Analysis of relationships between polymorphisms in the genes encoding the pentameric complex and neutralization of clinical cytomegalovirus isolates

IntroductionAs congenital cytomegalovirus (CMV) infection is one of the major causes of birth defects and developmental abnormalities, it is essential to develop vaccines and therapeutic antibodies against CMV. Clinical trials demonstrated that the subunit vaccine based on glycoprotein B, which had been believed to be the major target for neutralization, did not induce sufficient protective immunity. On the other hand, it has been reported that the immunization of animals with the Pentamer, the pentameric complex of gH/gL/UL128/UL130/UL131A, induced strong neutralizing antibodies. Here, we sought to clarify whether any polymorphic alterations present in the Pentamer of clinical isolates affect neutralization by anti-Pentamer antibodies.MethodsSequences of the genes encoding the Pentamer components of 25 Japanese clinical isolates were determined. Neutralization of infection by two seropositive sera and by anti-Pentamer serum was measured using a CMV reporter cell line based on ARPE-19.ResultsPolymorphisms of the amino acid sequence of UL128, UL130, and UL131A ORFs were limited and clustered into two major groups. The identified alterations, except UL128 I140T, were mapped outside of the reported regions recognized by neutralizing antibodies. Anti-Pentamer serum neutralized infection with all isolates to a similar degree and had no correlation with the polymorphic groups.ConclusionsOur findings indicate that Pentamer antigens prepared from Merlin Fix strain induce antibodies that neutralize infection with all isolates to a similar level and that anti-Pentamer antibodies neutralize CMV infection better than do human sera, suggesting that vaccines and therapeutic antibodies based on Pentamer as an antigen have some promise.     

乙型肝炎患者特异性CTL细胞数量的研究

目的 检测我国北方地区乙型肝炎患者表位特异性CTL细胞反应水平,研究急性自限性和慢性持续性乙型肝炎患者体内免疫反应的差异。方法 使用HBV抗原表位肽五聚体（HBcAg18-27）对急性和慢性乙型肝炎感染患者外周血染色后,用流式细胞仪检测HBV特异性CTL细胞。结果 在20例急性乙型肝炎的急性期,可以检测到高水平的HBcAg特异性的CTL细胞,而在急性乙型肝炎的恢复期,HBcAg特异性CTL细胞的水平可以明显下降。在20例慢性乙型肝炎患者中,可以检测到低水平的HBcAg特异性的CTL细胞。结论 HBcAg特异性CTL细胞在急性肝炎患者清除乙型肝炎病毒的过程中可能起到至关重要的作用。     

抗原肽HPV18 E77-15诱导产生特异性细胞毒T细胞的实验研究

目的探讨HLA—A2限制性表位HPV18E77—15的免疫原性。方法对抗原肽HPV18E7-15进行T2结合分析;通过体外细胞培养技术抗原肽诱导特异性CTL扩增;采用RPE标记的表位特异性五聚体和FITC标记的CD8＋抗体通过流式细胞仪检测抗原肽所诱导产生的特异性CTLs。结果抗原肽HPVl8E77—15可将T2细胞表面HLA—A＊0201分子的表达提高2．6倍;在流式细胞仪检测中,抗原肽HPV18E77—15刺激诱导产生CD8＋五聚体＋双阳性的抗原肽特异性CTLs为1．87％。结论抗原肽HPV18E77—15能够诱导HLA—A2阳性正常人外周血淋巴细胞产生特异性CTLs,表明HPV18E77-15具有一定的免疫原性。     

Vaccination against the human cytomegalovirus

The human cytomegalovirus (HCMV) is the most important infectious cause of congenital abnormalities and also of infectious complications of transplantation. The biology of the infection is complex and acquired immunity does not always prevent reinfection. Nevertheless, vaccine development is far advanced, with numerous candidate vaccines being tested, both live and inactivated. This article summarizes the status of the candidate vaccines.     

Structural and biochemical studies of HCMV gH/gL/gO and Pentamer reveal mutually exclusive cell entry complexes

Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and the leading viral cause of birth defects after congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are key targets of the human humoral response against HCMV and are required for HCMV entry into fibroblasts and endothelial/epithelial cells, respectively. We expressed and characterized soluble forms of gH/gL, gH/gL/gO, and Pentamer. Mass spectrometry and mutagenesis analysis revealed that gL-Cys144 forms disulfide bonds with gO-Cys351 in gH/gL/gO and with UL128-Cys162 in the Pentamer. Notably, Pentamer harboring the UL128-Cys162Ser/gL-Cys144Ser mutations had impaired syncytia formation and reduced interference of HCMV entry into epithelial cells. Electron microscopy analysis showed that HCMV gH/gL resembles HSV gH/gL and that gO and UL128/UL130/UL131A bind to the same site at the gH/gL N terminus. These data are consistent with gH/gL/gO and Pentamer forming mutually exclusive cell entry complexes and reveal the overall location of gH/gL-, gH/gL/gO-, and Pentamer-specific neutralizing antibody binding sites. Our results provide, to our knowledge, the first structural view of gH/gL/gO and Pentamer supporting the development of vaccines and antibody therapeutics against HCMV.Human cytomegalovirus (HCMV) is a member of the β-herpesvirus subfamily with >60% seropositivity in adults worldwide (1). HCMV infection is typically asymptomatic, but can cause severe disease or death in immunocompromised solid organ and hematopoietic stem cell transplant recipients. In addition, HCMV can infect the placenta and cross this barrier to infect developing fetuses, causing severe birth defects (2). Given the severity and importance of this disease, obtaining an effective vaccine is considered a public health priority (3).The ability of HCMV to cause disease in a wide range of organs and tissue types is reflected at the cellular level by the virus infecting epithelial cells, endothelial cells, fibroblasts, dendritic cells, hepatocytes, neurons, macrophages, and leukocytes (4). Similar to other herpesviruses, the envelope glycoproteins gB and gH/gL form the conserved fusion machinery required for viral entry (5, 6). Recent structural and mutagenesis analysis suggested that gB is responsible for mediating virus and host membrane fusion during viral entry (7, 8). The role of gH/gL in fusion is less clear because crystal structures of herpes simplex virus 2 (HSV-2), pseudo-rabies virus (PrV), and Epstein–Barr virus (EBV) gH/gL did not reveal any similarity to known viral fusion proteins (9–11). It has been proposed that gH/gL is involved in the entry process through activation of gB (12). In addition to gB and gH/gL, most herpesviruses encode additional glycoproteins that are able to interact with gH/gL and are capable of either mediating binding to specific cellular receptors or regulating the activity of the gH/gL–gB complex (5, 6).HCMV entry into both epithelial and endothelial cells requires a pentameric glycoprotein complex (Pentamer) formed between gH/gL and the UL128, UL130, and UL131A proteins (13, 14). Mutations in the UL131A–UL128 gene locus are sufficient to eliminate epithelial/endothelial tropism and occur spontaneously within only a few passages of wild-type (WT) HCMV in fibroblasts (15, 16). In addition, Pentamer cell surface overexpression interferes with HCMV entry into epithelial cells, but not into fibroblasts, suggesting the presence of a cell-type-specific Pentamer receptor (17).HCMV entry into fibroblasts is mediated by the gH/gL/gO complex at the cell surface at neutral pH (18–21). gO is a highly glycosylated protein and has been shown to covalently interact with gH/gL (22, 23). It has been proposed that gO might function as a molecular chaperone to promote gH/gL incorporation, but not gH/gL/gO, into the virion (21). However, it has been recently demonstrated that gH/gL/gO and Pentamer are much more abundant on the HCMV envelope than gH/gL alone (24).Highly potent HCMV-neutralizing monoclonal antibodies were isolated from the memory B-cell repertoire of HCMV-immune donors and shown to bind the Pentamer. These antibodies were capable of neutralizing HCMV infection of epithelial/endothelial cells, but not fibroblasts (25, 26). In addition, several studies have demonstrated that the Pentamer is the main target of the neutralizing humoral response to HCMV infection in epithelial/endothelial cells (27–29). Consistent with these observations, immunization with the Pentamer has been shown to elicit a strong neutralizing antibody response in mouse, rabbit, and rhesus macaque models (30–32). Together these data indicate that the Pentamer represents a key antigenic target for vaccine development against HCMV infection.Here we report the purification and biochemical characterization of HCMV gH/gL, gH/gL/gO, and Pentamer. In addition, we describe the architecture of these complexes by electron microscopy (EM) and characterize their interaction with MSL-109, a previously described HCMV-neutralizing antibody isolated from the spleen of a HCMV-seropositive individual (33, 34). Our data provide new insights into the structure and function of the HCMV gH/gL/gO and Pentamer complexes.     

Programmed death-1 expression is associated with the disease status in hepatitis B virus infection

AIM： TO define the potential role of programmed death-i/programmed death-ligand （PD-1/PD-L） pathway in different hepatitis B virus （HBV） infection disease status; we examined the expression of PD-1 on antigen specific CD8＋T cells in peripheral blood of patients with chronic hepatitis B （CriB） and acute exacerbation of hepatitis B （AEHB） infection.
METHODS： The PD-1 level on CD8＋ T lymphocytes and the number of HBV specific CD8＋ T lymphocytes in patients and healthy controls （HCs） were analyzed by staining with pentameric peptide-human leukocyte antigen2 （HLA2） complexes combined with flow cytometry. Real-time quantitative polymerase chain reaction （PCR） was used to measure the serum HBV- DNA levels.
RESULTS： The level of PD-1 expression on total CD8＋ T cells in CHB patients （13.86% ± 3.38%） was significantly higher than that in AEHB patients （6.80%± 2.19%, P 〈 0.01） and healthy individuals （4.63% ± 1.23%, P 〈 0.01）. Compared to AEHB patients （0.81% ± 0.73%）, lower frequency of HBV-specific CD8＋ T cells was detected in chronic hepatitis B patients （0.37% ± 0.43%, P 〈 0.05）. There was an inverse correlation between the strength of HBV-specific CD8＋ T-cell response and the level of PD-1 expression. Besides, there was a significant positive correlation between HBV viral load and the percentage of PD-1 expression on CD8＋ T cells in CriB and AEHB subjects （R = 0.541, P 〈 0.01）. However, PD-1 expression was not associated with disease flare-ups as indicated by alanine aminotransferase （ALT） levels （R = 0.066, P 〉 0.05）.
CONCLUSION： Our results confirm previous reports that HBV specific CD8＋T-cell response in the peripheral blood is more intense in patients with AEHB than in chronic hepatitis B wlth persistent viral infection. Moreover, there is a negative correlation between the level of PD-1 and the intensity of virus specific CD8＋ T cell response.     

