496
Response to Tetanus Toxoid Immunization after Allogeneic Bone Marrow Transplantation Per Ljungman, Mia Wiklund-Hammarsten, Viera Duraj, Lennart Hammarstrom, Berit LOnnqvist, Thomas Paulin, OUe Ringden, Margaret Sullivan Pepe, and Gosta Gahrton
From the Division of Hematology and Clinical Oncology, Department of Medicine, the Department of Clinical Immunology, and the Department of Transplantation Surgery, Karolinska Institute and Huddinge Hospital, Huddinge, Sweden, and Fred Hutchinson Cancer Research Center, Seattle, Washington
An ELISA was used to study long-term immunity and immunization responses to tetanus toxoid in 48 bone marrow transplant recipients. Among patients who were seropositive to tetanus before transplant, 51% had lost their seropositivity 1 year later. All patients who were not reimmunized with tetanus toxoid were seronegative 2 years after transplant. All patients who were seronegative before transplant remained seronegative 1 year later regardless of the donor's serologic status. There was no difference in the ability to remain seropositive to tetanus toxoid between patients with and without chronic graft-versus-host disease. Of 21 patients immunized with one dose of tetanus toxoid 1 year after transplant, 14 were seronegative at the time ofimmunization (response rate, 64%). At 1 year after immunization, 7 remained seropositive. Ten patients were reimmunized with two doses of tetanus toxoid. All responded and 90% remained seropositive 1 year later. When 21 patients were primarily immunized with three doses of tetanus toxoid, all patients seronegative at immunization responded and all tested patients remained seropositive 2 years later. The immunization responses were significantly superior in patients receiving three doses compared with those who received one. Reimmunization with tetanus toxoid of long-term survivors after marrow transplant seems necessary. A three-dose immunization schedule is recommended to obtain an adequate immune response.
Bone marrow transplantation (BMT) is increasingly used in treating an expanding number of disorders, mostly hematologic malignancies, aplastic anemias, and congenital enzyme defects. The immune system gradually reconstitutes after BMT [1-3]. In studies oftransfer of tetanus toxoid-specific immunity [4, 5], f\J50% ofBMT recipients retained antibody levels to tetanus toxoid at long-term follow-up but f\J50% are unprotected against tetanus. Thus, a reimmunization program is needed to ensure protection against tetanus. The aim of this study was to evaluate the response to immunization with tetanus toxoid after BMT.
Patients and Methods Bone marrow transplant recipients. Fifty-three consecutive patients who received BMT at Huddinge Hospital between September
Received 28 September 1989; revised 15 February 1990. Presented in part at the 15th annual meeting of the European Bone Marrow Transplantation Group, 27 February-2 March 1989, Badgarstein, Austria. Informed consent was obtained from patients or their guardians, and the guidelines for human experimentation at Huddinge Hospital and the Karolinska Institute were followed. Grant support: Swedish Society of Medicine, Swedish Medical Research Council, and National Institutes of Health (CA-15704). Reprints and correspondence: Dr. Per Ljungman, Department of Medicine, Huddinge Hospital, S-14186 Huddinge, Sweden. The Journal of Infectious Diseases 1990;162:496-500 © 1990 by The University of Chicago. All rights reserved. 0022-1899/90/6202-0031$01.00
1982 and September 1986 and who had a disease-free survival of after transplant were eligible for study. Four patients from whom adequate serum samples were not available and one patient who was seropositive for human immunodeficiency virus type 1 were excluded. The remaining 48 patients were studied: 11 had acute nonlymphoblastic leukemia, ·14 had acute lymphoblastic leukemia, 9 had chronic myelogenous leukemia, 7 had a aplastic anemia, 3 had multiple myeloma, and 1 each had Fanconi's anemia, non-Hodgkins lymphoma, Langerhans' cell histiocytosis, and combined Band T cell defect. Bone marrow transplant procedure. The procedure was as described in detail previously [6, 7]; 46 patients received matched allogeneic sibling grafts and 2 received syngeneic grafts. Methotrexate, cyclosporine, or both were given as acute graft-versus-host disease (GVHD) prophylaxis [8, 9]. One patient received marrow that was T cell-depleted with monoclonal antibodies [10]. Acute GVHD was graded according to Thomas· et al. [6] and treated with prednisone, initially 2 mg/kg and then tapered. Chronic GVHD was defined according to Shulman et al [11] and Schubert et al. [12] and verified by biopsies of the skin, liver, or oral mucosa. Chronic GVHD was treated with prednisone and azathioprine. Table 1 shows details of GVHD prophylaxis and acute and chronic GVHD in the patient groups. Serology. An enzyme-linked immunosorbent assay (ELISA) was used to determine antibody levels against tetanus toxoid as described previously [13, 14]. Briefly, after incubation of the serum samples (diluted 1:100-1:10,000) overnight on antigen-coated plates, a monoclonal mouse anti-IgG antibody (Bam 06; Seward Laboratories, London, UK) was added in previously determined optimal concentration. After 4 h of incubation at room temperature the plates were washed and rabbit anti-mouse immunoglobulin (Dakopatts, Glostrup, Den~ 2 years
JIO 1990;162 (August)
Tetanus Toxoid Immunization and BMT
497
2,0
Table 1. Acute and chronic graft-versus-host disease (GVHD) and GVHD prophylaxis in bone marrow transplant recipients immunized and not immunized with tetanus toxoid.
1,5
Group
E
c:
Characteristic GVHO prophylaxis Cyclosporine Methotrexate Cyclosporine and methotrexate T cell depletion None (syngeneic) GVHO Acute (grade n-IV) Chronic
.,.
