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Virus-specific antibodies to Epstein-Barr virus, varicellazoster virus and rubella virus in renal transplant patients with cytomegalovirus infections
ffournaloflnfectwn (x992) 24, 3ox-3o9 V i r u s - s p e c i f i c a n t i b o d i e s to E p s t e i n - B a r r virus, v a r i c e l l a z o s t e r v i r u s a n d r u b e l l a v i r u s in r e n a l t r a n s p l a n t p a t i e n t s with cytomegalovirus infections Hugh J. O'Neill and Praful V. Shirodaria
Regional Virus Laboratory, Department of Microbiology and Immunobiology, Royal Victoria Hospital and Department of Microbiology and Immunobiology, The Queen's University of Belfast, Belfast BTI2 6BN, U.K. Accepted for publication 23 October r99I Summary Renal transplant patients with primary and recurrent cytomegalovirus (CMV) infection had higher antibody titres to Epstein-Barr virus viral capsid antigen (EBVVCA-IgG) before and after transplantation than healthy blood donors. The geometric mean titres (GMT) of EBV-VCA-IgG were higher in renal transplant patients without CMV infection than in renal transplant patients with CMV infection. Fourfold or greater rises in EBV-VCA-IgG antibody were detected in six patients and a similar rise in antibody to EBV early antigen (EBV-EA-IgG) was detected in one other patient. IgM antibody to EBV-VCA (EBV-VCA-IgM) was detected in only three of these patients. EBV-EA-IgG was present in 39 % patients and in 30 % control subjects. IgG titres to varicella zoster virus (VZV-IgG) and rubella virus (rubeUa HI) were higher in patients without CMV infection compared to the patients with CMV infection. Raised titres were detected to VZV in five patients and to rubella virus in three patients. Reductions in antibody titre of four-fold or more were also detected in EBV-EA-IgG (one patient) and to rubella virus (one patient). Raised antibody titres to EBV, VZV, and rubella virus in renal transplant patients may indicate reactivation of these viruses without any symptoms.
Introduction I n renal transplant patients, infection with C M V is c o m m o n while reactivation of other latent viruses such as EBV, V Z V and herpes simplex virus (HSV) has also been reported. 1-5 Moreover, it is n o t u n c o m m o n for renal transplant patients to have a n t i b o d y responses to two or more viruses after transplantation. Some investigators have f o u n d a correlation between C M V infections and the a n t i b o d y responses to other viruses in renal transplant patients.2' 8 Similarly, raised titres o f rubella and other virus-specific antibodies have also been f o u n d in various a u t o i m m u n e diseases. 6'7 T h e principal objective o f this study was to measure viral-specific antibodies to EBV, VZV, and rubella virus and to examine the relationship between changes in these antibodies and C M V infection after renal transplantation.
Materials and methods S e r u m samples were collected before a n d after transplantation f r o m 20 patients with p r i m a r y C M V infection and 16 patients with recurrent C M V infection. T h e groups o f patients were selected according to the presence or absence of o6t3-4453/92/o3o3oi +09 $03.00/0
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antibodies to CMV which were detected by complement fixation (CFT) and immunofluorescence (IFA) as described previously. 8 T h e samples tested were collected within 3 months after transplantation. T h e y coincided with CMV infection and showed increased antibody titres to CMV. T h e samples were sex and age matched, within a range of 2 years, with samples collected from healthy blood donors. In addition, serum samples were also obtained from I5 patients who were sero-negative for C M V before transplantation and within 3 months after transplantation. All serum samples from patients and control subjects were stored at - 2 0 ~ and tested in a single batch for each virus antibody. In each batch of tests appropriate positive and negative controls of known titre were included. Immunoglobulin G (IgG) antibodies to EBV VCA (EBV-VCA-IgG), EBV EA (EBV-EA-IgG) and VZV (VZV-IgG) were determined by the indirect immunofluorescence technique2 EBV producing P 3 H R - I cells 1~were used to detect EBV-VCA-IgG. T h e cells were grown in R P M I medium supplemented with IO % heat-inactivated fetal bovine serum. T h e cells were washed three times in PBS and their concentration adjusted to i x ioT/ml. Each 8 m m well of Io-well multiwell slides (Flow Laboratories, Irvine, Scotland) was seeded with 5 #1 of this suspension. T h e slides were allowed to dry at room temperature, fixed in acetone for IO rain then stored at - 2 0 ~ until required. In order to detect EBV-EA-IgG, multiwell slides were prepared with EBV genome-carrying non-producing Raji cells. 