刚地弓形虫缓殖子期特异抗原BSR4的重组表达与免疫特性的初步研究

目的重组表达刚地弓形虫(Toxoplasma gondii)Prugniaud(PRU)株缓殖子期特异抗原(BSR4)基因,并检测其免疫特性。方法将已构建的重组表达质粒pET28a(+)-BSR4转化至大肠埃希菌(E.coli)BL21中,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达并纯化。分别以慢性感染PRU株刚地弓形虫小鼠血清和健康小鼠血清为一抗,用蛋白质印迹(Western blottting)鉴定BSR4重组蛋白的免疫反应性。用3H-TdR掺入法检测慢性感染PRU株刚地弓形虫小鼠脾淋巴细胞对不同浓度BSR4重组蛋白的特异性增殖水平,计算刺激指数(SI)。以BSR4重组蛋白为抗原,用ELISA法检测急性(抗弓形虫IgG-IgM+)和慢性(抗弓形虫IgG+IgM-)弓形虫病患者血清,以及健康人血清,各20份,评判该蛋白的免疫反应性。结果重组质粒pET28a(+)-BSR4经IPTG诱导后表达重组蛋白BSR4,经变性、复性和纯化后获得相对分子质量(Mr)45 000的可溶性蛋白。Western blotting分析结果显示,该蛋白能被慢性弓形虫感染小鼠血清识别。脾淋巴细胞增殖试验表明,1、5和25μg/ml重组蛋白BSR4刺激感染弓形虫小鼠脾淋巴细胞的SI分别为1.13、0.88和1.17,显著高于未感染组(分别为0.46、0.24和0.49,均P<0.01)。ELISA检测结果显示,BSR4抗原能被慢性弓形虫病患者血清(IgG+IgM-)特异性识别(20/20),而不能被急性弓形虫病患者血清(IgG-IgM+)识别(0/20)。结论重组BSR4蛋白具有特异的免疫原性和免疫反应性。     

蛋白质及多肽类药物长效化研究进展

随着分子生物学技术和基因重组技术的发展,蛋白质与多肽类药物凭借其独特的优势已逐渐成为一种重要的临床药物类型;但稳定性差,半衰期短的缺点也极大限制了其在临床上的广泛应用。本文针对目前延长蛋白质和多肽类药物半衰期的具体方法及其注意事项进行了综述。     

Immunogenicity of rituximab in patients with severe pemphigus

Pemphigus is a life-threatening autoimmune bullous disease associated with autoantibodies to desmoglein 1 and/or 3. The anti-CD20 chimeric mouse monoclonal antibody rituximab has previously been successfully applied in more than 130 reported pemphigus patients with severe and/or refractory disease. Since antibodies against other therapeutics such as IFNα and β, erythropoietin, and TNFα antagonists had led to decreased efficacy of these drugs, we determined anti-rituximab antibodies in 11 patients with pemphigus before rituximab administration as well as 3, 9, and 15 months thereafter. For this purpose, a novel, affinity capture elution assay was established using rabbit IgG against the F(ab)₂ fragment of rituximab. In addition, serum levels of rituximab were determined by a competition ELISA. In 2 of 11 pemphigus patients, antibodies to rituximab were detected. In both patients, only a partial remission was observed under treatment. In addition, when followed over a longer period of time, the occurrence of anti-rituximab antibodies paralleled an increase in disease activity. Of the 9 patients without development of antiantibodies to rituximab, in 5, all lesions healed and in 4, partial remissions were seen. These observations show that antibodies to rituximab are generated in some patients during rituximab treatment and may be associated with a less favourable response to treatment.     

