Journal of Medical Virology 3660-64 (1992)
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Antibodies to Epstein-Barr Virus and Cytomegalovirus in Relation to CD4 Cell Number in Human Immunodeficiency Virus 1 Infection A. Quesnel, B. Pozzetto, F. Touraine, P. Moja, F. Lucht, G. De The, J.L. Touraine, 0. Gaudin, and C. Genin Laboratory of Research Ln Immunology (A Q , P.M , C.G ) and Laboratory of VLrology ( B P ,F L , 0 G ) UnLverslty of Saint-Etzenne, Samt-Etienne, INSERM U 80, Lyon (F T , J L T ), and Pasteur Institute, Paris (G D T ), France Interaction between herpesviruses and human immunodeficiency virus (HIV)I is postulated in the progression of HIV disease. In order t o evaluate the specific antibody responses directed t o Epstein-Barr virus (EBV) and cytomegalovirus (CMV) and t o provide serological evidence suggesting reactivation of these viruses able to accelerate the immunodeficiency, w e studied IgA and IgG titres t o EBV and CMV in the serum of HIV positive patients in relation t o the CD4 cell number. The titres of IgG antibodies t o EBV and the prevalence of IgG t o CMV were significantly higher i n HIV positive patients compared t o control high risk HIV negative subjects. In HIV infected patients, anti-VCA IgG antibodies increased and anti-EBNA IgG antibodies decreased progressively in relation t o the decline of CD4 cell number whereas anti-CMV IgG antibodies did not varied significantly at the same time. Anti-VCA IgA and anti-EA IgG antibodies were found uncommonly and with l o w titres. IgA antibodies t o EA and CMV were not detected in any patient. The variations in EBV antibody response that we describe i n HIV infection were previously reported in other immunodeficiency states and could be distinctive of these diseases.
KEY WORDS: CD4 cells, herpes viruses, imm u nodef ici ency
INTRODUCTION Herpesvirus infections with Epstein-Barr virus (EBV) or cytomegalovirus (CMV) occur frequently in human immunodeficiency virus 1 (HIV) seropositive patients as the consequence of the immunodeficiency caused by this virus [Quinnan et al., 1984; Jacobson and Mills, 19881. Herpesviruses can induce the development of a n immunodeficiency state in HIV seroneg0 1992 WILEY-LISS, INC.
ative subjects as homosexual men [Detels et al., 1984; Drew et al., 19851 and it has been suggested that they may serve as cofactors in HIV infection by increasing HIV viral replication and expression or by accelerating the disease process by modulation of the host immune system [Hirsch et al., 1984; Holmberg et al., 1988; Webster et al., 1989; Clouse et al., 1989; McKeating et al., 19901. Several investigators focused their studies on the interaction between herpesviruses and HIV in patients with AIDS or AIDS-related complex (ARC) [Sumaya et al., 1986; Halbert e t al., 1986; Quinn et al., 19871. Our aim was to study this interaction during the time period preceding the development of the severe immunodeficiency state. In order to evaluate the specific antibody responses directed to EBV and CMV and to provide serological evidence suggesting reactivation of these viruses able to initiate or accelerate the immunodeficiency, IgA and IgG titres to EBV and CMV were studied in the serum of HIV positive patients and these observations were related to the absolute numbers of CD4+ lymphocytes in the blood. It was found t h a t the antibody titres to EBV and the prevalence of antibodies to CMV are significantly higher in HIV positive patients compared to control high risk HIV negative subjects. IgG antibodies to EBV viral capsid antigen (VCA) increase and IgG antibodies to Epstein-Barr nuclear antigen (EBNA) decrease progressively in relation to the decline of CD4 cell number whereas anti-CMV antibodies do not change significantly during these phases of HIV-1 infection.
SUBJECTS AND METHODS Subjects Fifty-six adult men with sexually or drug transmitted HIVl infection were divided into 3 groups on the basis of CD4 cell number in serum: Accepted for publication July 23, 1991. Address reprint requests to C. Genin, Laboratoire de Recherche en Immunologie, Faculte de Medecine, 15 rue Ambroise Pare, 42023 Saint-Etienne cedex 02, France.
