| Infection of human papillomavirus(HPV) is very common in human and has been identified as a risk factor in some malignant tumors. Lesions in recurrent or chronic infection patients can develop to precancerous lesions or tumors. Among high risk type in over 100 HPV genotypes, HPV16 plays a major role in the pathogenesis of condyloma acumilatum(CA), cervical cancer and cervical intraepithelial neoplasia. With the traditional treatments to this disease, such as chemotherapy, iatrophysics or surgical intervention, many patients have to face the problem of high recurrence. So that we urgently sought to a more effective therapies. Recent investigations emphasized that the disorder of host's immunological function, especially antigen-presenting environment in HPV-related epithelium, which is an important factor in recurrence and tumorigenesis. To improve antigen-presenting condition in HPV-related lesions and enhance immune response will effectively eradicate virus-infected cells. At the same time, recurrence and tumorigenesis will be decreased. Dendritic cells(DCs) are professional antigen-presenting cells, which are able to initiate primary immune reponse to antigens by naive T cell. HPV16 E7 protein is attractive target for immunotherapy because it is in close relation with cell transformation and cancerization. Therefore, we use HPV16 E7 peptide and DCs to elicit antigen-specific cellular immune response and investigate the role of DCs-based immunotherapy in infection of HPV16. Firstly, HPV16 E749-57(RAHYNIVTF) peptide was synthesized by solid-phase method, purified by reverse-phase HPLC and identified with mass spectrometry. This peptide was identified to be HLA-A2-restricted CTL epitope.The purity of synthesized peptide was 98.73%. Its molecular weight measured with mass spectrometry was identical with theoretical value. The study lay the foundation for experimental and clinical study of therapeutic peptide vaccines on HPV infection.Secondly, DCs were generated from peripheral blood mononuclear cells(PBMC) in thepresence of granulocyte/macrophage colony stimulating factor(GM-CSF)and interleukin-4(IL-4). HPV16 E7 peptide was used to activate DCs. The surface antigen of DCs were analyzed by flow cytometic analysis. The stimulatory capacity of DCs wes determined by homogenic mixed lymphocyte reaction.The killing activity of cytotoxic T lymphocytes(CTL) induced by DCs was detected by LDH release assay. Results indicated that E7-pulsed DCs showed typical shapes under light microscope. The CD1a(58.4±6.7)%,CD80(70.6±3.4)%,CD83(71.2±5.4)% and HLA-DR(74.8±4.2)% were highly expressed on the surface of isolated DCs, which are able to induce the proliferation of T cells. Antigen-special CTL induced by E7-pulsed DCs can kill Caski cells specifically. It suggested that DCs loaded with E7 peptide can elicit high efficient and specific cellular immune response in vitro.Lastly, we investigate the treatment effect of E7-pulsed DCs vaccine in recurrent CA with HPV16 who had HLA-A2. 11 cases were treated by DCs and 21 cases treated with interferon. DCs were injected subcutaneously in lesion and vicinity. Vaccinations were administered time a week. All patients were followed up for 6 months. By comparison of before and after 3 times of vaccinations the peripheral T-cell subpopulations of the patients showed no significant difference. At the same time, the size of the lesions became smaller after treatment.The infiltrated lymphocytes were founded in the lesions and koilocytotic cells also significantly decreased. The recurrent rate in the treatment group descedn to 18.2%, while that of control group was 61.9%. On the basis of study mentioned above, we draw a conclusion that E7-pulsed DCs can elicit antigen specific cellular immunity. E7-pulsed DCs may be an useful way for the clinical immunotherapy to infection of HPV16.
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