HLA-B宽特异性抗原表位Bw4对HCV特异性T细胞反应影响分析

目的 探讨HLA-B分子宽特异性抗原表位Bw4对丙型肝炎病毒(HCV)特异性T细胞反应的影响.方法 以有偿献血途径感染HCV患者86例为研究对象,采用聚合酶链反应-序列特异性引物(PCR-SSP)方法,进行HLA分型.采用ELISPOT技术观察HCV非结构蛋白NS3、NS4及NS5诱导T细胞分泌IFN-γ反应.结果 86例HCV感染者中Bw4/4纯合子患者29例(33.7％)、Bw4/6杂合子患者38例(44.2％)、Bw6/6纯合子患者19例(22.1％).Bw4/4纯合子、Bw4/6杂合子、Bw6/6纯合子HCV病毒载量分别为(3.98±0.32) Log(IU/ml)、(5.22±0.29) Log(IU/ml)、(5.04±0.38) Log(IU/ml),3组比较,HCV病毒载量差异具有统计学意义(P=0.0153).24例患者进行HCV非结构蛋白(NS3、NS4、NS5)诱导T细胞分泌IFN-γ反应,Bw4/4纯合子组HCV特异性T细胞反应率为50％ (5/10),反应强度中位数为70 SFU/106 PBMC(0～2020 SFU/106 PBMC),非Bw4/4纯合子组中,HCV特异性T细胞反应率为14.28％ (2/14),反应强度中位数为0 SFU/106 PBMC (0～200SFU/106 PBMC).两组比较,Bw4/4纯合子组反应强度明显高于非Bw4/4纯合子组,差异有统计学意义(P=0.0450),前者HCV特异性T细胞反应频率高于后者,差异接近有统计学意义(P=0.069).在Bw4/4纯合子组,HCV以NS5诱导的T细胞反应为主体,其反应频率为50％ (5/10),非Bw4/4纯合子组,NS5反应频率仅为7.14％(2/14),两者比较差异接近具有统计学意义(P=0.050).结论 携带Bw4/4纯合子HCV感染者,病毒载量较低,HCV特异性T细胞反应较强.Bw4可能通过增强HCV特异性T细胞免疫,进而产生对抗HCV复制的作用.     

Induction in vitro of a primary human antiviral cytotoxic T cell response

We report herein the successful priming of human anti-viral cytotoxic T cells (CTL) in vitro using two induction strategies based on the stimulation of peripheral blood mononuclear cells isolated from uninfected donors with synthetic viral peptides. The peptides used contain HLA-A2 binding motifs and have been identified as HLA-A2-restricted CTL epitopes in patients infected by the hepatitis B and C viruses. One approach uses repetitive long-term stimulation and the other uses bulk cultures containing large numbers of naive peripheral blood mononuclear cells. Both approaches successfully induce HLA-A2-restricted CTL specific for several viral epitopes. Some CTL recognize endogenously synthesized antigen on target cells infected with recombinant vaccinia virus expressing the corresponding viral proteins. This simple technique permits easy analysis of the primary human CTL repertoire, and may be exploitable for production of specific CTL effector cells for adoptive immunotherapy and dissection of the cellular and molecular requirements for priming of naive human CTL.     

Long-term T cell memory to human leucocyte antigen-A2 supertype epitopes in humans vaccinated against smallpox

