Engineering superior DNA vaccines: MHC class I single chain trimers bypass antigen processing and enhance the immune response to low affinity antigens

It is commonly believed that delivery of antigen into the class I antigen presentation pathway is a limiting factor in the clinical translation of DNA vaccines. This is of particular concern in the context of cancer vaccine development as many immunodominant peptides derived from self tumor antigens are not processed and presented efficiently. To address this limitation, we have engineered completely assembled peptide/MHC class I complexes whereby all three components (class I heavy chain, β₂m, and peptide) are attached by flexible linkers and expressed as a single polypeptide (single chain trimers or SCT). In this study, we tested the efficacy of progressive generations of SCT DNA vaccines engineered to (1) enhance peptide binding, (2) enhance interaction with the CD8 coreceptor, and/or (3) activate CD4⁺ helper T cells. Disulfide trap SCT (dtSCT) have been engineered to improve peptide binding, with mutations designed to create a disulfide bond between the class I heavy chain and the peptide linker. dtSCT DNA vaccines dramatically enhance the immune response to model low affinity antigens as measured by ELISPOT analysis and tumor challenge. SCT engineered to enhance interaction with the CD8 coreceptor have a higher affinity for the TCR/CD8 complex, and are associated with more robust CD8⁺ T cell responses following vaccination. Finally, SCT constructs that coexpress a universal helper epitope PADRE, dramatically enhance CD8⁺ T cell responses. Taken together, our data demonstrate that dtSCT DNA vaccines coexpressing a universal CD4 epitope are highly effective in generating immune responses to poorly processed and presented cancer antigens.     

Specific humoral and cellular immune responses in cancer patients undergoing chronic immunization with a VEGF-based therapeutic vaccine

CIGB-247 is a cancer therapeutic vaccine, based on recombinant modified human vascular endothelial growth factor (VEGF) as antigen, in combination with the adjuvant VSSP, a bacterially-derived adjuvant. The vaccine have demonstrated efficacy in several murine malignancy models. These studies supported the rationale for a phase I clinical trial where safety, tolerance, and immunogenicity of CIGB-247 was studied in patients with advanced solid tumors at three antigen dose level. Surviving individuals of this clinical trial were eligible to receive off-trial voluntary re-immunizations. The present work is focus in the immunological follow up of these patients after approximately three years of immunizations, without additional oncological treatments. Long term vaccination was feasible and safe. Our results indicated that after sustained vaccination most of the patients conserved their seroconversion status. The specific anti-VEGF IgG titer diminished, but in all the cases keeps values up from the pre-vaccination levels. Continued vaccination was also important to produce a gradual shift in the anti-VEGF IgG response from IgG1 to Ig4. Outstanding, our results indicated that long-term off-trial vaccination could be associated with the maintaining of one reserve of antibodies able to interfere with the VEGF/Receptor interaction and the production of IFNγ secretion in CD8⁺ cells. The results derived from the study of this series of patients suggest that long term therapeutic vaccination is a feasible strategy, and highlight the importance of continuing the clinical development program of this novel cancer therapeutic vaccine candidate. We also highlight the future clinical applications of CIGB-247 in cancer and explain knowledge gaps that future studies may address.Registration number and name of trial registry: RPCEC00000102. Cuban Public Clinical Trial Registry (WHO accepted Primary Registry). Available from: http://registroclinico.sld.cu/.     

Depletion of regulatory T cells by anti-ICOS antibody enhances anti-tumor immunity of tumor cell vaccine in prostate cancer

ICOS⁺Treg cells exert important immunosuppressive effects in tumor immunity. We adopt a combination approach of ICOS⁺Treg cells depletion with tumor cell vaccine to evaluate anti-tumor immunity in mouse prostate cancer model. Streptavidin (SA)-mGM-CSF surface-modified RM-1 cells were prepared as the vaccine and the mouse subcutaneous prostate tumor model was used to evaluate the immunity. Tumor growth, flow cytometry, immunohistochemistry, immunofluorescence and enzyme linked immunosorbent assay (ELISA) were performed to evaluate the therapeutic effects. Our results demonstrated that SA-mGM-CSF vaccine was prepared successfully and tumor growth was inhibited. The tumor size in the combination group was much smaller than that in the vaccine with IgG mAb group. The portions of dendritic cells, CD8⁺ and CD4⁺T cells in the mice blood and tumor tissues were increased after treatment with vaccine. There were more immune-suppressing Tregs infiltrated into tumor after treatment with tumor cell vaccine, and ICOS blocking could deplete the infiltrated Tregs, and T lymphocytes increased more dramatically in the combination therapy group. The concentrations of interferon-γ were increased in all vaccine group, the concentrations of Interleukin-10 and Interleukin-4 were much lower in the combination group. Our study demonstrated that ICOS blocking could deplete the tumor-infiltrated ICOS⁺Treg cells. Combining GM-CSF surface-modified RM-1 cell vaccine with Anti-ICOS antibody could induce better antitumor immunity than a vaccine alone.     