保存方法对同种异体软骨移植物免疫原性的影响★

背景：异体软骨移植是治疗关节软骨缺损主要手段，寻找软骨组织保存方法来降低移植软骨的免疫原性已成为临床上关键性的技术问题。 目的：比较慢速梯度降温法、玻璃化法与60Co射线照射+梯度降温法对同种异体骨软骨移植物免疫原性的影响。 方法：将关节软骨块随机分为4组：新鲜组不采取任何措施；慢速梯度降温组给予程序降温法保存关节软骨；玻璃化组利用玻璃化溶液处理后保存；60Co射线照射+梯度降温组给予60Co射线照射后，再按程序降温法保存关节软骨。保存8，15，30，60 d后关节软骨块经细胞培养及扩增后制备成细胞悬液。流式细胞仪检测关节软骨细胞表面主要组织相容性复合体Ⅰ，Ⅱ抗原表达率。 结果与结论：经过保存后，慢速梯度降温组、玻璃化组和60Co射线照射+梯度降温组细胞表面的主要组织相容性复合体Ⅰ和Ⅱ抗原表达率有大幅下降，与新鲜组相比有显著性差异，但前3组间没有显著性差异。各保存组主要组织相容性复合体Ⅰ类抗原和主要组织相容性复合体Ⅱ类抗原表达率均随着时间的推移下降，并且在30 d时3种不同保存方法保存的软骨细胞表面主要组织相容性复合体表达率就降低到最低。预示着经保存处理的关节软骨可能会提高同种异体骨软骨移植手术的成功率。     

A biomarker for tegumentary and visceral leishmaniasis based on a recombinant Leishmania hypothetical protein

The measures for leishmaniasis control include the precise diagnosis of disease. However, although several recombinant antigens have been tested with this biotechnological purpose, no effective product exists, which could detects patients with the active disease, as well as differentiates them from cured and treated patients. In this study, a conserved Leishmania hypothetical protein, which was identified in Leishmania infantum parasites, but evaluated to presents high homology in the amino acid sequences between distinct parasite species, was evaluated for the diagnosis of tegumentary and visceral leishmaniasis. In addition, PBMCs collected from treated and untreated mucosal leishmaniasis (ML) and visceral leishmaniasis (VL) patients, as well as in healthy subjects living in endemic region of disease, were in vitro stimulated, when IFN-γ, IL-4 and IL-10 levels were evaluated in the cell supernatant. Regarding the serological analyses, ELISA experiments using the recombinant protein (rLiHyL) and a human serological panel revealed high sensitivity and specificity values to detect both diseases, while control antigens showed worst results. Regarding the cellular response, results showed that rLiHyL-stimulated cells produced higher IFN-γ and lower IL-4 and IL-10 levels in the supernatants. Also, the anti-protein antibody production was evaluated in these patients, and data showed higher IgG2 and lower IgG1 levels found in the treated patients and healthy controls, demonstrating the stimulation of a Th1-type response induced by the rLiHyL protein. In conclusion, this hypothetical protein can be considered as antigenic in TL and VL, as well as a vaccine candidate to be tested in future studies to protect against disease.     

Comparable immune responsiveness but increased reactogenicity after subcutaneous versus intramuscular administration of tick borne encephalitis (TBE) vaccine

Evaluation of safety, immunogenicity and efficacy of vaccines during licensing studies is performed in relation to the selected vaccination route. For most adjuvanted vaccines, such as the TBE vaccine FSME-IMMUN, only intramuscular (i.m.) administration is licensed. Yet in certain situations, either because of medical indications, accidental application or due to a lack of sufficient muscular tissue, the vaccine might rather be applied subcutaneously (s.c.). With respect to the TBE vaccine there are currently however no data to support the use of the subcutaneous route of vaccination.In order to compare the reactogenicity and immune responsiveness upon i.m. and s.c. TBE vaccination 116 (58 females and 58 males) participants with a documented primary TBE vaccination course were randomized to receive either an i.m. or s.c. booster. Venous blood was collected before, 7 days, 1 month and 6 months after vaccination to determine antibody titer profiles. PBMC were isolated prior to and 7 days after booster to analyze lymphocyte subpopulations and cytokine production upon antigen restimulation. Subjects were monitored for the occurrence of side effects for 7 days post vaccination.Comparable levels of TBE specific neutralizing antibodies were induced after s.c. and i.m. vaccination. At the cellular level, IL-2, IFN gamma and IL-10 levels did not significantly differ using either route of vaccination and the distribution of T cell subsets was comparable along with a relative decrease of regulatory T-cells after both ways of administration.In contrast to the immunogenicity analyses, the data from safety diaries revealed a significantly higher rate of local, but not of systemic reactions after s.c. administration. In conclusion, this study demonstrates that both routes lead to comparable immune responses to the TBE antigen. The higher rate and intensity of local reactions, particularly among women, after s.c. vaccination however needs to be addressed during counseling.     

