Depletion of CD25^+CD4^+T cells (Tregs) enhances the HBV-specific CD8^+ T cell response primed by DNA immunization

AIM: Persistent hepatitis B virus (HBV) infection is characterized by a weak CD8+ T cell response to HBV. Immunotherapeutic strategies that overcome tolerance and boost these suboptimal responses may facilitate viral clearance in chronically infected individuals. Therefore, we examined whether CD25+CD4+ regulatory T (Treg) cells might be involved in a inhibition of CD8+T cell priming or in the modulation of the magnitude of the 'peak' antiviral CD8+ T cell response primed by DNA immunization. METHODS: B10.D2 mice were immunized once with plasmid pCMV-S. Mice received 500 μg of anti-CD25 mAb injected intraperitoneally 3 d before DNA immunization to deplete CD25+ cells. Induction of HBV-specific CD8+ T cells in peripheral blood mononuclear cells (PBMCs) was measured by S28-39 peptide loaded DimerX staining and their function was analyzed by intracellular IFN-γ staining. RESULTS: DNA immunization induced HBV-specific CD8+ T cells. At the peak T cell response (d 10), 7.1±2.0% of CD8+ T cells were HBV-specific after DNA immunization, whereas 12.7±3.2% of CD8+ T cells were HBV-specific in Treg-depleted mice, suggesting that DNA immunization induced more antigen-specific CD8+ T cells in the absence of CD25+ Treg cells (n = 6, P<0.05). Similarly, fewer HBV specific memory T cells were detected in the presence of these cells (1.3±0.4%) in comparison to Treg-depleted mice (2.6±0.9%) on d 30 after DNA immunization (n - 6, P<0.01). Both IFN-γ production and the avidity of the HBV-specific CD8+ T cell response to antigen were higher in HBV-specific CD8+ T cells induced in the absence of Treg cells. CONCLUSION: CD25+ Treg cells suppress priming and/or expansion of antigen-specific CD8+ T cells during DNA immunization and the peak CD8+ T cell response is enhanced by depleting this cell population. Furthermore, Treg cells appear to be involved in the contraction phase of the CD8+ T cell response and may affect the quality of memory T cell pools. The elimination of Treg cells or their inhibition may be important in immunotherapeutic strategies to control HBV infection by inducing virus-specific cytotoxic T lymphocyte responses in chronically infected subjects.

Study on DNA Immunization by Recombinants Encoding Japanese Encephalitis Virus prME and E Proteins

To study the expression characteristic of Japanese encephalitis virus (JEV) prME and E proteins and the efficacy of DNA immunization by different recombinant plasmids containing JEV prME (2001 bp) and E (1500 bp) genes, two recombinants (pJME and pJE) containing JEV prME and E genes fused with FLAG were constructed and then transfected into HepG2 and COS-1 cells by liposome fusion. The expression feature of FLAG-prME (about 72 kDa) and FLAG-E (about 54 kDa) proteins in transfected cells were analyzed by Western blot and two antibody systems (anti-FLAG and anti-E). BALB/c mice were immunized with 100 μg of two kinds of recombinants by intramuscular injection, and JEV JaGAr-01 strains (10 5 PFU/100 μl)were given to BALB/c mice by intraperioneal injection 3 wk after twice DNA immunization by a lethal virus challenge. BALB/c mice were observed for 21 days after challenge. 80% plaque reduction neutralization test was performed to titrate neutralization antibody before and after viral challenge. It was found that the expression of proteins associated with pJME and pJE was determined in transfected cells with anti-FLAG and a new protein of 11 kDa was detected in HepG2 and COS-1 cells transfected with pJME. Only E (53 kDa) protein was identified as transfected with pJME using anti-E. Higher level of neutralization antibodies and the efficacy of protective immunity were induced with pJME immunization, and were similar to those induced by inactivated Japanese encephalitis vaccine, but were better than those induced with pJE. It concludes that the expression level from prM to E proteins of JEV is different in vitro, and the in vitro expression efficiency of pJME was better than that of pJE. FLAG-prME protein expressed by pJME could be cleaved by peptidase from host. The efficacy of DNA immunization is correlated to the expression characterization of related proteins expressed in vitro.

