Little role of anti-gB antibodies in neutralizing activity of patient's sera with human cytomegalovirus (HCMV) infection

Human cytomegalovirus (HCMV) gB is known to play important roles in cell surface attachment, virion penetration, spread of infection from cell to cell, and provocation of neutralizing antibody. This study was performed to determine the role of anti-HCMV gB antibody in overall neutralizing response in patients with HCMV infection and healthy control with past infection. HCMV gB was stably expressed in 293 cells. With the stable cell line expressing gB as a specific immunosorbent, anti-gB antibody was removed from the current and past HCMV-infected sera and the remaining neutralizing activity was measured by plaque assay. It was shown that 19-50% of the total virus-neutralizing activity of sera with past HCMV infections was derived from anti-gB antibody, but anti-gB antibody had little effect on the total serum virus-neutralizing activity in patients currently infected with HCMV. This result suggests that neutralizing antibody to HCMV gB may reflect disease status.     

Human cytomegalovirus pp65 lower matrix protein: a humoral immunogen for systemic lupus erythematosus patients and autoantibody accelerator for NZB/W F1 mice

Both the infection of human cytomegalovirus (HCMV) and the immunization of its recombinant glycoprotein (gB) in mice have been known to induce autoimmunity, resulting in symptoms similar to those of human systemic lupus erythematosus (SLE). Research has also found that the murine cytomegalovirus (MCMV)-specific monoclonal antibody (mAb) is able to react with a human U1-70K-like autoantigen. To investigate HCMV involvement in autoimmunity, we analysed the humoral responses to HCMV by autoimmune patients and normal adults. Our studies show unambiguously that sera from SLE patients exhibited an elevated IgG titre to HCMV when compared with those observed in controls and other connective tissue disease (CTD) patients (P < 0.001). The IgM titres to HCMV and IgG to HBV were evaluated, and no significant differences were noted among all testing groups. In addition to initiating T cell activity, as reported by many investigators, we found that the HCMV pp65 antigen (also known as lower matrix protein) was able to induce humoral responses in SLE patients. Immunoblot assays showed that 82.56% of sera from SLE patients reacted with the HCMV pp65 antigen, but only 11.11%, 23.53% and 31.17% of patients from normal control, rheumatoid arthritis (RA) and CTD patients, respectively, reacted to it. Unlike HCMV pp65, HCMV pp150 induced B cell activity in most collected sera (92.22%-98.04%). Finally, female NZB/W F1 mice immunized with plasmids encoding HCMV pp65 open reading frame (pcDNApp65) developed an early onset of autoantibody activity and more severe glomerulonephritis. Thus, we conclude that the HCMV pp65 antigen triggers humoral immunity in SLE patients and autoimmune-prone mice and that it could very well exacerbate the autoimmune responses in susceptible animals.     

Functional antibody response to human cytomegalovirus in immunocompetent and HIV-1 infected individuals with antibodies that inhibit virus penetration into cells and intercellular transmission of viral infection.

Antibodies mediating post-attachment virus neutralisation (PN), inhibition of human cytomegalovirus (HCMV)-induced cell fusion in the glioblastoma cell line U373 (IF) and global neutralising activity (NA) were quantified in sera from healthy immunocompetent individuals, asymptomatic HIV-1-infected subjects and AIDS patients to further characterise the neutralising antibody response to HCMV in these population groups and to assess whether HIV-1-infected individuals exhibited an abnormal functional antibody profile. PN and IF antibodies accounted for a minor fraction of the NA activity of sera from all population groups. Sera from HIV-1-infected individuals (particularly AIDS patients) displayed higher levels of PN and IF antibodies than those from the healthy control group; however, the relative contribution of these antibodies to the global serum NA activity appeared to be lower in the former individuals than in immunocompetent controls. Serum antibodies preventing HCMV cell-to-cell spread (IP) were then measured to determine whether a specific deficiency could be detected in the HIV-1-infected group population. Serum IP antibody titres were significantly higher in HIV-1-infected individuals (particularly in AIDS patients) than in controls. The potential implications of the data for explaining the pathogenesis of HCMV infection are discussed.     