1~ EBV-EV was induced in these cells by supplementing the growth medium with 40 n g / m l I2-O-tetradecanoylphorbol-I3-acetate (TPA). Fixation of these cells in acetone for io min at room temperature allowed the detection of both the diffuse and restricted pattern of EBV-EA-IgG. For the detection of VZV-IgG, monolayers of normal uninfected h u m a n embryo lung (HEL) cells and monolayers of H E L cells infected with VZV were dispersed with 0"25% trypsin and then resuspended in growth medium at a concentration of i-2 x ioS/ml. T h e cells were mixed in a ratio of infected to uninfected cells of 3 : I and 50/zl of this suspension was placed in each well of multiwell slides. T h e slides were incubated on covered trays in a humidified 5 % COs atmosphere in air at 37 ~ for 48 h. At this time, when a 20 % cytopathic effect had developed, the slides were washed in PBS, fixed in acetone and stored as previously described until required. All serum samples were screened at a dilution of I in io by means of fluorescein isothiocyanate (FITC)-conjugated goat anti-human IgG (Atlantic Antibodies, Scarborough, ME, U.S.A.) which, after previous titration, was used at its optimal dilution (I in 40). Rubella antibody was measured by use of an established haemagglutination inhibition technique. 11 Briefy, test samples were absorbed with kaolin so as to remove non-specific inhibitors before being diluted from i in IO to I in 640 in microtitre trays with hepes saline albumin buffer. T h e n eight haemagglutinating units of antigen and 0.6 % trypsin-treated human group ' O ' cells were added to each dilution. T h e reciprocal of the highest dilution showing complete inhibition was recorded as the serum titre. Immunoglobulin M (IgM) antibodies to EBV-VCA (EBV-VCA-IgM) were determined by the indirect immunofluorescence technique. Serum samples were absorbed overnight with heat-aggregated gamma globulin and with
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protein A for 30 min at room temperature. Rubella IgM antibodies were detected with the Rubenostica IgM Microelisa System (Organon Teknika, Milton Road, Cambridge, U.K.). I g M antibodies to VZV (VZV-IgM) were determined by IFA. T h e VZV-infected cells were prepared on slides as described above and the serum samples screened at a dilution of I in IO by means of F I T C - c o n j u g a t e d goat anti-human IgM (Behring Diagnostics, Hoechst House, Salisbury Road, Hounslow, U.K.). T h e Wilcoxon matched-pairs signed-ranks test was used to compare the titres of EBV-VCA-IgG, EBV-EA-IgG, V Z V - I g G and rubella H I antibodies before and after transplantation in patients who developed CMV infection with samples from age- and sex-matched controls. T h e usual level of significance (P < o'o5) was used throughout. Results
T h e geometric mean antibody titres ( G M T ) of the renal transplant patients with and without CMV infection are shown in Table I. This table also shows the G M T of the age- and sex-matched controls for the patients with primary and recurrent CMV infection. In the three groups of renal transplant patients, the G M T of E B V - V C A - I g G was increased after transplantation. This rise in G M T was due to the increase in titre of E B V - V C A - I g G in four patients with primary CMV infection and in one patient without CMV infection. In addition, one patient with recurrent CMV infection sero-converted for EBV-VCA-IgG. E B V - V C A - I g M was detected post-transplantation in two of three patients tested. Serum was not available for IgM testing from the three other patients with raised titres of E B V - V C A - I g G (Table II). T h e EBVE A - I g G titres in most patients remained unchanged and therefore only small differences in the G M T were detected after transplantation (Table I). In one patient with primary CMV infection, however, the E B V - E A - I g G titre increased from < IO to I6O. E B V - V C A - I g M was also detected in this patient (Table II). In one patient without CMV infection the E B V - E A - I g G titre decreased from 16o to 2o (Table II). It is noteworthy that E B V - E A - I g G was present in 39 % patients and in 33 % controls. A small increase in the G M T of V Z V - I g G was detected after transplantation in all three groups of patients (Table I). In most patients, however, the titres remained unchanged or only a two-fold increase was detected. Four-fold rises in titres of V Z V - I g G were found in three patients with primary CMV infection and in two patients without CMV infection. V Z V - I g M was not detected in three of these five patients. Serum was not available for V Z V - I g M testing from the other two patients with raised V Z V - I g G titres (Table II). T h e r e was a small increase in the G M T of antibody to rubella virus (rubella HI) in patients with primary CMV infection but a small decline was detected in patients with recurrent CMV infection and in patients without CMV infection (Table I). Four-fold increases in titre of antibody to rubella virus were detected in two patients with primary CMV infection and in one patient with recurrent CMV infection. T h e r e was a greater than four-fold decrease in the rubella H I titre in a patient with recurrent CMV infection and also in a patient without CMV infection (Table II). Rubella virus IgM antibody was
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Table I I I Probabilities resulting from the comparison of viral antibody titres in
patients with C M V infections and their matched controls
Primary CMV
EBV-VCA-IgG before after transplantation
EBV-EA-IgG before after " transplantation
VZV-IgG before after transplantation
Rubella H I before after transplantation
0"030*
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0"87
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0"66
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0"97
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o.12
(n ~ 20
Recurrent CMV ( n - - I6)
* Indicates P < 0"05. Wilcoxon matched-pairs signed ranks test was used.