The immunogenicity in healthy infants and efficiency to prevent mother to child transmission of Hepatitis B virus of a 10 μg recombinant yeast-derived Hepatitis B vaccine (Hep-KSC)

Background and AimsTo evaluate immunogenicity and efficacy of a 10 μg recombinant Saccharomyces cerevisiae-derived hepatitis B vaccine (Kangtai Biological Products Co. Ltd, Shenzhen, China) (Hep-KSC) in newborns.MethodsOverall 1197 infants born to mothers negative for HBV markers (NM group) and 534 born to HBsAg-positive mothers (PM Group) were enrolled. Infants in NM group were given 10 μg Hep-KSC, 10 μg Engerix-B or 5 μg Hep-KSC and those in PM group received 10 μg Hep-KSC or 10 μg Engerix-B at 0, 1 and 6 months, with an additional 200 IU HBIG at birth for the latter.ResultsFor NM Group, 10 μg Hep-KSC paralleled 10 μg Engerix-B but outperformed 5 μg Hep-KSC regarding seroprotective rate (95.06% vs 94.83% vs 89.67%, p = 0.0077) and anti-HBs geometric mean concentration (GMC) (798.87 mIU/ml vs 790.16 mIU/ml vs 242.04 mIU/ml, p < 0.0001) at 7 months. The proportion of infants with anti-HBs greater than 1000 mIU/ml was higher in 10 μg Hep-KSC than 5 μg Hep-KSC group (45.77% vs 11.93%, p < 0.0001) at 7 and 12 months. For PM Group, the HBsAg positivity rate in 10 μg Hep-KSC and 10 μg Engerix-B group was 1.60% and 4.27% at 7 months, respectively. In 10 μg Hep-KSC group, 93.61% and 91.29% achieved seroprotection at 7 and 12 months, respectively, and correspondingly 90.24% and 86.96% in 10 μg Engerix-B group. The anti-HBs GMC was comparable between 10 μg Hep-KSC and 10 μg Engerix-B group at 7 and 12 months (575.31 mIU/ml vs 559.64 mIU/ml; 265.79 mIU/ml vs 264.48 mIU/ml).Conclusions10 μg Hep-KSC might be appropriate for neonatal immunization with good immunogenicity and efficacy, especially for infants born to HBsAg-positive mothers.     

Enhancing cellular immunogenicity of MVA-vectored vaccines by utilizing the F11L endogenous promoter

Modified vaccinia virus Ankara (MVA)-vectored vaccines against malaria, influenza, tuberculosis and recently Ebola virus are in clinical development. Although this vector is safe and immunogenic in humans, efforts remain on-going to enhance immunogenicity through various approaches such as using stronger promoters to boost transgene expression. We previously reported that endogenous MVA promoters such as pB8 and pF11 increased transgene expression and immunogenicity, as compared to the conventional p7.5 promoter. Here, we show that both promoters also rivalled the mH5 promoter in enhancing MVA immunogenicity. We investigated the mechanisms behind this improved immunogenicity and show that it was a result of strong early transgene expression in vivo, rather than in vitro as would normally be assessed. Moreover, keeping the TK gene intact resulted in a modest improvement in immunogenicity. Utilizing pB8 or pF11 as ectopic promoters at the TK locus instead of their natural loci also increased transgene expression and immunogenicity. In addition to a reporter antigen, the pF11 promoter was tested with the expression of two vaccine antigens for which cellular immunogenicity was significantly increased as compared to the p7.5 promoter. Our data support the use of the pF11 and pB8 promoters for improved immunogenicity in future MVA-vectored candidate vaccines.     