One initial dose;two subsequent doses (n = 21)
Three initial doses (n = 21)
Not immunized
13 7
7 4
3 0
1 0 0
7 1 2
3 0 0
5
1 3
5
12
II)
0
1,0
c
(n
0
0,5
= 6)
1
mark) was added. After another 4 h of incubation the plates were washed and alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin (Sigma Chemical, St. Louis) was added, after which the plates were incubated overnight. After additional washes, disodium p-nitrophenyl phosphate (Sigma) was added and the plates incubated for 10-20 min. Absorbance was measured at 405 om. Reference anti-tetanus toxoid antibodies. A positive reference serum (IgG fraction ofhyperimmune serum) containing 180 IU/mI anti-tetanus toxoid antibodies was purchased from the National Bacteriological Laboratory, Stockholm. Supernatant from a human anti-tetanus toxoid antibody-producing cell line was also used. The line (TT-l; IgG/k) previously has been extensively characterized [13]. Batches ofcrude culture supernatants contained 20-25 JLg/mI as determined by ELISA. The tetanus toxoid antigen was a gift of Dr. M. Fall-Persson (National Bacteriological Laboratory). The antigen (with an estimated purity of 80%) was kept in buffered saline at a concentration of 600 limes flocculation units/mI (1.2 mg/mI). Various amounts of antigen were coated onto polystyrene microtiter plates by using 0.05 M carbonate buffer, ph 9.6, and incubated for 4 h at 37°C. Binding assay was performed in ELISA using a 1:100 dilution of positive sera (reference serum and positive patient samples). Absorbance values leveled off at a coating concentration of 5 limes flocculation units (corresponding to 10 JLg/ml tetanus toxoid) and a coating concentration of 15 limes flocculation units was chosen to ensure antigen excess. An optical density (OD) of 0.5 at a serum dilution of 1:100 corresponding to an antibody concentration of rv 3 ng/mI was regarded as representative of seropositivity (figure 1). Studies were performed for all patients on serum samples taken before and 12 months after BMT. In nonimmunized patients these analyses were also done 24 months after BMT. Studies on immunized patients were done at least at 3 months and 1 year after immunization. Serum samples from 38 bone marrow donors were also analyzed; no sera were available from the other donors. A positive response to immunization was defined as a more than twofold increase in OD level. A few patients in both groups had antibody levels to tetanus toxoid that gave OD values of >1.0, corresponding to
0,0 -Q-T'"T'T......c;....,..l"'9ftIi'I;IIl=T-n-""""'..........,.."mr-TT'l.................TTn.... 1 0- 3 1 0- 2 1 0- 1 100 101 10 2 10 3 -8 -7 -6 -5 -4 -3 -2 10 10 10 10 10 10 10
ng/ml Dilution
Figure 1. Comparison on tetanus toxoid-coated plates between dilutions of positive hyperimmune reference serum containing 180 IU/mI anti-tetanus toxoid antibodies (0) and supernatants from tetanus toxoid antibody-producing cell line TT-l (.). Concentration of antibody in supernatant was previously determined by catch-up on an anti-IgG coated plate compared to a standard (WHO 67/97).
an antibody level of rv 20 ng/mI. These are defined' as high antibody levels concerning the response to immunization. Immunization. The first 21 patients were vaccinated with one dose of tetanus toxoid (National Bacteriological Laboratory, Stockholm) 12 months after BMT. A preliminary evaluation in 1984 showed that the antibody response to this single dose of vaccine was poor. Therefore 17 patients were reimmunized with two more doses of tetanus toxoid 24-36 months after BMT. The next 21 patients were immunized with three doses of vaccine given at 12, 13, and 14 months after BMT. Six patients who were not vaccinated at 1 year (three because of chronic GVHD and three by mistake) were studied at 18 months and 2 years. Statistics. The Mann-Whitney U test (two-sided) was used for comparing OD values after vaccination. The influence of pretransplant antibody levels of patients and donor on each patient's antibody level 1 year after transplant was analyzed using a multivariate linear regression [15].
Results
Serology. Thirty-seven (77 %) of 48 patients and 27 (71 %) of 38 examined donors were seropositive for tetanus toxoid before BMT. There was no difference in antibody levels between patients and donors before BMT. Eighteen patients (49 %) who were seropositive before BMT remained positive 1 year after transplant. There was no difference in antibody level before or 1 year after BMT between the patients primarily receiving one vaccine dose, those receiving three doses, or nonimmunized patients (figure 2). Among the 6 nonimmunized patients, 2 were seropositive 1 year and none were seropositive 2 years after BMT. No patient who was seronegative before BMT was seropositive at 1 year; 5 had seropositive donors, 5 had seronegative donors and 1 donor's antibody status was unknown. When the influences of pretransplant antibody levels of pa-
Ljungman et al.
498
JID 1990;162 (August)
Table 2. Correlation of serologic status 1 year after bone marrow transplantation in patients who were seropositive before transplantation with acute and chronic graft-versus-host disease (GVHD).
2,0
1,5
Status 1 year after transplantation
n.s.
E c
GYHD
.,
In 0
0
0
Seropositive
Acute Grade 0-1 Grade II-IV Chronic None
1,0
0,5
17 (52) 1 (25) 9 (47) 9 (45)
Seronegative 16 3 8 11
(48) (75) (53) (55)
NOTE. Data are no. (%) of patients. 18
12
24
30
36
The response rate to immunization was higher among patients receiving three doses than in those receiving one dose regardless of serologic status at the time of immunization. The strength of the antibody responses to one and three vaccine doses was significantly different 18 and 24 months after BMT (Mann-Whitney U test; figure 2). At 36 months there was no difference between groups. Among 15 patients receiving three vaccine doses from whom serum samples were available, all remained seropositive 2 years after immunization. Among nine seronegative patients who responded to one dose of vaccine, five had chronic GVHD and four did not. Conversely, three of five who did not respond had chronic GVHD and two did not. All seronegative patients responded after three vaccine doses regardless of whether they had chronic GVHD. Thus, chronic GVHD did not influence the immunization response to tetanus toxoid 1 year after BMT.
Months after BMT
Figure 2. Antibody levels (± SE) before and at different times after bone marrow transplantation (BMT) in patients receiving one dose of tetanus toxoid at 1 year followed by two subsequent doses «(!J), patients receiving three primary doses (D), and patients who were not immunized ( • ). Data were compared using MannWhitney U test.
tients and donors on the subsequent antibody level of the patient 1 year after BMT were analyzed, only the patient's pretransplant antibody level was predictive for posttransplant antibody level (P < .01 by linear regression; figure 3). There was no suggestion that the donor's serologic status influenced the patient's antibody level 12 months after transplant (P = .47). Serologic status and GVHD. Table 2 shows the probability of remaining seropositive 1 year after BMT in patients with and without GVHD. There was no difference in the probability of remaining seropositive at 1 year among patients who were seropositive before transplant whether or not they developed chronic GVHD. The number of patients who developed acute GVHD of grade II-IV was too small to allow analysis of an eventual impact on the serologic status at 1 year. Immunization. Table 3 shows the response to immunization in seropositive patients and table 4 in patients who were seronegative at the time of immunization. No major side effects were seen after immunization. One patient had a substantial local reaction after two doses and a third dose was not given.