Antibodies to EBV and CMV in HIV Disease Group I: control group of 20 HIV seronegative age matched subjects belonging to the same risk population. Group 11: 25 HIV infected patients with a number of CD4 cells higher than 600/mm3. Group 111: 20 HIV infected patients with a number of CD4 cells between 200 and 400/mm3. Group IV: 11HIV infected patients with a number of CD4 cells lower than 200/mm3.
Methods Imrnunofluorescence tests for Epstein-Barr virus serology. The antibody titres to EBV antigens were measured in serum specimens by immunofluorescence [Henle and Henle, 1966; Henle et al., 19741. All serum samples were tested simultaneously in order to minimize the influence of fluorescence reading. Lymphoblastoid B cells (P3HR1 for VCA, Raji cells for early antigen (EA))were fixed on glass slides and incubated with serial twofold dilutions of serum specimens ranging from 1/5to 1/1,280 in PBS for 45 min a t 37°C in a moist atmosphere. Then the smears were washed with PBS and incubated for 30 min at 37°C with fluorescein isothiocyanate conjugated rabbit antibodies to human IgG (Dakopatts, Copenhagen, Denmark) or goat antibodies to human IgA (Diagnostic Pasteur, Marnes la Coquette, France). For antibodies to EBNA, Raji cells were incubated with serial twofold dilutions of serum specimens ranging from 1/5 to 1/1,280 in PBS for 45 min a t 37°C in a moist atmosphere. After three washings in PBS, a n 1/20 dilution in Earle salts of human EBV-negative serum a s a source of complement was added for 30 rnin a t 37°C. After washing again as above, a rabbit antihuman C3c fluorescein-conjugated antibody (Dakopatts) was reacted with smears for 30 min a t 37°C. After final washing, the slides were mounted with buffered glycerin and read under a Leitz epifluorescence microscope under x 400 magnification. Antibody titres were expressed as the reciprocal of the serum end-point dilution exhibiting positive fluorescence. Titres less than 5 were considered as negative. Antibody-capture ELISA for CAW serology. CMV-specific IgG and IgM antibodies were detected by a commercial antibody-capture enzyme linked immunosorbent assay (ELISA) (Wellcome Laboratories, Dartford, England). The test was performed according to the manufacturer's instructions with the exception that serial twofold dilutions of serum specimens were used instead of a single dilution as recommended. Antibody titres were expressed as the reciprocal of the end-point dilution exhibiting the same absorbance as the low positive rate included in the assay. CMV-specific IgA serology was performed by antibody-capture ELISA. Plates (Nunc Immunoplate I, Roskilde, Denmark) were coated with F(ab'12 fragments of goat antihuman IgA (Cappel, Cochranville, PA) overnight at 4°C. In order to eliminate rheumatoid
61
factors, sera were absorbed onto sheep antibodies directed to human IgG (RF-absorbentBehringwerke AG, Marburg, Germany) overnight at 4°C. Supernatants diluted 1/16 from the original serum were added in each well and plates were incubated for 2 h r at room temperature. After three washing steps, they were incubated for 2 h r a t room temperature with peroxydase-conjugated purified CMV (Wellcome Laboratories). After washing again, orthophenylenediamine (1 mg/ml in 100 mM phosphate buffer [pH 6.01 containing 0.01% [w/v] H,Oz) was added for 30 min at 4°C. The absorbance was read at 492 nm with a Titertek Multiskan spectrophotometer (Flow Laboratories, McLean, VA). Serum specimens with absorbance 600) 5/25 (20) 10125 (40) Group I11 (CD4 200-400) 7/20 (35) 5/20 (25) 2/11 (18) 3/11 (27) Group IV (CD4 < 200)
group I V
(m< 2 0 0 ) Fig. 2. Anti-CMV IgG antibody titres in HIV positive patients in relation to CD4 cell number and in a n HIV negative control group. GMT (full line) ? SD (thin dashed lines) calculated for CMV seropositive individuals are indicated for each group. Thick dashed line represents the positivity threshold. No significant difference in titre was observed between the four groups.