DNA damage response-related immune activation signature predicts the response to immune checkpoint inhibitors: from gastrointestinal cancer analysis to pan-cancer validation

Objective: DNA damage response(DDR) deficiency has emerged as a prominent determinant of tumor immunogenicity. This study aimed to construct a DDR-related immune activation(DRIA) signature and evaluate the predictive accuracy of the DRIA signature for response to immune checkpoint inhibitor(ICI) therapy in gastrointestinal(GI) cancer.Methods: A DRIA signature was established based on two previously reported DNA damage immune response assays. Clinical and gene expression data from two published GI cancer cohorts were used to assess and validate the association between the DRIA score and response to ICI therapy. The predictive accuracy of the DRIA score was validated based on one ICI-treated melanoma and three pan-cancer published cohorts.Results: The DRIA signature includes three genes(CXCL10, IDO1, and IFI44L). In the discovery cancer cohort, DRIA-high patients with gastric cancer achieved a higher response rate to ICI therapy than DRIA-low patients(81.8% vs. 8.8%;P < 0.001), and the predictive accuracy of the DRIA score [area under the receiver operating characteristic curve(AUC) = 0.845] was superior to the predictive accuracy of PD-L1 expression, tumor mutational burden, microsatellite instability, and Epstein–Barr virus status. The validation cohort demonstrated that the DRIA score identified responders with microsatellite-stable colorectal and pancreatic adenocarcinoma who received dual PD-1 and CTLA-4 blockade with radiation therapy. Furthermore, the predictive performance of the DRIA score was shown to be robust through an extended validation in melanoma, urothelial cancer, and pan-cancer.Conclusions: The DRIA signature has superior and robust predictive accuracy for the efficacy of ICI therapy in GI cancer and pancancer, indicating that the DRIA signature may serve as a powerful biomarker for guiding ICI therapy decisions.

DNA immune responses induced by codelivery of IL-12 expression vectors with hepatitis C structural antigens

Objective: To demonstrate the utility of DNA vaccines for the tailored methods, the efficacy of enhanced immune responses, and the types of increased im- mune responses. Methods: Four recombinant plasmids constructed in- cluded the coding regions for the core protein (pC) and for the core, E_1 and E_2 together (pCE_1E_2), IL- 12 p35 and p40. These plasmids were transfected into mammalian cells to test their protein expression and were injected into the quadriceps muscles of BALB/ C mice for measurement of specific antibodies and cytotoxic T-lymphocyte (CTL) responses. Results: All the recombinant plasmids were shown to express specific antigens stably in mammalian cells. Codelivery of pIL-12 expression cassettes with pC and pCE_1E_2 in mice resulted in the enhancement of Ag-dependent CTL responses and the reduction of specific Ab response. The CTL activity was: pC= 18.65%±5.71%, pCE_1E_2=20.07%±11.11%, pC +pIL-12=60.11%±17.37%, pCE_1E_2+pIL-12= 67.48%±15.57%, respectively. The average A val- ues of anti-HCV were pC=0.415±0.127, pCE_1E_2= 0.358±0.096, pC+pIL-12=0.210±0.086, pCE_1E_2 +pIL-12=0.258±0.125. Conclusion: Codelivery of pIL-12 with plasmid DNA can enhance the efficacy of immune responses and shift the type of immune responses.

Construction and Expression of DNA Vaccine pIRES-Sj97-Sj14-Sj26 and Its Immunogenicity in Mice

To find a new preventive strategy for the infection of Schistosoma japonica, plasmid pIRES-Sj97-Sj 14-Sj26 that contains fatty binding protein （Sj 14）, GST （Sj26） and paramyocin （Sj97） that are expressed on the membrane, was constructed. RT-PCR was used to detect the expression of Sj 14 mRNA, Sj26 mRNA and Sj97 mRNA in the Hela cells, the indirect immunofluorescent test was employed for the detection of the expression of trans-membrane Sj26 after the plasmid was transfected into Hela cells. Fifty BALB/c mice were randomly divided into 5 groups and plRES-Sj97-SjI4-Sj26 plasmid DNA, plRES-Sj 14-Sj26 plasmid DNA, plRES-Sj26 plasmid DNA, plRES blank vector and normal saline were respectively injected into the quadriceps muscles of thigh Eight weeks after the immunization the mice were killed and significantly higher level of IgG was detected in the plRES-Sj97-Sj 14-Sj26 group as compared with the plRES blank vector, normal saline and plRES-Sj26 groups （P〈 0.01） and the plRES-Sj 14-Sj26（P〈0.05）. Single splenocyte suspension was prepared to detected the level of IFN-T by ELISA and the lymphocyte stimulating index （SI） by MTT. SI was significantly higher of in the plRES-Sj97-Sj 14-Sj26 group than in other groups （P〈 0.01）, while the IFN-T level was significantly higher the plRES-Sj97-Sj 14-Sj26 group than in plRES blank vector and normal saline groups （P〈0.01）, but no significant differences were found when compared with plRES-Sj 14-Sj26 and plRES-Sj26 groups. Flow cytometery showed that the percentages of CD4＋ and CD8＋ T cells were much higher in the plRES-Sj97-Sj 14-Sj26 group （P〈 0.01, P〈0.05）. It was concluded that plRES-Sj97-Sj 14-Sj26 vaccine may induce stronger immune response in BALB/c mice.

