Herpesvirus group antibodies in children with Hodgkin's disease

Summary During the period of three years (1972–1974), serum samples from 60 patients (children and adolescents) with lympho-hematopoietic system diseases were examined for antibodies to all four human herpesviruses. Among these were 26 active Hodgkin's disease (AHD) patients and 6 HD patients with a minimum five years' remission. Simultaneously matched controls (age, sex) of AHD patients were examined. Antibody levels against the viral capsid antigen of Epstein-Barr virus (EBV/VCA) in AHD patients were significantly higher, with overrepresentation of higher titres (1:160), than in matched controls. The lowest EBV/VCA antibody titres were in the leukemia-non-Hodgkin's lymphoma patients. We could not prove any significant relationship between cytomegalovirus or herpes simplex virus type 1 antibody titres and AHD or any other disease of lymphohematopoietic system. The varicella-zoster virus antibody titres in AHD patients were significantly higher than in matched controls. No significant differences in antibodies against EBV/VCA and the other human herpesviruses between the evolution and remission period of AHD patients could be detected. No differences in EBV/VCA antibody titres were observed between the healthy school-children aged 10 to 15 years who were and who were not in contact with a HD patient.     

Antibody response to Epstein-Barr virus antigens in patients with chronic viral infection

We tested antibody titres against Epstein-Barr virus (EBV) antigens in patients suffering from chronic viral disease and compared them with those determined in sex- and age-matched healthy controls. Patient sera showed signs of active EBV infection [antibodies against early antigen (EA) and/or viral capsid antigen (VCA) in the IgM or IgA classes] significantly more frequently than the control group. Correspondingly, geometric mean titres (GMT) of antibodies against all viral antigens were elevated in the patients. The strongest association with EBV was observed in patients whose clinical symptoms closely resembled infectious mononucleosis: 92% of the subjects in this subgroup possessed anti-EA and 41 and 25% had IgM and IgA anti-VCA antibody, respectively. In patients with signs of lymphoproliferation only and in those suffering from frequent respiratory infections the association with EBV was less marked but still significant. Patients with transient defects in humoral and cellular immunity mounted higher titres against VCA in the IgG class than those without immune defects.     

Presence of Epstein-Barr virus DNA in tonsillar tissues

Sera from 48 tonsillar carcinoma (TC) patients, 48 matched controls and 16 recurrent exudative tonsillitis (RET) patients were examined for the presence of Epstein-Barr virus (EBV) associated nuclear antigen (EBNA), early antigen (EA) and virus capsid antigen (VCA). Higher prevalence and significantly higher antibody titres against all three EBV-associated antigens were observed in TC patients in comparison with controls and RET patients. Patients suffering from anaplastic TC had higher titres of antibodies against VCA and EA than TC patients with other histological diagnoses. Five out of 11 TC biopsies obtained from 9 patients were positive for EBV DNA at levels of 0.17, 4 to 5, 15 to 18 and in two cases 3 EBV genome equivalents per cellular genome. Among 16 RET patients, 4 were found positive at levels not exceeding 2.17 EBV genome equivalents per cellular genome. Higher titres of antibody against all EBV antigens were found in TC and RET patients with EBV DNA-positive tonsillar tissue than in those with EBV DNA-negative tonsillar tissue.     

Thymus transplantation. Reconstitution of cellular immunity in a four-year-old patient with T-cell deficiency.

The case is reported of a 4-year-old girl affected with recurrent infections; anaemia, thrombocytopenia, haemorrhages and hepatosplenomegaly. Immunological investigations revealed a defect in cellular immunity related to the thymus-dependent system, hypergammaglobulinaemia (especially of class IgE), and very high titres of antibodies against Epstein-Barr virus (EBV). After foetal thymus transplantation, correction of the immunological defect and significant clinical improvement were noted, as well as a decrease of IgE and EBV antibody titres.     