MUC1 and survivin combination tumor gene vaccine generates specific immune responses and anti-tumor effects in a murine melanoma model

MUC1 and survivin are ideal tumor antigens. Although many cancer vaccines targeting survivin or MUC1 have entered clinical trials, no vaccine combining MUC1 and survivin have been reported. Due to tumor heterogeneity, vaccines containing a combination of antigens may have improved efficacy and coverage of a broader spectrum of cancer targets. Here, cellular responses and anti-tumor activities induced by a combination of DNA vaccine targeting MUC1 and survivin (MS) were evaluated. Results showed that CTL activity and inhibition of tumor growth were obviously enhanced in mice immunized with the combined vaccine in a protection assay. However, in order to enhance the therapeutic effect in the treatment assay, a recombinant adenovirus (rAd) vaccine expressing MUC1 and survivin (Ad-MS) was used as a booster following the DNA vaccine prime. Meanwhile, IL-2 promoting T cell proliferation was used as an immunoadjuvant for the DNA vaccine. Results showed that the CTL activity response to the DNA vaccine was enhanced nearly 200% when boosted by the rAd vaccine and was further enhanced by nearly 60% when combined with the IL-2 adjuvant. Therefore, DNA prime combined with rAd boost and IL-2 (MS/IL2/Ad-MS) adjuvant was considered as the best strategy and further evaluated. Multiple cytokines promoting cellular immune responses were shown to be greatly enhanced in mice immunized with MS/IL2/Ad-MS. Moreover, in the treatment assay, the tumor inhibition rate of MS/IL2/Ad-MS reached up to 50.1%, which may be attributed to the enhancement of immune responses and reduction of immunosuppressive factors in tumor-bearing mice. These results suggested that immunization with the combination vaccine targeting MUC1 and survivin using a DNA prime–rAd boost strategy along with IL-2 adjuvant may be an effective method for breaking through immune tolerance to tumors expressing these antigens with potential therapeutic benefits in melanoma cancer.     

Assessment of SARS-CoV-2 specific CD4(+) and CD8 (+) T cell responses using MHC class I and II tetramers

The success of SARS-CoV-2 (CoV-2) vaccines is measured by their ability to mount immune memory responses that are long-lasting. To achieve this goal, it is important to identify surrogates of immune protection, namely, CoV-2 MHC Class I and II immunodominant pieces/epitopes and methodologies to measure them. Here, we present results of flow cytometry-based MHC Class I and II QuickSwitch^TM platforms for assessing SARS-CoV-2 peptide binding affinities to various human alleles as well as the H-2 Kb mouse allele. Multiple SARS-CoV-2 potential MHC binders were screened and validated by QuickSwitch testing. The screen included 31 MHC Class I and 19 MHC Class II peptides predicted to be good binders by the IEDB web resource provided by NIAID. While several predicted peptides with acceptable theoretical Kd showed poor MHC occupancies, fourteen MHC class II and three MHC class I peptides showed promiscuity in that they bind to multiple MHC molecule types. In addition to providing important data towards the study of the SARS-CoV-2 virus and its presented antigenic epitopes, the peptides identified in this study can be used in the QuickSwitch platform to generate MHC tetramers. With those tetramers, scientists can assess CD4 + and CD8 + immune responses to these different MHC/peptide complexes.     