Identification of human leucocyte antigen (HLA) class I-restricted T cell epitopes is important to develop methods to track the evolution of T cell memory to new generation smallpox vaccines and allow comparison to older vaccinia virus preparations known to induce protection against smallpox. We evaluated the relative predictive values of four computational algorithms to identify candidate 9-mer HLA-A2 supertype epitopes that were confirmed to stimulate preferentially T cell interferon (IFN)-gamma responses by subjects last vaccinated with Dryvax 27-54 years previously. Six peptides encoded by I4L, G1L, A8R, I8R, D12L and H3L open reading frames that were identical for Vaccinia (Copenhagen), Variola major (Bangledesh 1975) and modified vaccinia Ankara strain preferentially stimulated IFN-gamma responses by healthy HLA-A2 supertype adults last given Dryvax 27-49 years earlier relative to remotely vaccinated non-HLA-A2 supertype and unvaccinated HLA-A2 supertype adults. Combining results from at least two computational algorithms that use different strategies to predict peptide binding to HLA-A2 supertype molecules was optimal for selection of candidate peptides that were confirmed to be epitopes by recall of T cell IFN-gamma responses. These data will facilitate evaluation of the immunogenicity of replication incompetent smallpox vaccines such as modified vaccinia Ankara and contribute to knowledge of poxvirus epitopes that are associated with long-lived T cell memory.     

Regulatory T-cell response and tumor vaccine-induced cytotoxic T lymphocytes in human melanoma

A role of CD4(+) cells in the regulation of immune responses has steadily gained renewed recognition. The understanding of these T-regulatory (T-reg) cells in the generation of antitumor cytolytic T lymphocyte (CTL) response is therefore important. It has been shown that immunization with specific peptides, DNA, or tumor lysate-based vaccines can induce CTL responses in vivo. We have immunized melanoma patients with major histocompatibility complex (MHC) class I restricted peptide- or melanoma tumor lysate-loaded antigen-presenting cell (APC)-based vaccines and have monitored the generation of CTL responses and T-reg cell responses, if any. Using tetramer staining and limiting dilution analyses as monitors of CTL responses, we found significant increases in the number of antigen-specific CTL in circulation after vaccination with the MART-1(27-35) peptide (AAGIGILTV)-pulsed autologous APC, the MAGE-1(161-169) peptide (EADPTGHSY)-pulsed APC, or with autologous tumor lysate-pulsed APC. The antigen-specific CTL reached the peak expansion by day 7 and then declined to the prevaccine levels by day 28. The decline in the CTL response was associated by a concomitant expansion of CD4(+) CD25(+)T cells. Analysis of postvaccine peripheral blood lymphocytes (PBL) from patients showed an increased amount of interleukin (IL)-10 secretion on in vitro stimulation with IL-2 after successive vaccination. Triple color flow cytometric analyses revealed cytoplasmic IL-10 in the CD4(+)CD25(+) T-cell fraction and the number of CD4(+)CD25(+) IL-10(+) T cells were found to increase significantly in postvaccine PBL. These observations have implications in tumor antigen and APC/dendritic cell (DC)-based cancer vaccine strategies.     

乳腺浸润性导管癌中HPV18、HPV16感染的研究

目的 :了解乳腺浸润性导管癌中HPV18、HPV16的感染情况 ,分析其是否是乳腺癌发生的危险因素及与临床病理的相关性。方法 :根据HPV16、HPV18的DNA序列 ,合成相应特异的寡核苷酸片段 ,用加尾标记法制备地高辛标记探针 ,用原位杂交法检测 5 1例乳腺浸润性导管癌、10例相应正常乳腺上皮及 15例良性乳腺病变中HPV18、HPV16的感染 ,并分析其与患者发病年龄、肿块大小及淋巴结转移的相关性。结果 :浸润性导管癌中HPV18或 16的总阳性率达 70 6 % ,其中HPV18与HPV16的阳性率分别为 5 8 8%、4 5 1% ,均明显高于正常乳腺上皮的感染率 (30 0 %、10 0 % ;P <0 0 5 ) ;乳腺良性病变的HPV18、16阳性率分别是 6 0 0 %、6 0 % ,其中HPV18的阳性率亦显著高于正常乳腺上皮 (P <0 0 5 )。结论 :(1)HPV16和18可能是乳腺浸润性导管癌发生的致病因子 ,HPV18尚可能与乳腺良性病变的发生有关。 (2 )HPV的感染与患者年龄、肿块大小及淋巴结转移无相关性。     