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Evidence for transfer of donor humoral immunity after allogeneic BMT has been found by several researchers [4, 5, 16-18]. Wahren et ale [16] showed in a sequential study that in most instances the transferred specific antibody production to viruses such as measles, mumps, and rubella, which presumably are not transferred by marrow graft, was finite and only rarely continued after 12 months. Lum et a1. [4] showed in nonsequential studies transfer of long-lasting donor immunity to measles and diphtheria antigens up to 15
1lI~
>c
.. z
Discussion
0.5
0 0 00
0.0 0.0
0
0 go 0 % 00
0 0
0
0.5
o
~
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~~
0
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o o
o o
::::e
00
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0.0 0.0
0.5
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2.5
donor antibody level before transplant
Figure 3. Influence of antibody levels of patients and donors before bone marrow transplantation (BMT) on anti-tetanus antibody levels of patients 12 months after BMT. Only pretransplant antibody level of patient had an influence on posttransplant antibody level (P < .01 by linear regression).
JID 1990;162 (August)
Tetanus Toxoid Immunization and BMT
Table 3. Results of immunization in patients who were seropositive at the time of immunization.
499
Table 4. Results of immunization in patients who were seronegative at the time of immunization.
Group
Group
One initial, two subsequent doses After first dose
Characteristic Immunized Responding With high antibody levels Seropositive 1 year after immunization Antibody titers 1 year after immunization
After "" subsequent doses
One initial, two subsequent doses Three initial doses
7 0
7 3 (43)
9* 6 (55)
4 (57)
4 (57)
3 (33)
7 (100)
7 (100)
9 (100)
1.06 (0.66-1.97) 1.47 (0.65-1.82) 1.65 (1.00-2.11)
NOTE. Data are no. (%) of patients except antibody titers, which are given as mean (range). High antibody levels are defined as optical density values >1.0 at the time of immunization that did not change during follow-up. • One patient developed a substantial local reaction after the second dose and did not receive the third dose.
months after transplant. We could not find any transfer of tetanus immunity to seronegative patients from their seropositive marrow donors, but the number of evaluable donor-recipient pairs was small. An unexpected finding was that patient's, but not the donor's, antibody level before BMT strongly influenced the antibody level 1 year after BMT. The mechanism of this influence is unknown, although persisting recipient lymphocytes have been detected close to a year after BMT [19]. Wimperis et al. [18] showed that when both donor and patient were immunized with tetanus toxoid before BMT the level of antibody measured after transplant was higher than when either donor or recipient alone were immunized. We previously showed that seropositive patients with seronegative donors may retain immunity to viral antigens up to 9 months after BMT [16, 19]. Continued presence of tetanus antigen is extremely unlikely, since in general the patients had not been immunized for several years before the transplant. Thus, persistence of antibodyproducing recipient cells may explain the influence of the recipient's antibody level on persisting immunity. However, there might also be other unknown influencing factors. A separate question is how many long-term survivors after BMT will remain seropositive and thus protected against important infectious agents such as tetanus. We showed that BMT recipients without chronic GVHD who were seropositive 2 years after transplant continued to lose immunity to measles, mumps, and rubella during the next 2 years of follow-up [20]. Here we confirm our previous data in that, although the number of patients who were not immunized was small, patients seropositive before BMT continued to lose specific immunity during long-term follow-up. Almost two-thirds ofthe BMT recipients studied were seronegative 1 year after transplant
After first dose
Characteristic Immunized Responding Seropositive 1 year after immunization Antibody titers 1 year after immunization
14
After subsequent doses
Three initial doses
9 (64)
10* 10 (100)
12 12 (100)
7 (50)
9 (90)
12 (100)
0.87 (0.13-1.82) 1.52 (0.75-2.38) 1.56 (0.77-2.48)
NOTE. Data are no. (%) of patients except antibody titers, which are given as mean (range). • Three patients were not reimmunized and one received immunoglobulin infusions and is not evaluable.
and no nonimmunized patient remained seropositive 2 years after transplant. This is comparable with findings by Lum et al. [5], who in a nonsequential study showed 47% seronegativity among patients studied 7 months to 10 years after BMT. Various posttransplant factors such as GVHD prophylaxis and presence of chronic GVHD are probably important in the duration of transferred and persistent immunity after BMT. Lum et al. [4] showed in long-term survivors a higher probability of retaining specific immunity to tetanus in patients without GVHD and in patients receiving T cell-depleted marrow grafts [5]. No such differences were seen for diphtheria and measles immunity [4]. In the present study we found no difference in the probability of remaining seropositive 1 year after transplant in patients with or without chronic GVHD. These data show that rv50 % oflong-term survivors are not seropositive to tetanus after BMT. The question of reimmunization of BMT recipients has been discussed [1, 17]. However, responses to immunization and immunization schedule needed to obtain adequate immunity are not known. Saxon et al. [17] showed in three patients that transferred donor cells can be restimulated by immunization 2-5 months after BMT. We previously showed that BMT recipients without chronic GVHD can be effectively and safely immunized with live measles, mumps, and rubella vaccine [20]. In the present study, the percentage of patients who responded to immunization and the chance of remaining seropositive after 1 year were lower in the group receiving one dose of vaccine. The immune system is not fully reconstituted 1 year after BMT. A single immunization performed at that time might therefore not be enough to elicit a durable immune response. Whether a different, more durable response could be obtained after a single immunization given a longer time after BMT when the immune system would be more completely reconstituted is unknown. This indicates that although immunity may be transferred from the marrow donor, the number of
500
Ljungman et a1.
