Identification of novel HLA-A*0201-restricted cytotoxic T lymphocyte epitopes from Zinc Transporter 8

Numerous evidences demonstrated that type 1 diabetes (T1D) is due to a loss of immune tolerance to islet antigens, and CD8⁺ T cells play an important role in the development of T1D. Zinc Transporter 8 (ZnT8) has emerged in recent years as a target of disease-associated autoreactive T cells in human T1D. However, ZnT8-associated CTL specific-peptides have not been identified. In this study, we predicted and identified HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) epitopes derived from ZnT8, and utilized it to immunize HLA-A2.1/Kb transgenic (Tg) mice. The results demonstrated that peptides of ZnT8 containing residues 107–115, 115–123 and 145–153 could elicit specific CTLs in vitro, and induce diabetes in mice. The results suggest that these specific peptides are novel HLA-A*0201-restricted CTL epitopes, and could have therapeutic potential in preventing of T1D disease.     

Hepatitis B virus (HBV)-derived DRB1*0101-restricted CD4 T-cell epitopes help in the development of HBV-specific CD8+ T cells in vivo

We previously identified two HLA-DRB1*0101-restricted epitopes in hepatitis B virus (HBV) X protein (HBx) and in HBV envelope proteins (preS2). To evaluated their help in the development of CD8+ T-cell responses, mice transgenic for human class I and class II HLA molecules were immunized with HBV-T helper constructs. The preS2 epitope favored a well-balanced response with CD4+ and CD8+ T cells producing IFN-γ, IL-2 and TNF-α. The response was focused on CD8+ T cells with the HBx epitope. Fine characterization of helper activities may meet clinical needs in terms of enhancing the potency of preventive or therapeutic polyepitope vaccines.     

Glutamic acid decarboxylase epitope protects against autoimmune diabetes through activation of Th2 immune response and induction of possible regulatory mechanism

Oral tolerance mediated by autoantigens has been applied successfully as a potential therapeutic strategy for preventing and treating autoimmune diseases. We previously showed cholera toxin B subunit (CTB) is an efficient mucosal carrier molecule for induction of systemic T cell tolerance to linked insulin antigens. In this study, we used an oral antigen consisting of a fusion protein composed of CTB and triple copies of glutamic acid decarboxylase 65 (GAD65) peptides 531–545 (3p531) to test its in vivo effect and investigate the mechanism of immune tolerance. Non-obese diabetic mice fed microgram quantities of the CTB-3p531 fusion protein showed a prominent reduction in pancreatic islet inflammation and a delay in the development of diabetes. Increased anti-GAD65 IgG1, serum IgA and unchanged IgG2a antibodies titers; together with an increase of IL-4, IL-10 production and a decrease of IFN-γ production suggested possible activation of GAD65-specific Th2 immune responses. Adoptive transfer of splenocytes indicated oral administration of CTB-3p531 fusion protein generated potent regulatory cells that can suppress diabetogenic T cells. This study demonstrates the CTB-3p531 fusion protein protects against autoimmune diabetes by generation of regulatory T cells and induction of immunological tolerance.     

Altered decamer and nonamer from an HLA-A0201-restricted epitope of Survivin differentially stimulate T-cell responses in different individuals

Survivin is a universal tumor antigen that is being currently targeted in vaccine approaches against cancer. Our study here examined the immunogenicity of a novel variant of an HLA-A0201-binding decamer peptide from region 95 to 104 of Survivin (ELMLGEFLKL) with a T → M modification at position 3 in the peptide. We found that this new modified 10-mer peptide had enhanced HLA-A0201 binding and induced a stronger T-cell response over its wild type counterpart peptide (ELTLGEFLKL) in select HLA-A0201⁺ normal donors. In addition, when compared to the previously characterized altered 96-104 peptide (LMLGEFLKL) from the same region of Survivin currently used in vaccine trials, we found that both peptides had similar immunogenicity, but donor T cells preferentially reacted strongly to either one or the other, but not strongly to both. These results suggest that these two closely related Survivin peptides yield distinct T-cell responses and that most individuals dominantly respond to one or the other altered peptide. We also found a novel association between positive reactivity to the new altered decamer Survivin peptide in some individuals and their expression of the HLA-C0701 allele along with HLA-A0201. Thus, vaccinating with both the 10-mer and 9-mer peptides would be required to immunize a maximum number of individuals in the HLA-A0201⁺ population and could lead to more consistent T-cell responses against this region of Survivin.     

