Correlative Detection of Human Immunodeficiency. Virus (HIV) Antigen p24 and Epstein-Barr Virus DNA

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1 Microbiol. Immunol. Vol. 36 (8), , 1992 Correlative Detection of Human Immunodeficiency Virus (HIV) Antigen p24 and Epstein-Barr Virus DNA ônh ô In Vitro ôns ô: Clinical Influence on HIV Infection Pilar LARDELLI,*,1 Diego MANZANO,1 Seth M. STEINBERG,2 Lucila MADARIAGA,1 Isabel ANT_??_N,1 and Ram_??_n CISTERNA1 1 Department of Microbiology and Immunology, School of Medicine, University of the Basque Country, P.O. Box 699, Bilbao, Spain, and 2Biostatistics and Data Management Section, National Cancer Institute, National Institutes of Health, 8601 Old Georgetown Road, Bethesda, Maryland, U.S.A. (Accepted for publication, March 23, 1992) ôgh ô Abstract ôgs ô The existence of molecular transactivations between EBV and HIV-1, as well as reactivations of EBV latent infections in AIDS patients, have been recently documented. In order to shed more light on the putative association between EBV and HIV, and its role in the evolution to AIDS, we have determined simultaneously p24 protein and EBV DNA in culture supernatants of peripheral blood mononuclear cells from 47 individuals suspected of having HIV infection. The results of the ônh ô in vitro ôns ô assays were correlated with the clinical stage of the individuals and their serologic status to EBV. Statistical analysis showed a concordance between HIV in fectionand ônh ôin vitro ôns ô detection of EBV DNA ( ônh ôp ôns ô<0.002); particularly, a strong correla tionbetween the presence of EBV DNA and p24 in culture was observed ( ônh ôp ôns ô<0.001). These results are consistent with the occurrence of viral interactions, manifested ônh ô in vitro ôns ô. However, in our series, the appearance of EBV DNA in culture was not con comitantwith an elevation of anti-vca IgG titers, anti-ea titers or the development of symptomatology, suggestive of a reactivation of a latent EBV infection or a progres sionof HIV infection, Therefore we conclude that, although interaction between both viruses may take place at the molecular level, there is no clear evidence of the reper cussionthat this event may have on the clinical course of HIV infection. The interaction between human immunodeficiency virus type 1 (HIV-1) and other opportunistic viruses, especially Epstein-Barr virus (EBV), is currently a matter of debate, as well as the contributory role of the latter in the development of AIDS or AIDS-related complex (ARC). In this sense, several authors have reported in vitro reciprocal transactivations and enhanced serological responses to EBV in HIV-infected individuals (6, 8), while other studies show no relationship between the progression of HIV-related disorders and EBV infection (7). In order to further investigate the in vitro interaction between these viruses and its eventual clinical repercussion, we have analyzed the coexpression of HIV antigen p24 (HIV Ag) and EBV DNA in culture supernatants of peripheral blood mono nuclearcells (PBMC) from 47 individuals under suspicion of HIV infection, all of 905