that these cells are needed for the maintenance of active CD8 population. The alteration in EBV antibody response previously reported in other immunologically compromised states as ataxia telangiectasia or Hodgkin’s disease [Berkel et al., 1979; Lange et al., 19781 is also typical of these diseases. Previous studies on serological responses to EBV and CMV in HIV positive patients demonstrated a high prevalence of specific antibodies and higher antibody titres as compared to HIV negative patients [Sumaya et al., 1986; Halbert et al., 19861. Nevertheless, these studies were based on the clinical assessment of patients and not on the absolute CD4 counts as prognostic indicators. High titres of CMV antibodies have been reported to be associated with subsequent AIDS [Polk et al., 19871 but these results have not been confirmed [Holmberg et al., 19881. Rahman et al. [1989] failed to demonstrate any significant change of EBV antibody level in ARC patients compared with HIV-seropositive
men who were asymptomatic or who had lymphadenopathy . We found IgA to VCA with a slight increased frequency in HIV positive compared to negative patients but no significant difference was noted. The percentage of patients with IgA antibodies to VCA did not increased when CD4 cell number declined. These results are in contrast with a previous study demonstrating a n increase of these antibodies as the stage of the disease worsened [Margalith et al., 19901. IgA antibodies to EA and to CMV could not be detected in any HIV infected patients, as previously reported [Sumaya et al., 1986; Nielsen et al., 19881. This study provides serological evidence for a n active infection with EBV and CMV early in the course of HIV disease. It suggests a parallel stimulation of humoral immunity to VCA in relation to the EBV reactivation and a decrease of immunity to EBNA in relation to the immunodeficiency, a s the number of CD4 cells declines. It is speculated th a t this persistent infection with viruses well-known to suppress cell-mediated immune functions accelerate the immunodeficiency disease but further experimental data are needed to elucidate the mechanism.
ACKNOWLEDGMENTS We wish to thank F. Duplat for technical assistance and E. Gagnaire for secretarial assistance. We acknowledge Wellcome Laboratories for providing peroxydase conjugated CMV antigen. This work was supported by a grant of “Fondation Merieux” and a grant of “Region RhBne-Alpes.”
REFERENCES Alsip GR, Sumaya CV, Boswell RN (1988): Increased Epstein-Barr Virus DNA in oropharyngeal secretions from patients with AIDS, AIDS-related complex, or asymptomatic human immunodeficiency virus infections. Journal of Infectious Diseases 157:1072-1076. Berkel AI, Henle W, Henle G, Klein G, Ersoy F, Sanal 0 (1979): Epstein-Barr virus related antibody patterns in ataxia-telangiectasia. Clinical Experimental Immunology 35:196-201. Birx DL, Redfield RR, Tosato G (1986): Defective regulation of Epstein-Barr virus infection in patients with acquired immunodeficiency syndrome (AIDS)or AIDS-related disorders. New England Journal of Medicine 314:874-879. Blumberg RS, Paradis T, Byington R, Henie W, Hirsch MS, Scooley RT (1987): Effects of human immunodeficiency virus on the cellular immune response to Epstein-Barr virus in homosexual men: characterization of the cytotoxic response and lymphokine production. Journal of Infectious Diseases 155:877-890. Clouse KA, Robbins PB, Fernie B, Ostrove JM, Fauci AS (1989):Viral
