Bone Marrow Transplantation, (1997) 20, 663–668  1997 Stockton Press All rights reserved 0268–3369/97 $12.00

Randomized comparison of early and late vaccination with inactivated poliovirus after allogeneic BMT

T Parkkali1, M Stenvik2, T Ruutu1, T Hovi2, L Volin1 and P Ruutu2

1Division of Haematology, Department of Medicine, Helsinki University Central Hospital and 2National Public Health Institute, Helsinki, Finland

Summary: immune memory, and antibodies to recall gradu- ally disappear over years. Forty-five adult HLA-matched sibling BMT recipients The proportion of allogeneic BMT recipients with were randomized to receive inactivated poliovirus vac- poliovirus immunity declines during long-term follow-up.3–6 cine (IPV) at 6, 8 and 14 months (early group, n = 23) The risk for infection is low in developed countries. or at 18, 20 and 26 months after BMT (late group, n = However, small outbreaks of poliomyelitis involving heal- 22). Ninety-five percent of the early group patients had thy adults and children have occurred during the last 12 protective antibody titres of у4 to poliovirus type 1 years.7,8 Inactivated poliovirus vaccine (IPV) has been (PV1), poliovirus type 2 (PV2) and poliovirus type 3 shown to be effective and safe among BMT recipients,3–5 (PV3) by a microneutralization assay prior to the first and recently the Infectious Diseases Working Party of the vaccination, at 6 months after BMT. The corresponding European Group for Blood and Marrow Transplantation has proportion for the late group patients was only 67% at recommended immunization of BMT recipients with IPV.9 18 months. The antibody responses 1 month after each In order to find out if early vaccination of adult allo- of the three IPV doses were similar in the two vacci- geneic BMT recipients against poliomyelitis is as effective nation groups, except that four-fold responses occurred as that given later, a prospective, randomized study with more frequently after the first dose to PV2 and PV3 in inactivated poliovirus vaccine was carried out, comparing the late group. All patients had a protective antibody two different vaccination schedules. titre to all poliovirus serotypes 1 and 22 months after the third vaccine dose, except one patient in the early group who lacked antibodies to PV3 at 22 months. Materials and methods Acute GVHD accelerated the decrease of poliovirus antibody titres prior to vaccination but had no influence Patients on vaccination response. Chronic GVHD neither influ- enced the patient’s ability to retain poliovirus antibodies Fifty-five consecutive adults (age у16 years) who had prior to vaccination nor impaired responses to vacci- received a bone marrow transplant from a sibling donor nations. A vaccination schedule consisting of three IPV between January 1985 and November 1989 at Helsinki Uni- doses was equally immunogenic when started at 6 or 18 versity Central Hospital were randomized into two vacci- months after allogeneic BMT. nation groups at 5 months after transplantation. Informed Keywords: inactivated poliovirus vaccine; BMT consent was obtained from the patients before randomiz- recipients ation. The vaccination program started either at 6 (early group) or 18 (late group) months after BMT. Eight patients died before the intended vaccinations started (two in the early vaccination group and six in the late group). One Immune reconstitution after allogeneic BMT from cells patient in the early group was treated at another hospital originating from the donor takes 1–2 years in healthy and could not be vaccinated, and one patient in the late recipients and even longer in patients with chronic group relapsed. Characteristics of the remaining 45 patients 1,2 GVHD. The numbers of different lymphocyte types in (23 in the early group and 22 in the late group) are blood involved in immunological functions usually nor- presented in Table 1. malize during the first 6–12 months after BMT. Serum Patients received a bone marrow graft from an HLA- immunoglobulin levels, except that of IgA, become normal identical, MLC-negative sibling donor. depletion was 2 during the 12–18 months after BMT. However, the con- not done. The recipients with haematological malignancy ditioning treatment prior to BMT causes loss of specific were conditioned with cyclophosphamide (60 mg/kg body weight intravenously on 2 consecutive days) and total body irradiation (total dose from 11 to 12 Gy in five or six frac- tions on 5 consecutive days) or cyclophosphamide and bus- Correspondence: Dr T Parkkali, Division of Haematology, Department of Medicine, Helsinki University Central Hospital, Haartmaninkatu 4, FIN- ulphan (4 mg/kg on 4 consecutive days). Four patients with 00290 Helsinki, Finland chronic myeloid leukaemia also received one infusion of Received 26 February 1997; accepted 23 June 1997 daunorubicin, 60 mg/m2, and splenic irradiation. The Response to poliovirus vaccine after allogeneic BMT T Parkkali et al 664 Table 1 Characteristics of the patients Early group