Clinical significance of variations in levels of Epstein-Barr Virus (EBV) antigen and adaptive immune response during chronic active EBV infection in children

Abstract

Pediatric patients were recruited to analyze differences in Epstein-Barr virus (EBV) copy numbers and adaptive immune reactions in children with chronic active vs acute EBV infection (CAEBVI vs AEBVI), as well as to examine the relationship between these parameters and the pathogenesis of CAEBVI. Fluorescent qPCR was used to assess EBV-DNA levels, while ELISA, antibody affinity, flow cytometry, and heterophil agglutination (HA) assays were used to evaluate patient EBV-adaptive humoral and cellular immunity. Lastly, ELISPOT was employed to assess interferon (IFN)-γ secretory functions of EBV-specific cytotoxic T-lymphocytes (CTL) as a marker of subject EBV-specific adaptive cellular immunity. The results indicated that, compared with AEBVI patients or normal children, there was a dramatic elevation in viral copy levels, viral capsid antigen (VCA)-IgA, early antigen (EA)-IgA, and EA-IgG, but a lack of EBV nuclear antigen (EBNA)-IgG and a negative HA in CAEBVI patients (p?<?0.01). These subjects also had decreased CD4⁺, CD8⁺ (naïve), CD8⁺CD38⁺, and effective memory T-lymphocyte levels compared with AEBVI patients (p?<?0.01), and decreased EBV-specific CTL function compared with normal children (p?<?0.01). These results suggest that there is a disturbance in EBV antigen availability and in both the adaptive humoral and cellular immune responses in patients with CAEBVI, and that these outcomes may be associated with the chronic active re-infection process itself associated with CAEBVI.     

Herpes simplex virus, cytomegalovirus and Epstein-Barr virus antibody titres in sera from schizophrenic patients

Serum antibody titres to herpes-simplex (HSV-1, 2), cytomegalovirus (CMV), and Epstein-Barr virus capsid antigen (EBV-VCA) were determined in 38 unrelated chronic schizophrenic patients, 11 nuclear families with at least 2 schizophrenic members, and 2 control groups. The distributions of antibody titres to herpes simplex and cytomegalovirus were similar among all groups. Patients had higher anti-EBV-VCA titres than non-hospitalized controls; however, hospital staff members in contact with the patients also had significantly higher antibody titres to EBV-VCA. Antibodies to EBV early antigen (EBV-EA) were also determined for 27 unrelated patients and 24 mental hospital employees. The schizophrenic patients had significantly higher antibody titres to EBV-EA than the hospital worker control group. These data do not support the hypothesis that herpes viruses are associated with the aetiology of schizophrenia. Although elevated anti-EBV early antigen titres may suggest persistent active EBV infection, it is unlikely to be related to the aetiology of the disorder, since discordance for EBV seropositivity was present among sibling pairs concordant for schizophrenia.     

Acute cytomegalovirus infection and the host immune response. I. Development and maintenance of cytomegalovirus (CMV) induced in vitro lymphocyte reactivity and its relationship to the production of CMV antibodies

Cytomegalovirus-induced lymphocyte reactivity (CMV-LR) of peripheral blood lymphocytes and antibodies against CMV-early (EA) and late (LA) antigens were studied in eighteen patients with documented acute CMV infections and seventy-six healthy persons. The development of a positive CMV-LR test lagged far behind the appearance of virus-specific antibodies. Positive CMV-LR tests were shown in all fourteen patients who could be tested twice or more. The median value of twenty-two tests in the acute phase (<50 days) was 269 d/min and the median of thirty tests in the post-illness phase (>50 days) was 1301 d/min (P<0·02). Once positive CMV-LR remained so during the follow-up period, up to 479 days after the onset of illness symptoms. In the meantime the LA and EA antibody response remained positive. Only two patients studied once showed negative CMV-LR responses while their serum contained high CMV-EA antibody titres. In CMV-LA seropositive healthy individuals who also possessed CMV-EA antibodies (LA+EA+) CMV-LR was higher (P<0·01) than in the LA seropositives who lacked EA antibodies (LA+EA−). The young LA+EA+ individuals, however, showed better (P<0·02) CMV−LR test results than the aged ones while their CMV antibody levels—especially the EA antibodies (P<0·02)—were lower. This phenomenon of increased CMV antibody production in relation to depressed CMV-LR is possibly caused by age-associated impairment of T lymphocyte function. The combined CMV-LR and EA antibody tests may provide useful means to study the specific immunological host/virus relationship in different clinical situations.     