Lack of association between the kinetics of human cytomegalovirus (HCMV) glycoprotein B (gB)-specific and neutralizing serum antibodies and development or recovery from HCMV active infection in patients undergoing allogeneic stem cell transplant

The kinetics of the gB-specific and neutralizing antibody responses to human cytomegalovirus (HCMV) were analyzed in 26 allogeneic stem-cell transplant recipients who either did (n = 20) or did not (n = 6) develop asymptomatic HCMV active infection during the study period. Antibody response profiles varied widely among individuals in both groups, irrespective of whether HCMV active infection did or did not occur. Development of HCMV active infection was not preceded by a decline in functional serum antibody levels. Neither the absence nor the presence of HCMV active infection correlated with either high or low serum levels of gB-specific and neutralizing antibodies, respectively. In most patients, episodes of HCMV replication were not followed by a marked increment in functional serum antibody titers. Therefore, resolution of an ongoing HCMV active infection was not associated with a vigorous antibody response to viral replication. In addition, this study supports previous data indicating that passive transfer of human immunoglobulins may result in an increment in gB-specific and neutralizing serum antibody levels, the magnitude of which varies among recipients; however, both patients with and without measurable increments in serum antibody levels developed HCMV active infections with comparable frequency.     

原发性高血压患者巨细胞病毒感染及与血管并发症关系 …

目的 探讨原发性高血压患者巨细胞病毒感染及与血管并发症之间的关系。方法 应用间接酶联免疫吸附试验（ＥＬＩＳＡ）检测了１０５例原发性高血压患者血清人巨细胞病毒（ＨＣＭＶ）特异性抗体。结论 研究结果提示原发性高血压患者存在较高的活动性巨细胞病毒感染，ＨＣＭＶ活动性感染似与血管并发症有一定关联。     

Gene cloning of the human cytomegalovirus (HCMV) antigen reactive with the serum from a HCMV-infected patient.

The human cytomegalovirus(HCMV) gene encoding the protein reactive with the sera of HCMV-infected patient was cloned and characterized. A reactive phage clone was screened from a lambda gt11 expression library of cDNA of HCMV AD169 strain using HCMV-infected patient sera. The recombinant protein was expressed as 138 kDa-fusion protein with beta-galactosidase, which was reactive with IgM or IgG HCMV antibody-positive sera, but not with anti-HCMV antibody-negative sera. A homology search of the DNA sequence of the cloned gene with HCMV AD169 sequences revealed that it was composed of 709 base pairs spanning between 0.174 and 0.177 map units of the UL32 region of the HCMV AD169 strain genome. This position corresponded to a part of the gene encoding 150 kDa phosphoprotein-(pp150), a major tegument protein, which was reported as an immunogenic protein which evoked strong and longstanding antibody response and had no sequence homology with the proteins of other herpesviruses. These results suggested that pp150 was an immunogenic protein in natural HCMV infection and therefore this clone was regarded as a useful candidate for developing an antigen for the serodiagnosis of HCMV.     

The DNA-binding protein P52 of human cytomegalovirus reacts with monoclonal antibody CCH2 and associates with the nuclear membrane at late times after infection.

Monoclonal antibody CCH2 is commonly used for the detection of human cytomegalovirus (HCMV) infected cells in tissue sections as well as in cultured cells. The specificity of CCH2 was determined by screening a recombinant lambda-gt11 cDNA gene bank from HCMV-infected fibroblasts. By sequencing a reactive clone, the antigen was identified to be the non-structural DNA binding protein p52 of HCMV (UL44 reading frame). The viral insert from the lambda clone was recloned in bacterial expression vectors. For this, a new vector, pRos-RS, was constructed. The resulting clones were tested in immunoblot analyses. They were reactive with CCH2 as well as with reconvalescent sera positive for antibodies against HCMV, by this proving the specificity of CCH2. Using this monoclonal antibody in confocal microscopy, the subcellular localization of p52 in infected cells was analyzed. In these analyses, p52 was found to be nuclear and to be associated with the nuclear membrane at late times after infection.     

巨细胞病毒感染与可溶性白细胞介素2受体的关系

应用酶联免疫吸附试验（ＥＬＩＳＡ）对１０４例育龄妇女的血清进行了巨细胞病毒（ＨＣＭＶ）ＩｇＧ、ＩｇＭ抗体的检测，同时用ＥＬＩＳＡ双抗体夹心法测定了不同感染状态下血清中可溶性白细胞介素２受体（ｓＩＬ－２Ｒ）的水平，并将ｓＩＬ－２Ｒ水平与未感染ＨＣＭＶ的正常育龄妇女进行了比较。结果，育龄妇女中抗－ＨＣＭＶＩｇＧ的阳性率为８９．４％，ＩｇＭ的阳性率为９．６％，感染ＨＣＭＶ的妇女血清中ｓＩＬ－２Ｒ水平均大于未感染的对照组（１７８．１±５７．３Ｕ／ｍｌ），Ｐ＜０．０５，其中ＩｇＭ阳性者和ＩｇＭ、ＩｇＧ同时阳性者血清中ｓＩＬ－２Ｒ水平最高，分别为９１０±４６５．６Ｕ／ｍｌ和９０５±３４７．８Ｕ／ｍｌ，两者间的差异无显著性意义（Ｐ＞０．０５），但均大于仅抗－ＨＣＭＶＩｇＧ阳性者（４４６．８±１５８．９Ｕ／ｍｌ），Ｐ均＜０．０５。表明，ＨＣＭＶ感染可致ｓＩＬ－２Ｒ水平升高，并且活动性感染者上升明显。提示：ｓＩＬ－２Ｒ可能参与了ＨＣＭＶ的免疫致病机制。     

