The effect of fluorescein labels on the affinity of antisera to small haptens

Small haptens such as methylamphetamine (MW 149) cannot, on their own, induce an immune response. It is also unlikely that they fill the binding site of any antibody that recognises them. Under such circumstances any attached label might be expected to enter the area of the binding site and exert an influence on overall binding. To investigate the possible influence of the label on binding, a range of fluorescein-labelled derivatives, differing in bridge length, were prepared. Antiserum binding of these labelled derivatives was then compared to that of the unlabelled drug. Evidence is presented which suggests that, with small haptens, the closeness of the fluorescein molecule can markedly influence antibody binding. Significant differences were found in titre, sensitivity, and assay kinetics. These overall effects appear to be brought about by the change in affinity of the antibody for the labelled hapten.     

The immunogenicity of cells derived from induced pluripotent stem cells

With their ability to undergo unlimited self-renewal in culture and to differentiate into all cell types in the body, human embryonic stem ceils （hESCs） hold great potential for the treatment of currently incurable diseases. Two hESC-based cell therapies for spinal cord injury and macular degeneration have been advanced into human clinical trials. Despite this rapid progress, one key challenge of hESC-based cell therapy is the allogeneic immune rejection of hESC-derived cells by recipients. This problem could be mitigated by a recent breakthrough in the technology of induced pluripotent stem cells （iPSCs） by nuclear reprogramming of patient-specific somatic cells with defined factors, which could become a renewable source of autologous ceils for cell therapy. However, recent studies revealing the abnormal epigenetics, genomic stability and immunogenicity of iPSCs have raised safety concerns over iPSC-based therapy. Recent findings related to the immunogenicity of iPSC derivatives will be summarized in this review.     

The provision of 125I-labelled tracers for radioimmunoassay of haptens: A general approach?

Use of 125iodinated progesterone 11 alpha-glucuronide-tyramine conjugate as radioligand with the majority of antisera raised against progesterone 11 alpha-hemisuccinate-bovine serum albumin is shown to produce steep and sensitive standard curves. Accurate precise and robust assays for progesterone which are clinically useful have been readily developed from such systems. Together with data from other heterologous-bridge systems, the results suggest a hypothesis concerning desirable structural features of the links in heterologous-bridge immunoassays for haptens. The hypothesis should facilitate the establishment of other adequately sensitive external-label assays, especially for gonadal steroids.     

Nuclear magnetic resonance controlled method for coupling of fenoterol to a carrier and enzyme

Fenoterol is a phenethanolamine with β‐adrenergic agonist activity. The development of an enzyme immunoassay for fenoterol requires coupling to a carrier (bovine serum albumin, BSA) and an enzyme (horseradish peroxidase, HRP). 1,4‐Butanediol diglycidyl ether was used as the coupling agent, providing for a 12‐atom spacer. During the coupling procedure of the spacer to the protein and fenoterol to the spacer, the coupling yield was monitored by nuclear magnetic resonance (NMR). This ensured a coupling at desired hapten: protein ratios. The average coupling ratios obtained using this method were 30 moles of fenoterol to 1 mole of BSA and 1 mole of fenoterol to 1 mole of HRP. In practice, this method can be used for the coupling of compounds containing phenolic, anilinic, primary amine and thiol groups to proteins. Its major advantage is the real‐time NMR quantification of coupling efficiency and the option to interrupt the coupling reaction to obtain the desired hapten: protein ratio.     

NetMHCstab – predicting stability of peptide–MHC‐I complexes; impacts for cytotoxic T lymphocyte epitope discovery