not detected in three of these patients. Serum from the other two patients was not available for I g M testing. Comparison of the viral antibody titres in patients with primary C M V infection and recurrent C M V infection with those of their respective age- and sex-matched controls is shown in Table III. T h e r e were significant differences in the titres of E B V - V C A - I g G before transplantation (P < o'o5) between the patients with primary C M V infection as well as those with recurrent C M V infection and their respective age- and sex-matched controls. T h e s e differences were more significant after transplantation in patients with increases of EBVV C A - I g G titres. T h e r e was no significant difference between the patients and controls as regards the titres o f E B V - E A - I g G , V Z V - I g G and rubella H I either before or after transplantation. It is noteworthy that antibody titres were generally higher in patients without C M V infection than in those with C M V infection (Table I). Discussion
T h e renal transplant patients with primary and recurrent C M V infection were selected on the presence or absence of C M V antibodies as determined by the relatively insensitive C F T . T h e results were confirmed, however, by the more sensitive I F A technique, s Renal transplant patients with primary and recurrent C M V infection had higher E B V - V C A - I g G titres before transplantation than healthy blood donor controls. Although age- and sex-matched controls were not available for the group of patients without C M V infection, it is worth noting that they had a higher E B V - V C A - I g G G M T than patients with either primary or recurrent C M V infection. T h e increase in the G M T of E B V - V C A - I g G after transplantation in the three groups of patients was caused mainly by greater than four-fold rises in antibody titres in a few individual patients rather than by a general increase in the titres of all the patients in each group. Although four-fold or greater rises in E B V - V C A - I g G antibody titres were found in five patients, and another patient sero-converted to E B V - V C A - I g G , there was no corresponding rise in the titres of E B V - E A - I g G in these patients. Moreover, there was no significant difference between the incidence of antibody to E B V - E A - I g G in the patients (39 %) compared to normal control
Virus-specific antibodies
3o7
subjects (33 %). This contrasts with a previous study 12 which found that EBVE A - I g G was present in Ioo % renal transplant patients compared to only 25 % control subjects. In the present study, a greater than four-fold increase in the E B V - E A - I g G titre was detected in one patient. This suggests reactivation of EBV infection. 13 T h e presence of E B V - V C A - I g M in this patient and in two others is further evidence of current or active EBV infection. 14 In contrast, specific-IgM antibody was not found in patients with raised titres of V Z V - I g G or of antibody to rubella virus. Reactivation of VZV is not always accompanied by the presence of V Z V - I g M antibodies. 15 It is worth noting that higher titres of V Z V - I g G and rubella antibody were found in patients without CMV infection compared to the patients with CMV infection. Raised viral antibody titres have previously been demonstrated in the serum of renal patients after transplantation. 1-5,1~ Furthermore, primary EBV infection has been found in conjunction with primary CMV infection. 5 Concurrent infection with CMV, VZV or HSV has also been demonstrated, e, * In addition, raised antibody titres to measles virus and adenovirus have been reported in conjunction with CMV infection in renal transplant patients. 2 Although raised antibody titres to rubella virus have been observed in various autoimmune diseases, 6'7 the present study is the first to investigate this response in renal transplant patients. T h e results show that the increase in antibody titres to rubella virus, like the increased titres to EBV and VZV, was concurrent with CMV infection. T h e increased antibody titres to more than one viral antigen in renal transplant patients may be due to genuine dual infections. Significant rises in antibody titres to EBV and VZV in renal transplant patients and which were accompanied by typical clinical symptoms have been reported. 1,2,5 Latency and reactivation of EBV and VZV are well recorded is' 19 as is reinfection with rubella virus, e~ It is also known that, after congenital and post natally-acquired rubella infection, the virus may persist and cause chronic infection of the central nervous systemfl I In the present study, however, the patients with significant four-fold rises in antibody titres to EBV, VZV and rubella virus did not show any symptoms normally associated with infections caused by these viruses (Dr McGeown, Renal Unit, Belfast City Hospital, personal communication). Significant reductions in antibody titres to EBV-VCA have been previously reported in renal transplant patients. 16 Falling titres of EBV-VCA-IgG or V Z V - I g G were not found in the present study but reductions in titre of fourfold or more were detected in E B V - E A - I g G and in antibody to rubella virus. T h e r e is no obvious explanation for this. It should be emphasised, however, that these patients in the Renal Unit, Belfast City Hospital were treated with low doses of steroid after transplantation. 22 It may be that this form of treatment accounts for the fluctuations in the antibody titres since it is known that long-term, low-dose steroid therapy may result in hypogammaglobulinaemia. ~ Alternatively, the serological responses may be due to an alteration in the immunoregulatory mechanism. Hence, in some patients this may lead to polyclonal activation of antibody-producing cells. It is known that CMV replicates and persists in cells of the immune system such as B and T lymphocytes and macrophages ~4 and that during C M V infection the number 14-2
308
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o f h e l p e r T - c e l l s falls. T h e c y t o t o x i c T - c e l l r e s p o n s e m a y b e d e c r e a s e d also. 2s A d e f e c t in t h e T - c e l l f u n c t i o n h a s also b e e n o b s e r v e d in p a t i e n t s w i t h diseases o f u n k n o w n a e t i o l o g y s u c h as c h r o n i c a c t i v e h e p a t i t i s , r h e u m a t o i d a r t h r i t i s , s y s t e m i c l u p u s e r y t h e m a t o s i s a n d m u l t i p l e sclerosis. 26-2a I n a d d i t i o n , a n t i b o d y r e s p o n s e s to m o r e t h a n o n e v i r u s h a v e also b e e n o b s e r v e d in s u c h patients.e. 730-32 F u r t h e r i n v e s t i g a t i o n will b e n e c e s s a r y in o r d e r to d e t e r m i n e w h a t c a u s e s a n i n c r e a s e o r d e c r e a s e in a n t i b o d y r e s p o n s e s to m o r e t h a n o n e v i r u s in r e n a l t r a n s p l a n t p a t i e n t s w i t h a n d w i t h o u t C M V i n f e c t i o n .