antigen-specific B cells present 1 year after transplant is too low to mediate an adequate immune response. This is supported by in vitro antibody production studies in which peripheral blood lymphocytes from BMT recipients failed to produce tetanus toxoid-specific antibodies when cultured at cell numbers adequate for antibody production in normal control subjects [21]. Patients primarily receiving three vaccine doses had less chronic GYHO than did patients receiving one dose. However, this difference cannot explain the poor immunization response after one vaccine dose, since chronic GYHO did not influence the ability to respond to immunization. We conclude that reimmunization with tetanus toxoid of long-term survivors after BMT is necessary and that a schedule with three doses is needed to obtain protective specific antibody levels. Whether BMT recipients lose immunity more rapidly than do normal individuals is unknown. However on the basis ofthe present study it is likely that 3 years after BMT, patients who do not have chronic GYHO will not differ significantly from normal individuals. Further studies are needed to determine if and when follow-up immunizations need to be given. References 1. Lum LG. The kinetics of immune reconstitution after human marrow transplantation. Blood 1987;69:369-81 2. Witherspoon RP, Matthews 0, Storb R, Atkinson K, Cheever M, Deeg HJ, Doney K, Kalbfleisch J, Noel D, Prentice R, Sullivan KM, Thomas ED. Recovery of in vivo cellular immunity after human marrow grafting. Influence of time postgrafting and acute graft-versus-host disease. Transplantation 1984;37:145-150 3. Paulin T, Ringden 0, Nilsson B. Immunological recovery after bone marrow transplantation: role of age, graft-versus-host disease, prednisolone treatment and infections. Bone Marrow Transplant 1987;1:317-328 4. Lum LG, Munn NA, Schanfield MS, Storb R. The detection of specific antibody formation to recall antigens after human bone marrow transplantation. Blood 1986;67:582-587 5. Lum LG, Noges JE, Beatty P, Martin PJ, Deeg J, Doney KC, Loughran T, Sullivan KM, Witherspoon RP, Thomas ED, Storb R. Transfer of specific immunity in marrow recipients given HLA-mismatched, T cell-depleted, or HLA-identical marrow grafts. Bone Marrow Transplant 1988;3:399-406 6. Thomas ED, Storb R, Clift RA, Fefer A, Johnson FL, Neiman PE, Lerner KG, Glucksberg H, Buckner CD. Bone marrow transplantation. N Engl J Med 1975;292:832-843, 895-902 7. Ringden 0, Lonnqvist B, Lundgren G, Gahrton G, Groth CG, Moller E, Baryd I, Johansson B, Pihlstedt P, Gullbring B. Experience with a cooperative bone marrow transplantation program in Stockholm. Transplantation 1982;33:500-504
JID 1990;162 (August)
8. Ringden 0, Backman L, LOnnqvist B, Heimdahl A, Lindholm A, Bolme P, Gahrton G. A randomized trial comparing use of cyclosporin and methotrexate for graft-versus-host disease prophylaxis in bone marrow transplant recipients with haematologic malignancies. Bone Marrow Transplant 1986;1:41-51. 9. Storb R, Deeg HJ, Whitehead J, Farewell V, Appelbaum FR, Beatty P, Bensinger W, Buckner CD, Clift RA, Doney K, Hansen JA, Hill R, Lum LG, Martin P, McGuffin R, Sanders JE, Singer J, Stewart P, Sullivan KM, Witherspoon RP, Thomas ED. Marrow transplantation for leukemia and aplastic anemia: two controlled trials of a combination of methotrexate and cyclosporine versus cyclosporine alone or methotrexate alone for prophylaxis of acute graft-v-host disease. Transplant Proc 1987;19:2608-2613 10. Prentice HG, Blacklock HA, Janossy G, Gilmore MJML, Price-Jones L, Tidman N, Trejdosiewicz LK, Skeggs DBL, Panjwani 0, Ball S, Graphakos S, Patterson J, Ivory K, Hoftbrand AV. Depletion of Tlymphocytes in donor marrow prevents significant graft-versus-host disease in matched allogeneic leukaemic marrow transplant recipients. Lancet 1984;1:472-476 11. Shulman HM, Sullivan KM, Weiden PL, McDonald GB, Striker GE, Sale GE, Hackman R, Tsoi MS, Storb R, Thomas ED. Chronic graftversus-host syndrome in man. A long time clinicopathologic study of 20 Seattle patients. Am J Med 1980;69:204-217 12. Schubert MM, Sullivan KM, Morton TH, Izutsu KT, Peterson DE, Flournoy N, Truelove EL, Sale GE, Buckner CD, Storb R, Thomas ED. Oral manifestations ofchronic graft-v-host disease. Arch Intern Med 1984;144:1591-1595 13. Tiebout RF, Stricker EAM, Hagenaars R, Zeijlemaker WP. Human lymphoblastoid cell line producing protective monoclonal IgGl, K antitetanus toxin. Eur J ImmimolI984;14:399-404 14. Persson MAA, HammarstrOm L, Smith CIE. Enzyme-linked immunosorbent assay for subclass distribution of human IgG and IgA antigenspecific antibodies. J Immunol Methods 1985;78:109-121 15. Draper N, Smith H. Applied Regression Analyses. 2nd ed. New York: John Wiley & Sons, 1981 16. Wahren B, Gahrton G, Linde A, Ljungman P, LOnnqvist B, Ringden 0, Sundqvist VA. Transfer and persistence of viral antibody-producing cells in bone marrow transplantation. J Infect Dis 1984;150:358-365 17. Saxon A, Mitsuyasu R, Stevens R, Champlin RE, Kimata H, Gale RP. Designed transfer of specific immune responses with bone marrow transplantation. J Clin Invest 1986;78:959-967 18. Wimperis JZ, Brenner MK, Prentice HG, Reittie JE, Karayiannis P, Griffiths PD, Hoftbrand AV. Transfer of a functioning humoral immune system in transplantation of T-Iymphocyte-depleted bone marrow. Lancet 1986;1:339-343 19. Ljungman P, Gahrton G, Ringden 0. Wahren B. Donor and recipient origin of herpesvirus-reactive lymphocytes after bone marrow transplantation. Arch VirolI985;83:117-122 20. Ljungman P, Fridell E, LOnnqvist B. Bolme P, BOttiger M, Gahrton G, Linde A. Ringden 0, Wahren B. Efficacy and safety of vaccination of marrow transplant recipients with a live attenuated measles, mumps, and rubella vaccine. J Infect Dis 1989;159:610-615 21. Lum LG, Seigneuret MC, Storb R. The transfer of antigen-specific humoral immunity from marrow donors to marrow recipients. J Clin ImmunolI986;6:389-396