Major histocompatibility complex class I (MHC‐I) molecules play an essential role in the cellular immune response, presenting peptides to cytotoxic T lymphocytes (CTLs) allowing the immune system to scrutinize ongoing intracellular production of proteins. In the early 1990s, immunogenicity and stability of the peptide–MHC‐I (pMHC‐I) complex were shown to be correlated. At that time, measuring stability was cumbersome and time consuming and only small data sets were analysed. Here, we investigate this fairly unexplored area on a large scale compared with earlier studies. A recent small‐scale study demonstrated that pMHC‐I complex stability was a better correlate of CTL immunogenicity than peptide–MHC‐I affinity. We here extended this study and analysed a total of 5509 distinct peptide stability measurements covering 10 different HLA class I molecules. Artificial neural networks were used to construct stability predictors capable of predicting the half‐life of the pMHC‐I complex. These predictors were shown to predict T‐cell epitopes and MHC ligands from SYFPEITHI and IEDB to form significantly more stable MHC‐I complexes compared with affinity‐matched non‐epitopes. Combining the stability predictions with a state‐of‐the‐art affinity predictions NetMHCcons significantly improved the performance for identification of T‐cell epitopes and ligands. For the HLA alleles included in the study, we could identify distinct sub‐motifs that differentiate between stable and unstable peptide binders and demonstrate that anchor positions in the N‐terminal of the binding motif (primarily P2 and P3) play a critical role for the formation of stable pMHC‐I complexes. A webserver implementing the method is available at www.cbs.dtu.dk/services/NetMHCstab .     

The potential of polymeric nanoparticles to increase the immunogenicity of the hapten clenbuterol

Micro‐ and nanoparticles prepared from the biodegradable and biocompatible polymers poly(lactide‐co‐glycolide) (PLGA) and polymethylmethacrylate (PMMA) have been successfully used as immunopotentiating antigen delivery systems. In our study, this approach was used to improve polyclonal antibody production to clenbuterol (CBL), a model hapten. PLGA and PMMA nanoparticles were loaded with either CBL alone or with a clenbuterol‐transferrin conjugate (CBL—Tfn) and administered subcutaneously to mice. PLGA nanoparticles were administered with or without the saponin adjuvant Quil A. The anti‐CBL titres present in experimental sera were determined by an enzyme immunoassay (ELISA). CBL‐Tfn‐loaded PLGA nanoparticles co‐administered with Quil A had obvious advantages immunologically over the currently used method of raising antibodies to CBL (the positive control). The combined adjuvanticity of Quil A and PLGA nanoparticles resulted in a positive response in all four of the mice tested and in higher antibody titres than were seen in the positive control group. Furthermore, the sustained release of immunogen from the nanoparticles permitted a reduction in immunizing frequency over the 15‐week study period.     

Therapeutic implications of peptide interactions with G‐protein‐coupled receptors in diabetic vasculopathy

The dramatic worldwide increase in the prevalence of diabetes has generated an attempt by the scientific community to identify strategies for its treatment and prevention. Vascular dysfunction is a hallmark of diabetes and frequently leads to the development of atherosclerosis, coronary disease‐derived myocardial infarction, stroke, peripheral arterial disease and diabetic ‘triopathy’ (retinopathy, nephropathy and neuropathy). These vascular complications, developing in an increasingly younger cohort of patients with diabetes, contribute to morbidity and mortality. Despite the development of new anti‐diabetic or anti‐hyperglycaemic drugs, vascular complications remain to be a problem. This warrants a need for new therapeutic strategies to tackle diabetic vasculopathy. There is a growing body of evidence showing that peptide‐binding G‐protein‐coupled receptors (peptide‐binding GPCRs) play an important role in the pathophysiology of vascular dysfunction during diabetes. Thus, in this review, we discuss some of the peptide‐binding GPCRs involved in the regulation of vascular function that have potential to be a therapeutic target in the treatment of diabetic vasculopathy.     

Understanding and measuring human B‐cell tolerance and its breakdown in autoimmune disease

The maintenance of immunological tolerance of B lymphocytes is a complex and critical process that must be implemented as to avoid the detrimental development of autoreactivity and possible autoimmunity. Murine models have been invaluable to elucidate many of the key components in B‐cell tolerance; however, translation to human homeostatic and pathogenic immune states can be difficult to assess. Functional autoreactive, flow cytometric, and single‐cell cloning assays have proven to be critical in deciphering breaks in B‐cell tolerance within autoimmunity; however, newer approaches to assess human B‐cell tolerance may prove to be vital in the further exploration of underlying tolerance defects. In this review, we supply a comprehensive overview of human immune tolerance checkpoints with associated mechanisms of enforcement, and highlight current and future methodologies which are likely to benefit future studies into the mechanisms that become defective in human autoimmune conditions.     

