LOS诱导的特异性抗体分泌细胞的ELISPOT法检测

目的 :动态测定卡他性莫拉氏菌 (Moraxellacatarrhalis,M .cat)脱毒脂寡糖 (dLOS)蛋白质结合疫苗诱导的抗体分泌细胞的应答状态。方法 :以M .catdLOS蛋白质结合疫苗滴鼻免疫BALB/c小鼠。应用酶联免疫斑点试验 (ELISPOT)检测免疫小鼠不同免疫诱导部位和免疫效应部位 ,包括 :鼻相关淋巴组织 (NALT)、脾脏、颈部淋巴结、鼻内容物、肺脏和派伊尔氏结的特异性抗体分泌细胞 ,并同时测定血清、鼻冲洗液、肺泡灌洗液、唾液及粪便提取液中特异性IgA、IgG和IgM的水平。结果 :M .catdLOS蛋白质结合疫苗免疫小鼠的NALT、脾脏、颈部淋巴结、鼻内容物、肺脏和派伊尔氏结中 ,均测出分泌LOS特异抗体的抗体分泌细胞 ,以鼻内容物中IgA分泌细胞的数目最多 ,其次是在NALT和肺脏中 ,这与特异性抗体测定的结果相一致。结论 :M .catdLOS蛋白质结合疫苗经滴鼻免疫 ,能刺激产生LOS特异的黏膜和全身抗体分泌细胞的应答。ELISPOT试验具有快速、灵敏、特异的优点 ,为动态分析单个抗体分泌细胞应答规律提供了新方法。     

Cellular immunity induced by the recombinant Plasmodium falciparum malaria vaccine, RTS,S/AS02, in semi-immune adults in The Gambia

Vaccination of malaria-naive humans with recombinant RTS,S/AS02, which includes the C-terminus of the circumsporozoite protein (CS), has been shown to induce strong T cell responses to both the whole protein antigen and to peptides from CS. Here we show that strong T cell responses were also observed in a semi-immune population in The Gambia, West Africa. In a Phase I study, 20 adult male volunteers, lifelong residents in a malaria-endemic region, were given three doses of RTS,S/AS02 at 0, 1 and 6 months. Responses to RTS,S, hepatitis B surface antigen and peptides from CS were tested using lymphocyte proliferation, interferon (IFN)-gamma production in microcultures, and IFN-gamma ex vivo and cultured ELISPOT, before and after vaccination. Cytotoxic responses were tested only after vaccination and none were detected. Before vaccination, the majority of the volunteers (15/20) had detectable responses in at least one of the tests. After vaccination, responses increased in all assays except cytotoxicity. The increase was most marked for proliferation; all donors responded to RTS,S after the third dose and all except one donor responded to at least one peptide after the second or third dose. There was a lack of close association of peptide responses detected by the different assays, although in microcultures IFN-gamma responses were found only when proliferative responses were high, and responses by cultured ELISPOT and proliferation were found together more frequently after vaccination. We have therefore identified several peptide-specific T cell responses induced by RTS,S/AS02 which provides a mechanism to investigate potentially protective immune responses in the field.     

Clinical and immunological efficacy of intradermal vaccine plus lamivudine with or without interleukin-2 in patients with chronic hepatitis B

To evaluate therapeutic immunostimulation nine chronic hepatitis B patients received six monthly intradermal vaccinations with HBsAg in combination with daily lamivudine. Another five patients received six doses of the vaccine and daily lamivudine together with daily Interleukin-2 (IL-2) s.c. within 3 months in an open-labeled trial. Clinical efficacy was assessed by alanine transaminase levels and HBV serology. The induction of specific T and B cell responses was analyzed serially by 3H-thymidine uptake, ELISA and ELISPOT assays. After the therapy was stopped, seven of nine vaccine/lamivudine and two of five vaccine/lamivudine/IL-2 recipients did not have detectable HBV DNA. Four complete responders cleared the virus and had normalized ALT levels, however, one of these patients showed transient disease reactivation followed by spontaneous viral clearance and normal ALT five months later. Low frequencies of anti-HBs producing B cells and HBV specific T helper cells secreting predominantly interferon-gamma were induced by i.d. vaccine therapy. The ELISPOT technique demonstrated transient induction of HBV peptide specific cytotoxic T cells in seven HLA-A2 positive chronic HBV carriers. The preliminary data from this study demonstrate that the HBV surface antigen vaccine in combination with antiviral or immunomodulating drugs induced antiviral immune responses and consequently viral elimination may be achieved in patients with unfavorable prognosis.     

几种不同检测方法对结核性胸膜炎诊断价值分析

目的探讨几种不同检测方法对结核性胸膜炎诊断价值。方法检测47例胸腔积液患者,其中37例经组织病理学或病原学明确诊断的结核性胸膜炎,10例诊断为恶性胸水。检测全部患者胸水中ADA活性、抗结核分枝杆菌抗体、结核分支杆菌特异性ELISPOT检测,经统计学处理后,评价各项指标对结核性胸膜炎诊断的灵敏度、特异度及临床诊断符合率。结果结核性胸水中腺苷脱氨酶（ADA）活性、结核分枝杆菌抗体,结核分支杆菌特异性ELISPOT检测,与恶性胸水组比较差别有统计学意义,P〈0.05。以45U/L为临界值,胸水中ADA对结核性胸膜炎诊断的灵敏度为70.2%（26/37）,特异度45%（9/20）,临床诊断符合率为74.5%（35/47）;胸水中结核分枝杆菌抗体检测对结核性胸膜炎诊断的灵敏度为62.2%（23/37）,特异度为33.3%（7/21）,临床诊断符合率为63.8%（30/47）;以胸水中致敏T淋巴细胞含量40个为临界值,对结核性胸膜炎诊断的灵敏度为97.3%（36/37）,特异度为90%（9/10）。临床诊断符合率为95.7%（45/47）。结论胸水中ADA活性、结核分枝杆菌抗体检测可提高结核性胸膜炎的诊断效能,而结核分支杆菌特异性ELISPOT检测可以达到更好的快速诊断效果。     