Response to Tetanus Toxoid Immunization after Allogeneic Bone Marrow Transplantation Per Ljungman, Mia Wiklund-Hammarsten, Viera Duraj, Lennart Hammarstrom, Berit LOnnqvist, Thomas Paulin, OUe Ringden, Margaret Sullivan Pepe, and Gosta Gahrton
From the Division of Hematology and Clinical Oncology, Department of Medicine, the Department of Clinical Immunology, and the Department of Transplantation Surgery, Karolinska Institute and Huddinge Hospital, Huddinge, Sweden, and Fred Hutchinson Cancer Research Center, Seattle, Washington
An ELISA was used to study long-term immunity and immunization responses to tetanus toxoid in 48 bone marrow transplant recipients. Among patients who were seropositive to tetanus before transplant, 51% had lost their seropositivity 1 year later. All patients who were not reimmunized with tetanus toxoid were seronegative 2 years after transplant. All patients who were seronegative before transplant remained seronegative 1 year later regardless of the donor's serologic status. There was no difference in the ability to remain seropositive to tetanus toxoid between patients with and without chronic graft-versus-host disease. Of 21 patients immunized with one dose of tetanus toxoid 1 year after transplant, 14 were seronegative at the time ofimmunization (response rate, 64%). At 1 year after immunization, 7 remained seropositive. Ten patients were reimmunized with two doses of tetanus toxoid. All responded and 90% remained seropositive 1 year later. When 21 patients were primarily immunized with three doses of tetanus toxoid, all patients seronegative at immunization responded and all tested patients remained seropositive 2 years later. The immunization responses were significantly superior in patients receiving three doses compared with those who received one. Reimmunization with tetanus toxoid of long-term survivors after marrow transplant seems necessary. A three-dose immunization schedule is recommended to obtain an adequate immune response.
Bone marrow transplantation (BMT) is increasingly used in treating an expanding number of disorders, mostly hematologic malignancies, aplastic anemias, and congenital enzyme defects. The immune system gradually reconstitutes after BMT [1-3]. In studies oftransfer of tetanus toxoid-specific immunity [4, 5], f\J50% ofBMT recipients retained antibody levels to tetanus toxoid at long-term follow-up but f\J50% are unprotected against tetanus. Thus, a reimmunization program is needed to ensure protection against tetanus. The aim of this study was to evaluate the response to immunization with tetanus toxoid after BMT.
Patients and Methods Bone marrow transplant recipients. Fifty-three consecutive patients who received BMT at Huddinge Hospital between September
Received 28 September 1989; revised 15 February 1990. Presented in part at the 15th annual meeting of the European Bone Marrow Transplantation Group, 27 February-2 March 1989, Badgarstein, Austria. Informed consent was obtained from patients or their guardians, and the guidelines for human experimentation at Huddinge Hospital and the Karolinska Institute were followed. Grant support: Swedish Society of Medicine, Swedish Medical Research Council, and National Institutes of Health (CA-15704). Reprints and correspondence: Dr. Per Ljungman, Department of Medicine, Huddinge Hospital, S-14186 Huddinge, Sweden. The Journal of Infectious Diseases 1990;162:496-500 © 1990 by The University of Chicago. All rights reserved. 0022-1899/90/6202-0031$01.00
1982 and September 1986 and who had a disease-free survival of after transplant were eligible for study. Four patients from whom adequate serum samples were not available and one patient who was seropositive for human immunodeficiency virus type 1 were excluded. The remaining 48 patients were studied: 11 had acute nonlymphoblastic leukemia, ·14 had acute lymphoblastic leukemia, 9 had chronic myelogenous leukemia, 7 had a aplastic anemia, 3 had multiple myeloma, and 1 each had Fanconi's anemia, non-Hodgkins lymphoma, Langerhans' cell histiocytosis, and combined Band T cell defect. Bone marrow transplant procedure. The procedure was as described in detail previously [6, 7]; 46 patients received matched allogeneic sibling grafts and 2 received syngeneic grafts. Methotrexate, cyclosporine, or both were given as acute graft-versus-host disease (GVHD) prophylaxis [8, 9]. One patient received marrow that was T cell-depleted with monoclonal antibodies [10]. Acute GVHD was graded according to Thomas· et al. [6] and treated with prednisone, initially 2 mg/kg and then tapered. Chronic GVHD was defined according to Shulman et al [11] and Schubert et al. [12] and verified by biopsies of the skin, liver, or oral mucosa. Chronic GVHD was treated with prednisone and azathioprine. Table 1 shows details of GVHD prophylaxis and acute and chronic GVHD in the patient groups. Serology. An enzyme-linked immunosorbent assay (ELISA) was used to determine antibody levels against tetanus toxoid as described previously [13, 14]. Briefly, after incubation of the serum samples (diluted 1:100-1:10,000) overnight on antigen-coated plates, a monoclonal mouse anti-IgG antibody (Bam 06; Seward Laboratories, London, UK) was added in previously determined optimal concentration. After 4 h of incubation at room temperature the plates were washed and rabbit anti-mouse immunoglobulin (Dakopatts, Glostrup, Den~ 2 years
JIO 1990;162 (August)
Tetanus Toxoid Immunization and BMT
497
2,0
Table 1. Acute and chronic graft-versus-host disease (GVHD) and GVHD prophylaxis in bone marrow transplant recipients immunized and not immunized with tetanus toxoid.
1,5
Group
E
c:
Characteristic GVHO prophylaxis Cyclosporine Methotrexate Cyclosporine and methotrexate T cell depletion None (syngeneic) GVHO Acute (grade n-IV) Chronic
.,.