Rheumatoid factor as a cause of positive reactions in tests for Epstein–Barr virus-specific IgM antibodies

Sera from twenty-eight patients with rheumatoid arthritis (RA) were titrated in indirect immunofluorescence tests for Epstein–Barr virus (EBV) specific antibodies. All had IgG antibodies to viral capsid antigen (VCA), 64% at titres [unk] 320, and 71% reacted also in tests for VCA-specific IgM antibodies at titres ranging from 20 to 640. The reactions observed in the IgM test were not due to VCA-specific IgM antibodies, however, but rather to rheumatoid factor (RF) usually an IgM antibody to the Fc regions of IgG. The titres recorded in the anti-VCA IgM test correlated significantly with the RF titres and both reactivities were abolished by adsorption onto IgG coated latex particles. In addition, they clearly depended upon the height of the IgG antibody titre to VCA, indicating that the more VCA-specific IgG molecules are present the more likely it is that RF will combine with them in sufficient quantity before or after their attachment to VCA-positive test cells so as to become detectable by the fluorescent antibodies to human IgM. Results comparable in every aspect were obtained with those sera from patients with Hodgkin's disease, nasopharyngeal or cervical carcinomas which reacted in the anti-VCA IgM test. Sera from patients with infectious mononucleosis may also contain RF, but in such cases its removal by adsorption onto IgG-coated latex particles did not generally reduce the VCA-specific IgM antibody titre. Removal of RF from any of the sera studied did not affect the titres of VCA-specific IgG and, where applicable, IgA or heterophil antibody titres. These results re-emphasize the pitfall created by RF noted previously in tests for virus-specific IgM antibodies.     

Humoral response to defined epitopes of tubercle bacilli in adult pulmonary and child tuberculosis

In order to investigate the humoral response to tuberculosis in different categories of patients, serum antibody levels to six epitopes ofMycobacterium tuberculosis in adult pulmonary and child tuberculosis were determined. Serum antibody titres were determined by competitive inhibition with radio-labelled murine monoclonal antibodies in 67 adults and 85 children with tuberculosis and in 79 age-matched controls. BCG vaccination (n=39) and self-healed tuberculosis (n=11) in adults gave rise to higher antibody titres to TB68, TB23 and TB72 epitopes (all p<0.003) when compared to non-vaccinated controls (n=18). TB68 titres were higher (p=0.006) in self-healed than in vaccinated adults. Adult sputum-negative patients (n=15) had higher titres to TB71 (p=0.015) and ML34 (p=0.02) epitopes compared to BCG-vaccinated healthy controls, while sputum-positive patients (n=41) had higher titres to all epitopes tested (all p<10^–4). The diagnostic sensitivity, with a 95 % specificity, was best with the combination of probes TB23, TB68, TB72 for sputum-positive (85 %) and TB78, ML34 (53 %) for sputum-negative patients. Antibody titres in children with tuberculosis were lower than in adult patients; diagnostic sensitivity in histologically or microbiologically proven cases (n=18) was only 44 %, while that in mediastinal lymphadenitis (n=67) was 13.5 %. This study suggests that the magnitude and specificity of the humoral response to tubercle bacilli varies with site and severity of infection; the implications for pathogenesis or protective immunity are discussed.     

Relationship of in vitro immune responses to Epstein-Barr herpesvirus and severity of infectious mononucleosis

Immune responses to Epstein-Barr herpesvirus (EBV) and EBV-related antigens were studied serially in 18 patients with heterophil antibody-positive infectious mononucleosis and in 18 control subjects. Enhanced cellular immune responses to EBV particles and to EBV intracellular soluble antigens were found in the patients at convalescence, suggesting that the development of specific cellular immune responses was associated with apparent control of the virus infection. In addition, a correlation between severity of disease and specific cellular immune response was found. Patients with severe clinical signs were found to have a more active cellular immune response to EBV intracellular soluble antigens early in the infection compared with patients with mild disease. This suggests that an increased immune reactivity to intracellular antigens during the early part of the illness is related to the severity of clinical manifestations in infectious mononucleosis. Serum antibody to viral capsid antigen and early antigen was not related to the severity of clinical disease.     

