Induction of multiple cytotoxic T lymphocyte responses in mice by a multiepitope DNA vaccine against dengue virus serotype 1.

Dengue virus (DENV) is still a major threat to human health in most tropical and subtropical countries and regions. In the present study, a multi-epitope DNA vaccine that encodes 15 immunogenic and conserved HLA-A*0201-, HLA-A*1101-, HLA-A*2402-restricted CTL epitopes from DENV serotype 1 (DENV-1) was constructed based on the eukaryotic expressing plasmid pcDNATM 3.1/myc-His(-) A. Immunization of HLA-A*0201, HLA-A*1101 and HLA-A*2402 transgenic mice with the recombinant plasmid pcDNATM 3.1/myc-His(-) A-DENV-1-Meg resulted in significantly greater IFN-γ-secreting T-cell responses against most (14/15) CTL epitopes than occurred in mice immunized with the empty plasmid pcDNATM 3.1/myc-His(-) A. Additionally, the epitope-specific T cells directed to some epitopes secreted not only IFN-γ but also IL-6 and/or TNF-α. Finally, the induced epitope-specific T cells also efficiently lysed epitope-pulsed splenocytes and DENV-1-infected splenic monocytes. The present study confirms the immunogenicity of multi-epitope DENV vaccine, suggesting that it may contribute to the development of a universal DENV vaccine.

Identification of conserved and HLA-A*2402-restricted epitopes in Dengue virus serotype 2.

In this study, we set out to identify dengue virus serotype 2 (DENV-2)-specific HLA-A*2402-restricted epitopes and determine the characteristics of T cells generated to these epitopes. We screened the full-length amino-acid sequence of DENV-2 to find potential epitopes using the SYFPEITHI algorithm. Twelve putative HLA-A*2402-binding peptides conserved in hundreds of DENV-2 strains were synthesized, and the HLA restriction of peptides was tested in HLA-A*2402 transgenic mice. Nine peptides (NS4b(228-237), NS2a(73-81), E(298-306), M(141-149), NS4a(96-105), NS4b(159-168), NS5(475-484), NS1(162-171), and NS5(611-620)) induced high levels of peptide-specific IFN-γ-secreting cells in HLA-A*2402 transgenic mice. Apart from IFN-γ, NS4b(228-237-), NS2a(73-81-) and E(298-306)-specific CD8(+) cells produced TNF-α and IL-6 simultaneously, whereas M(141-149-) and NS5(475-484-) CD8(+) cells produced only IL-6. Moreover, splenic mononuclear cells (SMCs) efficiently recognized and killed peptide-pulsed splenocytes. Furthermore, each of nine peptides could be recognized by splenocytes from DENV-2-infected HLA-A*2402 transgenic mice. The SMCs from HLA-A*2402 transgenic mice immunized with nine immunogenic peptides efficiently killed DENV-2-infected splenic monocytes. The present identified epitopes have the potential to be new diagnostic tools for characterization of T-cell immunity in DENV infection and may serve as part of a universal epitope-based vaccine.

[Studies on the establishment of a method to detect HPV 6b E7-specific antibodies of serum and cervical secretion from patients with condyloma acuminatum and its epidemiological significance].

OBJECTIVE: To establish a method for detection of the human papi11omavirus (HPV) 6b E7-specific antibodies in serum and cervical secretion from patients with condyloma acuminatum (CA). METHODS: A full-length HPV 6b E7 gene was amplified by PCR from the CA tissue to construct the recombinant plasmid pET32a(+)/HPV 6b E7. The expression of prokaryotic protein was analyzed by SDS-PAGE and Western blot, then purified with Ni-NTA Agarose affinity column and used as an diagnostic antigen for establishing indirect ELISA method, to detect specific serum IgG and specific cervical secretion sIgA from 56 CA patients, 81 healthy control. Sera from 43 cervical cancer was served as control. HPV 6b DNA from 56 CA patients was identified by PCR. RESULTS: Data showed that the nucleotide homology of cloned sequence was 99.5%, compared to the standard sequences of HPV 6b E7 gene (GenBank accession number: NC001355). A high level expression of E7 fusion protein was obtained in prokaryotic expression system (40 µg/ml). Based on HPV 6b E7 fusion protein being used as coating antigen, results from ELISA showed that the absorbance rates (A) of serum IgG from CA, cervix cancer and healthy control groups were 1.82 ± 0.48, 1.36 ± 0.39 and 1.39 ± 0.27, respectively. The level of IgG antibody in the serum of CA group was significantly higher than that in cervix cancer group and healthy control (P < 0.05). The A values of cervical secretion sIgA in CA and healthy control groups were 0.63 ± 0.26 and 0.53 ± 0.06, respectively, while the level of sIgA antibody in the cervical secretion of CA group was also significantly higher than that in healthy controls (P < 0.05). The positive rate of HPV 6b E7 DNA in CA tissue was 78.6% (44/56) by PCR method. When compared the results measured by PCR, the HPV 6b E7-specific IgG and sIgA antibodies by ELISA used to detect the patients infected with HPV 6b infection, showed that the sensitivity rates were 68.2% (30/44) and 54.6% (24/44) respectively, and the specificity were all 100% (12/12). CONCLUSION: Based on the serum and cervical secretion specific HPV 6b E7 antibodies from patients with CA to diagnose HPV 6b infection, results showed medium sensitivity and high specificity, and could further be used to investigate the epidemiological characteristics of HPV 6b infection.

Investigation of HGV and TTV infection in sera and saliva from non-hepatitis patients with oral diseases.

AIM: To determine the frequencies of HGV and TTV infections in serum and saliva samples of non-hepatitis patients with oral diseases in Hangzhou area, and to understand the correlation between detected results of HGV RNA and/or TTV DNA in sera and in saliva from the same patients. METHODS: RT-nested PCR for HGV RNA detection and semi-nested PCR for TTV DNA detection were performed in the serum and saliva samples from 226 non-hepatitis patients with oral diseases, and nucleotide sequence analysis. RESULTS: Twenty-seven (11.9 %) and 21 (9.3 %) of the 226 serum samples were only positive for HGV RNA and TTV DNA, respectively. 10 (4.4 %) and 9 (3.9 %) of the 226 saliva samples were only positive for HGV RNA and TTV DNA, respectively. And 7 (3.1 %) of the serum samples and 2 (0.9 %) of the saliva samples showed the positive amplification results for both HGV RNA and TTV DNA. 12 saliva samples from the 34 patients (35.3 %) with HGV or HGV/TTV viremia and 11 saliva samples from the 28 patients (39.3 %) with TTV or HGV/TTV viremia were HGV RNA detectable, respectively, including two patients positive for both HGV RNA and TTV DNA in serum and saliva samples. No saliva samples from the 226 patients were found to be HGV RNA or TTV DNA detectable while their serum samples were negative for HGV or TTV. Homologies of the nucleotide sequences of HGV and TTV amplification products from the serum and saliva samples of the two patients compared with the reported sequences were 88.65-91.49 % and 65.32-66.67 %, respectively. In comparison with the nucleotide sequences of amplification products between serum and from saliva sample from any one of the two patients, the homologies were 98.58 % and 99.29 % for HGV, and were 98.65 % and 98.20 % for TTV, respectively. CONCLUSION: Relatively high carrying rates of HGV and/or TTV in the sera of non-hepatitis patients with oral diseases in Hangzhou area are demonstrated. Parts of the carriers are HGV and/or TTV positive in their saliva. The results of this study indicate that dentists may be one of the populations with high risk for HGV and/or TTV infection, and by way of saliva HGV and TTV may be transmitted among individuals.