DNA vaccine expressing herpes simplex virus 1 glycoprotein C and D protects mice against herpes simplex keratitis

AIM： To investigate whether DNA vaccine encoding herpes simplex virus 1（HSV-1） glycoprotein C（g C） and glycoprotein D（g D） will achieve better protective effect against herpes simplex keratitis（HSK） than DNA vaccine encoding gD alone. METHODS： DNA vaccine expressing gD or gC combined g D（g D.g C） were constructed and carried by chitosan nanoparticle. The expression of fusion protein gD and gC were detected in DNA/nanoparticle transfected 293 T cells by Western-blot. For immunization, mice were inoculated with DNA/nanoparticle for 3 times with 2 wk interval, and two weeks after the final immunization, the specific immune responses and clinical degrees of primary HSK were evaluated. RESULTS： Fusion protein g D.g C could be expressed successfully in cultured 293 T cells. And, p RSC-g C.g DIL21 DNA/chitosan nanoparticle could effectively elicit strongest humoral and cellular immune response in primary HSK mice evidenced by higher levels of specific neutralizing antibody and s Ig A production, enhanced cytotoxicities of splenocytes and nature killer cells（NK）,when compared with those of gD alone or mocked vaccine immunized mice. As a result, gC-based vaccine immunized mice showed least HSK disease. CONCLUSION： gC-based DNA vaccine could effectively prevent the progress of primary HSK, suggesting that this DNA vaccine could be a promising vaccine for HSK treatment in the future.

Protective effect of DNA-mediated immunization with a combination of SAG1 and IL-2 gene adjuvant against infection of Toxoplasma gondii in mice

To characterize the immune response induced by SAG1 encoding plasmid combined with IL 2 gene adjuvant in mice and to assess the protective effect of this vaccination against toxoplasmosis Methods Mice were co injected intramuscularly with plasmid encoding Toxoplasma gondii SAG1 plus murine IL 2 expression vector at a dose of 100 μg Booster immunizations were employed 2 more times at 3 week interval As controls, mice were inoculated with PBS or empty plasmid pcDNA3 Humoral and cellular responses were assayed using ELISA for the determination of Ab, Ab isotype and IFN γ, as well as IL 4 To detect the integration and dissemination of DNA in the injected mice, PCR and in situ hybridization were performed All mice were then infected with highly virulent RH tachyzoites of Toxoplasma gondii intraperitoneally Results Significant increases in specific IgG levels were observed in mice after immunization three times with SAG1 expression plasmid With respect to the IgG isotype, co inoculation of IL 2 expression plasmid enhanced the level of IgG2a and the production of IFN γ Challenging mice by vaccinating with combined plasmids with RH tachyzoites resulted in prolonged survival Conclusion Humoral and cytokine responses elicited by SAG1 DNA immunization can be modulated by co inoculation with IL 2 expression plasmid The use of DNA vaccine in combination with an appropriate cytokine gene to prevent T gondii infection warrants further investigation

Eukaryotic expression of outer membrane protein Gpd from Treponema pallidum and preliminary studies on its immune response in rabbits

The Gpd gene was amplified from the genomic DNA of Treponema the appropriate site of pcDNA3.1 （ ＋ ） vector. The expression of pcDNA3. I was tested with Western blotting and technology of immunoeytochemisty. New munized with the eukaryotic expression recombinant pcDNA3, 1 （ ＋ ）-Gpd pallidum and cloned into （ ＋ ）-Gpd in Hel,a cells Zealand rabbits were imA fusion protein of C, pd with 4.1 kDa has been effectively expressed in HeLa cells, which were detected bv Western blotting and the immunocytochemistry techniques. The New Zealand rabbits were able to elicit the specific antibody after immunization with the nucleic acid vaccine. The antibody titer could reach as high as 1 ： 1024 after 2 weeks of the third injection; and the splenocytes proliferated evidently due to the Gpd protein stimulation. Both the antibody titer and the splenocytes proliferation were higher substantially than those of controls （ P 〈 0.01 ）. All above data will contribute to an experimental basis of further study of the biological function of Gpd protein as well as DNA vaccine for syphilis.