Inhibition of tumor angiogenesis in lung cancer by T4 phage surface displaying mVEGFR2 vaccine

Vascular endothelial growth factor (VEGF) has been known as a potential vasculogenic and angiogenic factor and its receptor (VEGFR2) is a major receptor to response to the angiogenic activity of VEGF. The technique that to break the immune tolerance of “self-antigens” associated with angiogenesis is an attractive approach for cancer therapy with T4 phage display system. In this experiment, mouse VEGFR2 was constructed on T4 phage nanometer-particle surface as a recombinant vaccine. T4-mVEGFR2 recombinant vaccine was identified by PCR and western blot assay. Immunotherapy with T4-mVEGFR2 was confirmed by protective immunity against Lewis lung carcinoma (LLC) in mice. The antibody against mVEGFR2 was detected by ELISPOT, ELISA and Dot ELISA. The inhibitive effects against angiogenesis were studied using CD31 and CD105 via histological analysis. VEGF-mediated endothelial cells proliferation and tube formation were inhibited in vitro by immunoglobulin induced by T4-mVEGFR2. The antitumor activity was substantiated from the adoptive transfer of the purified immunoglobulin. Antitumor activity and autoantibody production of mVEGFR2 could be neutralized by the depletion of CD4+T lymphocytes. These studies strongly suggest that T4-mVEGFR2 recombinant vaccine might be a promising antitumor approach.     

CA125及HE4蛋白在卵巢癌组织及血清中的表达及其临床意义

目的:探讨HE4(人类附睾蛋白4)、CA125在卵巢癌组织及血清中的表达及其临床意义.方法:选择2010年12月～2012年7月海南医学院附属医院收治的卵巢癌60例、良性卵巢肿瘤60例和正常卵巢组织35例,采用免疫组化法检测不同卵巢组织中的HE4和CA125的蛋白表达情况;应用酶联免疫吸附法检测术前、术后血清HE4及CA125的浓度,综合分析各参数之间的临床相关性.结果:HE4及CA125的蛋白表达卵巢癌组高于良性卵巢肿瘤组和正常卵巢组(P＜0.000 1);HE4及CA125的血清水平卵巢癌组均高于良性卵巢肿瘤组和正常卵巢组(P＜0.000 1).血清HE4和CA125联合检测对卵巢癌的诊断敏感性均高于两者单项检测,为90％,特异性高于CA125单项检测,低于HE4单项检测.卵巢癌组术前血清CA125及HE4的表达水平明显高于术后,差异有统计学意义(P ＜0.000 1).结论:HE4、CA125在卵巢癌组织中的蛋白表达与血清水平联合检测是卵巢癌早期诊断、疗效评价及预后评估的敏感综合指标,可为临床个性化防治提供科学实验依据.     

血清人附睾蛋白4和癌抗原125在预测卵巢癌发病风险中的价值

目的 探讨血清人附睾蛋白4(HE4)和癌抗原125(CA125)单独和联合使用在预测卵巢癌发病风险中的价值.方法 75例卵巢癌患者(卵巢癌组)分别应用酶联免疫吸附法和电化学发光法检测血清HE4和CA125水平,并与132例良性卵巢疾病患者(良性卵巢疾病组)以及30例健康女性(对照组)进行比较,并计算两者单独和联合预测卵巢癌发病风险的特异度和灵敏度,绘制受试者工作特征(ROC)曲线.结果 卵巢癌组血清HE4和CA125水平明显高于良性卵巢疾病组和对照组(P＜0.05).两者都可以预测卵巢癌的发生,HE4在预测卵巢癌发病风险方面较CA125具有较明显优势(ROC曲线下面积分别为0.966和0.905).HE4在浆液性腺癌中阳性表达率最高,为100.0%(42/42).HE4阳性诊断卵巢癌的灵敏度为86.7%(65/75),HE4和/或CA125阳性诊断卵巢癌的灵敏度为93.3%(70/75),两者比较差异无统计学意义(P＞0.05).结论 血清HE4比CA125能够更好地预测卵巢癌的发生,HE4可以作为预测卵巢癌发病风险的指标,HE4联合CA125不能够明显提高卵巢癌的检出率.