Comparison of the basal and glucocorticoid-inducible activities of the upstream regulatory regions of HPV18 and HPV31 in multiple epithelial cell lines

Steroid hormone receptors have been shown to bind to response elements in the upstream regulatory region (URR) of human papillomavirus (HPV) in a ligand-dependent manner to affect viral promoter activity. To better understand how the enhancer activity of the URR differs between high risk HPV types, we chose to compare the basal and glucocorticoid-dependent activities of the URRs of HPV18 and HPV31. We found that the URR of HPV18 is a stronger enhancer than the URR of HPV31 in six different cell lines of epithelial origin. Furthermore, the activity of the URR of HPV31 was not inducible by the synthetic glucocorticoid dexamethasone (dex) in any cell line tested, while the URR of HPV18 was dex-inducible in the majority of these lines. These studies indicate significant differences between the URRs of high risk HPV types.     

食管鳞状细胞癌中人乳头状瘤病毒感染的意义

探讨人乳头状瘤病毒感染在食管鳞状细胞癌发生发展听意义。方法采用原位杂交和免疫组化ＬＳＡＢ法检测３３例ＥＳＣＣ中的ＨＰＶＤＮＡ，ＨＰＶ属特异性结构抗原和增殖细胞核抗原。     

Detection of HPV 16 and HPV 18 DNA in the blood of patients with cervical cancer

Persistent infection of the uterine cervix with high-risk human papillomaviruses (HPV) is causally associated with cancer of the cervix. A few studies have reported the presence of HPV DNA in the blood of women with cervical neoplasia. The aim of this study was to determine if HPV DNA could be detected in whole blood of women with a range of cervical pathologies and with HPV 16 or 18 cervical infections and if there is a correlation between cervical lesion grade and the appearance of HPV DNA in the circulatory system. Forty-five women with histologically graded cervical cancer were confirmed to have cervical HPV 16 or 18 infections. Eleven (24.4%) of these women had detectable HPV 16 or 18 in their blood. The HPV types detected in the blood matched those detected at the cervix. No HPV 16 or 18 DNA was detected in the blood of 32 women with pre-cursor cervical lesions or normal cervical pathology but who had cervical HPV 16 or 18 infections. One of 77 women with normal cervical pathology and no cervical HPV infection was positive for HPV 16 DNA in her blood. The results indicate that HPV DNA can be detected in the blood of women with more advanced cervical carcinomas but not in the blood of women with pre-cursor cervical lesions. The results of our study indicate that the role of HPV DNA in the circulatory system appears not be of diagnostic significance and HPV DNA is only detectable in women with more advanced cervical cancers.     

Epitope analysis of the T cell response to a complex antigen: Proliferative responses to human rhinovirus capsids

Understanding the factors which regulate the repertoire of a T cell response is important when selecting T helper cell epitopes for inclusion in synthetic viral vaccines. In this study we have examined the T cell response to human rhinovirus (HRV) type 1A in a mouse model system, using a comprehensive set of synthetic peptides which span all four of the proteins which make up the HRV capsid. This constitutes the first study to use a set of peptides covering the entire sequence of all structural proteins of any virus. This study identifies the major proliferative (CD4) T cell epitopes within the minor receptor group HRV 1 A, and analyzes these epitopes with relation to their location within the three-dimensional structure of the virus. The proliferative response to HRV is highly selective, with strong responses to only a very small number of epitopes, many of which are grouped together within restricted areas of the primary structure of the HRV proteins. The repertoire of the response is almost entirely specific to the major histocompatibility complex haplotype of the host. The major T cell epitopes are spatially distinct from the sites of the major antibody recognition sites, and are buried within the viral capsid. In striking contrast to the antibody responses, the T cell responses are highly cross-reactive against a wide variety of viral serotypes.     