References
I. Spencer ES, Andersen HK. Antibodies to the Epstein-Barr virus in kidney transplant recipients. Acta Med Scand 1972; 191: lO7-11o. 2. Fiala M, Payne JE, Berne TV et al. Epidemiology of cytomegalovirus infection after transplantation and immunosuppression. J Infect Dis 1975; 132: 421-433. 3. Balfour H H Jr, Slade MS, Kalis JM, Howard RJ, Simmons RL, Narjarian JS. Viral infections in renal transplant donors and their recipients: a prospective study. Surgery 1977; 8I: 487-492. 4- Ho M. Virus infections after transplantation in man. Arch Virol 1975; 55: 1-24. 5- Marker SC, Ascher NL, Kalis JM, Simmons RL, Narjarian JS, Balfour H H Jr. Epstein-Barr virus antibody responses and clinical illness in renal transplant recipients. Surgery 1979; 85 : 433-44o6. Triger DR, MacCallum FO, Kurtz JB, Wright R. Raised antibody titres to measles and rubella viruses in chronic active hepatitis. Lancet 1972; i: 665-667. 7. Kalliomaki JL, Halonen P, Salmi A. Virus antibodies in serum and synovial fluid of patients with rheumatoid arthritis and other connective tissue diseases. Ann Rheum Dis 1975; 34: 43-47. 8. O'Neill HJ, Shirodaria PV, Connolly JH, Simpson D I H , McGeown MG. Cytomegalovirus-specific antibody responses in renal transplant patients with primary and recurrent CMV infections. J Med Virol 1988; 24: 461-47o. 9. Shirodaria PV, McMillan SA, Callender ME et al. Antibodies specific for measles virus envelope antigens and autoantibodies in patients with chronic active hepatitis, ff Clin Pathol 1985; 38: 1281-1288. IO. Hudewewtz J, Bornkamm GW, Zur hausen H. Effect of the Diterpene Ester TPA on Epstein-Barr virus antigen and DNA synthesis in producer and non-producer cell lines. Virology 198o; 1oo : 175-178. I I. Grist NR, Bell EJ, Follet EAC, Urquhart GED. Diagnostic methods in clinical virology. 3rd ed. Oxford: Blackwell Scientific Publications, 1975 : 116-128. 12. Sumaya CV. Infectious mononucleosis and other EBV infections : diagnostic factors. Lab Manag 1986; 24: 37-46. 13. Sumaya CV. Serological testing of Epstein-Barr virus : developments in interpretation. J Infect Dis 1985; I51: 984-987. 14. Lamy ME, Fauart AM, Leclercq M K et aL Epstein-Barr virus VCA IgM and E B N A antibodies altered by immunofluorescence in microplates. Med Microbiol Immunol 1982; 17o: 247-253. 15. Warier JL, Weller TH. VariceUa-Zoster virus. In: Schmidt NJ, Emmons RW, Eds. Diagnostic procedures for viral, rickettsial and chlamydial infections. Washington, D.C. : American Public Health Association, 1989: 379-406. 16. Armstrong JA, Evans AS, Rao N, Ho M. Viral infections in renal transplant recipients. Infect Immun 1976; 14: 970-975. 17. Walker DP, Longson M, Mallick NP, Johnson RWG. A prospective study of cytomegalovirus and herpes simplex virus disease in renal transplant recipients, ff Clin Pathol 1982; 35: 1190-1193. 18. Henle G, Henle W. Immunofluorescence in cells derived from Burkitt's lymphoma. J Bacteriol 1966; 91: 1248-1256.
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19. Heath RB. Varicella zoster. In: Zukerman AJ, Banatvala JE, Pattison JR, Eds. Principles and practice of clinical virology. London: John Wiley & Sons, 199o: 51-73. 2o. Cradock-Watson JE. Laboratory diagnosis of rubella: past, present and future. Epidemiol Infect I99I; xo7: I-I5. 21. Stroop WG, Beringer JR. Persistent, slow and latent viral infections. Prog Med Virol 1982; 2 8 : I-32. 22. McGeown MG, Douglas JF, Donaldson RA, Hill CM, Kennedy JA, Loughridge WGG. Ten-year results of renal transplantation with azathioprine and prednisolone as only immunosuppression. Lancet 1988; i: 983-985. 23. Lee RJE, Fay AC. Hypogammaglobulinaemia associated with long term, low dose steroid therapy. Postgrad Med J I985 ; 6I: 523-524 . 24. Hamilton JC. Cytomegalovirus and immunity. In : Melnick JL, Ed. Monograph in virology. Basel: Karger, 1982 : Vol. I2. 25. Ho M. Immunology of cytomegalovirus : immunosuppressive effects during infections. In: Plotkin SA, Michelson S, Pagano JS, Rapp F, Eds. C M V : Pathogenesis and prevention of human infection. New York: AR Liss (Birth defects: Original article series), 1984: 2o: I3I-I47. 26. Antel JP, Arnason HGW, Medof KE. Suppressor cell function in multiple sclerosis: correlation with clinical disease activity. Ann Neurol I979; 5: 338-342. 27. Alexander GJM, Nouri-Aria KT, Eddleston A L W F , Williams R. Contrasting relations between suppressor-cell function and suppressor-cell number in chronic liver disease. Lancet I983; i: I29I-I293. 28. Egeland T, Lea T, Mellbye OJ. T-cell immunoregulatory functions in rheumatoid arthritis patients. Scand J Immunol 1983; I8: 355-362. 29. Sakane T, Kotani H, Takada S, Murakawa Y, Veda Y. A defect in the suppressor circuits among OKT4 cell populations in patients with systemic lupus erythematosus occurs independently of a defect in the OKT8 suppressor T cell function. J Immunol 1983; I3X : 753-761. 3o. Phillips PE, Christian CL. Virus antibodies in systemic lupus erythematosus and other connective tissue diseases. Ann Rheum Dis 1973 ; 32:450-456. 31. Fraser KB. Population serology. In: Davidson AN, Humphrey JH, Liversedge WI, McDonald WI, Porterfield JS, Eds. Multiple Sclerosis Research (Proceedings of a joint
conference held by the medical research council and the Multiple Sclerosis Society of Great Britain and Northern Ireland, 17-18 October 1974). London: HMSO, 1975: 53-7932. Haire M. Significance of virus antibodies in multiple sclerosis. Br Med Bull 1977; 33: 40-44 .