Evaluation of IgM anti human cytomegalovirus antibody

An anti-human cytomegalovirus (HCMV) antibody in 330 sera from patients and normal subjects was examined by ELISA. The test results were compared with the results of CF test and there was 99.1% agreement. Specific anti-HCMV IgM antibody was searched for and only 4 sera were positive. This makes it unlikely that screening for IgM anti-HCMV will effectively prevent posttransfusion HCMV infections.     

Detection of cytotoxic CD13-specific autoantibodies in sera from patients with ulcerative colitis and Crohn's disease

Recent evidence suggests an association between inflammatory bowel disease (IBD) and human cytomegalovirus (HCMV) infection, but the exact pathogenic role of HCMV in this disease remains unclear. HCMV infection has for a long time been known to be associated with various autoimmune manifestations and the formation of autoantibodies. Previous studies from our group have shown that HCMV is associated with a human protein, CD13 (aminopeptidase N) and that autoantibodies against this protein are frequently found in HCMV infected bone marrow transplant patients with chronic graft versus host disease. We have recently observed that 90% of IBD patients have an active HCMV infection. In this study, we examined the presence and cytotoxicity of CD13-specific autoantibodies in sera obtained from 28 patients with ulcerative colitis and 26 patients with Crohn's disease, and in sera obtained from healthy blood donors by using flow cytometric assays against mouse cells transfected with human CD13 or a microcytotoxicity assay against different CD13 positive human cells. Cytotoxic CD13-specific autoantibodies were identified in 66% of the sera obtained from HCMV-IgG positive patients with ulcerative colitis and in 58% of the sera obtained from HCMV-IgG positive patients with Crohn's disease, but not in control individuals. These cytotoxic autoantibodies may interfere with biological cell functions and could thereby contribute to the chronic inflammation in patients with IBD.     

Development and evaluation of a capture ELISA for IgM antibody to the human cytomegalovirus major DNA binding protein.

A new capture ELISA (ELAb) for determination of the IgM antibody response to the human cytomegalovirus major DNA binding protein (p52) was developed by using a p52-specific monoclonal antibody. As a reference test, a capture ELISA using in parallel viral- and cell-control labeled antigens (ELA) was employed. General specificity, which was determined on 180 unselected IgM-negative sera from an adult population was 100%; stringent specificity, which was evaluated on 108 potentially interfering sera from patients with Epstein-Barr virus infectious mononucleosis, autoimmune diseases, rheumatoid factor or treated with radioimmunotherapy, was 96.3%; finally, clinical specificity, determined on 79 IgM-negative sera drawn prior to onset of primary HCMV infection, was 100%. Thus, the overall specificity was 98.9% (363/367 IgM negative tested sera). Sensitivity assayed on 277 IgM-positive sera was 100%. The study of the kinetics of the IgM antibody response in sequential blood samples from 9 immunocompetent and 9 heart transplanted patients showed that, while in the immunocompetent p52-specific IgM titer fell sharply 2-3 months after onset and was virtually undetectable 12 months after onset, in the immunocompromised the IgM response persisted for longer than a year. Recurrent HCMV infections were associated with a high titer IgM response in 6 (30%), and with a low IgM response in another 6 (30%) heart transplanted patients within a group of 20 patients sequentially examined. Finally, IgM antibodies were detected in all 4 infants with congenital infection and in 5 of 6 infants with neonatal infection. The results show that the HCMV p52-specific IgM antibody response parallels that obtained by ELA, thus representing a major component of it. ELAb is highly sensitive, specific and reproducible. It represents a major advance among capture ELISA techniques, allowing detection of IgM antibody reactive to a specific viral protein.     