Isolation and characterization of human monoclonal antibodies against hepatitis C virus envelope glycoproteins

The isolation and characterization of human monoclonal antibodies (humAbs) against the hepatitis C Virus (HCV) glycoproteins E1 and E2 are described. B-cells from blood donors with anti-HCV were transformed with Epstein-Barr virus. The supernatants of the resulting lymphoblastoid clones were screened by ELISA with an extract of cells infected with a recombinant vaccinia virus RMPA95 expressing the envelope proteins E1 and E2 of an HCV genotype 1a virus (H strain). Positive clones were fused to the heteromyeloma cell line K6H6/B5. Fifteen heterohybridoma cell lines have been established. The specificity of the isolated humAbs was determined both by ELISA and Western blot assays. Several recombinant extracts expressing either the E1 or E2 protein or truncated forms were used in an attempt to map the epitopes on the viral glycoproteins. Some of the humAbs were used successfully for immunofluorescence investigation of transfected cells. Seven specific anti-E2 humAbs, which react with the envelope protein 2 of genotype 1a and 1b isolates, were characterized. J. Med. Virol. 55:28–34, 1998. © 1998 Wiley-Liss, Inc.     

声带息肉中凹空样细胞和HPV的相关性

目的；通过形态学和分子生物学技术来探讨声带息肉与人类乳头状瘤病毒（ｈｕｍａｎ ｐａｐｉｌｌｏｍａ ｖｉｒｕｓ，ＨＰＶ）的关系。方法：对９９例声带息肉ＨＥ染色标本，进行形态学光镜研究；同时，随机选择其中４０例标本采用聚合酶链反应技术行ＨＰＶＤＮＡ检测。结果：９９例标本中有５５例声带息肉复层鳞状上皮棘细胞层中出现凹空样细胞，阳性率为５５．５％。     

Effect of herpes simplex virus on the DNA of human papillomavirus 18

Some types of human papillomaviruses (HPV) appear to be associated with carcinoma of the cervix or other tissues, but patients infected with HPV do not necessarily develop carcinoma. Some epidemiological studies of risk factors for cervical carcinoma have indicated the involvement of herpes simplex virus (HSV). To study the effect of HSV on the genome of HPV, total DNAs were extracted and analyzed after HeLa cells, or A431 cells, transiently transfected with HPV18 DNA, were infected with HSV-1 or -2 for 24 hours. In HeLa cells, integrated HPV18 DNA was amplified almost threefold. In A431 cells, HPV 18 DNA fragments, sensitive to the restriction enzyme Mbo I, indicated newly replicated DNA. Replication intermediates were detected when the DNA was resolved by two-dimensional gel electrophoresis. This study showed that HSV caused some amplification of HPV and indicated the possibility of HSV involved in the integration and amplification of HPV in host cells. J. Med. Virol. 53:4–12, 1997. © 1997 Wiley-Liss, Inc.     

Epitope specific T‐cell responses against influenza A in a healthy population

Pre‐existing human CD4⁺ and CD8⁺ T‐cell‐mediated immunity may be a useful correlate of protection against severe influenza disease. Identification and evaluation of common epitopes recognized by T cells with broad cross‐reactivity is therefore important to guide universal influenza vaccine development, and to monitor immunological preparedness against pandemics. We have retrieved an optimal combination of MHC class I and class II restricted epitopes from the Immune Epitope Database ( www.iedb.org ), by defining a fitness score function depending on prevalence, sequence conservancy and HLA super‐type coverage. Optimized libraries of CD4⁺ and CD8⁺ T‐cell epitopes were selected from influenza antigens commonly present in seasonal and pandemic influenza strains from 1934 to 2009. These epitope pools were used to characterize human T‐cell responses in healthy donors using interferon‐γ ELISPOT assays. Upon stimulation, significant CD4⁺ and CD8⁺ T‐cell responses were induced, primarily recognizing epitopes from the conserved viral core proteins. Furthermore, the CD4⁺ and CD8⁺ T cells were phenotypically characterized regarding functionality, cytotoxic potential and memory phenotype using flow cytometry. Optimized sets of T‐cell peptide epitopes may be a useful tool to monitor the efficacy of clinical trials, the immune status of a population to predict immunological preparedness against pandemics, as well as being candidates for universal influenza vaccines.     