生殖器疱疹患者HSVⅡ抗原CD8^＋细胞毒性T细胞检测

目的研究生殖器疱疹患者外周血中HSVⅡ抗原特异性CD8^＋细胞毒性T细胞（CTL）在发病中的作用。方法采用荧光标记MHCⅠ类分子-多肽五聚体技术，以流式细胞仪定量检测20例生殖器疱疹患者初发和复发双份外周血中HSVⅡ抗原特异性CD8^＋细胞毒性T细胞。并进行比较分析。结果生殖器疱疹患者初发和复发双份外周血中HSVⅡ抗原特异性CD8^＋细胞毒性T细胞百分数分别为0．73％±0．29％和0．35％±0．24％，正常对照组为0.31％±0．27％，初发组与复发组、正常对照两组结果相比较P〈0．001；复发组和正常对照组结果相比较P〉0．05。结论初发生殖器疱疹患者外周血HSVⅡ抗原特异性CD8^＋细胞毒性T细胞活性增强，复发时患者CTL的活性减弱。五聚体技术具有快速、敏感、高效和特异的特点。     

Cholera toxin B subunit pentamer reassembled from Escherichia coli inclusion bodies for use in vaccination

The cholera toxin B subunit (CTB) is secreted in its pentameric form from Escherichia coli if its leader peptide is replaced with one of E. coli origin. However, the secretion of the pentamer is generally severely impaired when the molecule is mutated or fused to a foreign peptide. Therefore, we attempted to regenerate pentameric CTB from the inclusion bodies (IBs) of E. coli. Stepwise dialysis of the IBs solubilized in guanidine hydrochloride predominantly generated soluble high-molecular-mass (HMM) aggregates and only a small fraction of pentamer. Three methods to reassemble homogeneous pentameric molecules were evaluated: (i) using a pentameric coiled-coil fusion partner, expecting it to function as an assembly core; (ii) optimizing the protein concentration during refolding; and (iii) eliminating contaminants before refolding. Coiled-coil fusion had some effect, but substantial amounts of HMM aggregates were still generated. Varying the protein concentration from 0.05 mg/mL to 5 mg/mL had almost no effect. In contrast, eliminating the contaminants before refolding had a robust effect, and only the pentamer was regenerated, with no detectable HMM aggregates. Surprisingly, the protein concentration at refolding was up to 5 mg/mL when the contaminants were removed, with no adverse effects on refolding. The regenerated pentamer was indistinguishable in its biochemical and immunological characteristics from CTB secreted from E. coli or choleragenoid from Vibrio cholerae. This study provides a simple but very efficient strategy for pentamerizing CTB with a highly homogeneous molecular conformation, with which it may be feasible to engineer CTB derivatives and CTB fusion antigens.     

Repeat-directed isolation of a novel gene preferentially expressed from the maternal allele in human placenta

Using a repetitive sequence of tandemly arrayed pentanucleotides in the human H19 3′-flanking region, we isolated a phage clone (λ PEN11) which localized to chromosome 11p15.5. The λ PEN11 phage encodes a 2.3-kb cDNA consisting of seven exons at least. The gene was mainly expressed in brain and pancreas (and less abundantly in testis), and demonstrated differential allele usage, with maternal expression being predominant in placenta, which indicates the gene is an atypical imprinted gene. While the pentamer repeat might contribute to this effect, it is also possible that the differential allele usage might reflect the local chromosomal structure known as the imprinting domain. Received: July 24, 1998 / Accepted: August 31, 1998     

Multimer monitoring of CMV-specific T cells in research and in clinical applications

Multimer monitoring has become a standard technique for detection of antigen-specific T cells. The term “multimer” refers to a group of reagents based on the multimerisation of molecules in order to raise avidity and thus stabilize binding to their ligand. Multimers for detection of antigen-specific T-cell responses are based on major histocompatibility complex class I peptide complexes. Multimer staining enables fast and direct visualization of antigen-specific T cells; thus, it is widely applied to assess antiviral immunity, e.g., monitor patients in vaccination trials or confirm purity of cell products for adoptive transfer. Assessment of T-cell immunity against persistent pathogens like cytomegalovirus (CMV) is of major importance in immunosuppressed patients. Recent advancements of multimers facilitate reversible labeling and allow isolation of epitope-specific T cells for adoptive transfer. Here, we give an overview on the different multimers and their applications, with an emphasis on CMV-specific T-cell responses.