Prokaryotic expression of human cytomegalovirus pUS22 and its reactivity with human antibody

Summary.  This work demonstrates that antibodies to the product of the recombinant pUS22 of human cytomegalovirus (HCMV) are present in human sera during natural infection. US22 gene product has been identified as a member of the US22 family which may be secreted from infected cells. It is an early protein of 593 amino acids, 76 Kd in molecular weight. US22 seems to be an antigen which stimulates a good IgG response. In fact specific IgGs were found in approximately 40% of the CMV positive sera irrespective of their anti-CMV IgG titer. Specific IgM antibodies to pUS22 were observed exclusively during primary infection and in the sera with a high anti-CMV IgM titer. pUS22 could be considered for inclusion in a cocktail of CMV recombinant proteins to determine seropositivity to CMV and also to diagnose an active CMV infection. Received May 29, 1998 Accepted July 29, 1998     

Cytomegalovirus DNA detection in sera from patients with active cytomegalovirus infections.

Using a specific and sensitive polymerase chain reaction method, we detected reliably the presence of human cytomegalovirus (HCMV) DNA directly in serum samples collected at an early stage of HCMV infection, even before immunoglobulin M (IgM) antibodies were measurable. HCMV DNA was detected in serum from all patients with active HCMV infection; in 91% of these patients, HCMV DNA was found in the acute-phase serum. In 13 of 44 patients, HCMV DNA was found in serum before HCMV-specific IgM. For four kidney transplant recipients, the occurrence of HCMV DNA in serum, virus isolation from urine and leukocytes, and HCMV IgG and IgM serology were determined. We found a correlation between HCMV DNA in serum and positive virus isolation from leukocytes. In three of five congenitally infected infants, HCMV DNA and HCMV IgM were detected in the same sample. Two other infants were HCMV DNA positive, although no HCMV IgM antibodies were measurable. HCMV was found in urine from these infants either by virus isolation or with the polymerase chain reaction. Serum from one of the 22 healthy HCMV-seropositive blood donors was HCMV polymerase chain reaction positive.     

Comparison of the neutralizing and ELISA antibody titres to human cytomegalovirus (HCMV) in human sera and in gamma globulin preparations

Administration of anti-cytomegalovirus gamma globulin has been reported to prevent acute human cytomegalovirus (HCMV) infection in immunocompromised patients. Most likely the gamma globulin acts by its neutralizing activity. However, the antibody titer to HCMV in hyperimmune gamma globulin is usually detected by an enzyme immunoassay (ELISA). We, therefore, compared the ELISA titres with the neutralizing antibody titres in the sera of blood donors and in hyperimmune gamma globulins. A poor correlation between both titres was found. Gamma globulin preparations with identical ELISA titres clearly differed in the neutralizing titre. If neutralization is the important mechanism by which the hyperimmune gamma globulins work, our results would favour a routine testing of the neutralizing antibody titre in HCMV hyperimmune gamma globulins.     

Human cytomegalovirus infection: diagnostic potential of recombinant antigens for cytomegalovirus antibody detection.

Recombinant antigen-based enzyme immunoassays (EIAs) for the detection of human cytomegalovirus (HCMV) specific antibody are believed to yield a higher sensitivity and specificity than virus lysate EIAs. The aim of the present study was to evaluate the accuracy of newly established HCMV assays (Copalis CMV Multiplex, Sorin; Cobas Core CMV IgG and IgM EIAs, Roche Diagnostics; Anti-HCMV recombinant IgG, gB-IgG, IgM and IgA, Biotest; and ETI-CYTOK-G PLUS and M reverse PLUS, Sorin) based on recombinant antigens and/or virus lysate for laboratory diagnosis of HCMV infection. For the assessment of sensitivity, follow-up samples from patients suffering from active HCMV infection were tested. Testing a large number of potentially interfering samples challenged the specificity of the assays. There was no statistically significant difference in the performance of HCMV IgG assays. The results were more heterogeneous for the detection of serological markers of active infection (HCMV IgM, HCMV IgA and anti-CM2). The sensitivities of the different assays ranged between 40.5 and 71.4%. A variable number (17.8-1.7%) of false-positive results were obtained among potentially interfering serum samples. Two of the recombinant antigen based assays showed a high degree of interference with EBV VCA-IgM-positive sera. The best performance was achieved with ETI-CYTOK-M reverse PLUS since it combined the highest sensitivity with specificity. Commercially available assays based on recombinant antigens showed, overall, a poorer performance than the virus lysate EIA.     