One initial dose;two subsequent doses (n = 21)
Three initial doses (n = 21)
Not immunized
13 7
7 4
3 0
1 0 0
7 1 2
3 0 0
5
1 3
5
12
II)
0
1,0
c
(n
0
0,5
= 6)
1
mark) was added. After another 4 h of incubation the plates were washed and alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin (Sigma Chemical, St. Louis) was added, after which the plates were incubated overnight. After additional washes, disodium p-nitrophenyl phosphate (Sigma) was added and the plates incubated for 10-20 min. Absorbance was measured at 405 om. Reference anti-tetanus toxoid antibodies. A positive reference serum (IgG fraction ofhyperimmune serum) containing 180 IU/mI anti-tetanus toxoid antibodies was purchased from the National Bacteriological Laboratory, Stockholm. Supernatant from a human anti-tetanus toxoid antibody-producing cell line was also used. The line (TT-l; IgG/k) previously has been extensively characterized [13]. Batches ofcrude culture supernatants contained 20-25 JLg/mI as determined by ELISA. The tetanus toxoid antigen was a gift of Dr. M. Fall-Persson (National Bacteriological Laboratory). The antigen (with an estimated purity of 80%) was kept in buffered saline at a concentration of 600 limes flocculation units/mI (1.2 mg/mI). Various amounts of antigen were coated onto polystyrene microtiter plates by using 0.05 M carbonate buffer, ph 9.6, and incubated for 4 h at 37°C. Binding assay was performed in ELISA using a 1:100 dilution of positive sera (reference serum and positive patient samples). Absorbance values leveled off at a coating concentration of 5 limes flocculation units (corresponding to 10 JLg/ml tetanus toxoid) and a coating concentration of 15 limes flocculation units was chosen to ensure antigen excess. An optical density (OD) of 0.5 at a serum dilution of 1:100 corresponding to an antibody concentration of rv 3 ng/mI was regarded as representative of seropositivity (figure 1). Studies were performed for all patients on serum samples taken before and 12 months after BMT. In nonimmunized patients these analyses were also done 24 months after BMT. Studies on immunized patients were done at least at 3 months and 1 year after immunization. Serum samples from 38 bone marrow donors were also analyzed; no sera were available from the other donors. A positive response to immunization was defined as a more than twofold increase in OD level. A few patients in both groups had antibody levels to tetanus toxoid that gave OD values of >1.0, corresponding to
0,0 -Q-T'"T'T......c;....,..l"'9ftIi'I;IIl=T-n-""""'..........,.."mr-TT'l.................TTn.... 1 0- 3 1 0- 2 1 0- 1 100 101 10 2 10 3 -8 -7 -6 -5 -4 -3 -2 10 10 10 10 10 10 10
ng/ml Dilution
Figure 1. Comparison on tetanus toxoid-coated plates between dilutions of positive hyperimmune reference serum containing 180 IU/mI anti-tetanus toxoid antibodies (0) and supernatants from tetanus toxoid antibody-producing cell line TT-l (.). Concentration of antibody in supernatant was previously determined by catch-up on an anti-IgG coated plate compared to a standard (WHO 67/97).
an antibody level of rv 20 ng/mI. These are defined' as high antibody levels concerning the response to immunization. Immunization. The first 21 patients were vaccinated with one dose of tetanus toxoid (National Bacteriological Laboratory, Stockholm) 12 months after BMT. A preliminary evaluation in 1984 showed that the antibody response to this single dose of vaccine was poor. Therefore 17 patients were reimmunized with two more doses of tetanus toxoid 24-36 months after BMT. The next 21 patients were immunized with three doses of vaccine given at 12, 13, and 14 months after BMT. Six patients who were not vaccinated at 1 year (three because of chronic GVHD and three by mistake) were studied at 18 months and 2 years. Statistics. The Mann-Whitney U test (two-sided) was used for comparing OD values after vaccination. The influence of pretransplant antibody levels of patients and donor on each patient's antibody level 1 year after transplant was analyzed using a multivariate linear regression [15].
Results
Serology. Thirty-seven (77 %) of 48 patients and 27 (71 %) of 38 examined donors were seropositive for tetanus toxoid before BMT. There was no difference in antibody levels between patients and donors before BMT. Eighteen patients (49 %) who were seropositive before BMT remained positive 1 year after transplant. There was no difference in antibody level before or 1 year after BMT between the patients primarily receiving one vaccine dose, those receiving three doses, or nonimmunized patients (figure 2). Among the 6 nonimmunized patients, 2 were seropositive 1 year and none were seropositive 2 years after BMT. No patient who was seronegative before BMT was seropositive at 1 year; 5 had seropositive donors, 5 had seronegative donors and 1 donor's antibody status was unknown. When the influences of pretransplant antibody levels of pa-
Ljungman et al.
498
JID 1990;162 (August)
Table 2. Correlation of serologic status 1 year after bone marrow transplantation in patients who were seropositive before transplantation with acute and chronic graft-versus-host disease (GVHD).
2,0
1,5
Status 1 year after transplantation
n.s.
E c
GYHD
.,
In 0
0
0
Seropositive
Acute Grade 0-1 Grade II-IV Chronic None
1,0
0,5
17 (52) 1 (25) 9 (47) 9 (45)
Seronegative 16 3 8 11
(48) (75) (53) (55)
NOTE. Data are no. (%) of patients. 18
12
24
30
36
The response rate to immunization was higher among patients receiving three doses than in those receiving one dose regardless of serologic status at the time of immunization. The strength of the antibody responses to one and three vaccine doses was significantly different 18 and 24 months after BMT (Mann-Whitney U test; figure 2). At 36 months there was no difference between groups. Among 15 patients receiving three vaccine doses from whom serum samples were available, all remained seropositive 2 years after immunization. Among nine seronegative patients who responded to one dose of vaccine, five had chronic GVHD and four did not. Conversely, three of five who did not respond had chronic GVHD and two did not. All seronegative patients responded after three vaccine doses regardless of whether they had chronic GVHD. Thus, chronic GVHD did not influence the immunization response to tetanus toxoid 1 year after BMT.
Months after BMT
Figure 2. Antibody levels (± SE) before and at different times after bone marrow transplantation (BMT) in patients receiving one dose of tetanus toxoid at 1 year followed by two subsequent doses «(!J), patients receiving three primary doses (D), and patients who were not immunized ( • ). Data were compared using MannWhitney U test.
tients and donors on the subsequent antibody level of the patient 1 year after BMT were analyzed, only the patient's pretransplant antibody level was predictive for posttransplant antibody level (P < .01 by linear regression; figure 3). There was no suggestion that the donor's serologic status influenced the patient's antibody level 12 months after transplant (P = .47). Serologic status and GVHD. Table 2 shows the probability of remaining seropositive 1 year after BMT in patients with and without GVHD. There was no difference in the probability of remaining seropositive at 1 year among patients who were seropositive before transplant whether or not they developed chronic GVHD. The number of patients who developed acute GVHD of grade II-IV was too small to allow analysis of an eventual impact on the serologic status at 1 year. Immunization. Table 3 shows the response to immunization in seropositive patients and table 4 in patients who were seronegative at the time of immunization. No major side effects were seen after immunization. One patient had a substantial local reaction after two doses and a third dose was not given.