Regional Virus Laboratory, Department of Microbiology and Immunobiology, Royal Victoria Hospital and Department of Microbiology and Immunobiology, The Queen's University of Belfast, Belfast BTI2 6BN, U.K. Accepted for publication 23 October r99I Summary Renal transplant patients with primary and recurrent cytomegalovirus (CMV) infection had higher antibody titres to Epstein-Barr virus viral capsid antigen (EBVVCA-IgG) before and after transplantation than healthy blood donors. The geometric mean titres (GMT) of EBV-VCA-IgG were higher in renal transplant patients without CMV infection than in renal transplant patients with CMV infection. Fourfold or greater rises in EBV-VCA-IgG antibody were detected in six patients and a similar rise in antibody to EBV early antigen (EBV-EA-IgG) was detected in one other patient. IgM antibody to EBV-VCA (EBV-VCA-IgM) was detected in only three of these patients. EBV-EA-IgG was present in 39 % patients and in 30 % control subjects. IgG titres to varicella zoster virus (VZV-IgG) and rubella virus (rubeUa HI) were higher in patients without CMV infection compared to the patients with CMV infection. Raised titres were detected to VZV in five patients and to rubella virus in three patients. Reductions in antibody titre of four-fold or more were also detected in EBV-EA-IgG (one patient) and to rubella virus (one patient). Raised antibody titres to EBV, VZV, and rubella virus in renal transplant patients may indicate reactivation of these viruses without any symptoms.
Introduction I n renal transplant patients, infection with C M V is c o m m o n while reactivation of other latent viruses such as EBV, V Z V and herpes simplex virus (HSV) has also been reported. 1-5 Moreover, it is n o t u n c o m m o n for renal transplant patients to have a n t i b o d y responses to two or more viruses after transplantation. Some investigators have f o u n d a correlation between C M V infections and the a n t i b o d y responses to other viruses in renal transplant patients.2' 8 Similarly, raised titres o f rubella and other virus-specific antibodies have also been f o u n d in various a u t o i m m u n e diseases. 6'7 T h e principal objective o f this study was to measure viral-specific antibodies to EBV, VZV, and rubella virus and to examine the relationship between changes in these antibodies and C M V infection after renal transplantation.
Materials and methods S e r u m samples were collected before a n d after transplantation f r o m 20 patients with p r i m a r y C M V infection and 16 patients with recurrent C M V infection. T h e groups o f patients were selected according to the presence or absence of o6t3-4453/92/o3o3oi +09 $03.00/0
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H. J . O ' N E I L L
AND P.V. SHIRODARIA
antibodies to CMV which were detected by complement fixation (CFT) and immunofluorescence (IFA) as described previously. 8 T h e samples tested were collected within 3 months after transplantation. T h e y coincided with CMV infection and showed increased antibody titres to CMV. T h e samples were sex and age matched, within a range of 2 years, with samples collected from healthy blood donors. In addition, serum samples were also obtained from I5 patients who were sero-negative for C M V before transplantation and within 3 months after transplantation. All serum samples from patients and control subjects were stored at - 2 0 ~ and tested in a single batch for each virus antibody. In each batch of tests appropriate positive and negative controls of known titre were included. Immunoglobulin G (IgG) antibodies to EBV VCA (EBV-VCA-IgG), EBV EA (EBV-EA-IgG) and VZV (VZV-IgG) were determined by the indirect immunofluorescence technique2 EBV producing P 3 H R - I cells 1~were used to detect EBV-VCA-IgG. T h e cells were grown in R P M I medium supplemented with IO % heat-inactivated fetal bovine serum. T h e cells were washed three times in PBS and their concentration adjusted to i x ioT/ml. Each 8 m m well of Io-well multiwell slides (Flow Laboratories, Irvine, Scotland) was seeded with 5 #1 of this suspension. T h e slides were allowed to dry at room temperature, fixed in acetone for IO rain then stored at - 2 0 ~ until required. In order to detect EBV-EA-IgG, multiwell slides were prepared with EBV genome-carrying non-producing Raji cells. 1~ EBV-EV was induced in these cells by supplementing the growth medium with 40 n g / m l I2-O-tetradecanoylphorbol-I3-acetate (TPA). Fixation of these cells in acetone for io min at room temperature allowed the detection of both the diffuse and restricted pattern of EBV-EA-IgG. For the detection of VZV-IgG, monolayers of normal uninfected h u m a n embryo lung (HEL) cells and monolayers of H E L cells infected with VZV were dispersed with 0"25% trypsin and then resuspended in growth medium at a concentration of i-2 x ioS/ml. T h e cells were mixed in a ratio of infected to uninfected cells of 3 : I and 50/zl of this suspension was placed in each well of multiwell slides. T h e slides were incubated on covered trays in a humidified 5 % COs atmosphere in air at 37 ~ for 48 h. At this time, when a 20 % cytopathic effect had developed, the slides were washed in PBS, fixed in acetone and stored as previously described until required. All serum samples were screened at a dilution of I in io by means of fluorescein isothiocyanate (FITC)-conjugated goat anti-human IgG (Atlantic Antibodies, Scarborough, ME, U.S.A.) which, after previous titration, was used at its optimal dilution (I in 40). Rubella antibody was measured by use of an established haemagglutination inhibition technique. 