The immunogenicity of CpG-antigen conjugates

Compared to “live” vaccines the immunogenicity of “split” vaccines is notably poor, because exogeneous antigens (Ag) insufficiently access the MHC class I processing pathway needed for cross-presentation. Here we review our evidence that targeting ligands of endosomally expressed Toll-like Receptors (TLRs), together with exogeneous Ag to endosomes of dendritic cells (DCs) conveys immunogenicity to Ag similar in magnitude as “live” vaccines that produce Ag. We explored the consequences of enforced endocytosis of “Ag plus TLR ligands” either by crosslinking Ag and CpG-Oligonucleotides (CpG-Ag conjugates) or by co-encapsulating Ag plus endosomally expressed TLR ligands in biodegradable microspheres (MP). While both approaches equally well yielded in effective cross-priming of MHC class I restricted CD8 T effector cells, our data recommend MP as a generally applicable endosomal delivery device to vaccinate for protective and therapeutic CD4 and CD8 T cell immunity. Furthermore, our data suggest that functional inactivation of Foxp3⁺ regulatory T cells further enhances the immunogenicity of “split vaccines”.     

口服Ag85A DNA疫苗表达产物在脾脏的分布

目的 检测口服Ag85A DNA疫苗表达产物在脾脏内的分布,为阐明口服DNA疫苗可诱导全身性免疫应答的机制提供依据。方法 将本实验室构建的pCDNA3．1^＋-Ag85A真核表达重组质粒转化感受态大肠杆菌DH5α进行扩增,无内毒素抽提纯化,进一步用脂质体包裹制成口服重组Ag85A DNA疫苗。将C57BL／6小鼠随机分为2组,即生理盐水组和DNA疫苗组。分别将生理盐水和Ag85A DNA疫苗以灌胃方式投给各组小鼠,共免疫3次,每次间隔14d,末次免疫后14d处死小鼠,取脾,免疫组化、免疫荧光法检测Ag85A表达产物在脾脏的分布情况。结果 Ag85A重组DNA疫苗的表达产物在小鼠脾脏白髓、边缘区和红髓的脾索处有广泛分布,在边缘区及红髓的脾索处的检出强度高于白髓。免疫组化结果中边缘区与白髓比较t=3．039,P〈0．05;红髓的脾索与白髓比较t=3．068,P〈0．05;边缘区与红髓的脾索比较t=1．750,P〉0．05。免疫荧光结果中边缘区与白髓比较t=3．144,P〈0．05;红髓的脾索与白髓比较t=3．098,P〈0．05;边缘区与红髓的脾索比较t=1．369,P〉0．05。结论口服脂质体包裹的DNA疫苗的表达产物存在于脾脏,表明经口途径接种的DNA疫苗可能会在脾脏诱导全身性免疫应答的产生。     

Randomised clinical trial of Hydrofiber dressing with silver versus povidone-iodine gauze in the management of open surgical and traumatic wounds

This prospective, randomised clinical trial compared pain, comfort, exudate management, wound healing and safety with Hydrofiber dressing with ionic silver (Hydrofiber Ag dressing) and with povidone-iodine gauze for the treatment of open surgical and traumatic wounds. Patients were treated with Hydrofiber Ag dressing or povidone-iodine gauze for up to 2 weeks. Pain severity was measured with a 10-cm visual analogue scale (VAS). Other parameters were assessed clinically with various scales. Pain VAS scores decreased during dressing removal in both groups, and decreased while the dressing was in place in the Hydrofiber Ag dressing group (n = 35) but not in the povidone-iodine gauze group (n = 32). Pain VAS scores were similar between treatment groups. At final evaluation, Hydrofiber Ag dressing was significantly better than povidone-iodine gauze for overall ability to manage pain (P < 0.001), overall comfort (P < or = 0.001), wound trauma on dressing removal (P = 0.001), exudate handling (P < 0.001) and ease of use (P < or = 0.001). Rates of complete healing at study completion were 23% for Hydrofiber Ag dressing and 9% for povidone-iodine gauze (P = ns). No adverse events were reported with Hydrofiber Ag dressing; one subject discontinued povidone-iodine gauze due to adverse skin reaction. Hydrofiber Ag dressing supported wound healing and reduced overall pain compared with povidone-iodine gauze in the treatment of open surgical wounds requiring an antimicrobial dressing.     

Electrocatalytic effects in the electrochemical reduction of pyrazine on single crystal IB metal electrodes

Due to the presence of the second nitrogen atom, pyrazine has an increased affinity for electrons and lower energies of the empty orbitals than pyridine. For this reason the electrochemical reduction of pyrazine takes place at considerably less negative potentials than that of pyridine. The reduction of pyrazine on Cu(111), Ag(111) and Au(111) electrodes in neutral aqueous media has been found to take place in one step as previously reported for Hg. The potential region and the current densities associated with this process were, however, observed to depend significantly on the electrode nature. Pyrazine was also found to be involved in a chemical interaction with the hydrogen atoms resulting in the water electro-reduction process occurring on Au(111) and Cu(111). The specific interaction of water with the single crystal electrodes turned out to be an important factor for developing the reduction of the organic molecule, both chemically and electrochemically.     