Epstein-Barr virus (EBV) antibodies in children with non-Hodgkin's lymphomas

Antibody titres against Epstein-Barr virus (EBV) antigens in children suffering from non-Hodgkin's lymphoma (NHL) were determined. IgG antibody titres against the viral capsid antigen (VCA) and early antigen (EA) exceeded those found in healthy control subjects. On the other hand, antibody titres against EBV-determined nuclear antigen (EBNA complex) were generally lower than in the control group. The most striking phenomenon observed in the patient group was the frequent activation of latent virus infection as revealed by the periodical appearance of anti-EA and IgM class anti-VCA antibodies. Antibody titres against EBV antigens were generally lower among patients with progressing disease than in those with a more favourable course of the illness. The closest relation to EBV based on serological findings, was detected in lymphoblastic lymphomas of Burkitt-type histology, poorly differentiated lymphocytic lymphomas, and in lymphomas localized in the abdomen. The question whether EBV might be involved in a certain proportion of the cases examined is discussed and further approaches to elucidate this problem are suggested.     

Effect of n-butyrate on cellular and viral DNA synthesis in cells latently infected with Epstein-Barr virus

Cellular DNA synthesis and Epstein-Barr virus (EBV)-specific DNA synthesis was examined after n-butyrate treatment of the producer line P3HR-1. Butyrate was found to effectively inhibit cellular DNA synthesis. This was followed by activation of the viral cycle as seen by marked increase in the expression of the EBV early antigen (EA), viral capsid antigen, and EBV-specific DNA synthesis. In spite of efficient induction of EA in the nonproducer Raji line, there was no increase in EBV-specific DNA synthesis above the level of the untreated control. It is likely that the cellular inhibitory effects of n-butyrate are responsible for the activation of EBV in producer lines and that different cellular controls, not affected by n-butyrate, are responsible for keeping the virus latent in nonproducer cells.     

Maternal immunity against avian influenza H5N1 in chickens: limited protection and interference with vaccine efficacy

After avian influenza (AI) vaccination, hens will produce progeny chickens with maternally derived AI-specific antibodies. In the present study we examined the effect of maternal immunity in young chickens on the protection against highly pathogenic AI H5N1 virus infection and on the effectiveness of AI vaccination. The mean haemagglutination inhibition antibody titre in sera of 14-day-old progeny chickens was approximately eight-fold lower than the mean titre in sera of vaccinated hens. After H5N1 infection at the age of 14 days, chickens with maternal antibody titres lived a few days longer than control chickens. However, only a low proportion of chickens with maternal immunity survived challenge with H5N1. In most progeny chickens with maternal immunity, high virus titres (>10⁴ median embryo infective dose) were present in the trachea during the first 4 days after H5N1 infection. In the cloaca, only low virus titres were present in most chickens. In 14-day-old progeny chickens with maternal immunity, the induction of antibody titres by vaccination was severely inhibited, with only a few chickens showing responses similar to the control chickens. It is concluded that high maternal antibody titres are required for clinical protection and reduction of virus titres after infection of chickens, whereas low antibody titres already interfere with vaccine efficacy.     

Immunization of Ferrets against Influenza: A Comparison of Killed Ferret Grown and Egg Grown Virus

Both killed unadjuvanted ferret and egg grown A/Moscow/1019/65 (H₂N₂) influenza virus failed to immunize ferrets against challenge with homologous virus; the preparations were given in 2 doses, 2 weeks apart, distributed intranasally, intramuscularly and intraperitoneally. However, small doses (<2 HA units) of both preparations induced immunity in ferrets previously “primed” with a live heterologous virus (A/FM/1/47 (H₁N₁)) according to the method of Potter et al. (1973a, b). Although no difference in immunizing activity was detected between ferret and egg grown virus, the former induced greater HI titres than the latter. There was no correlation between HI titres in serum and protection to challenge; in fact, some protection seemed to be afforded by the “priming” virus in the absence of HI antibody to the challenge virus.These results are discussed in relation to the possibility that a previously unrecognized antigen different from haemagglutinin and neuraminidase may contribute to immunity to influenza.     