64 antigen stimulation o f the production of' human moniikines capable of regulating H I V l expression. Journal of Immunology 143 470-475. 1)avis G , Kenncy SC, Kaminr J. Pagano JS. Huang ES (1987): Immediate-early gene region of human cytomegalovirus transactivates the promoter of human immunodeficiency virus. Proceedings of the National Academy of Sciences of the United States 01' America 84:8642-8646. Drtels R , Visscher BR, Fahey JL. Schwartz K , Grcene RS, Madden Dl., Sever JL. Gottlieb MS 19641:The relation of cytomega1oviru.i and Etxtein-Barr virus antibodies to T-cell subsets in homosexually active men. Journal of the American Medical Association 251:1719-1722. Diaz-Mitoma F, Ruiz A, Flowerdew G, Houston S, Romanowski B, Kovithavongs T, Preiksaitis J , Tyrrell D (1990): High levels of Epstein-Barr virus in the oropharynx: a predictor of disease progression in human immunodeficiency virus infection. Journal of Medical Virology 31:69-75. Drew WL, Mills J, Levy J , Dylewski J , Casavant C, Ammann AJ, Brodie H, Merigan T (1985): Cytomegalovirus infection and abnormal T-lymphocyte subset ratios in homosexual men. Annals of Internal Medecine 103:61-63. Fiala M, Cone LA, Chang CM, Mocarski ES (1986): Cytomegalovirus viremia increases with progressive immune deficiency in patients infected with HTLV-111. AIDS Research 2:175-181. Halbert SP, Kiefer DJ, Friedman-Kien AE, Poiesz B (19861: Antibody levels for cytomegalovirus, herpes simplex virus ans rubella in patients with acquired immune deficiency syndrome. Journal of Clinical Microbiology 23:318-321. Henle G, Henle W (1966): Imniunofluorescence in cells derived from Burkitt's lymphoma. Journal of Bacteriology 91:1248-1256. Henle W, Henle G (1981): Epstein-Barr virus-specific serology in immunologically compromised individuals. Cancer Research 41:42224225. Henle G, Henle W, Horwitz CA (1974): Antibodies to Epstein-Barr virus-associated nuclear antigen in infectious mononucleosis. Journal of Infectious Diseases 130:231-239. Hirsch MS, Schooley RT, Ho DD, Kaplan J C (1984): Possible viral interactions in the acquired immunodeficiency syndrome (AIDS). Reviews of Infectious Diseases 6:726-731. Holmberg SD, Gerber AR, Stewart J A , Lee FK, OMalley P, Nahmias AJ (1988):Herpes viruses as co-factors in AIDS. Lancet 2:746747. Jacobson MA, Mills J (1988): Serious cytomegalovirus disease in the acquired immunodeficiency syndrome (AIDS).Annals of Internal Medicine 108:585-594. Lange B, Arbeter A, Hewetson J , Henle W (1978):Longitudinal study of Eustein-Barr virus antibodv titers and excretion in uediatric patients with Hodgkin's disease. International Journal df Cancer 22:521-527. Margalith M, Sarov B, Sarov I, Rinaldo C, Detels R, Phair J, Kaslow R, Ginsberg H, Saah A (1990): Serum IgG and IgA antibodies
Quesnel et al. specific to Epstein-Barr virus capsid antigen in a longitudinal study of human immunodeficiency virus infection and disease progression in homosexual men. AIDS Research and Human Retroviruses 6:607-616. McKeating JA, Griffiths PD, Weiss RA (1990): HIV susceptibility conferred to human fibroblasts by cytomegalovirus-induced Fc receptor. Nature 343:659-661. Nelson JA, Reynolds-Kohler C, Oldstone MBA, Wiley CA (1988):HIV and HCMV coinfect brain cells in patients with AIDS. Virology 165:286290. Nielsen SL, Sorensen I, Andersen HK (1988): Kinetics of specific immunoglobulins M, E, A and G in congenital, primary, and secondary cytomegalovirus infection studied by antibody-capture Enzyme-linked immunosorbent assay. Journal of Clinical Microbiology 26:654-661. Pagano JS, Kenney S, Markovitz D, Kamine J (1988):Epstein-Barr virus and interactions with human retroviruses. Journal of Virological Methods 21:229-239. Polk BF, Fox R, Brookmeyer R, Kanchanaraksa S, Kaslow R, Visscher B, Rinaldo C, Phair J (19871: Predictors of the acquired immunodeficiency syndrome developing in a cohort of seropositive homosexual men. New England Journal of Medicine 316:61-66. Quinn TC, Piot P, Mc Cormick