Abstract：

Objective To explore the value of serum human epididymis protein 4 (HE4) and cancer antigen 125 (CA125) in the prediction of ovarian cancer onset risk. Methods Petected the expression of HE4 by enzyme linked immunosorbent assay (ELISA) and CA 125 by electrochemiluminescence (ECL) in serum of 75 samples of ovarian cancer (ovarian cancer group),132 samples of ovarian benign disease (ovarian benign disease group) and 30 samples of healthy women (control group),calculated the sensitivity and specificity of HE4 and CA125 in the prediction of ovarian cancer onset risk,and drew receiver operating characteristic (ROC) curve. Results The expression of serum HE4 and CA125 were both higher in ovarian cancer group than those in ovarian benign disease group and control group (P ＜ 0.05 ). HE4 was a better index in the prediction of ovarian cancer onset risk than CA 125 (ROC area under curve was 0.966 and 0.905 respectively). The expression of HE4 was various in different pathological types,serous ovarian adenocarcinoma had the highest expression of HE4 (100.0% ,42/42). The sensitivity of diagnosing ovarian cancer by HE4 was 86.7%(65/75), 93.3%(70/75) by HE4 combining and/or CA125,there was no significant difference(P＞0.05).Conclusion Serum HE4 could be a good index of predicting the onset risk of ovarian cancer,the combination of HE4 and/or CA125 could not increase the sensitivity of prediction of ovarian cancer significantly.     

XPO4、MAP2K4及ZIC1在子宫内膜癌组织表达及其对子宫内膜癌患者预后评估价值

目的探讨输出蛋白(XPO)4、丝裂原活化蛋白激酶(MAP2K)4及小脑锌指结构蛋白(ZIC)1在子宫内膜癌(EC)组织中的表达及其对EC患者的预后评估价值。 方法选择2017年6月至2018年6月,无锡市妇幼保健院、复旦大学附属中山医院青浦分院收治的147例EC患者为研究对象。采用免疫组化染色法检测患者EC组织及癌旁正常组织XPO4、MAP2K4、ZIC1表达水平。采用χ²检验,对患者EC组织及癌旁正常组织XPO4、MAP2K4、ZIC1阳性率,以及不同临床病理特征患者EC组织XPO4、MAP2K4、ZIC1阳性率进行比较。采用Two-stage检验,对EC组织XPO4、MAP2K4、ZIC1呈阳性与呈阴性患者的3年总体生存(OS)率进行比较。本研究遵循的程序经过无锡市妇幼保健院、复旦大学附属中山医院青浦分院伦理委员会批准(审批文号：20160803、20160502)。 结果①本组患者EC组织XPO4、MAP2K4阳性率分别为34.7%(51/147)、38.1%(56/147),显著低于癌旁正常组织的66.7%(98/147)与72.8%(107/147),并且差异均有统计学意义(χ²＝30.060、35.812,P<0.001)。本组患者EC组织ZIC1阳性率为72.1%(106/147),显著高于癌旁正常组织的40.1%(59/147),并且差异亦有统计学意义(χ²＝30.512、P<0.001)。②不同年龄、EC直径及绝经与否患者的EC组织XPO4、MAP2K4、ZIC1阳性率比较,差异均无统计学意义(P>0.05)。不同国际妇产科联盟(FIGO)临床分期、组织病理学分级、子宫肌层侵犯程度及淋巴结转移与否患者EC组织XPO4、MAP2K4、ZIC1阳性率分别比较,差异均有统计学意义(P<0.05)。③本组EC组织XPO4、MAP2K4呈阴性患者的3年OS率分别为42.7%、38.5%,均显著低于XPO4、MAP2K4呈阳性者的68.6%、71.4%,并且差异均有统计学意义(χ²＝8.960、P＝0.003,χ²＝15.077、P<0.001)。EC组织ZIC1呈阳性患者3年OS率为35.8%,显著低于ZIC1呈阴性者的68.3%,并且差异亦有统计学意义(χ²＝12.579、P<0.001)。 结论在EC组织中,XPO4、MAP2K4呈低表达,而ZIC1则呈高表达。XPO4、MAP2K4、ZIC1表达水平与患者组织病理学分级、淋巴结转移、FIGO临床分期、子宫肌层侵犯及3年OS率显著相关,其在评估EC患者发病、进展及预后中具有一定价值。     

Delayed-type hypersensitivity and hepatitis B vaccine responses,in vivo markers of cellular and humoral immune function,and the risk of AIDS or death