Novel HLA-A2-restricted human metapneumovirus epitopes reduce viral titers in mice and are recognized by human T cells

Human metapneumovirus (HMPV) is a major cause of morbidity and mortality from acute lower respiratory tract illness, with most individuals seropositive by age five. Despite the presence of neutralizing antibodies, secondary infections are common and can be severe in young, elderly, and immunocompromised persons. Preclinical vaccine studies for HMPV have suggested a need for a balanced antibody and T cell immune response to enhance protection and avoid lung immunopathology. We infected transgenic mice expressing human HLA-A*0201 with HMPV and used ELISPOT to screen overlapping and predicted epitope peptides. We identified six novel HLA-A2 restricted CD8⁺ T cell (T_CD8) epitopes, with M_39–47 (M39) immunodominant. Tetramer staining detected M39-specific T_CD8 in lungs and spleen of HMPV-immune mice. Immunization with adjuvant-formulated M39 peptide reduced lung virus titers upon challenge. Finally, we show that T_CD8 from HLA-A*0201 positive humans recognize M39 by IFNγ ELISPOT and tetramer staining. These results will facilitate HMPV vaccine development and human studies.     

CD8 T cell response in HLA-A*0201 transgenic mice is elicited by epitopes from SARS-CoV S protein

Cytotoxic CD8⁺ T lymphocytes (CTLs) play an important role in antiviral immunity. Several human HLA-A*0201 restricted CTL epitopes of severe acute respiratory syndrome (SARS) spike (S) protein have been identified in HLA-A*0201 transgenic (Tg) mice, but the mechanisms and properties of immune responses are still not well understood. In this study, HLA-A*0201 Tg mice were primed intramuscularly with SARS S DNA and boosted subcutaneously with HLA-A*0201 restricted peptides. The lymphocytes from draining lymph nodes, spleens and lungs were stimulated with the cognate peptides. Three different methods (ELISA, ELISPOT and FACS) were used to evaluate the immune responses during short and long periods of time after immunization. Results showed that peptide-specific CD8⁺ T cells secreted IFN-γ, TNF-α and IL-2 and expressed CD107a/b on cell surface. IFN-γ⁺CD8⁺ T cells and CD107a/b⁺CD8⁺ T cells distributed throughout the lymphoid and non-lymphoid tissues, but the frequency of peptide-specific CD8⁺ T cells was higher in lungs than in spleens and lymph nodes. The phenotype of the CD8⁺ T cells was characterized based on the expression of IFN-γ. Most of the HLA-A*0201 restricted peptide-specific CD8⁺ T cells represented a memory subset with CD45RB^high and CD62L^low. Taken together, these data demonstrate that immunization with SARS S DNA and HLA-A*0201 restricted peptides can elicit antigen-specific CD8⁺ T cell immune responses which may have a significant implication in the long-term protection. We provide novel information in cellular immune responses of SARS S antigen-specific CD8⁺ T cells, which are important in the development of vaccine against SARS-CoV infection.     

Construction of a novel vaccine by conjugating a B-cell epitope of DPP4 to peptide IA2(5)-P2-1 to significantly control type 1 diabetes in NOD mice

