (64.1%) and HLA-DRA(81.5%)were high, whereas CD14,specific for monocytes ,was relative low( 7.3% ). It was suggested that large amount of mature DC could be generated from human moncytes in presence of GM-CSF ,IL-14 and TNF- a , which could be the foundation of the immunotherapy with DC.(2) Effects of transfection with HPV-11 E7 recombinant adenovirus ondendritic cellsDC were transfected with pAD-E7 recombinant adenovirus at different multiplicity of infection(MOI).The expression of E7 protein on transfected mature DC was analyses by FCM, and the dead cells were counted by typan blue staining. The alteration of surface markers on mature DC including CDla, CD83, CD40, CD80 and HLA-DR was detected by means of FCM before and after transfection. Meanwhile, the function including stimulating allogeneic T cell proliferation and secreting cytokines IL-10, IL-12, IFN-Y on co-culture supernatant of DC and T cell IL-12 was measured by a method of 3H-thymidine uptake and ELISA respectively. The results showed MOI 100 is the optimal MOI. With MOI 100 transfection, the above 89% of mature DC expressed E7 protein and above 93% of the cells were viable. No significant changes in the surface markers and the cytomorphosis of mature DC were examined during the transfection. Transfected DC still has strong potential to stimulate the proliferation of allergenic T cell. The level of cytokines IL-12, IFN- y in supernatant of co-culture cell is increased and IL-10 is on change. It was suggested that pAD-E7 recombinant adenovirus could be transferred into DC more efficiently. The function of mature DC was not affected significantly by the transfection of pAD-E7, and could induce Thl...