11 Briefy, test samples were absorbed with kaolin so as to remove non-specific inhibitors before being diluted from i in IO to I in 640 in microtitre trays with hepes saline albumin buffer. T h e n eight haemagglutinating units of antigen and 0.6 % trypsin-treated human group ' O ' cells were added to each dilution. T h e reciprocal of the highest dilution showing complete inhibition was recorded as the serum titre. Immunoglobulin M (IgM) antibodies to EBV-VCA (EBV-VCA-IgM) were determined by the indirect immunofluorescence technique. Serum samples were absorbed overnight with heat-aggregated gamma globulin and with
Virus-specific antibodies
303
protein A for 30 min at room temperature. Rubella IgM antibodies were detected with the Rubenostica IgM Microelisa System (Organon Teknika, Milton Road, Cambridge, U.K.). I g M antibodies to VZV (VZV-IgM) were determined by IFA. T h e VZV-infected cells were prepared on slides as described above and the serum samples screened at a dilution of I in IO by means of F I T C - c o n j u g a t e d goat anti-human IgM (Behring Diagnostics, Hoechst House, Salisbury Road, Hounslow, U.K.). T h e Wilcoxon matched-pairs signed-ranks test was used to compare the titres of EBV-VCA-IgG, EBV-EA-IgG, V Z V - I g G and rubella H I antibodies before and after transplantation in patients who developed CMV infection with samples from age- and sex-matched controls. T h e usual level of significance (P < o'o5) was used throughout. Results
T h e geometric mean antibody titres ( G M T ) of the renal transplant patients with and without CMV infection are shown in Table I. This table also shows the G M T of the age- and sex-matched controls for the patients with primary and recurrent CMV infection. In the three groups of renal transplant patients, the G M T of E B V - V C A - I g G was increased after transplantation. This rise in G M T was due to the increase in titre of E B V - V C A - I g G in four patients with primary CMV infection and in one patient without CMV infection. In addition, one patient with recurrent CMV infection sero-converted for EBV-VCA-IgG. E B V - V C A - I g M was detected post-transplantation in two of three patients tested. Serum was not available for IgM testing from the three other patients with raised titres of E B V - V C A - I g G (Table II). T h e EBVE A - I g G titres in most patients remained unchanged and therefore only small differences in the G M T were detected after transplantation (Table I). In one patient with primary CMV infection, however, the E B V - E A - I g G titre increased from < IO to I6O. E B V - V C A - I g M was also detected in this patient (Table II). In one patient without CMV infection the E B V - E A - I g G titre decreased from 16o to 2o (Table II). It is noteworthy that E B V - E A - I g G was present in 39 % patients and in 33 % controls. A small increase in the G M T of V Z V - I g G was detected after transplantation in all three groups of patients (Table I). In most patients, however, the titres remained unchanged or only a two-fold increase was detected. Four-fold rises in titres of V Z V - I g G were found in three patients with primary CMV infection and in two patients without CMV infection. V Z V - I g M was not detected in three of these five patients. Serum was not available for V Z V - I g M testing from the other two patients with raised V Z V - I g G titres (Table II). T h e r e was a small increase in the G M T of antibody to rubella virus (rubella HI) in patients with primary CMV infection but a small decline was detected in patients with recurrent CMV infection and in patients without CMV infection (Table I). Four-fold increases in titre of antibody to rubella virus were detected in two patients with primary CMV infection and in one patient with recurrent CMV infection. T h e r e was a greater than four-fold decrease in the rubella H I titre in a patient with recurrent CMV infection and also in a patient without CMV infection (Table II). Rubella virus IgM antibody was
H. J.O'NEILL AND P. V. SHIRODARIA
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Table I I I Probabilities resulting from the comparison of viral antibody titres in
patients with C M V infections and their matched controls
Primary CMV
EBV-VCA-IgG before after transplantation
EBV-EA-IgG before after " transplantation
VZV-IgG before after transplantation
Rubella H I before after transplantation
0"030*
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0"87
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0"97
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(n ~ 20
Recurrent CMV ( n - - I6)
* Indicates P < 0"05. Wilcoxon matched-pairs signed ranks test was used.