The immunological properties of haptens coupled to thymus-independent carrier molecules. III. The role of the immunogenicity and mitogenicity of the carrier in the induction of primary IgM anti-hapten responses

Hapten (DNP-lys) conjugates of two putatively nonimmunogenic polymers, hyaluronic acid and poly-γ-D-glutamic acid, induce significant primary IgM anti-DNP responses in C3H mice. Preparations of various immunogenic (Type 3 pneumococcal polysaccharide (SIII), levan, E. coli lipopolysaccharide) and nonimmunogenic (hyaluronic acid and poly-glutamic acid) polymers were tested for their ability to act as polyclonal mitogens in vitro. In serum-containing spleen cell cultures, only lipopolysaccharide stimulated substantial cell proliferation. In serum-free medium, and using high specific activity [³H]thymidine, lipopolysaccharide, levan, SIII and to a lesser degree hyaluronic acid induced significant thymidine incorporation. However, under the latter conditions cell survival and proliferation were much less impressive. There was no apparent correlation between the capacity of various polymers to induce lymphocyte proliferation and their ?potency”? as carriers for the generation of a primary IgM anti-DNP response. Furthermore while low doses of lipopolysaccharide elicited ?polyclonal”? antibody formation in vivo, high doses of SIII, levan and hyaluronic acid did not. These results indicate that T cell-independent B cell triggering is dependent on the polymeric nature of the antigen, and that polymers need not be immunogenic or mitogenic to act as carriers for the induction of primary IgM anti-hapten antibody responses.     

Influence of Escherichia coli chaperone DnaK on protein immunogenicity

The production of anti‐drug antibodies can impact significantly upon the safety and efficacy of biotherapeutics. It is known that various factors, including aggregation and the presence of process‐related impurities, can modify and augment the immunogenic potential of proteins. The purpose of the investigations reported here was to characterize in mice the influence of aggregation and host cell protein impurities on the immunogenicity of a humanized single‐chain antibody variable fragment (scFv), and mouse albumin. Host cell protein impurities within an scFv preparation purified from Escherichia coli displayed adjuvant‐like activity for responses to the scFv in BALB/c strain mice. The 70 000 MW E. coli chaperone protein DnaK was identified as a key contaminant of scFv by mass spectrometric analysis. Preparations of scFv lacking detectable DnaK were spiked with recombinant E. coli DnaK to mimic the process‐related impurity. Mice were immunized with monomeric and aggregated preparations, with and without 0·1% DnaK by mass. Aggregation alone enhanced IgM and IgG2a antibody responses, but had no significant effect on total IgG or IgG1 responses. The addition of DnaK further enhanced IgG and IgG2a antibody responses, but only in the presence of aggregated protein. DnaK was shown to be associated with the aggregated scFv by Western blot analysis. Experiments with mouse albumin showed an overall increase in immunogenicity with protein aggregation alone, and the presence of DnaK increased the vigour of the IgG2a antibody response further. Collectively these data reveal that DnaK has the potential to modify and enhance immunogenicity when associated with aggregated protein.     

国产流行性感冒病毒疫苗免疫原性研究的meta分析

目的 评价国产流行性感冒(简称流感)病毒疫苗的免疫原性.方法 通过文献检索搜集2012年9月前发表的有关国产与进口流感病毒疫苗免疫原性比较研究的文献,利用Stata10.0软件进行meta分析.结果 共有20篇符合标准的文献纳入本研究.结果显示:国产流感病毒疫苗B型抗体阳转率显著高于进口流感病毒疫苗(P=0.036,RR=1.036,95％ CI:1.002～1.070),未发现国产流感病毒疫苗3个亚型的保护率及H1N1和H3N2抗体阳转率与进口流感病毒疫苗存在显著差异(P＞0.05).进一步的亚组分析发现:国产流感全病毒灭活疫苗B型抗体阳转率(P=0.024,RR=1.040,95％CI:1.005～ 1.077)及保护率(P=0.031,RR =1.059,95％CI:1.005～1.116)、H3N2抗体(P=0.019,RR=1.053,95％ CI:1.009 ～1.098)保护率均显著高于进口流感病毒疫苗.结论 国产流感病毒疫苗具有较好的免疫原性.     