Evaluation of the interferon-γ ELISPOT-assay for quantification of peptide specific T lymphocytes from peripheral blood

ELISPOT assays for detection of antigen specific HLA class I restricted T lymphocytes have recently been developed. Reliable quantitation of T cell frequencies is crucial for the monitoring of specific cancer vaccines. We have evaluated the accuracy of quantitative results obtained with the IFN-γ ELISPOT assay and both sensitivity and specificity obtained with several pairs of antibodies was compared. The detection system was tested with IFN-γ coupled latex beads and the quantitation of influenza specific T cells with several sets of dilution experiments. The reproducibility of the quantitative results was established. Only one pair of monoclonal antibodies had an acceptable specificity. In a serial dilution, almost 100% of IFN-γ-coated beads could be detected. Furthermore, the number of influenza-peptide specific spots correlated closely with (a) the number of antigen specific T cells derived from an influenza-peptide specific T cell line diluted in PBMC, and (b) the number of CD8+ T cells diluted in autologous CD8-depleted PBMC. In three healthy individuals and three cohorts of healthy volunteers, the number of influenza-peptide specific spots was highly reproducible. The data presented here demonstrate that the frequency of peptide specific CD8+ T cells can be reliably determined from peripheral blood with the IFN-γ ELISPOT assay.     

Monitoring of cellular responses after vaccination against tetanus toxoid: comparison of the measurement of IFN-gamma production by ELISA, ELISPOT, flow cytometry and real-time PCR

One of the challenges for immunomonitoring in clinical trials is to detect an antigen specific T cell-mediated immune response. In an attempt to define the most suitable assay, tetanus toxoid was used to compare the capacity of 4 different methods to detect cytokine responses, before and after recall vaccination, in peripheral blood mononuclear cells (PBMC) of 14 healthy volunteers. ELISA, ELISPOT, intracytoplasmic detection and real-time RT-PCR were chosen to measure IFN-gamma production before and after vaccination. As far as the detection of memory T cell status (before vaccination) was concerned, we found that ELISPOT was the most sensitive method to discriminate TT-induced from spontaneous responses. On the other hand, intracytoplasmic cytokine detection was the most efficient method to detect the restimulating effect of TT vaccination.     

Poorly soluble peptides can mimic authentic ELISPOT responses

The ELISPOT assay is a specific, sensitive, quantitative assay for assessing cell-mediated immune responses to a variety of antigens including HIV-1 peptides. In an interferon (IFN)-gamma-ELISPOT assay, peripheral blood mononuclear cells (PBMC) from two HIV-1 exposed seronegative (ESN) individuals appeared to respond strongly to an HIV Gag peptide. Analysis of this peptide revealed that it was incompletely dissolved and induced non-specific spot formation, even in the absence of cells. In subsequent experiments, the peptide was found to interact with avidin and the ELISPOT membrane. Filtering the peptide prevented non-specific spot formation. These findings underscore the need for appropriate controls and proper peptide preparation in order to reduce the risk of false-positive ELISPOT responses.     

Skin reactions to human papillomavirus (HPV) 16 specific antigens intradermally injected in healthy subjects and patients with cervical neoplasia

We have tested the safety and feasibility of a synthetic long peptide-based HPV16-specific skin test to detect cellular immune responses to HPV16 E2, E6 and E7 in vivo. Women with cervical neoplasia (n = 11) and healthy individuals (n = 19) were intradermally challenged with 8 different pools of HPV16 E2, E6 and E7 peptides. The skin test was safe as the injections were perceived as mildly painful and no adverse events were observed. The majority of skin reactions appeared significantly earlier in HPV16+ patients (<8 days) than in healthy subjects (8-25 days). The development of late skin reactions in healthy subjects was associated with the appearance of circulating HPV16-specific T cells and the infiltration of both HPV16-specific CD4+ Th1/Th2 and CD8+ T cells into the skin. These data show that the intradermal injection of pools of HPV16 synthetic long peptides is safe and results in the migration of HPV16-specific T cells into the skin as well as in an increase in the number of circulating HPV16-specific T cells. The use of this test to measure HPV16-specific immunity is currently tested in a low resource setting for the measurement of spontaneously induced T-cell responses as well as in our HPV16 vaccination trials for the detection of vaccine-induced immunity.     

Polyclonal B cell activation for accurate analysis of pre‐existing antigen‐specific memory B cells

The enzyme‐linked immunospot (ELISPOT) assay is a widely used tool for enumeration of antigen‐specific memory B cells in several disciplines, such as vaccination, cancer immunotherapy and transplantation. For the accurate estimation of antigen‐specific memory B cell frequencies, a well‐defined B cell activation protocol is pivotal. In this study, we aimed to characterize a polyclonal B cell activation protocol to facilitate optimal monitoring of antigen‐specific memory B cell frequencies. Total, naive and memory B cells were activated polyclonally with an α‐CD40 monoclonal antibody, cytosine–phosphate–guanine (CPG) oligodeoxynucleotide (ODN) 2006, interleukin (IL)‐2, IL‐10 and IL‐21. Polyclonal activation of B cells resulted in equal cell death ratios in naive and memory B cells. When tested in an antigen‐specific system, immunoglobulin (Ig)G spots were detected only in the memory fraction. There was no change in B cell polyclonality due to in‐vitro activation. Our data show that the current polyclonal activation protocol may be used reliably to estimate the frequency of memory B cells in ELISPOT assays.