Type 1 diabetes is a chronic organ-specific autoimmune disease in which selective destruction of insulin-producing β cells leads to impaired glucose metabolism and its attendant complications. IA2(5)P2-1, a potent immunogenic carrier which designed by our laboratory, can induce high titer specific antibodies when carry a B cell epitope, such as B cell epitopes of DPP4, xanthine oxidase, and Urate transporter protein. In this report, we describe a novel multi-epitope vaccine composing a peptide of DPP4, an anti-diabetic B epitope of Insulinoma antigen-2(IA-2) and a Th2 epitope (P2:IPALDSLTPANED) of P277 peptide in human heat shock protein 60 (HSP60). Immunization with the multi-epitope vaccine in non-obese diabetic (NOD) mice successfully induced specific anti-DPP4 antibody, inhibited plasma DPP4 activity, and increased serum GLP-1 level. Moreover, this antibody titer was correlated with the dose of immunization (20μg, 100μg). Inoculation of this vaccine in NOD mice significantly control blood glucose level, improved glucose excursion and increased insulin level in vivo. Consistent with a lower diabetic and insulitis incidence, a induced splenic T cells proliferation and tolerance were observed. IFN-γ secretion reduced and IL-10 increased significantly in the D41-IA2(5)-P2-1 treated mice compared to P277 and control group due to the potential immunomodulatory effect of the epitope in the vaccine. Immunohistochemical analysis and cytometry showed a rebalance of Th1/Th2 in NOD mice. Our results demonstrate that this multi-epitope vaccine may serve as a promising therapeutic approach for type 1 diabetes.     

Contributions of neutrophils to the adaptive immune response in autoimmune disease

Neutrophils are granulocytic cytotoxic leukocytes of the innate immune system that activate during acute inflammation. Neutrophils can also persist beyond the acute phase of inflammation to impact the adaptive immune response during chronic inflammation. In the context of the autoimmune disease, neutrophils modulating T and B cell functions by producing cytokines and chemokines, forming neutrophil extracellular traps, and acting as or priming antigen presentation cells. Thus, neutrophils are actively involved in chronic inflammation and tissue damage in autoimmune disease. Using rheumatoid arthritis as an example, this review focuses on functions of neutrophils in adaptive immunity and the therapeutic potential of these cells in the treatment of autoimmune disease and chronic inflammation.     

2型糖尿病的全基因组关联研究进展

2型糖尿病是环境和遗传因素共同作用的结果,遗传因素的研究在2006年以前主要是基于连锁分析和候选基因策略,近年来全基因组关联研究的出现使2型糖尿病遗传易感性的研究进入了一个崭新的阶段,发现了很多新的易感基因或区域,为其病因学研究及防治提供新的思路.     

Vaccine generated immunity targets an HPV16 E7 HLA-A2.1-restricted CD8(+) T cell epitope relocated to an early gene or a late gene of the cottontail rabbit papillomavirus (CRPV) genome in HLA-A2.1 transgenic rabbits

The newly established HLA-A2.1 transgenic rabbit model has proven useful for testing the immunogenicity of well known and computer-predicted A2-restricted epitopes. In the current study we compared the protective immunity induced to a preferred HPV16 E7 A2-restricted epitope that has been relocated to positions within the CRPV E7 gene and the CRPV L2 gene. Epitope expression from both the E7 protein and the L2 protein resulted in increased protection against viral DNA challenge of the HLA-A2.1 transgenic rabbits as compared to control-vaccinated rabbit groups. These data indicate that proteins expressed at both early and late time points during a natural papillomavirus infection can be targeted by epitope-specific immunity and indicate this immunity is increased to early rather than late expressed proteins of papillomaviruses. This study also highlights the broad utility of the HLAA2.1 transgenic rabbit model for testing numerous immunological factors involved in vaccine generated protective immunity.     

2型糖尿病患者并发外周动脉疾病的危险因素

目的分析外周动脉疾病(PAD)在伴有危险因素的2型糖尿病(T2DM)患者中的发病率及PAD的危险因素。方法401例T2DM患者中,男201例,女200例,平均年龄(63.4±8.8)岁,双下肢任一侧踝肱指数(ABI)≤0.9诊断为PAD。结果T2DM患者PAD发病率为19.4%,性别差异无统计学意义。单变量分析提示年龄、高血压、收缩压、低密度脂蛋白-胆固醇和(T2DM)病程为其危险因素,多因素Logistic回归分析提示年龄、收缩压、低密度脂蛋白-胆固醇和T2DM病程为其独立危险因素,各自OR值及95%可信限(CI)分别为:年龄1.94(1.20~2.60),收缩压1.12(0.88~1.43),低密度脂蛋白-胆固醇1.32(0.97~1.63),T2DM病程1.27(0.96~1.73)。结论T2DM患者PAD发病率约为19.4%,其主要危险因素是年龄、收缩压、低密度脂蛋白-胆固醇和T2DM病程。这提示尽早良好控制血压、血脂和血糖对延缓或阻止PAD发生非常重要。     