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Evidence for transfer of donor humoral immunity after allogeneic BMT has been found by several researchers [4, 5, 16-18]. Wahren et ale [16] showed in a sequential study that in most instances the transferred specific antibody production to viruses such as measles, mumps, and rubella, which presumably are not transferred by marrow graft, was finite and only rarely continued after 12 months. Lum et a1. [4] showed in nonsequential studies transfer of long-lasting donor immunity to measles and diphtheria antigens up to 15
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0.5
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Figure 3. Influence of antibody levels of patients and donors before bone marrow transplantation (BMT) on anti-tetanus antibody levels of patients 12 months after BMT. Only pretransplant antibody level of patient had an influence on posttransplant antibody level (P < .01 by linear regression).
JID 1990;162 (August)
Tetanus Toxoid Immunization and BMT
Table 3. Results of immunization in patients who were seropositive at the time of immunization.
499
Table 4. Results of immunization in patients who were seronegative at the time of immunization.
Group
Group
One initial, two subsequent doses After first dose
Characteristic Immunized Responding With high antibody levels Seropositive 1 year after immunization Antibody titers 1 year after immunization
After "" subsequent doses
One initial, two subsequent doses Three initial doses
7 0
7 3 (43)
9* 6 (55)
4 (57)
4 (57)
3 (33)
7 (100)
7 (100)
9 (100)
1.06 (0.66-1.97) 1.47 (0.65-1.82) 1.65 (1.00-2.11)
NOTE. Data are no. (%) of patients except antibody titers, which are given as mean (range). High antibody levels are defined as optical density values >1.0 at the time of immunization that did not change during follow-up. • One patient developed a substantial local reaction after the second dose and did not receive the third dose.
months after transplant. We could not find any transfer of tetanus immunity to seronegative patients from their seropositive marrow donors, but the number of evaluable donor-recipient pairs was small. An unexpected finding was that patient's, but not the donor's, antibody level before BMT strongly influenced the antibody level 1 year after BMT. The mechanism of this influence is unknown, although persisting recipient lymphocytes have been detected close to a year after BMT [19]. Wimperis et al. [18] showed that when both donor and patient were immunized with tetanus toxoid before BMT the level of antibody measured after transplant was higher than when either donor or recipient alone were immunized. We previously showed that seropositive patients with seronegative donors may retain immunity to viral antigens up to 9 months after BMT [16, 19]. Continued presence of tetanus antigen is extremely unlikely, since in general the patients had not been immunized for several years before the transplant. Thus, persistence of antibodyproducing recipient cells may explain the influence of the recipient's antibody level on persisting immunity. However, there might also be other unknown influencing factors. A separate question is how many long-term survivors after BMT will remain seropositive and thus protected against important infectious agents such as tetanus. We showed that BMT recipients without chronic GVHD who were seropositive 2 years after transplant continued to lose immunity to measles, mumps, and rubella during the next 2 years of follow-up [20]. Here we confirm our previous data in that, although the number of patients who were not immunized was small, patients seropositive before BMT continued to lose specific immunity during long-term follow-up. Almost two-thirds ofthe BMT recipients studied were seronegative 1 year after transplant
After first dose
Characteristic Immunized Responding Seropositive 1 year after immunization Antibody titers 1 year after immunization
14
After subsequent doses
Three initial doses
9 (64)
10* 10 (100)
12 12 (100)
7 (50)
9 (90)
12 (100)
0.87 (0.13-1.82) 1.52 (0.75-2.38) 1.56 (0.77-2.48)
NOTE. Data are no. (%) of patients except antibody titers, which are given as mean (range). • Three patients were not reimmunized and one received immunoglobulin infusions and is not evaluable.
and no nonimmunized patient remained seropositive 2 years after transplant. This is comparable with findings by Lum et al. [5], who in a nonsequential study showed 47% seronegativity among patients studied 7 months to 10 years after BMT. Various posttransplant factors such as GVHD prophylaxis and presence of chronic GVHD are probably important in the duration of transferred and persistent immunity after BMT. Lum et al. [4] showed in long-term survivors a higher probability of retaining specific immunity to tetanus in patients without GVHD and in patients receiving T cell-depleted marrow grafts [5]. No such differences were seen for diphtheria and measles immunity [4]. In the present study we found no difference in the probability of remaining seropositive 1 year after transplant in patients with or without chronic GVHD. These data show that rv50 % oflong-term survivors are not seropositive to tetanus after BMT. The question of reimmunization of BMT recipients has been discussed [1, 17]. However, responses to immunization and immunization schedule needed to obtain adequate immunity are not known. Saxon et al. [17] showed in three patients that transferred donor cells can be restimulated by immunization 2-5 months after BMT. We previously showed that BMT recipients without chronic GVHD can be effectively and safely immunized with live measles, mumps, and rubella vaccine [20]. In the present study, the percentage of patients who responded to immunization and the chance of remaining seropositive after 1 year were lower in the group receiving one dose of vaccine. The immune system is not fully reconstituted 1 year after BMT. A single immunization performed at that time might therefore not be enough to elicit a durable immune response. Whether a different, more durable response could be obtained after a single immunization given a longer time after BMT when the immune system would be more completely reconstituted is unknown. This indicates that although immunity may be transferred from the marrow donor, the number of
500
Ljungman et a1.