Quantitative Analysis of Epstein-Barr Virus Load by Using a Real-Time PCR Assay

To measure the virus load in patients with symptomatic Epstein-Barr virus (EBV) infections, we used a real-time PCR assay to quantify the amount of EBV DNA in blood. The real-time PCR assay could detect from 2 to over 10⁷ copies of EBV DNA with a wide linear range. We estimated the virus load in peripheral blood mononuclear cells (PBMNC) from patients with symptomatic EBV infections. The mean EBV-DNA copy number in the PBMNC was 10^3.7 copies/μg of DNA in patients with EBV-related lymphoproliferative disorders, 10^4.1 copies/μg of DNA in patients with chronic active EBV infections, and 10^2.2 copies/μg of DNA in patients with infectious mononucleosis. These numbers were significantly larger than those in either posttransplant patients or immunocompetent control patients without EBV-related diseases. In a patient with infectious mononucleosis, the virus load decreased as the symptoms resolved. The copy number of EBV DNA in PBMNC from symptomatic EBV infections was correlated with the EBV-positive cell number determined by the in situ hybridization assay (r = 0.842; P < 0.0001). These results indicate that the real-time PCR assay is useful for diagnosing symptomatic EBV infection and for monitoring the virus load.     

Evaluation of a multianalyte profiling assay and an enzyme-linked immunosorbent assay for serological examination of Epstein-Barr virus-specific antibody responses in diagnosis of nasopharyngeal carcinoma

Assessment of antibody responses to Epstein-Barr virus (EBV) antigens has been used to assist in nasopharyngeal carcinoma (NPC) diagnosis by several methods. In this study, we evaluated an in-house Luminex multianalyte profiling (xMAP) technology and commercial enzyme-linked immunosorbent assay (ELISA) kits for serological examination of EBV-specific antibody responses in 135 NPC patients and 130 healthy controls. Four EBV biomarkers were measured: immunoglobulin A (IgA) against viral capsid antigen (VCA), EBV nuclear antigen 1 (EBNA1), diffused early antigen (EA-D), and IgG against EA-D. The sensitivities and specificities of the four markers ranged between 71.5 and 90% for xMAP assays and 80 and 92% for ELISA. Logistic regression analysis revealed that the combined markers in the xMAP assay had overall sensitivity and specificity values of 82% and 92%, respectively. The correlation coefficient (r) values for the xMAP assay and ELISA were lowest for IgA-VCA (0.468) and highest for IgA-EBNA1 (0.846); for IgA-EA-D and IgG-EA-D, the r values were 0.719 and 0.798, respectively. The concordances of the two methods for NPC discrimination were good (79 to 88%). Our results suggest that both the xMAP assay and ELISA are satisfactory for EBV antibody evaluation when multiple antigens are included.     

Antibodies to Epstein-Barr virus in nasopharyngeal carcinoma, tonsillar carcinoma and control groups

In the sera of 17 patients with nasopharyngeal carcinoma (NPC) and of 19 patients with tonsillar carcinoma (TC) the titres of IgA, IgG and IgM antibodies to EBV VCA (viral capsid antigen) and of IgG antibodies to EBV EA (early antigen) were determined by the indirect immunofluorescence (IF) method. Significant difference was observed in the frequency of IgA antibodies to EBV VCA and IgG antibodies to EBV EA between NPC patients and controls. There was also a significant difference between the frequency of IgM antibody to EBV VCA and EBV EA antibody titres in TC patients and controls. The geometric mean titre (GMT) of IgG antibodies to EBV VCA was significantly higher in the NPC and TC patients as compared to controls.     

Immune responses to Epstein-Barr virus lytic proteins in patients with nasopharyngeal carcinoma