Human cytomegalovirus infection is not increased in common variable immunodeficiency

It has been postulated that human cytomegalovirus (HCMV) infection may have a role in the pathogenesis of common variable immunodeficiency (CVID). Many patients have a lymphocyte phenotype similar to that seen in HCMV infection, HCMV mononucleosis may precipitate hypogammaglobulinaemia, and a previous small study of common variable immunodeficient patients reported a high rate of active HCMV infection. This study investigated the presence and activity of HCMV infection in 102 CVID patients. Buffy coats were examined for the presence of HCMV IE and glycoprotein B genes using highly sensitive nested PCR. 30 blood donors of known HCMV serologic status were used as controls. There was no significant difference in HCMV positivity by PCR between patients and controls. Enrichment for mononuclear cells prior to PCR had no effect on sensitivity. Twenty-five patients were also examined for HCMV antigenaemia by staining buffy coat cytospins with monoclonal antibodies directed against the HCMV pp65 lower matrix protein, a technique widely used for diagnosis of active HCMV disease. Only one patient was positive (and also positive by PCR). Whilst these results do not exclude prior infection contributing to antibody deficiency in a small proportion of CVID patients, this study refutes the previously reported increase in active HCMV infection in CVID.     

Human cytomegalovirus genetic variability in strains isolated from Japanese children during 1983-2003

The genetic variability of 74 human cytomegalovirus (HCMV) clinical isolates from 60 Japanese infants and children during 1983-2003 was investigated, and the relevance to their clinical course was studied. The patients consisted of 10 asymptomatic congenitally infected babies, 45 infected perinatally or postnatally resulting in HCMV mononucleosis/hepatitis and 5 immunocompromised hosts. The hypervariable region of the HCMV genome, that is the a sequence and UL144 region was analyzed using the polymerase chain reaction (PCR) and unrooted phylogenetic trees. HCMV glycoprotein B (gB) polymorphism was also studied. Unrooted phylogenetic trees of a sequence and UL144 allowed the isolates to be grouped to 5 and 3 clades, respectively. Three gB genotypes were also determined. However, there was no correlation between specific genotypes of these three genes and clinical forms, except for congenital infection which fell into one of three clades of the UL144 gene. In addition, the variability of the three genes had no correlation with each other. This implies that study of a single gene is insufficient for investigating the molecular epidemiology of HCMV. This study provides basic data on the genetic variability of HCMV in an Asian population and should help to determine the strains for vaccine candidates.     

先天性HCMV感染新生儿尿液中病毒载量与疾病严重度的关系探讨

目的探讨新生儿尿液中人巨细胞病毒（HCMV）病毒载量与先天性感染的关系及与HCMV所致疾病严重程度的关系。方法采集98例经PCR方法确诊的有症状及无症状的先天性HCMV感染新生儿尿液标本,用实时荧光定量PCR法（FQ—PCR）检测尿液中巨细胞病毒载量。结果98例先天性HCMV感染的新生儿中85例在出生后有临床症状（86％）。无症状感染和有症状感染的新生儿尿液中平均HCMV病毒载量分别是1．4×10^5拷贝／ml和3．1×10^6拷贝／ml,P〈0．01,差异有统计学意义。结论先天性HCMV感染的新生儿中无症状感染者尿液中病毒载量显著低于有症状感染者,提示病毒载量与疾病严重程度相关。     

Human cytomegalovirus (HCMV)-specific immunoglobulin E as a serologic marker for HCMV infection in immunocompromised patients

Summary An antibody capture assay using an enzyme-linked human cytomegalovirus (HCMV) antigen for the detection of specific immunoglobulin E (IgE) was established. IgG, M, and E responses to HCMV were studied in 497 sera obtained from 44 renal transplant recipients and 51 acquired immunodeficiency syndrome (AIDS) patients. The results were compared with those obtained from 58 HCMV-seropositive healthy individuals. HCMV-specific IgE was detected in 11 (91.7%) renal transplant recipients with primary HCMV infection. In contrast, antibodies of the IgG and IgM classes were detected in only 6 (50.0%) of these patients. Specific IgE was detected in 10 (90.9%) out of 11 renal allograft recipients suffering from secondary HCMV infection. Significant IgG titer rises and IgM were detected in 2 (18.2%) and 6 (54.6%) of these patients, respectively. IgG titer rises and IgM and IgE antibodies were seen in 5 (12.2%), 1 (2.4%) and 18 (43.9%) AIDS patients respectively. All healthy immunocompetent HCMV-seropositive individuals were tested IgE negative. The results obtained in our study indicate that IgE against HCMV is a more reliable serologic marker for primary and secondary HCMV infection than IgM in immunocompromised individuals, especially in organ transplant recipients, since it is not affected by the prophylactic application of HCMV hyperimmune globulin preparations.Abbreviations AIDS acquired immunodeficiency syndrome - BAL bronchoalveolar lavage - CDC Centers for Disease Control, Atlanta, USA - ELISA enzyme-linked immunosorbent assay - HCMV human cytomegalovirus - HIV human immunodeficiency virus - Ig immunoglobulin - PBS phosphate buffered saline - RTR renal transplant recipients