BackgroundDelayed-type hypersensitivity (DTH) test responsiveness is associated with HIV disease progression; however it is unknown whether other immune markers, such as hepatitis B virus (HBV) vaccine seroresponse, also predict HIV outcomes.MethodsEligible participants received HBV vaccine after HIV diagnosis, had non-anergic DTH testing at the time of last HBV vaccination, and available post-vaccine HBV antibody responses. The risk of progression to AIDS or death from the time of last HBV vaccination was evaluated.ResultsOf 369 eligible participants with non-anergic DTH responses, 148 (40%) were HBV vaccine responders. In a multivariate model adjusted for age, CD4 count, viral load, and number of vaccinations, HBV vaccine non-responders had an increased risk of progression to AIDS or death (HR 1.81; 95% CI, 1.03–3.19).ConclusionsHBV vaccine seroresponses were independent of DTH responses which suggest that non-response to HBV vaccine is not solely due to cell-mediated immune dysfunction in HIV-infected persons.     

Universal influenza vaccine based on conserved antigens provides long-term durability of immune responses and durable broad protection against diverse challenge virus strains in mice

Current influenza vaccines rely on inducing antibody responses to the rapidly evolving hemagglutinin (HA) and neuraminidase (NA) proteins, and thus need to be strain-matched. However, predictions of strains that will circulate are imperfect, and manufacturing of new vaccines based on them takes months. As an alternative, universal influenza vaccines target highly conserved antigens. In proof of concept studies of universal vaccine candidates in animal models challenge is generally conducted only a short time after vaccination, but protective immunity lasting far longer is important for the intended public health impact. We address the challenge of providing long-term protection. We demonstrate here broad, powerful, and long-lasting immune protection for a promising universal vaccine candidate. A single intranasal dose of recombinant adenoviruses (rAd) expressing influenza A nucleoprotein (A/NP) and matrix 2 (M2) was used. Extending our previous studies of this type of vaccine, we show that antibody and T-cell responses persist for over a year without boosting, and that protection against challenge persists a year after vaccination and remains broad, covering both group 1 and 2 influenza A viruses. In addition, we extend the work to influenza B. Immunization with influenza B nucleoprotein (B/NP)-rAd also gives immune responses that last a year without boosting and protect against challenge with influenza B viruses of mismatched HA lineages. Despite host immunity to adenoviral antigens, effective readministration is possible a year after primary vaccination, as shown by successful immunization to a transgene product the animals had not seen before. Protection against challenge with divergent and highly pathogenic A/H7N9 virus was weaker but was enhanced by a second dose of vaccine. Thus, this mucosal vaccination to conserved influenza antigens confers very long-lasting immune protection in animals against a broad range of influenza A and B viruses.     

Generation of CTL responses against pancreatic cancer in vitro using dendritic cells co-transfected with MUC4 and survivin RNA

Pancreatic cancer (PC) is one of the most devastating human malignancies without effective therapies. Tumor vaccine based on RNA-transfected dendritic cells (DCs) has emerged as an alternative therapeutic approach for a variety of human cancers including advanced PC. In the present study we compared the cytotoxic T lymphocyte (CTL) responses against PC cells in vitro, which were induced by DCs co-transfected with two mRNAs of tumor associated-antigens (TAA) MUC4 and survivin, versus DCs transfected with a single mRNA encoding either MUC4 or survivin. DCs co-transfected with two TAA mRNAs were found to induce stronger CTL responses against PC target cells in vitro, compared with the DCs transfected with a single mRNA. Moreover, the antigen-specific CTL responses were MHC class I-restricted. These results provide an experimental foundation for further clinical investigations of DC vaccines encoding multiple TAA epitopes for metastatic PC.     

Production of NY-ESO-1 peptide/DRB1*08:03 tetramers and ex vivo detection of CD4 T-cell responses in vaccinated cancer patients