Early group Late group

Total No. of patients 23 22 BMT 6 8 1415 20 24 36 Age of patients months Median (years) 31 30 Late group Range (years) 16–48 18–49 Age of donors Median (years) 29 31 BMT 6 12 18 20 2627 32 36 48 Range (years) 12–49 20–59 months Sex Male 12 12 Female 11 10 = Blood sample Primary disease AML 11 9 = Inactivated poliovirus vaccine ALL 5 2 CML 2 8 Figure 1 Schedule for blood samples and vaccinations after BMT. Multiple myeloma 3 2 Burkitt’s lymphoma 0 1 Lymphoblastic lymphoma 1 0 Severe aplastic anaemia 1 0 (Pneumovax; Merck, Sharp and Dohme, West Point, Conditioning regimen PA,USA), and with meningococcal polysaccharide vaccine CY + TBI 18 19 (Mencevax ACW Y; Smith Kline RIT, Rixensart, CY + BU 4 3 135 Cy + total nodal irradiation 1 Belgium). GVHD prophylaxis CsA 5 8 Serum samples CsA + a short course of MTX 17 13 Corticosteroids + MTX 1 1 Blood samples were collected from 42 of the 45 donors, Acute GVHD 14 (61%) 13 (59%) from 41 of the 45 patients before BMT, and from the Chronic GVHD 13 (57%) 9 (41%) patients after BMT according to the schedule in Figure 1. During the vaccination program and the first year after the third vaccine dose, blood specimens taken within 1 week prior to or 3 weeks after the scheduled time-point were patient transplanted for aplastic anaemia was conditioned included in the analyses. During the second year after com- with cyclophosphamide 50 mg/kg on 4 consecutive days pleting the vaccination schedule blood samples taken one and total nodal irradiation 8.0 Gy in four fractions on 2 month before or after the scheduled time were considered consecutive days. Acute GVHD was defined according to adequate. One of the late group patients was vaccinated Thomas et al10 and treated with methylprednisolone, start- against polio between 6 and 12 months after BMT. Speci- ing with a dose of 2 mg/kg/day or 10–15 mg/kg/day, mens drawn from this patient after vaccination were depending on the severity of GVHD. Chronic GVHD was excluded from the analysis of poliovirus antibodies. defined according to Shulman et al11 and treated with methylprednisolone alone or in combination with azathio- Neutralizing antibodies to poliovirus prine or thalidomide. The standard microneutralization assay used was based on the standard procedures described by Albrecht et al.12 Vaccination Serum specimens were heat inactivated (30 min at 56°C) The 23 patients in the early group were vaccinated at 6, 8 and serially diluted on microtitre plates (Nunclon Microtest and 14 months and the 22 patients in the late group at 18, 96-well plates). An equal volume of pretitrated virus 20 and 26 months after BMT with inactivated trivalent dilution (corresponding to 100 TCID50) was added, and the poliovirus vaccine (Polio Novum, RIVM, Bilthoven, The plates were sealed. The following virus strains were used: Netherlands) (Figure 1). One 1.0 ml dose of the vaccine poliovirus type 1 strain Mahoney (PV1), poliovirus type 2 contained at least 40–8-32 D- units for poliovirus strain MEF-1 (PV2) and poliovirus type 3 strain Saukett types 1, 2 and 3, respectively. The vaccine was adminis- (PV3). After overnight incubation at +36°C, 15 000 Vero tered subcutaneously. Three doses of tetanus toxoid vaccine cells were added per well, and the resealed plates were (manufactured by National Public Health Institute, Hel- incubated at 36°C. Virus growth was determined by sinki, Finland) were given with the same schedule as the microscopy at 6 days. The highest dilution with complete polio vaccine. At the time of the first dose of polio vaccine inhibition of cytopathogenic effect was considered the end- the patients received Haemophilus influenzae type b poly- point titre. A standard serum preparation, calibrated accord- saccharide–diphtheria toxoid conjugate vaccine (ProHIBIT; ing to an international reference serum, was included as a Connaught Laboratories, Swiftwater, PA, USA). At the reference in each assay. For statistical calculations, titres time of the second dose of polio vaccine the recipients were Ͻ4 were given an arbitrary value of 2 and titres Ͼ4096 a immunized with pneumococcal polysaccharide vaccine value of 8192. Poliovirus antibody titres у4 were con- Response to poliovirus vaccine after allogeneic BMT T Parkkali et al 665 sidered protective. A four-fold or higher increase in anti- of the poliovirus antibodies between the groups during the body titre was used as the criterion for response. 