Conservation of HIV-1 T cell epitopes across time and clades: Validation of immunogenic HLA-A2 epitopes selected for the GAIA HIV vaccine

HIV genomic sequence variability has complicated efforts to generate an effective globally relevant vaccine. Regions of the viral genome conserved in sequence and across time may represent the “Achilles’ heel” of HIV. In this study, highly conserved T-cell epitopes were selected using immunoinformatics tools combining HLA-A2 supertype binding predictions with relative global conservation. Analysis performed in 2002 on 10,803 HIV-1 sequences, and again in 2009, on 43,822 sequences, yielded 38 HLA-A2 epitopes. These epitopes were experimentally validated for HLA binding and immunogenicity with PBMCs from HIV-infected patients in Providence, Rhode Island, and/or Bamako, Mali. Thirty-five (92%) stimulated an IFNγ response in PBMCs from at least one subject. Eleven of fourteen peptides (79%) were confirmed as HLA-A2 epitopes in both locations. Validation of these HLA-A2 epitopes conserved across time, clades, and geography supports the hypothesis that such epitopes could provide effective coverage of virus diversity and would be appropriate for inclusion in a globally relevant HIV vaccine.     

脑血管疾病合并2型糖尿病的肠内营养支持

目的:观察脑血管疾病合并2型糖尿病病人使用不同的肠内营养(EN)配方后对血糖(Glu)、血清清蛋白(ALB)、淋巴细胞总数(LYM)、血清钾(K )、钠(Na )、氯(Cl-)、三酰甘油(TG)、胆固醇(TC)等的影响. 方法:对40例脑血管疾病合并2型糖尿病病人分别用普通配方、糖尿病配方进行相同热量的EN支持,并监测Glu、ALB、LYM、K 、Na 、Cl-、TG、TC. 结果:鼻饲普通配方病人的血糖明显上升,需加用胰岛素或口服降糖药.而鼻饲糖尿病配方病人的血糖无明显升高,无需加用或减少胰岛素用量(P＜0.05). 结论:脑血管疾病合并2型糖尿病病人选用合适的EN配方,能较好地控制血糖,提高疗效.     

2型糖尿病患者检测血清中IL-6和尿样中8-OHdG的变化

目的探讨白介素-6(IL-6)在2型糖尿病患者糖尿病发生、发展过程中的变化。了解健康人群尿样中8-羟基脱氧鸟苷(8-OHdG)含量的正常范围,探索尿样中8-OHdG含量水平能否作为反映2型糖尿病患者DNA氧化损伤程度的直接指标。方法用(酶联免疫吸附法)ELISA分析法检测40例2型糖尿病人及40例健康检查者IL-6水平;采用ELISA检测健康人和2型糖尿病患者尿样中8-OHdG的含量。结果 2型糖尿病患者血浆中IL-6明显高于正常对照组(P<0.01)。2型糖尿病患者尿样中的8-OHdG含量为(23.58±5.42)ng/mgCr,明显高于健康人的(14.54±5.10)ng/mg Cr(P<0.01)。结论 IL-6在糖尿病的发生、发展过程中发挥作用,观察其浓度变化对探讨糖尿病的发病机制、预防及指导用药均有重要价值;同时2型糖尿病患者的DNA氧化损伤水平均高于健康人群。     

The burden of type 2 diabetes in Serbia and the cost-effectiveness of its management

The purpose of this study was to analyse whether the nationwide application of the national Serbian guideline for diabetes mellitus (NSGDM) would save a relevant amount of disability-adjusted life years (DALYs) and/or reduce the medical cost of treating diabetic patients in Serbia, as compared to the present situation. Disability-adjusted life years were calculated for Serbia and the cost-effectiveness was analysed in eight population groups under ideal and present conditions; prevalent and incident cases were each split up for patients with blood glucose that was well controlled and that was uncontrolled. Under ideal conditions, i.e., according to the NSGDM, 8,031 DALYs could be saved with a potential cost reduction at the same time of approximately 19 million Euros. The implementation of the NSGDM in clinical practice bears a great potential to save lives and reduce years lived with lowered quality of life, but in addition it may reduce costs by about a quarter.     