JB, Feinsod FM, Taelman H, Kapita B, Stevens W, Fauci AS (1987):Serologic and immunologic studies in patients with AIDS in North America and Africa. Journal of the American Medical Association 257:2617-2621. Quinnan GV, Masur H, Rook AH, Armstrong G, Frederick WR, Epstein J , Manischewitz J F , Macher AM, Jackson L, Ames J , Smith HA, Parker M, Pearson GR, Parrillo J, Mitchell C, Straus SE (1984): Herpesvirus infections in the acquired immune deficiency syndrome. Journal of the American Medical Association 252:72-77. Rahman MA, Kingsley LA, Breinig MK, Ho M, Armstrong JA, Atchison RW, Lyter DW, Rinaldo CR (1989): Enhanced antibody responses to Epstein-Barr virus in HIV-infected homosexual men. Journal of Infectious Diseases 159:472-478. Skolnik PR, Kosloff BR, Hirsch MS (1988):Bidirectional interactions between human immunodeficiency virus type 1 and cytomegalovirus. Journal of Infectious Diseases 157508-514. Sumaya CV, Boswell RN, Ench Y, Kisner DL, Hersh EM, Reuben JM, Mansell PWA (1986): Enhanced serological and virological findings of Epstein-Barr virus in patients with AIDS and AIDS-related complex. Journal of Infectious Diseases 154:864-870. Webster A, Cook DG, Emery VC, Lee CA, Grundy J E , Kernoff PBA (1989): Cytomegalovirus infection and progression towards AIDS in haemophiliacs with human immunodeficiency virus infection. Lancet 2:63-66. Yarchoan R, Redfield RR, Broder S (1986): Mechanisms of B cell activation in patients with acquired immunodeficiency syndrome and related disorders. Journal of Clinical Investigation 78:439447.
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Antibodies to Epstein-Barr Virus and Cytomegalovirus in Relation to CD4 Cell Number in Human Immunodeficiency Virus 1 Infection A. Quesnel, B. Pozzetto, F. Touraine, P. Moja, F. Lucht, G. De The, J.L. Touraine, 0. Gaudin, and C. Genin Laboratory of Research Ln Immunology (A Q , P.M , C.G ) and Laboratory of VLrology ( B P ,F L , 0 G ) UnLverslty of Saint-Etzenne, Samt-Etienne, INSERM U 80, Lyon (F T , J L T ), and Pasteur Institute, Paris (G D T ), France Interaction between herpesviruses and human immunodeficiency virus (HIV)I is postulated in the progression of HIV disease. In order t o evaluate the specific antibody responses directed t o Epstein-Barr virus (EBV) and cytomegalovirus (CMV) and t o provide serological evidence suggesting reactivation of these viruses able to accelerate the immunodeficiency, w e studied IgA and IgG titres t o EBV and CMV in the serum of HIV positive patients in relation t o the CD4 cell number. The titres of IgG antibodies t o EBV and the prevalence of IgG t o CMV were significantly higher i n HIV positive patients compared t o control high risk HIV negative subjects. In HIV infected patients, anti-VCA IgG antibodies increased and anti-EBNA IgG antibodies decreased progressively in relation t o the decline of CD4 cell number whereas anti-CMV IgG antibodies did not varied significantly at the same time. Anti-VCA IgA and anti-EA IgG antibodies were found uncommonly and with l o w titres. IgA antibodies t o EA and CMV were not detected in any patient. The variations in EBV antibody response that we describe i n HIV infection were previously reported in other immunodeficiency states and could be distinctive of these diseases.
KEY WORDS: CD4 cells, herpes viruses, imm u nodef ici ency
INTRODUCTION Herpesvirus infections with Epstein-Barr virus (EBV) or cytomegalovirus (CMV) occur frequently in human immunodeficiency virus 1 (HIV) seropositive patients as the consequence of the immunodeficiency caused by this virus [Quinnan et al., 1984; Jacobson and Mills, 19881. Herpesviruses can induce the development of a n immunodeficiency state in HIV seroneg0 1992 WILEY-LISS, INC.