22 months after completing the vaccinations (Figure 3). The first dose of poliovirus vaccine induced a four-fold or Statistical methods higher increase in antibody titre to PV2 and PV3 in a larger proportion in the late group than in the early group patients Fisher’s exact test was used to compare the frequency of (Table 3). After the subsequent doses, the difference in the individuals in the two vaccination groups with indicated cumulative frequencies of patients with at least a four-fold antibody titres to poliovirus serotypes 1–3 and fold response diminished dose after dose. responses. t-test for two independent samples with log- Prior to vaccination, 21 out of 22 early group patients transformed data was used to compare the geometric mean (95%) and 14 out of 21 late group patients (67%) had a titres (GMT) of the poliovirus antibodies between the two poliovirus antibody titre у4 to all three poliovirus sero- vaccination groups. The paired t-test was used when the types (P = 0.04). At one and 22 months after the third vac- GMTs pre- and post-vaccination were compared. cine dose, all patients had a poliovirus titre у4 to all poliovirus types, except one patient to PV3 in the early group at 22 months. The GMTs measured 6 months after BMT for PV1 and Results PV2 were significantly higher in patients without preceding acute GVHD than in those with it (401 and 434, 78 and Prior to transplantation, the GMTs of the antibodies to 132, respectively; P = 0.003 and P = 0.02, respectively) poliovirus types 1–3 were similar in patients and donors but showed no difference for PV3. The patients without (707 and 1023 for PV1, 1029 and 898 for PV2, and 180 acute GVHD had fewer decreases in antibody titres to PV3 and 318 for PV3, respectively). Forty out of 41 patients than the patients with acute GVHD (a decrease to at least (98%) and all 42 donors with serum samples prior to BMT a quarter of the titre prior to BMT in six out of 18 without had an antibody titre у4 to PV1, all patients and donors vs 18 out of 22 patients with acute GVHD, P = 0.003). to PV2, and 38 patients (93%) and 40 (95%) donors to PV3. One year after BMT, the GMTs to PV1, PV2 and PV3 in During the first 6 months after BMT the antibody titres the late group patients, not yet vaccinated, were 121, 163 against PV1, PV2 and PV3 continuously decreased (Figure and 42 in those without acute GVHD and 21, 32 and 10 2), at least to a quarter of the titre prior to BMT in 58, 63 = = = and 61% of the patients, respectively. By 18 months after in those with acute GVHD (P 0.03, P 0.07 and P BMT 14 (74%), 15 (79%) and 14 (74%) out of 19 late 0.12). Preceding acute GVHD did not impair the vacci- group patients showed a decrease to a quarter of the initial nation response in either group. antibody titre to PV1, PV2 and PV3, respectively. Prior to Chronic GVHD did not influence patients’ ability to the first vaccinations, the GMTs of the antibodies to PV1, retain poliovirus antibodies prior to vaccination or PV2 and PV3 were higher in the early group at 6 months responses to vaccinations. GMTs of the poliovirus anti- after BMT than in the late group at 18 months after BMT bodies at 6 and 10 months after completing the vaccination (Table 2). After each of the three vaccine doses the GMTs schedule were similar in patients with and without chronic were not significantly different in the two vaccination GVHD. However, 22 months after the third vaccine dose, groups, although there was a tendency towards higher patients in the early group without chronic GVHD had sig- GMTs in the late group for PV2 (P = 0.064) and PV3 (P nificantly or almost significantly higher geometric mean = 0.11) after the third vaccine dose. After each vaccination antibody titres to PV1 (1933 vs 214, P = 0.06), PV2 (1351 the GMT was significantly higher than the respective pre- vs 186, P = 0.05) or PV3 (4705 vs 235, P = 0.02) than vaccination titre, except for the second vaccine dose to PV1 patients with chronic GVHD. In the late group this could and the first and second vaccine doses to PV2 in the early not be assessed, as only one patient had chronic GVHD group. There were no significant differences in the GMTs and serum samples Ͼ36 months after BMT.