结核杆菌Edman株Ag85C基因的克隆表达与鉴定

目的克隆人结核杆菌Ag85C基因并在大肠杆菌中表达,研究其对结核病预防和治疗过程中的免疫保护作用。方法设计一对特异性引物,PCR扩增人结核杆菌Edman株Ag85C基因开放阅读框并定向克隆到原核表达载体pQE30,通过酶切、PCR和测序鉴定重组质粒,转化大肠杆菌,IPTG诱导表达,SDS-PAGE和Western blot鉴定表达的重组蛋白。结果构建了pQE30-Ag85C重组质粒;在大肠杆菌中表达了一分子量约30kDa的重组蛋白。结论重组质粒pQE30-Ag85C构建成功并在大肠杆菌中高效表达,为Ag85C保护性抗原位点分析和新型疫苗的研制奠定了基础。     

Immunogenicity and therapeutic effects of recombinant Ag85AB fusion protein vaccines in mice infected with Mycobacterium tuberculosis

The immune function of tuberculosis (TB) patients is disordered. By using immune regulators to assist chemotherapy for TB the curative effect might be improved. In this study, a vaccine containing Mycobacterium tuberculosis (M. tuberculosis) recombinant Ag85AB fusion protein (rAg85AB) was constructed and evaluated. The mice were immunized intramuscularly three times at two-week intervals with Ag85AB fusion protein combined with Corynebacterium parvum adjuvant (rAg85AB+CP). In comparison to control mice that received either CP alone or saline, the mice that received rAg85AB+CP had significantly higher number of T cells secreting IFN-γ and higher levels of specific antibodies of IgG, IgG1 and IgG2a isotypes in sera. The specific antibodies also had higher ratios of IgG2a to IgG1, indicating a predominant Th1 immune response. To test for immunotherapy of TB, M. tuberculosis infected mice were given three intramuscular doses of 20 μg, 40 μg or 60 μg of rAg85AB in rAg85AB+CP, or phosphate-buffered saline (PBS), or CP or Mycobacterium phlei (M. Phlei) F.U.36. Compared with the PBS group, 20 µg, 40 µg and 60 µg rAg85AB+CP and M. phlei F.U.36 groups reduced the pulmonary bacterial loads by 0.13, 0.15, 0.42 and 0.40 log₁₀, and the liver bacterial loads by 0.64, 0.64, 0.53 and 0.61 log₁₀, respectively. Pathological changes of lungs were less, and the lesions were limited to a certain extent in 40 µg and 60 µg rAg85AB+CP and M. phlei F.U.36 groups. These results showed that rAg85AB+CP had immunotherapeutic effect on TB, significantly increasing the cellular immune response, and inhibiting the growth of M. tuberculosis.     

急性脑梗塞患者血浆t—PA,PAI及vWF：Ag的检测及其与血脂的关系

本文同时检测了22例急性期脑梗塞患者血浆组织纤溶酶原激活物(t-PA)抑制物(PAI)活性及因子Ⅷ相关抗原(vWF:Ag)含量,结果示三指标与对照组比较,均有显著的增高;并对患者组上述指标与血脂间进行了相关性分析,认为血脂与纤溶系统间的相互联系对动脉粥样硬化、脑血栓形成具有重要意义。     

结核分支杆菌Ag85B基因在大肠杆菌中的高效表达

目的 通过结核分支杆菌Ag85B蛋白编码基因在大肠杆菌中稳定表达，以获得大量纯化的Ag85B蛋白。方法 采用DNA重组技术构建结核分支杆菌Ag85B基因的表达载体，双酶切和聚合酶链反应(PCR)鉴定重组子，阳性重组子转化大肠杆菌，并诱导表达外源蛋白。十二烷基磺酸钠聚丙烯酰胺凝胶电泳(SDSPAGE)鉴定Ag85B蛋白抗原在大肠杆菌中的表达，对染色的凝胶扫描，以测定目的蛋白表达水平。结果 菌体蛋白经SDSPAGE，含阳性重组质粒的菌体蛋白中出现一条新蛋白带，表达量占菌体总蛋白的33％～38％。Ag85B蛋白抗原在大肠杆菌中表达方式主要是包涵体形式。结论 构建的大肠杆菌重组体能高效表达结核分支杆菌Ag85B蛋白抗原。     

A multistage-polyepitope vaccine protects against Mycobacterium tuberculosis infection in HLA-DR3 transgenic mice

Mycobacterium tuberculosis (Mtb) is responsible for almost 2 million deaths annually. BCG, currently the only TB vaccine, induces variable protection and does not protect against reactivation of latent TB. Thus, efficient vaccines to supplement BCG are required urgently. Since Mtb's proteome differs qualitatively and quantitatively during bacterial replication stages from that expressed during dormancy, improved TB vaccines should drive immune responses to Mtb antigens expressed during multiple stages of infection. Consequently, such “multistage” vaccines should be composed of (immunodominant) antigens expressed during different phases of Mtb infection. As a concept multistage vaccine, we constructed a polyepitope by fusing five HLA-DR3-restricted T-cell epitopes derived from different Mtb proteins either expressed highly by replicating bacteria (Ag85B, hsp65, 19 kDa lipoprotein), or abundantly expressed by dormant bacilli and recognized preferentially by TST⁺ individuals (hsp16, Rv1733c). PBMC of HLA-DR3⁺ but not HLA-DR3⁻ cured TB patients and TST⁺ individuals responded well to the multistage-polyepitope in vitro. The in vivo immunogenicity and protective efficacy of the multistage-polyepitope were analyzed using HLA-DR3 transgenic mice lacking endogenous murine class II as a model. Immunization with the multistage-polyepitope adjuvanted with CpG generated high IgG levels as well as polyfunctional CD4⁺ T-cells producing IFN-γ, TNF and IL-2, specific for these HLA-DR3-restricted epitopes. Importantly, multistage-polyepitope immunization reduced the number of bacilli in the lungs after Mtb challenge when administered as prophylactic vaccine. Given the extensive repertoire of potential Mtb antigens available for immune recognition, the data of our model demonstrate the potential of multistage-polyepitope vaccines to protect against TB.