Factors determining anti-poliovirus type 3 antibodies among orally immunised Indian infants

BackgroundAmong the three poliovirus serotypes, the lowest responses after vaccination with trivalent oral polio vaccine (tOPV) are to serotype 3. Although improvements in routine immunisation and supplementary immunisation activities have greatly increased vaccine coverage, there are limited data on antibody prevalence in Indian infants.MethodsChildren aged 5–11 months with a history of not having received inactivated polio vaccine were screened for serum antibodies to poliovirus serotype 3 (PV3) by a micro-neutralisation assay according to a modified World Health Organization (WHO) protocol. Limited demographic information was collected to assess risk-factors for a lack of protective antibodies. Student’s t-test, logistic regression and multilevel logistic regression (MLR) model were used to estimate model parameters.ResultsOf 8454 children screened at a mean age of 8.3 (standard deviation [SD]-1.8) months, 88.1% (95% confidence interval (CI): 87.4–88.8) had protective antibodies to PV3. The number of tOPV doses received was the main determinant of seroprevalence; the maximum likelihood estimate yields a 37.7% (95% CI: 36.2–38.3) increase in seroprevalence per dose of tOPV. In multivariable logistic regression analysis increasing age, male sex, and urban residence were also independently associated with seropositivity (Odds Ratios (OR): 1.17 (95% CI: 1.12–1.23) per month of age, 1.27 (1.11–1.46) and 1.24 (1.05–1.45) respectively).ConclusionSeroprevalence of antibodies to PV3 is associated with age, gender and place of residence, in addition to the number of tOPV doses received. Ensuring high coverage and monitoring of response are essential as long as oral vaccines are used in polio eradication.     

Human Phase 1 trial of low-dose inactivated seasonal influenza vaccine formulated with Advax™ delta inulin adjuvant

Influenza vaccines are usually non-adjuvanted but addition of adjuvant may improve immunogenicity and permit dose-sparing, critical for vaccine supply in the event of an influenza pandemic. The aim of this first-in-man study was to determine the effect of delta inulin adjuvant on the safety and immunogenicity of a reduced dose seasonal influenza vaccine. Healthy male and female adults aged 18–65 years were recruited to participate in a randomized controlled study to compare the safety, tolerability and immunogenicity of a reduced-dose 2007 Southern Hemisphere trivalent inactivated influenza vaccine formulated with Advax™ delta inulin adjuvant (LTIV + Adj) when compared to a full-dose of the standard TIV vaccine which does not contain an adjuvant. LTIV + Adj provided equivalent immunogenicity to standard TIV vaccine as assessed by hemagglutination inhibition (HI) assays against each vaccine strain as well as against a number of heterosubtypic strains. HI responses were sustained at 3 months post-immunisation in both groups. Antibody landscapes against a large panel of H3N2 influenza viruses showed distinct age effects whereby subjects over 40 years old had a bimodal baseline HI distribution pattern, with the highest HI titers against the very oldest H3N2 isolates and with a second HI peak against influenza isolates from the last 5–10 years. By contrast, subjects >40 years had a unimodal baseline HI distribution with peak recognition of H3N2 isolates from approximately 20 years ago. The reduced dose TIV vaccine containing Advax adjuvant was well tolerated and no safety issues were identified. Hence, delta inulin may be a useful adjuvant for use in seasonal or pandemic influenza vaccines.Australia New Zealand Clinical Trial Registry: ACTRN12607000599471