Induction of human papillomavirus type 16-specific immunologic responses in a normal and an human papillomavirus-infected populations

Human papillomavirus (HPV) infection, especially with the oncogenic genotypes, is the most important risk factor for developing cervical cancer. We focused on generating HPV16 E7-specific cytotoxic CD8(+) T lymphocytes and evaluating HPV16 E7-specific immune responses in HPV16-infected and uninfected populations. Peripheral blood mononuclear cells (PBMCs) were first collected from an uninfected group with an human lymphocyte antigen (HLA) A2 haplotype (four volunteers). Mature monocyte-derived dendritic cells (DCs) were generated from the PBMCs and pulsed with one of two HLA-A2-restricted E7 peptides, aa 11-20 [YMLDLQPETT] and aa 86-93 [TLGIVCPI], as antigen presenting cells. The autologous naive or cultured PBMCs were then cultured with peptide-pulsed DCs to detect the HPV16 E7-specific immune responses by a variety of techniques such as enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISPOT) assay and cytotoxic T lymphocyte assay. Interferon-gamma (IFN-gamma) from E7-specific cytotoxic CD8(+) T lymphocytes stimulated with the respective peptide was detected by ELISA. Using ELISPOT analysis, a marked increase in the number of IFN-gamma-secreting CD8(+) E7-specific lymphocytes was observed following peptide stimulation. Cultured CD8(+) T lymphocytes were highly cytotoxic against the CaSki cells. PBMCs were then collected from an HPV16-infected population of the HLA-A2 haplotype, including four persons of HPV16 infection only, four with cervical intraepithelial neoplasia (CIN) lesions, and four cervical cancer patients. We then compared the immunologic responses to E7 between HPV16-infected and uninfected populations by ELISA and ELISPOT assay. The E7-specific immunologic responses of the HPV16-infected populations were significantly higher than those of the uninfected population. In addition, persons with an HPV16 infection only or those with CIN lesions generated higher E7-specific immunologic responses than cervical cancer patients. Our results demonstrate methods for evaluating E7-specific immunologic responses and reflect the biological responses of HPV16-infected people during different periods of cervical disease.     

Sourcing of the WHO human papillomavirus type 18 international standards for HPV antibody levels

BackgroundHPV serology is important for studies of vaccine immunogenicity, but can not be performed in a comparable manner without international standardisation.ObjectivesTo find suitable candidate sera from naturally infected persons for use as International Standards (IS) for antibodies to high-risk HPVs, with priority for HPV-18.Study design946 healthy Thai women (median age 44, range 18–83) and 61 cervical cancer patients were screened using an HPV pseudovirion-Luminex assay to detect antibodies to genital (HPV-6,-11,-16,-18,-31,-33,-45,-52,-58,-68) and non-genital HPV types (HPV-5,-15,-32,-38 and -76). Suitable candidate sera should ideally be mono-specific (have reactivity against only one genital HPV) and have high antibody levels that are stable over time.ResultsSeroprevalences of HPV-16,-31,-52 and -58 were at least twice as high among cancer patients compared to healthy individuals. Thirteen healthy women who met the IS inclusion criteria in initial testing also consented to blood-bag donations. Donations from 2 women with high HPV-18 Ab titers were pooled to the HPV-18 candidate IS, later established as the WHO official IS for HPV antibodies. Sera that could potentially be used as candidate IS for other oncogenic HPVs have also been identified.ConclusionsIn the Thai population, seroepidemiology implicated HPV types HPV-16,-31,-52 and -58 as particularly associated with cervical cancer. A well characterized cohort study has allowed sourcing of materials for an IS for HPV-18 antibodies and could conceivably be used for IS for other HPV types as well.