not detected in three of these patients. Serum from the other two patients was not available for I g M testing. Comparison of the viral antibody titres in patients with primary C M V infection and recurrent C M V infection with those of their respective age- and sex-matched controls is shown in Table III. T h e r e were significant differences in the titres of E B V - V C A - I g G before transplantation (P < o'o5) between the patients with primary C M V infection as well as those with recurrent C M V infection and their respective age- and sex-matched controls. T h e s e differences were more significant after transplantation in patients with increases of EBVV C A - I g G titres. T h e r e was no significant difference between the patients and controls as regards the titres o f E B V - E A - I g G , V Z V - I g G and rubella H I either before or after transplantation. It is noteworthy that antibody titres were generally higher in patients without C M V infection than in those with C M V infection (Table I). Discussion
T h e renal transplant patients with primary and recurrent C M V infection were selected on the presence or absence of C M V antibodies as determined by the relatively insensitive C F T . T h e results were confirmed, however, by the more sensitive I F A technique, s Renal transplant patients with primary and recurrent C M V infection had higher E B V - V C A - I g G titres before transplantation than healthy blood donor controls. Although age- and sex-matched controls were not available for the group of patients without C M V infection, it is worth noting that they had a higher E B V - V C A - I g G G M T than patients with either primary or recurrent C M V infection. T h e increase in the G M T of E B V - V C A - I g G after transplantation in the three groups of patients was caused mainly by greater than four-fold rises in antibody titres in a few individual patients rather than by a general increase in the titres of all the patients in each group. Although four-fold or greater rises in E B V - V C A - I g G antibody titres were found in five patients, and another patient sero-converted to E B V - V C A - I g G , there was no corresponding rise in the titres of E B V - E A - I g G in these patients. Moreover, there was no significant difference between the incidence of antibody to E B V - E A - I g G in the patients (39 %) compared to normal control
Virus-specific antibodies
3o7
subjects (33 %). This contrasts with a previous study 12 which found that EBVE A - I g G was present in Ioo % renal transplant patients compared to only 25 % control subjects. In the present study, a greater than four-fold increase in the E B V - E A - I g G titre was detected in one patient. This suggests reactivation of EBV infection. 13 T h e presence of E B V - V C A - I g M in this patient and in two others is further evidence of current or active EBV infection. 14 In contrast, specific-IgM antibody was not found in patients with raised titres of V Z V - I g G or of antibody to rubella virus. Reactivation of VZV is not always accompanied by the presence of V Z V - I g M antibodies. 15 It is worth noting that higher titres of V Z V - I g G and rubella antibody were found in patients without CMV infection compared to the patients with CMV infection. Raised viral antibody titres have previously been demonstrated in the serum of renal patients after transplantation. 1-5,1~ Furthermore, primary EBV infection has been found in conjunction with primary CMV infection. 5 Concurrent infection with CMV, VZV or HSV has also been demonstrated, e, * In addition, raised antibody titres to measles virus and adenovirus have been reported in conjunction with CMV infection in renal transplant patients. 2 Although raised antibody titres to rubella virus have been observed in various autoimmune diseases, 6'7 the present study is the first to investigate this response in renal transplant patients. T h e results show that the increase in antibody titres to rubella virus, like the increased titres to EBV and VZV, was concurrent with CMV infection. T h e increased antibody titres to more than one viral antigen in renal transplant patients may be due to genuine dual infections. Significant rises in antibody titres to EBV and VZV in renal transplant patients and which were accompanied by typical clinical symptoms have been reported. 1,2,5 Latency and reactivation of EBV and VZV are well recorded is' 19 as is reinfection with rubella virus, e~ It is also known that, after congenital and post natally-acquired rubella infection, the virus may persist and cause chronic infection of the central nervous systemfl I In the present study, however, the patients with significant four-fold rises in antibody titres to EBV, VZV and rubella virus did not show any symptoms normally associated with infections caused by these viruses (Dr McGeown, Renal Unit, Belfast City Hospital, personal communication). Significant reductions in antibody titres to EBV-VCA have been previously reported in renal transplant patients. 16 Falling titres of EBV-VCA-IgG or V Z V - I g G were not found in the present study but reductions in titre of fourfold or more were detected in E B V - E A - I g G and in antibody to rubella virus. T h e r e is no obvious explanation for this. It should be emphasised, however, that these patients in the Renal Unit, Belfast City Hospital were treated with low doses of steroid after transplantation. 22 It may be that this form of treatment accounts for the fluctuations in the antibody titres since it is known that long-term, low-dose steroid therapy may result in hypogammaglobulinaemia. ~ Alternatively, the serological responses may be due to an alteration in the immunoregulatory mechanism. Hence, in some patients this may lead to polyclonal activation of antibody-producing cells. It is known that CMV replicates and persists in cells of the immune system such as B and T lymphocytes and macrophages ~4 and that during C M V infection the number 14-2
308
H. J. O ' N E I L L AND P. V. S H I R O D A R I A
o f h e l p e r T - c e l l s falls. T h e c y t o t o x i c T - c e l l r e s p o n s e m a y b e d e c r e a s e d also. 2s A d e f e c t in t h e T - c e l l f u n c t i o n h a s also b e e n o b s e r v e d in p a t i e n t s w i t h diseases o f u n k n o w n a e t i o l o g y s u c h as c h r o n i c a c t i v e h e p a t i t i s , r h e u m a t o i d a r t h r i t i s , s y s t e m i c l u p u s e r y t h e m a t o s i s a n d m u l t i p l e sclerosis. 26-2a I n a d d i t i o n , a n t i b o d y r e s p o n s e s to m o r e t h a n o n e v i r u s h a v e also b e e n o b s e r v e d in s u c h patients.e. 730-32 F u r t h e r i n v e s t i g a t i o n will b e n e c e s s a r y in o r d e r to d e t e r m i n e w h a t c a u s e s a n i n c r e a s e o r d e c r e a s e in a n t i b o d y r e s p o n s e s to m o r e t h a n o n e v i r u s in r e n a l t r a n s p l a n t p a t i e n t s w i t h a n d w i t h o u t C M V i n f e c t i o n .