人免疫缺陷病毒1型(HIV-1)gag基因疫苗的免疫原性

目的 :检测人免疫缺陷病毒 1型 (HIV 1)gag基因疫苗的免疫原性。方法 :分别以ELISA、荧光抗体染色和乳酸脱氢酶释放法 ,检测免疫小鼠血清抗体滴度、脾T细胞亚群的数量和淋巴细胞杀伤效应。结果 :血清抗体滴度、脾T细胞亚群的数量及淋巴细胞杀伤效应 ,重组质粒pVAXGAG免疫组与空载体pVAX1对照组相比较差异显著(分别为P <0 .0 5和P <0 .0 1)。结论 :HIV 1DNA疫苗质粒pVAXGAG在BALB/c小鼠中不仅可诱导特异性体液免疫 ,而且可诱导特异性细胞免疫。     

Reflections on the immunogenicity of therapeutic proteins

Therapeutic proteins are now established as a major class of medication with proven efficacy in treating a wide range of diseases. The administration of such proteins to patients can lead to the development of antibodies that are able to bind and eventually neutralise the protein administered. Many advances have been made in understanding the immunogenicity of therapeutic proteins, but opinions are often conflicting. Although it is important to understand more about the antibodies generated against therapeutic proteins, the need for future research must not be neglected, so that other relevant factors can be identified and addressed to maximise treatment benefits for patients.     

Anti‐CD3 treatment up‐regulates programmed cell death protein‐1 expression on activated effector T cells and severely impairs their inflammatory capacity

T cells play a key role in the pathogenesis of type 1 diabetes, and targeting the CD3 component of the T‐cell receptor complex provides one therapeutic approach. Anti‐CD3 treatment can reverse overt disease in spontaneously diabetic non‐obese diabetic mice, an effect proposed to, at least in part, be caused by a selective depletion of pathogenic cells. We have used a transfer model to further investigate the effects of anti‐CD3 treatment on green fluorescent protein (GFP)⁺ islet‐specific effector T cells in vivo. The GFP expression allowed us to isolate the known effectors at different time‐points during treatment to assess cell presence in various organs as well as gene expression and cytokine production. We find, in this model, that anti‐CD3 treatment does not preferentially deplete the transferred effector cells, but instead inhibits their metabolic function and their production of interferon‐γ. Programmed cell death protein 1 (PD‐1) expression was up‐regulated on the effector cells from anti‐CD3‐treated mice, and diabetes induced through anti‐PD‐L1 antibody could only be reversed with anti‐CD3 antibody if the anti‐CD3 treatment lasted beyond the point when the anti‐PD‐L1 antibody was washed out of the system. This suggests that PD‐1/PD‐L1 interaction plays an important role in the anti‐CD3 antibody mediated protection. Our data demonstrate an additional mechanism by which anti‐CD3 therapy can reverse diabetogenesis.     