ative subjects as homosexual men [Detels et al., 1984; Drew et al., 19851 and it has been suggested that they may serve as cofactors in HIV infection by increasing HIV viral replication and expression or by accelerating the disease process by modulation of the host immune system [Hirsch et al., 1984; Holmberg et al., 1988; Webster et al., 1989; Clouse et al., 1989; McKeating et al., 19901. Several investigators focused their studies on the interaction between herpesviruses and HIV in patients with AIDS or AIDS-related complex (ARC) [Sumaya et al., 1986; Halbert e t al., 1986; Quinn et al., 19871. Our aim was to study this interaction during the time period preceding the development of the severe immunodeficiency state. In order to evaluate the specific antibody responses directed to EBV and CMV and to provide serological evidence suggesting reactivation of these viruses able to initiate or accelerate the immunodeficiency, IgA and IgG titres to EBV and CMV were studied in the serum of HIV positive patients and these observations were related to the absolute numbers of CD4+ lymphocytes in the blood. It was found t h a t the antibody titres to EBV and the prevalence of antibodies to CMV are significantly higher in HIV positive patients compared to control high risk HIV negative subjects. IgG antibodies to EBV viral capsid antigen (VCA) increase and IgG antibodies to Epstein-Barr nuclear antigen (EBNA) decrease progressively in relation to the decline of CD4 cell number whereas anti-CMV antibodies do not change significantly during these phases of HIV-1 infection.
SUBJECTS AND METHODS Subjects Fifty-six adult men with sexually or drug transmitted HIVl infection were divided into 3 groups on the basis of CD4 cell number in serum: Accepted for publication July 23, 1991. Address reprint requests to C. Genin, Laboratoire de Recherche en Immunologie, Faculte de Medecine, 15 rue Ambroise Pare, 42023 Saint-Etienne cedex 02, France.
Antibodies to EBV and CMV in HIV Disease Group I: control group of 20 HIV seronegative age matched subjects belonging to the same risk population. Group 11: 25 HIV infected patients with a number of CD4 cells higher than 600/mm3. Group 111: 20 HIV infected patients with a number of CD4 cells between 200 and 400/mm3. Group IV: 11HIV infected patients with a number of CD4 cells lower than 200/mm3.
Methods Imrnunofluorescence tests for Epstein-Barr virus serology. The antibody titres to EBV antigens were measured in serum specimens by immunofluorescence [Henle and Henle, 1966; Henle et al., 19741. All serum samples were tested simultaneously in order to minimize the influence of fluorescence reading. Lymphoblastoid B cells (P3HR1 for VCA, Raji cells for early antigen (EA))were fixed on glass slides and incubated with serial twofold dilutions of serum specimens ranging from 1/5to 1/1,280 in PBS for 45 min a t 37°C in a moist atmosphere. Then the smears were washed with PBS and incubated for 30 min at 37°C with fluorescein isothiocyanate conjugated rabbit antibodies to human IgG (Dakopatts, Copenhagen, Denmark) or goat antibodies to human IgA (Diagnostic Pasteur, Marnes la Coquette, France). For antibodies to EBNA, Raji cells were incubated with serial twofold dilutions of serum specimens ranging from 1/5 to 1/1,280 in PBS for 45 min a t 37°C in a moist atmosphere. After three washings in PBS, a n 1/20 dilution in Earle salts of human EBV-negative serum a s a source of complement was added for 30 rnin a t 37°C. After washing again as above, a rabbit antihuman C3c fluorescein-conjugated antibody (Dakopatts) was reacted with smears for 30 min a t 37°C. After final washing, the slides were mounted with buffered glycerin and read under a Leitz epifluorescence microscope under x 400 magnification. Antibody titres were expressed as the reciprocal of the serum end-point dilution exhibiting positive fluorescence. Titres less than 5 were considered as negative. Antibody-capture ELISA for CAW serology. CMV-specific IgG and IgM antibodies were detected by a commercial antibody-capture enzyme linked immunosorbent assay (ELISA) (Wellcome Laboratories, Dartford, England). The test was performed according to the manufacturer's instructions with the exception that serial twofold dilutions of serum specimens were used instead of a single dilution as recommended. Antibody titres were expressed as the reciprocal of the end-point dilution exhibiting the same absorbance as the low positive rate included in the assay. CMV-specific IgA serology was performed by antibody-capture ELISA. Plates (Nunc Immunoplate I, Roskilde, Denmark) were coated with F(ab'12 fragments of goat antihuman IgA (Cappel, Cochranville, PA) overnight at 4°C. In order to eliminate rheumatoid