antigen-specific B cells present 1 year after transplant is too low to mediate an adequate immune response. This is supported by in vitro antibody production studies in which peripheral blood lymphocytes from BMT recipients failed to produce tetanus toxoid-specific antibodies when cultured at cell numbers adequate for antibody production in normal control subjects [21]. Patients primarily receiving three vaccine doses had less chronic GYHO than did patients receiving one dose. However, this difference cannot explain the poor immunization response after one vaccine dose, since chronic GYHO did not influence the ability to respond to immunization. We conclude that reimmunization with tetanus toxoid of long-term survivors after BMT is necessary and that a schedule with three doses is needed to obtain protective specific antibody levels. Whether BMT recipients lose immunity more rapidly than do normal individuals is unknown. However on the basis ofthe present study it is likely that 3 years after BMT, patients who do not have chronic GYHO will not differ significantly from normal individuals. Further studies are needed to determine if and when follow-up immunizations need to be given. References 1. Lum LG. The kinetics of immune reconstitution after human marrow transplantation. Blood 1987;69:369-81 2. Witherspoon RP, Matthews 0, Storb R, Atkinson K, Cheever M, Deeg HJ, Doney K, Kalbfleisch J, Noel D, Prentice R, Sullivan KM, Thomas ED. Recovery of in vivo cellular immunity after human marrow grafting. Influence of time postgrafting and acute graft-versus-host disease. Transplantation 1984;37:145-150 3. Paulin T, Ringden 0, Nilsson B. Immunological recovery after bone marrow transplantation: role of age, graft-versus-host disease, prednisolone treatment and infections. Bone Marrow Transplant 1987;1:317-328 4. Lum LG, Munn NA, Schanfield MS, Storb R. The detection of specific antibody formation to recall antigens after human bone marrow transplantation. Blood 1986;67:582-587 5. Lum LG, Noges JE, Beatty P, Martin PJ, Deeg J, Doney KC, Loughran T, Sullivan KM, Witherspoon RP, Thomas ED, Storb R. Transfer of specific immunity in marrow recipients given HLA-mismatched, T cell-depleted, or HLA-identical marrow grafts. Bone Marrow Transplant 1988;3:399-406 6. Thomas ED, Storb R, Clift RA, Fefer A, Johnson FL, Neiman PE, Lerner KG, Glucksberg H, Buckner CD. Bone marrow transplantation. N Engl J Med 1975;292:832-843, 895-902 7. Ringden 0, Lonnqvist B, Lundgren G, Gahrton G, Groth CG, Moller E, Baryd I, Johansson B, Pihlstedt P, Gullbring B. Experience with a cooperative bone marrow transplantation program in Stockholm. Transplantation 1982;33:500-504
JID 1990;162 (August)
8. Ringden 0, Backman L, LOnnqvist B, Heimdahl A, Lindholm A, Bolme P, Gahrton G. A randomized trial comparing use of cyclosporin and methotrexate for graft-versus-host disease prophylaxis in bone marrow transplant recipients with haematologic malignancies. Bone Marrow Transplant 1986;1:41-51. 9. Storb R, Deeg HJ, Whitehead J, Farewell V, Appelbaum FR, Beatty P, Bensinger W, Buckner CD, Clift RA, Doney K, Hansen JA, Hill R, Lum LG, Martin P, McGuffin R, Sanders JE, Singer J, Stewart P, Sullivan KM, Witherspoon RP, Thomas ED. Marrow transplantation for leukemia and aplastic anemia: two controlled trials of a combination of methotrexate and cyclosporine versus cyclosporine alone or methotrexate alone for prophylaxis of acute graft-v-host disease. Transplant Proc 1987;19:2608-2613 10. Prentice HG, Blacklock HA, Janossy G, Gilmore MJML, Price-Jones L, Tidman N, Trejdosiewicz LK, Skeggs DBL, Panjwani 0, Ball S, Graphakos S, Patterson J, Ivory K, Hoftbrand AV. Depletion of Tlymphocytes in donor marrow prevents significant graft-versus-host disease in matched allogeneic leukaemic marrow transplant recipients. Lancet 1984;1:472-476 11. Shulman HM, Sullivan KM, Weiden PL, McDonald GB, Striker GE, Sale GE, Hackman R, Tsoi MS, Storb R, Thomas ED. Chronic graftversus-host syndrome in man. A long time clinicopathologic study of 20 Seattle patients. Am J Med 1980;69:204-217 12. Schubert MM, Sullivan KM, Morton TH, Izutsu KT, Peterson DE, Flournoy N, Truelove EL, Sale GE, Buckner CD, Storb R, Thomas ED. Oral manifestations ofchronic graft-v-host disease. Arch Intern Med 1984;144:1591-1595 13. Tiebout RF, Stricker EAM, Hagenaars R, Zeijlemaker WP. Human lymphoblastoid cell line producing protective monoclonal IgGl, K antitetanus toxin. Eur J ImmimolI984;14:399-404 14. Persson MAA, HammarstrOm L, Smith CIE. Enzyme-linked immunosorbent assay for subclass distribution of human IgG and IgA antigenspecific antibodies. J Immunol Methods 1985;78:109-121 15. Draper N, Smith H. Applied Regression Analyses. 2nd ed. New York: John Wiley & Sons, 1981 16. Wahren B, Gahrton G, Linde A, Ljungman P, LOnnqvist B, Ringden 0, Sundqvist VA. Transfer and persistence of viral antibody-producing cells in bone marrow transplantation. J Infect Dis 1984;150:358-365 17. Saxon A, Mitsuyasu R, Stevens R, Champlin RE, Kimata H, Gale RP. Designed transfer of specific immune responses with bone marrow transplantation. J Clin Invest 1986;78:959-967 18. Wimperis JZ, Brenner MK, Prentice HG, Reittie JE, Karayiannis P, Griffiths PD, Hoftbrand AV. Transfer of a functioning humoral immune system in transplantation of T-Iymphocyte-depleted bone marrow. Lancet 1986;1:339-343 19. Ljungman P, Gahrton G, Ringden 0. Wahren B. Donor and recipient origin of herpesvirus-reactive lymphocytes after bone marrow transplantation. Arch VirolI985;83:117-122 20. Ljungman P, Fridell E, LOnnqvist B. Bolme P, BOttiger M, Gahrton G, Linde A. Ringden 0, Wahren B. Efficacy and safety of vaccination of marrow transplant recipients with a live attenuated measles, mumps, and rubella vaccine. J Infect Dis 1989;159:610-615 21. Lum LG, Seigneuret MC, Storb R. The transfer of antigen-specific humoral immunity from marrow donors to marrow recipients. J Clin ImmunolI986;6:389-396