小鼠神经干细胞免疫原性的研究

目的:探讨小鼠神经干细胞(NSCs)免疫原性,以利于解决神经干细胞移植中的免疫排斥问题。方法:(1)首先将30只C57BL/6近交系小鼠随机分成两组,以BALB/c近交系小鼠的NSCs及肝细胞(作为体细胞对照组)分别进行腹腔主动免疫,然后将NSCs及肝细胞分别与各自免疫后小鼠的T淋巴细胞进行单向混合淋巴细胞培养,通过液闪计数仪检测各自T淋巴细胞增殖程度,从而比较两者免疫原性的强弱。(2)利用异硫氰酸荧光素标记抗体(FITC)及Phycoery-thrin(PE)抗体分别标记BALB/c小鼠NSCs主要组织相容性抗原复合物Ⅰ、Ⅱ(MHC-Ⅰ、MHC-Ⅱ),然后通过流式细胞术(FCM)分别检测BALB/c小鼠NSCs及其肝细胞的MHC-Ⅰ和MHC-Ⅱ阳性细胞比例,从而推断小鼠NSCs的免疫原性。结果:(1)NSC组液闪计数仪检测所得cpm值为16592.8±2865.3,肝细胞组为27815.0±2416.3,NSC组明显低于肝细胞组,差别有统计学意义(P0.01)。(2)小鼠NSCs实验组中,约(87.55±3.65)%的增殖期神经干细胞没有检测到MHC-Ⅰ和MHC-Ⅱ分子表达;而作为对照组的小鼠肝细胞组中,仅有约(27.45±1.86)%的肝细胞未表达MHC-Ⅰ和MHC-Ⅱ分子。且NSC组MHC-Ⅰ和MHC-Ⅱ阴性细胞比例明显高于肝细胞组,差别有统计学意义(P0.01)。结论:小鼠神经干细胞具有较弱的免疫原性,且其免疫原性明显低于同一个体的肝细胞。     

A final report on safety and immunogenicity of a bivalent aqueous subunit HBV vaccine

The bivalent form of an aqueous formalin-inactivated hepatitis B vaccine was evaluated for safety and immunogenicity in chimpanzees. To evaluate safety five animals were inoculated intravenously with vaccine containing 500 micrograms HBsAg and two animals with 50 micrograms. None of these animals developed hepatitis or any serologic marker indicative of the presence of residual live virus in the vaccine. Twenty-four animals were used to evaluate immunogenicity and protective efficacy. Seven of these immunized animals produced weak or no anti-HBs responses. Two doses of 50 micrograms HBsAg given subcutaneously 1 month apart protected each of four animals that were challenged with 10(3.5) CID50 HBV at 6 and 12 months after immunization and protected three of four animals challenged at 24 months against development of hepatitis or HBsAg. Three of 4 animals in each group immunized with two doses of 20, 10, or 5 micrograms HBsAg were similarly protected when challenged 6 months after immunization. Thirteen of 20 immunized animals that did not develop HBsAg after challenge with HBV developed anamnestic anti-HBs or anti-HBc responses between 2 and 18 months after challenge, indicating minimal replication of challenge virus. The time of onset and frequency of occurrence of these delayed responses was related to the titer of anti-HBs at the time of challenge. False positive Ausab test results were observed in quarantined chimpanzees. These were neither preceded by appearance of HBsAg nor accompanied by development of anti-HBc. In most cases these reactions were due to a reactant having a sedimentation coefficient and an electrophoretic mobility resembling that of IgM. This reactant generally did not appear to confer resistance to challenge with HBV. The humoral immune response was characterized as being entirely of the IgM class 2 weeks after immunization and switched entirely into the IgG class by 10-12 weeks after vaccine administration. At the time of challenge all animals with antibody had anti-HBs of subtype a.     

Single‐cell based high‐throughput sequencing of full‐length immunoglobulin heavy and light chain genes

Single‐cell PCR and sequencing of full‐length Ig heavy (Igh) and Igk and Igl light chain genes is a powerful tool to measure the diversity of antibody repertoires and allows the functional assessment of B‐cell responses through direct Ig gene cloning and the generation of recombinant mAbs. However, the current methodology is not high‐throughput compatible. Here we developed a two‐dimensional bar‐coded primer matrix to combine Igh and Igk/Igl chain gene single‐cell PCR with next‐generation sequencing for the parallel analysis of the antibody repertoire of over 46 000 individual B cells. Our approach provides full‐length Igh and corresponding Igk/Igl chain gene‐sequence information and permits the accurate correction of sequencing errors by consensus building. The use of indexed cell sorting for the isolation of single B cells enables the integration of flow cytometry and Ig gene sequence information. The strategy is fully compatible with established protocols for direct antibody gene cloning and expression and therefore advances over previously described high‐throughput approaches to assess antibody repertoires at the single‐cell level.