61
factors, sera were absorbed onto sheep antibodies directed to human IgG (RF-absorbentBehringwerke AG, Marburg, Germany) overnight at 4°C. Supernatants diluted 1/16 from the original serum were added in each well and plates were incubated for 2 h r at room temperature. After three washing steps, they were incubated for 2 h r a t room temperature with peroxydase-conjugated purified CMV (Wellcome Laboratories). After washing again, orthophenylenediamine (1 mg/ml in 100 mM phosphate buffer [pH 6.01 containing 0.01% [w/v] H,Oz) was added for 30 min at 4°C. The absorbance was read at 492 nm with a Titertek Multiskan spectrophotometer (Flow Laboratories, McLean, VA). Serum specimens with absorbance 600) 5/25 (20) 10125 (40) Group I11 (CD4 200-400) 7/20 (35) 5/20 (25) 2/11 (18) 3/11 (27) Group IV (CD4 < 200)
group I V
(m< 2 0 0 ) Fig. 2. Anti-CMV IgG antibody titres in HIV positive patients in relation to CD4 cell number and in a n HIV negative control group. GMT (full line) ? SD (thin dashed lines) calculated for CMV seropositive individuals are indicated for each group. Thick dashed line represents the positivity threshold. No significant difference in titre was observed between the four groups.
that these cells are needed for the maintenance of active CD8 population. The alteration in EBV antibody response previously reported in other immunologically compromised states as ataxia telangiectasia or Hodgkin’s disease [Berkel et al., 1979; Lange et al., 19781 is also typical of these diseases. Previous studies on serological responses to EBV and CMV in HIV positive patients demonstrated a high prevalence of specific antibodies and higher antibody titres as compared to HIV negative patients [Sumaya et al., 1986; Halbert et al., 19861. Nevertheless, these studies were based on the clinical assessment of patients and not on the absolute CD4 counts as prognostic indicators. High titres of CMV antibodies have been reported to be associated with subsequent AIDS [Polk et al., 19871 but these results have not been confirmed [Holmberg et al., 19881. Rahman et al. [1989] failed to demonstrate any significant change of EBV antibody level in ARC patients compared with HIV-seropositive
men who were asymptomatic or who had lymphadenopathy . We found IgA to VCA with a slight increased frequency in HIV positive compared to negative patients but no significant difference was noted. The percentage of patients with IgA antibodies to VCA did not increased when CD4 cell number declined. These results are in contrast with a previous study demonstrating a n increase of these antibodies as the stage of the disease worsened [Margalith et al., 19901. IgA antibodies to EA and to CMV could not be detected in any HIV infected patients, as previously reported [Sumaya et al., 1986; Nielsen et al., 19881. This study provides serological evidence for a n active infection with EBV and CMV early in the course of HIV disease. It suggests a parallel stimulation of humoral immunity to VCA in relation to the EBV reactivation and a decrease of immunity to EBNA in relation to the immunodeficiency, a s the number of CD4 cells declines. It is speculated th a t this persistent infection with viruses well-known to suppress cell-mediated immune functions accelerate the immunodeficiency disease but further experimental data are needed to elucidate the mechanism.
ACKNOWLEDGMENTS We wish to thank F. Duplat for technical assistance and E. Gagnaire for secretarial assistance. We acknowledge Wellcome Laboratories for providing peroxydase conjugated CMV antigen. This work was supported by a grant of “Fondation Merieux” and a grant of “Region RhBne-Alpes.”