We established CD4 T-cell clones, Mz-1B7, and Ue-21, which recognized the NY-ESO-1 121–138 peptide from peripheral blood mononuclear cells (PBMCs) of an esophageal cancer patient, E-2, immunized with an NY-ESO-1 protein and determined the NY-ESO-1 minimal epitopes. Minimal peptides recognized by Mz-1B7 and Ue-21 were NY-ESO-1 125–134 and 124–134, respectively, both in restriction to DRB1*08:03. Using a longer peptide, 122–135, and five other related peptides, including either of the minimal epitopes recognized by the CD4 T-cell clones, we investigated the free peptide/DR recognition on autologous EBV-B cells as APC and peptide/DR tetramer binding. The results showed a discrepancy between them. The tetramers with several peptides recognized by either Mz-1B7 or the Ue-21 CD4 T-cell clone did not bind to the respective clone. On the other hand, unexpected binding of the tetramer with the peptide not recognized by CD4 T-cells was observed. The clone Mz-1B7 did not recognize the free peptide 122–135 on APC, but the peptide 122–135/DRB1*08:03 tetramer bound to the TCR on those cells. The failure of tetramer production and the unexpected tetramer binding could be due to a subtly modified structure of the peptide/DR tetramer from the structure of the free peptide/DR molecule. We also demonstrated that the NY-ESO-1 123–135/DRB1*08:03 tetramer detected ex vivo CD4 T-cell responses in PBMCs from patients after NY-ESO-1 vaccination in immunomonitoring.     

MyD88 signal is required for more efficient induction of Ag-specific adaptive immune responses and antitumor resistance in a human papillomavirus E7 DNA vaccine model

The function of MyD88 signals for induction of adaptive immunity is still controversial. Here we investigate using a human papillomavirus (HPV) 16 E7 DNA vaccine on MyD88 knock out mouse model whether MyD88 signals are required for induction of Ag-specific antibody and cellular responses, as well as antitumor resistance. When injected intramuscularly with E7 DNA vaccines, MyD88 deficient mice displayed antitumor protective responses to tumor cell challenges while having far lower responses than wild type mice. A similar finding was observed in antitumor therapeutic models by intramuscular-electroporation of E7 DNA vaccines. E7 DNA vaccines induced Ag-specific humoral and CD8+ CTL responses in MyD88 deficient mice. However, the levels were much less than those of wild type mice. These data suggest that the immune stimulatory sequence of E7 DNA vaccines and its signaling through MyD88 are not absolutely essential for induction of adaptive immune responses. However, MyD88 deficient mice co-delivered with MyD88 cDNA plus E7 DNA vaccines showed a recovery of Ag-specific IgG and CTL responses, and antitumor immunity to the levels of wild type mice, highlighting the importance of MyD88 signals for augmenting an adaptive immune response. Thus, these data clearly show that MyD88 signals are required only for more efficient induction of Ag-specific humoral and antitumor CD8+ CTL responses in this model.     

真核细胞转录起始因子4E表达与乳腺癌血管生成及淋巴结转移的关系

目的　探讨乳腺癌中真核细胞转录起始因子 4E(eIF - 4E)基因表达与血管生成及转移的关系。方法　以Western-blot法检测 5 7例手术切除乳癌标本匀浆中eIF - 4E基因表达 ,免疫组化法检测乳癌组织微血管密度(MVD)表达 ,结合临床资料进行分析。结果　 34例淋巴结阳性组织中eIF - 4E水平上调 10 .11± 0 .30 ,MVD升高32 .2 8± 7.4 2 ,与淋巴结转移阴性组 (5 .94± 0 .13,2 4 .6 7± 8.12 )比较差异显著 (t=4 .37、2 .5 1,P <0 .0 5 )。相关分析表明 ,eIF - 4E表达与乳癌血管生成及淋巴结转移正相关。结论　eIF - 4E基因通过调控乳癌血管生成参与淋巴结转移。eIF - 4E表达可作为乳癌淋巴结转移的判定因素     

Evidence for induction of humoral and cytotoxic immune responses against devil facial tumor disease cells in Tasmanian devils (Sarcophilus harrisii) immunized with killed cell preparations

Tasmanian devils (Sarcophilus harrisii) risk extinction from a contagious cancer, devil facial tumour disease (DFTD) in which the infectious agent is the tumor cell itself. Because devils are unable to produce an immune response against the tumor cells no devil has survived ‘infection’. To promote an immune response we immunized healthy devils with killed DFTD tumor cells in the presence of adjuvants. Immune responses, including cytotoxicity and antibody production, were detected in five of the six devils. The incorporation of adjuvants that act via toll like receptors may provide additional signals to break ‘immunological ignorance’. One of these devils was protected against a challenge with viable DFTD cells. This was a short-term protection as re-challenge one year later resulted in tumor growth. These results suggest that Tasmanian devils can generate immune responses against DFTD cells. With further optimization of immune stimulation it should be possible to protect Tasmanian devils against DFTD with an injectable vaccine.     