References
I. Spencer ES, Andersen HK. Antibodies to the Epstein-Barr virus in kidney transplant recipients. Acta Med Scand 1972; 191: lO7-11o. 2. Fiala M, Payne JE, Berne TV et al. Epidemiology of cytomegalovirus infection after transplantation and immunosuppression. J Infect Dis 1975; 132: 421-433. 3. Balfour H H Jr, Slade MS, Kalis JM, Howard RJ, Simmons RL, Narjarian JS. Viral infections in renal transplant donors and their recipients: a prospective study. Surgery 1977; 8I: 487-492. 4- Ho M. Virus infections after transplantation in man. Arch Virol 1975; 55: 1-24. 5- Marker SC, Ascher NL, Kalis JM, Simmons RL, Narjarian JS, Balfour H H Jr. Epstein-Barr virus antibody responses and clinical illness in renal transplant recipients. Surgery 1979; 85 : 433-44o6. Triger DR, MacCallum FO, Kurtz JB, Wright R. Raised antibody titres to measles and rubella viruses in chronic active hepatitis. Lancet 1972; i: 665-667. 7. Kalliomaki JL, Halonen P, Salmi A. Virus antibodies in serum and synovial fluid of patients with rheumatoid arthritis and other connective tissue diseases. Ann Rheum Dis 1975; 34: 43-47. 8. O'Neill HJ, Shirodaria PV, Connolly JH, Simpson D I H , McGeown MG. Cytomegalovirus-specific antibody responses in renal transplant patients with primary and recurrent CMV infections. J Med Virol 1988; 24: 461-47o. 9. Shirodaria PV, McMillan SA, Callender ME et al. Antibodies specific for measles virus envelope antigens and autoantibodies in patients with chronic active hepatitis, ff Clin Pathol 1985; 38: 1281-1288. IO. Hudewewtz J, Bornkamm GW, Zur hausen H. Effect of the Diterpene Ester TPA on Epstein-Barr virus antigen and DNA synthesis in producer and non-producer cell lines. Virology 198o; 1oo : 175-178. I I. Grist NR, Bell EJ, Follet EAC, Urquhart GED. Diagnostic methods in clinical virology. 3rd ed. Oxford: Blackwell Scientific Publications, 1975 : 116-128. 12. Sumaya CV. Infectious mononucleosis and other EBV infections : diagnostic factors. Lab Manag 1986; 24: 37-46. 13. Sumaya CV. Serological testing of Epstein-Barr virus : developments in interpretation. J Infect Dis 1985; I51: 984-987. 14. Lamy ME, Fauart AM, Leclercq M K et aL Epstein-Barr virus VCA IgM and E B N A antibodies altered by immunofluorescence in microplates. Med Microbiol Immunol 1982; 17o: 247-253. 15. Warier JL, Weller TH. VariceUa-Zoster virus. In: Schmidt NJ, Emmons RW, Eds. Diagnostic procedures for viral, rickettsial and chlamydial infections. Washington, D.C. : American Public Health Association, 1989: 379-406. 16. Armstrong JA, Evans AS, Rao N, Ho M. Viral infections in renal transplant recipients. Infect Immun 1976; 14: 970-975. 17. Walker DP, Longson M, Mallick NP, Johnson RWG. A prospective study of cytomegalovirus and herpes simplex virus disease in renal transplant recipients, ff Clin Pathol 1982; 35: 1190-1193. 18. Henle G, Henle W. Immunofluorescence in cells derived from Burkitt's lymphoma. J Bacteriol 1966; 91: 1248-1256.
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19. Heath RB. Varicella zoster. In: Zukerman AJ, Banatvala JE, Pattison JR, Eds. Principles and practice of clinical virology. London: John Wiley & Sons, 199o: 51-73. 2o. Cradock-Watson JE. Laboratory diagnosis of rubella: past, present and future. Epidemiol Infect I99I; xo7: I-I5. 21. Stroop WG, Beringer JR. Persistent, slow and latent viral infections. Prog Med Virol 1982; 2 8 : I-32. 22. McGeown MG, Douglas JF, Donaldson RA, Hill CM, Kennedy JA, Loughridge WGG. Ten-year results of renal transplantation with azathioprine and prednisolone as only immunosuppression. Lancet 1988; i: 983-985. 23. Lee RJE, Fay AC. Hypogammaglobulinaemia associated with long term, low dose steroid therapy. Postgrad Med J I985 ; 6I: 523-524 . 24. Hamilton JC. Cytomegalovirus and immunity. In : Melnick JL, Ed. Monograph in virology. Basel: Karger, 1982 : Vol. I2. 25. Ho M. Immunology of cytomegalovirus : immunosuppressive effects during infections. In: Plotkin SA, Michelson S, Pagano JS, Rapp F, Eds. C M V : Pathogenesis and prevention of human infection. New York: AR Liss (Birth defects: Original article series), 1984: 2o: I3I-I47. 26. Antel JP, Arnason HGW, Medof KE. Suppressor cell function in multiple sclerosis: correlation with clinical disease activity. Ann Neurol I979; 5: 338-342. 27. Alexander GJM, Nouri-Aria KT, Eddleston A L W F , Williams R. Contrasting relations between suppressor-cell function and suppressor-cell number in chronic liver disease. Lancet I983; i: I29I-I293. 28. Egeland T, Lea T, Mellbye OJ. T-cell immunoregulatory functions in rheumatoid arthritis patients. Scand J Immunol 1983; I8: 355-362. 29. Sakane T, Kotani H, Takada S, Murakawa Y, Veda Y. A defect in the suppressor circuits among OKT4 cell populations in patients with systemic lupus erythematosus occurs independently of a defect in the OKT8 suppressor T cell function. J Immunol 1983; I3X : 753-761. 3o. Phillips PE, Christian CL. Virus antibodies in systemic lupus erythematosus and other connective tissue diseases. Ann Rheum Dis 1973 ; 32:450-456. 31. Fraser KB. Population serology. In: Davidson AN, Humphrey JH, Liversedge WI, McDonald WI, Porterfield JS, Eds. Multiple Sclerosis Research (Proceedings of a joint
conference held by the medical research council and the Multiple Sclerosis Society of Great Britain and Northern Ireland, 17-18 October 1974). London: HMSO, 1975: 53-7932. Haire M. Significance of virus antibodies in multiple sclerosis. Br Med Bull 1977; 33: 40-44 .