REFERENCES Alsip GR, Sumaya CV, Boswell RN (1988): Increased Epstein-Barr Virus DNA in oropharyngeal secretions from patients with AIDS, AIDS-related complex, or asymptomatic human immunodeficiency virus infections. Journal of Infectious Diseases 157:1072-1076. Berkel AI, Henle W, Henle G, Klein G, Ersoy F, Sanal 0 (1979): Epstein-Barr virus related antibody patterns in ataxia-telangiectasia. Clinical Experimental Immunology 35:196-201. Birx DL, Redfield RR, Tosato G (1986): Defective regulation of Epstein-Barr virus infection in patients with acquired immunodeficiency syndrome (AIDS)or AIDS-related disorders. New England Journal of Medicine 314:874-879. Blumberg RS, Paradis T, Byington R, Henie W, Hirsch MS, Scooley RT (1987): Effects of human immunodeficiency virus on the cellular immune response to Epstein-Barr virus in homosexual men: characterization of the cytotoxic response and lymphokine production. Journal of Infectious Diseases 155:877-890. Clouse KA, Robbins PB, Fernie B, Ostrove JM, Fauci AS (1989):Viral
64 antigen stimulation o f the production of' human moniikines capable of regulating H I V l expression. Journal of Immunology 143 470-475. 1)avis G , Kenncy SC, Kaminr J. Pagano JS. Huang ES (1987): Immediate-early gene region of human cytomegalovirus transactivates the promoter of human immunodeficiency virus. Proceedings of the National Academy of Sciences of the United States 01' America 84:8642-8646. Drtels R , Visscher BR, Fahey JL. Schwartz K , Grcene RS, Madden Dl., Sever JL. Gottlieb MS 19641:The relation of cytomega1oviru.i and Etxtein-Barr virus antibodies to T-cell subsets in homosexually active men. Journal of the American Medical Association 251:1719-1722. Diaz-Mitoma F, Ruiz A, Flowerdew G, Houston S, Romanowski B, Kovithavongs T, Preiksaitis J , Tyrrell D (1990): High levels of Epstein-Barr virus in the oropharynx: a predictor of disease progression in human immunodeficiency virus infection. Journal of Medical Virology 31:69-75. Drew WL, Mills J, Levy J , Dylewski J , Casavant C, Ammann AJ, Brodie H, Merigan T (1985): Cytomegalovirus infection and abnormal T-lymphocyte subset ratios in homosexual men. Annals of Internal Medecine 103:61-63. Fiala M, Cone LA, Chang CM, Mocarski ES (1986): Cytomegalovirus viremia increases with progressive immune deficiency in patients infected with HTLV-111. AIDS Research 2:175-181. Halbert SP, Kiefer DJ, Friedman-Kien AE, Poiesz B (19861: Antibody levels for cytomegalovirus, herpes simplex virus ans rubella in patients with acquired immune deficiency syndrome. Journal of Clinical Microbiology 23:318-321. Henle G, Henle W (1966): Imniunofluorescence in cells derived from Burkitt's lymphoma. Journal of Bacteriology 91:1248-1256. Henle W, Henle G (1981): Epstein-Barr virus-specific serology in immunologically compromised individuals. Cancer Research 41:42224225. Henle G, Henle W, Horwitz CA (1974): Antibodies to Epstein-Barr virus-associated nuclear antigen in infectious mononucleosis. Journal of Infectious Diseases 130:231-239. Hirsch MS, Schooley RT, Ho DD, Kaplan J C (1984): Possible viral interactions in the acquired immunodeficiency syndrome (AIDS). Reviews of Infectious Diseases 6:726-731. Holmberg SD, Gerber AR, Stewart J A , Lee FK, OMalley P, Nahmias AJ (1988):Herpes viruses as co-factors in AIDS. Lancet 2:746747. Jacobson MA, Mills J (1988): Serious cytomegalovirus disease in the acquired immunodeficiency syndrome (AIDS).Annals of Internal Medicine 108:585-594. Lange B, Arbeter A, Hewetson J , Henle W (1978):Longitudinal study of Eustein-Barr virus antibodv titers and excretion in uediatric patients with Hodgkin's disease. International Journal df Cancer 22:521-527. Margalith M, Sarov B, Sarov I, Rinaldo C, Detels R, Phair J, Kaslow R, Ginsberg H, Saah A (1990): Serum IgG and IgA antibodies
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