CD4+ and CD8+ mediated cellular immune response to recombinant influenza nucleoprotein

The stimulatory properties of soluble recombinant influenza nucleoprotein (NP) on purified CD4(+) and CD8(+) T cells from young and elderly individuals were studied. Recombinant influenza NP failed to induce proliferation of resting CD4(+) and CD8(+) T cells in the absence of IL-2. Addition of small amounts of IL-2, however, led to strong proliferation of resting CD4(+) and CD8(+) T cells from young and elderly donors. NP-reactive CD4(+) and CD8(+) T cell lines from both age groups grew equally well under long-term culture conditions. T cell lines raised to live influenza virus could recognize recombinant influenza NP and showed a substantial proliferative response. Stimulation of CD8(+) T cells is presumably due to cross-presentation, as EBV-transformed MHC class I-positive cell lines, which are incapable of antigen processing, stimulated live influenza virus-reactive CD8(+) T cell lines when loaded with NP-derived immunodominant peptides but not following loading with the whole NP molecule. Vaccines containing recombinant influenza NP might confer cross-protective immunity and could therefore be especially useful in cases of major epidemics or pandemics.     

浙江省2013年2型糖尿病伤残调整寿命年分析

目的 定量估计浙江省人群2013年2型糖尿病的疾病负担。方法 使用2010年全球疾病负担研究(GBD 2010)方法,利用浙江省人群2型糖尿病发病率、患病率和死亡率数据,借助DISMODⅡ软件计算伤残调整寿命年(DALY)、伤残损失寿命年(YLD)和损失寿命年(YLL)。结果 2013年浙江省2型糖尿病每千人DALY值为5.36人年,女性(5.49人年)高于男性(5.24人年),城市(5.47人年)高于农村(5.42人年);DALY强度主要集中在老年组,其中以80～84岁人群最高(32.63人年/1 000人)。2型糖尿病主要引起伤残负担,YLL/YLD=0.62。结论 浙江省2型糖尿病平均YLL率高于全国水平。     

A prospective,placebo controlled study on the humoral immune response to and safety of tetanus revaccination in myasthenia gravis

ObjectiveTo investigate the humoral immune response to and safety of a tetanus revaccination in patients with myasthenia gravis or Lambert-Eaton myasthenic syndrome.MethodsA tetanus revaccination was administered to 66 patients. Before and 4 weeks after revaccination a blood sample and clinical outcome scores were obtained. Anti-tetanus IgG total, IgG1 and IgG4 titres were measured with an ELISA and disease-specific antibody titres (AChR, MuSK or VGCC) with a radio-immunoprecipitation assay. A historic healthy control group was used for comparing tetanus antibody titres with that of our patients. A placebo (saline) vaccination group was used to investigate the variability of clinical outcome scores with a 4 weeks interval.ResultsIn 60 of 65 patients, a significant increase of the anti-tetanus antibody response was measured. Thymectomy did not have an impact on this responsiveness. Patients with immunosuppressive medication had a significantly lower pre and post titre compared to healthy controls, but their response was still significant. The titres of disease-specific antibodies were unchanged 4 weeks after revaccination. The clinical outcome scores showed no exacerbation of symptoms of the disease.ConclusionA tetanus revaccination in patients with myasthenia gravis or Lambert-Eaton myasthenic syndrome is safe and induces a significant immune response, irrespectively of their immunosuppressive medication. We observed neither immunological nor clinical relevant exacerbations associated with the tetanus revaccination.Clinical trial registryThe tetanus trial is listed on clinicaltrialsregister.eu under 2014-004344-35. The placebo AChR MG group was part of another clinical trial, investigating influenza vaccination in myasthenic patients. This trial is listed on clinicaltrialsregister.eu under 2016-003138-26.