Construction of a recombinant plasmid harbouring the rhoptry protein 1 gene of Toxoplasma gondii and preliminary observations on DNA immunity

Objective To observe the immune responses elicited in BALB/c mice by a DNA vaccine. A gene encoding rhoptry protein 1 (ROP1) from Toxoplasma gondii (T. gondii) was cloned into vector pcDNA3. Methods Amplifyied gene fragments coding for ROP1 from the genomic DNA of T.gondii ZS2 were inserted into cloning vector, pUC18, and sub-cloned into pcDNA3. Mice were injected at a dosage of 100?μg recombinant plasmid DNA by intramuscular injection and boosted after 2 weeks. pcDNA3 and normal saline were used as control. 30, 50 and 70 days after the second immunization, NK cell activity, T lymphocyte proliferation and sub-clusters and serum IgG antibody were assayed.Results The specific gene fragment coding for ROP1 was amplified and a pcROP1 recombinant was constructed. At 30 days after immunization, the spleens of the mice were obviously enlarged evidently. NKC activity and the proliferation of spleen T lymphocytes seen on MTT assay were higher in pcROP1 group than in the controls. The number of CD4+ T cells exhibited no obvious increase compared with that of the control, but CD8+ T cells were obviously increased (P<0.05). At 90 days after vaccination, the titer of IgG antibody in the serum of vaccinated mice was positive (1∶100). Conclusion pcROP1 was constructed and it could elicit both cellular and humoral immune responses in immunized mice.

Induction of immune responses in mice by vaccination with Liposome-entrapped DNA complexes encoding Toxoplasma gondii SAG1 and ROP1 genes

Objective To evaluate the immune responses induced by experimental DNA construct encoding Toxoplasma gondii ( T.gondii ) surface antigen1 (SAG1) and rhoptry protein 1 (ROP1) in mice as a hybrid gene. Methods Truncated SAG1 and ROP1 DNA fragments were amplified using polymerase chain reaction (PCR) and inserted into pEGFP-N3 vector to construct recombinant plasmid pSAG1-ROP1. NIH3T3 mammalian cells were transiently transfected with the DNA construct. Female BALB/c mice were given three intramuscular injections of 10 μg plasmid DNA entrapped in liposome. Four weeks after the final booster injection,blood samples were collected and subjected to enzyme-linked immuno sorbent assay (ELISA) to investigate humoral and cell-mediated immune responses. Reversal transcript-polymerase chain reaction (RT-PCR) was used to evaluate the transcription of inoculated DNA-liposome complex in the injected site. Dot-blot hybridization was employed in order to detect whether or not the injected DNA was incorporated into the genomic DNA of the immunized mice. Results Green fluorescence was observed in pSAG1-ROP1-transfected cells. Western blot analysis showed antibody recognition of the expressed SAG1-ROP1 was between 58 kDa and 75 kDa. No expression was observed in blank control plasmid-transfected cells. The sera of immunized mice exhibited antibodies to T.gondii tachyzoites and primarily interferon-γ and interlukin-2. RT-PCR showed that the duration of transcribed inoculated liposome entrapped DNA in the injected muscular tissue was at least ten days post the first injection. Dot-blot hybridization revealed that the presence of foreign DNA in the splenocytes and peripheral blood leukocytes was transient and that no foreign DNA had inserted into the genomic DNA of mice immunized with pSAG1-ROP1. Conclusions Immunization with a liposome-encapsulated DNA construct encoding the T.gondii SAG1 and ROP1 can induce humoral and cell-mediated immune responses.

Immune responses against Schistosoma japonicum after vaccinating mice with a multivalent DNA vaccine encoding integrated membrane protein Sj23 and cytokine interleukin-12

Background The vaccination of mice with DNA encoding single candidate antigens has failed to induce significant protection against Schistosoma japonicum (S. japonicum) challenge infections In this study, we evaluated the feasibility of using a multivalent DNA vaccine which co expressed S japonicum integral membrane protein Sj23 and murine cytokine IL 12 to induce protective immune responses Methods The plasmid pVIVO2 IL12 Sj23, a eukaryotic expression vector expressing Sj23 and murine IL 12 simultaneously, was constructed, identified, and tested for expression in vitro Its ability to protect against S japonicum challenge infections was analyed according to worm reduction rate and egg reduction rate after vaccination of BALB/c mice The serum levels of specific IgG antibody were determined by enzyme linked immuno sorbent assay (ELISA) and Western blot analysis Using cultured spleen cells, IFN γ and IL 4 post stimulation were quantified by ELISA The phenotypes of splenocyte populations were analyzed by flow cytometry (FCM) Results The plasmid DNA pVIVO2 IL12 Sj23 was proven to express well in vitro by transient transfection of HEK 293 cells Immunization resulted in a worm reduction rate of 45 53% and egg reduction rate of 58 35% ELISA and Western blot analysis indicated that immunized mice generated specific IgG against Sj23 Spleen cells showed significant increases in IFN γ but decreases in IL 4 No significant differences in CD4 + and CD8 + subgroup ratios were observed after the challenges Conclusions The multivalent DNA vaccine pVIVO2 IL12 Sj23 is sufficient to elicit moderate but highly significant levels of protective immunity against challenge infections Cytokine IL 12, as a gene adjuvant, was able to enhance the Th1 responses and, hence, the protective immunity