2 906 P. LARDELLI ET AL them having a past contact with EBV. The results of the in vitro assay were corre latedwith the patients' symptomatology and immune status against EBV. Forty-seven individuals at high risk of HIV infection (intravenous drugs abusers, HIV-infected sexual partners, history of contaminated wounds) were selected for our study on the basis of having a previous contact with EBV assessed by titration of IgG antibodies to the Epstein-Barr viral capsid (VCA). Sixteen of them pre sentedwith systemic symptomatology at entry. Co-cultures were established following methods previously described (2, 4); briefly, Ficoll-Hypaque separated lymphocytes, at a density of 2 ~106 cells/ml, were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 of human purified interleukin-2, 2ƒÊg/ml polybrene, 5ƒÊg/ml hydrocortisone and 5ƒÊg/ml of phytohemagglutinin (PHA). Three days later, healthy donor lymphocytes (106 cells/ml), previously stimulated with PHA (5ƒÊg/ml) for 48hr, were added. Co-cultures were kept over a period of 30 to 40 days and supernatants were collected every four days. HIV Ag and EBV DNA were analyzed in parallel from samples of every supernatant. p24 antigen was determined by ELISA test and Western blot, and EBV DNA was detected by dot-blot hybridization of every sample after its denaturation and immobilization on nitrocellulose filters. Bio tinylatedbamhi W cloned fragment of EBV DNA was used as a probe, and an alka linephosphatase method was employed for signal detection and development. Positive and negative controls were run in every assay. Serum anti-hiv antibodies were determined by ELISA and Western blot. Titers of IgG and IgM antibodies to VCA and to the diffuse component of the early antigen of Epstein-Barr virus (EA) were measured by indirect immunofluorescence. Titers of anti nuclear antigen (EBNA) antibodies were determined by anti-complement immunofluorescence. Correlations between the in vitro findings, serologic data and clinical parameters were studied by both chi square and Wilcoxon rank sum tests. In the present series, a correlation between HIV infection and in vitro expression of EBV DNA was observed. As mentioned above, previous contact with EBV was demonstrated in all 47 individuals (anti-vca IgG titer range: 1/40-1/10,240), and 36 patients were also HIV infected (76.6%), on the basis of their seropositivity for anti-p24 antigen and the detection of HIV Ag in culture supernatants. Pres enceof EBV DNA, demonstrated by dot-blot hybridization of supernatants (Fig. 1), was found in 34 patients, 32 of whom were HIV positive, and in only 2 of the HIVuninfected cases, while Epstein-Barr viral DNA failed to appear in culture super natantsof 9 HIV-1-negative individuals (Chi square test, P<0.002). Furthermore, Chi square test disclosed a strong correlation between the in vitro detection of EBV DNA and p24 HIV Ag (P<0.001). In addition, a parallelism between the super natantsamples in which both markers (EBV DNA and p24) started to appear was observed (data not shown). These findings may reflect the existence of an association between EBV and HIV-1, at least under in vitro conditions. Nevertheless, taking into account the serological data of the studied series, the clinical manifestation of this putative association were not so evident. Tables 1 and 2 show the correlations of anti-vca

3 NOTES 907 Fig. 1. Dot-blot hybridization of lymphocyte culture supernatants to BamHI W cloned fragment of Epstein-Barr virus. Lanes 1 to 4 correspond to hybridization of 8 consecutive samples (A-H) of four different cases; case 1 is negative for EBV DNA; in cases 2, 3 and 4 EBV DNA started to be detected in the fifth, second and seventh samples, respectively. Table 1. Correlation of VCA IgG titers with in vitro and clinical parameters a) Wilcoxon rank sum test. Table 2. Relationship between VCA IgG titers to various parameters a) Chi square test.