Immune responses to three Epstein-Barr virus (EBV) lytic proteins, DNase, thymidine kinase (TK), and BMRF-1 gene products (50/52 kDa diffused early antigen, EA-D complex) were determined in EBV-infected control individuals and patients with nasopharyngeal carcinoma (NPC). Immunofluorescence assays (IFA) were used to detect their humoral immune responses using recombinant EBV lytic proteins expressed in a baculovirus system as antigens. Cell proliferation assays were performed to evaluate their cellular immune responses by monitoring 3H-thymidine incorporation. Seventy patients with NPC and 32 non-cancer controls were analyzed. The results of IFA showed antibody titers to all three EBV lytic proteins to be higher in the patients with NPC especially for the IgA class. Positivity rates of the three IgA antibodies also were higher in the patients with NPC population. Furthermore, the profiles of the IgA antibodies correlated with those to total early antigens (EA) expressed in the early phase and viral capsid antigen (VCA) expressed in the late phase, of EBV replication. The most interesting finding was that antibody titers to the three EBV lytic proteins were associated significantly with metastases of cervical lymph nodes in patients with NPC. As for cellular immunity to the EA-D complex and DNase, weak responses were observed in the cell proliferation assays. Peripheral blood cells from most individuals could not be stimulated to proliferate, except for a few patients with NPC whose antibody titers against the EA-D complex and DNase also were very high.     

Deletions in the Epstein-Barr virus latent membrane protein-1 oncogene in Hodgkin''s disease

Aims—To analyse the latent membrane protein-1 (LMP-1) gene in a series of patients with Epstein-Barr virus (EBV) positive LMP expressing ordinary and HIV associated Hodgkin's disease to detect possible genetic alterations and particularly the existence of deletions near the 3′ end of the gene.     

Evaluation of an Immunofiltration Assay That Detects Immunoglobulin M Antibodies against the ZEBRA Protein for the Diagnosis of Epstein-Barr Virus Infectious Mononucleosis in Immunocompetent Patients

The performance of an immunofiltration assay (IMFA) that detects immunoglobulin M (IgM) antibodies to the Epstein-Barr virus (EBV) ZEBRA (BamHI Z EBV replication activator) protein was evaluated for the diagnosis of EBV infectious mononucleosis (IM) in immunocompetent patients. The test panel consisted of 47 sera displaying an EBV-specific antibody profile compatible with an acute primary EBV infection from patients with clinical and biological features of EBV IM, 20 sera from healthy individuals either with a past EBV infection or who were EBV seronegative, 20 sera displaying an equivocal EBV antibody pattern (viral capsid antigen IgG positive [VCA IgG⁺], VCA IgM⁺, and EBV nuclear antigen-1 IgG⁺), and 15 sera obtained from patients with a mononucleosis-like syndrome owing to cytomegalovirus, human herpesvirus 6, or parvovirus B19. Overall, the sensitivity and the specificity of the assay were found to be 92.5%, and 97.3%, respectively. The sensitivity of the assay for the diagnosis of heterophile antibody-negative EBV IM was 86.2%. The IMFA is rapid, easy to perform, and, thus, suitable for point-of-care testing, and it may be used as a first-line test for the diagnosis of acute EBV IM in immunocompetent patients.Diagnosis of Epstein-Barr virus (EBV) infectious mononucleosis (IM) is commonly made on the basis of characteristic clinical manifestations and the detection of heterophile antibodies (HA). Nevertheless, HA may be absent, particularly in young children (14) but also in as many as 20% of adults with EBV IM (7). In these cases, demonstration of the presence of EBV viral capsid antigen (VCA) immunoglobulin G (IgG) and/or IgM antibodies, along with the absence of IgG antibodies to EBV nuclear antigen-1 (EBNA-1), allows the diagnosis of EBV primary infection (9). Detection of EBV-specific antibodies is accomplished by the use of commercial enzyme immunoassays, indirect immunofluorescence assays, line blot immunoassays (9), or, as established more recently, a multiplexed bead assay (3). These methods have long turnaround times, are labor-intensive, or require specific instruments or skilled technologists for their performance. In addition, interpretation of EBV VCA IgG/IgM and EBNA-1 IgG reactivity profiles is not always straightforward (9).The ZEBRA (BamHI Z EBV replication activator) protein is encoded by the immediate early BZLF1 gene. ZEBRA is expressed during the lytic cycle in EBV-permissive cells and plays a critical role in transactivating several immediate early, early, and late EBV genes (5). Antibodies against ZEBRA are produced during primary EBV infection (11, 15, 18), and thus, the detection of ZEBRA-specific IgMs may allow an early diagnosis of EBV IM. In the present study, we evaluated a rapid and easy-to-perform immunofiltration assay (IMFA) detecting IgMs to the EBV ZEBRA protein for the biological diagnosis of IM in immunocompetent patients.