Further analysis of protection induced by the MIC3 DNA vaccine against T. gondii: CD4 and CD8 T cells are the major effectors of the MIC3 DNA vaccine-induced protection,both Lectin-like and EGF-like domains of MIC3 conferred protection

The present study was conducted mainly to evaluate the contribution of the cellular and the humoral responses in protection conferred by the MIC3 DNA vaccine (pMIC3i) that was proved as a potent vaccine against toxoplasmosis. We performed the adoptive transfer of CD4⁺ and CD8⁺ T lymphocytes from pMIC3i immunized mice to naive ones and the role of humoral immunity was evaluated by in vitro invasion assays. We also constructed plasmids encoding the EGF-like domains and the Lectin-like domain of MIC3, to define which domains of MIC3 are involved in the protection. Furthermore, the adjuvant effect of the GM-CSF-expressing vector (granulocyte-macrophage colony-stimulating factor) required the precise temporal and spatial codelivery of GM-CSF with antigen, thus, we constructed a bicistronic plasmid expressing MIC3 and GM-CSF. In conclusion, the protection induced by pMIC3i was mainly mediated by CD4⁺ and CD8⁺ T lymphocytes and both EGF and Lectin domains of MIC3 conferred protection. Furthermore, the codelivery of GM-CSF by a bicistronic plasmid appeared to be a most effective way for enhancing the adjuvant properties of GM-CSF.     

CD4~+ CD25~+调节性T细胞在上皮性卵巢癌患者外周血中的表达及临床意义

目的:研究上皮性卵巢癌患者外周血中CD4+ CD25+调节性T细胞数量及其相关基因Foxp3 mRNA的表达特点,探讨CD4+CD25+调节性T细胞在诱导肿瘤免疫耐受中的作用。方法:采用流式细胞术检测卵巢浆液性囊腺癌和粘液性囊腺癌患者外周血中CD4+ CD25+调节性T细胞数量变化;采用半定量RT-PCR方法检测卵巢浆液性囊腺癌和粘液性囊腺癌患者外周血PBMC中Foxp3 mRNA表达水平。结果:卵巢浆液性囊腺癌和粘液性囊腺癌患者外周血中CD4+CD25+调节性T细胞数量均明显升高(P<0.05);Foxp3 mRNA在卵巢浆液性囊腺癌和粘液性囊腺癌患者外周血PBMC中表达明显增高(P<0.05)。结论:CD4+ CD25+调节性T细胞可能在卵巢癌免疫耐受中发挥重要作用。     

A multi-trimeric fusion of CD40L and gp100 tumor antigen activates dendritic cells and enhances survival in a B16-F10 melanoma DNA vaccine model

Vaccination with tumor-associated antigens can induce cancer-specific CD8+ T cells. A recent improvement has been the targeting of antigen to dendritic cells (DC) using antibodies that bind DC surface molecules. This study explored the use of multi-trimers of CD40L to target the gp100 melanoma tumor antigen to DC. The spontaneously-multimerizing gene Surfactant Protein D (SPD) was used to fuse gp100 tumor antigen and CD40L, creating the recombinant protein SPD-gp100-CD40L. This “third generation” DC-targeting vaccine was designed to both target antigen to DC and optimally activate dendritic cells by aggregating CD40 trimers on the DC membrane surface. SPD-gp100-CD40L expressed as a 110 kDa protein. Analytical light scattering analysis gave elution data corresponding to 4-trimer and multi-trimer SPD-gp100-CD40L oligomers. The protein was biologically active on dendritic cells and induced CD40-mediated NF-κB signaling. DNA vaccination with SPD-gp100-CD40L plasmid, together with plasmids encoding IL-12p70 and GM-CSF, significantly enhanced survival and inhibited tumor growth in a B16-F10 melanoma model. Expression of gp100 and SPD-CD40L as separate molecules did not enhance survival, highlighting the requirement to encode gp100 within SPD-CD40L for optimal vaccine activity. These data support a model where DNA vaccination with SPD-gp100-CD40L targets gp100 to DC in situ, induces activation of these DC, and generates a protective anti-tumor response when given in combination with IL-12p70 and GM-CSF plasmids.