4 908 P. LARDELLI ET AL IgG titers with the detection of EBV DNA and p24 in vitro and with the presence of systemic symptomatology, obtained by Wilcoxon sum test and Chi square test, respectively. According to current investigations, a reactivation of EBV infection is considered when either anti-vca antibodies (IgG or IgA) are significantly elevated or anti-ea antibodies are present (8). In the present series, in spite of the persistent EBV DNA detection in culture supernatants of HIV-infected patients, none of these two criteria was fulfilled. Thus, although some cases presented high anti-vca IgG values, contrarily to what was expected, elevated titers of IgG to EBV VCA did not correlate with the presence of in vitro EBV DNA and were not associated with the development of AIDS, ARC or systemic symptomatology in relation to reac tivationof a latent EBV infection (Tables 1 and 2). In addition, although a certain positive correlation between VCA IgG values and HIV infection was observed (Table 1) (Wilcoxon sum test: P=0.039), Chi square test did not achieve statistical significance (Table 2). Likewise, seroprevalence to EA in the whole series was low, as 53.2% of the patients were EA negative (<1/10) and only one patient reached a value of 1/80. Moreover, similar to VCA IgG, EA titers were independent of the presence of in vitro EBV DNA, as well as of the stage of the disease in HIV patients (P=0.92) and symptomatology (P=0.76). Data corresponding to the rest of the parameters investigated were inconclusive. The observed concomitant proliferation of EBV and HIV-1 in vitro, could be due to the existence of reciprocal or simultaneous activations of both viruses at the molecular level, triggered and/or enhanced by culture conditions but, regarding the serological and clinical data of our series, it seems to have little clinical repercus sion.in fact, the majority of studies in this field show striking in vitro findings but contradictory clinical correlations (1, 5), and large epidemiological reports have demonstrated that AIDS acquisition and progression are independent of EBV status (7). According to these analyses, jointly with the controversial current knowledge about EBV reactivation, chronic infection and immune status (3), we conclude that, regarding the relationship between Epstein-Barr virus and HIV-1, attention has to be mainly focused on the development of polyclonal proliferations induced by EBV, favored by the immunodepressed status of HIV patients (6) or other EBV-specific lesions, such as hairy leukoplakia, indicative of progression to AIDS (9), rather than to the hypothetical coadjutant role of EBV in the pathogenesis of AIDS; yet these studies have to be completed with electron microscopy and Southern blot hybridi zation,and other EBV probes have to be tested. Also, further in vitro and in vivo investigations about the molecular interactions between both viruses in HIV patients who develop malignant lymphoma are required. REFERENCES 1) Alsip, G.R., Ench, Y., Sumaya, C.V., and Boswell, R.N Increased Epstein-Barr virus DNA in oropharingeal secretions from patients with AIDS, AIDS-related complex or asymp tomatichuman immunodeficiency infections. J. Infect. Dis. 157:

5 NOTES 909 2) Barr_??_-Sinoussi, F., Mathur-Wagh, B., Rey, F., Brun-Vezinet, F., Yancovitz, S.R., Rouzioux, C., Mantaigner, L., Mildvan, D., and Chermann, J.C Isolation of lymphadenopathy-as sociatedvirus (LAV) and detection of LAV antibodies from US patients with AIDS. JAMA 253: ) Buchwald, D., Sullivan, J. L., and Komaroff, A.L Frequency of echronic Epstein-Barr virus infection f in a general medical practice. JAMA 257: ) Ehrnst, A., Sonneborg, A., Bergdahl, S., and Strannegard, O Efficient isolation of HIV from plasma during different stages of HIV infection. J. Med. Virol. 26: ) Hamilton-Dutoit, S.J., Pallesen, G., Franzmann, M.B., Karkov, J., Black, F., Skinhoj, P., and Pedersen, C AIDS-related lymphoma. Histopathology, immunophenotype, and associa tionwith Epstein-Barr virus as demonstrated by in situ nucleic acid hybridization. Am. J. Pathol. 138: ) Kenney, S., Kamine, J., Markovitz, D., Fenrick, R. and Pagano, J An Epstein-Barr virus immediate early gene product transactivates gene expression from the human immunodeficiency virus type 1 and cytomegalovirus. J. Infect. Dis. 157: ) Lang, D.J., Kovacs, A.S., Zaia, J.A., Doelkin, F., Niland, J.C., Aledorf, L., Azen, S.P., Fletcher, M.A., Gauderman, J., Gjerset, G.J., Lusher, J., Operalski, E.A., Parker, J.W., Pegelow, C., Vyas, G.N., Mosley, J.W., and the Transfusion Safety Study Group. 1989, Seroepidemiological studies of cytomegalovirus and Epstein-Barr virus infection in relation to human immunodeficiency virus type 1 infection in selected recipient population. J. AIDS 2: ) Rahman, M.A., Kingsley, L.A., Breinig, M.K., Ho, M., Armstrong, J.A., Atchison, R.W., Lyter, D.W., and Rinaldo, C.E Enhanced antibody responses to Epstein-Barr virus in HIV infected homosexual men. J. Infect. Dis. 169: ) Souza de, Y.G., Freese, U.K., Greenspan, D., and Greenspan, J.S Diagnosis of Epstein- Barr virus infection in hairy leukoplakia by using nucleic acid hybridization and non-invasive techniques. J. Clin. Microbiol. 28: (Received for publication, January 10, 1992; in revised form, March 16, 1992)