abc 10000 10000 10000

1000 1000 1000

100 100 100 Titre Titre Titre

10 10 10

1 1 1 pre-BMT 2 4 6 12 18 pre-BMT 2 4 6 12 18 pre-BMT 2 4 6 12 18 Months Months Months

Figure 2 The geometric mean titres and their 95% confidence intervals of antibodies to poliovirus type 1 (a), type 2 (b) and type 3 (c) before and after BMT until vaccination. (b) Early group; (ć) late group. Response to poliovirus vaccine after allogeneic BMT T Parkkali et al 666 Table 2 Geometric mean titres of poliovirus antibodies in allogeneic BMT recipients before and one month after immunization with inactivated poliovirus vaccine

First vaccine dose Second vaccine dose Third vaccine dose

Before (n) After (n) Before (n) After (n) Before (n) After (n)

Poliovirus type 1 Early group 271a (22) 765 (23) 608 (23) 972 (22) 613 (19) 2163 (17) Late group 38 (21) 916 (15) 526 (20) 1695 (18) 896 (16) 3764 (17) Poliovirus type 2 Early group 275b (22) 475 (23) 549 (23) 769 (22) 429 (19) 1260 (17) Late group 47 (21) 959 (15) 518 (20) 1250 (18) 1142 (16) 3100 (17) Poliovirus type 3 Early group 51c (22) 424 (23) 426 (23) 1094 (22) 630 (19) 2418 (17) Late group 15 (21) 402 (15) 330 (20) 1984 (18) 922 (16) 5057 (17)

When geometric mean titres between the two groups were compared: aP = 0.0016; bP = 0.0042; cP = 0.036.

100000 abc100000 100000

10000 10000 10000

1000 1000 1000

Titre 100 Titre 100 Titre 100

10 10 10

1 1 1 pre-vac 1 6 10 22 pre-vac 1 6 10 22 pre-vac 1 6 10 22 Months Months Months

Figure 3 The geometric mean titres and their 95% confidence intervals of antibodies to poliovirus type 1 (a), type 2 (b) and type 3 (c) before starting and 1, 6, 10 and 22 months after completing the three-dose vaccination schedule. (b) Early group; (ć) late group.

Table 3 Responses to vaccinations with inactivated poliovirus vaccine vated poliovirus vaccine (IPV) at 6, 8, and 14 or at 18, after allogeneic BMT 20 and 26 months after transplantation. Both vaccination schedules elicited satisfactory antibody responses, and, 1 Early group % Late % P month after completing the three-dose vaccination program, group all patients had protective antibody titres to all poliovirus serotypes. High antibody titres were measured at least 2 Polio type 1 1st dose 6/22a 27 9/15 60 0.087 years after the third vaccination. 2nd dose 9/21 43 13/18 72 0.106 The present study included only adult allogeneic BMT 3rd dose 10/17 59 14/17 82 0.259 recipients with transplantations carried out without T cell Polio type 2 depletion. The three previous polio vaccination trials 1st dose 5/22 23 10/15 67 0.016 among BMT recipients have included children,3–5 and one 2nd dose 9/21 43 13/18 72 0.106 of them only T cell-depleted allogeneic and autologous 3rd dose 9/17 53 13/17 76 0.282 transplantations.3 After intensive chemotherapy CD4- Polio type 3 positive T cells recover sooner in younger than in older 1st dose 10/22 45 12/15 80 0.047 patients,13 and younger BMT recipients have better 2nd dose 13/21 62 16/18 89 0.074 3rd dose 13/17 76 16/17 94 0.34 immunocompetence following BMT due to the greater capacity of the thymus to generate new T lymphocytes.14 aCumulative No. of patients with at least a four-fold increase in antibody Thus, the vaccination responses to IPV might show better titre/No. of patients tested. results in paediatric than in adult BMT recipients. In this study, as in two previous poliovirus immuno- genicity trials among BMT patients,3,4 a microneutraliz- Discussion ation assay was used. Li Volti et al5 also used tissue culture plates containing Vero cell monolayers. Engelhard et al3 In the present study adult HLA-matched sibling BMT and ourselves considered a titre у4 and Li Volti et al5 a recipients were randomized prospectively to receive inacti- titre Ͼ4 protective. Ljungman et al4 expressed their results Response to poliovirus vaccine after allogeneic BMT T Parkkali et al 667 as seropositivity and seronegativity without a definitive comparable to those seen in the present early group statement on the protective level of antibodies. These dif- patients.4 ferences must be taken into account when comparing the The early responses in the present study differ from the proportions of patients with protective antibody titres findings of previous studies according to which T cell- between different studies. dependent antigens elicit antibody responses to recall pro- A nationwide vaccination campaign against polioviruses tein antigens late, у1 year after BMT,4,17–19 but not early was organized in Finland between 9 February and 15 March after unmanipulated20 or T cell-depleted21 BMT. If the 1985, due to an outbreak of poliomyelitis.7 Most of the donor or both the donor and the recipient have been immun- BMT recipients whose haematological diagnosis was estab- ized with another protein antigen, tetanus toxoid, before lished only after this campaign and their donors probably BMT, the patient has been able to respond to one to two received oral poliovirus vaccine during the campaign. doses of the same vaccine during the first year after Those who had their haematological disease diagnosed earl- BMT.20–22 It is possible that the high level of immunity of ier were vaccinated with one dose of the new IPV (Polio the present donors against polio is reflected in the better Novum, RIVM) before BMT, as were also their household response rate of the early group compared to other BMT contacts. The recipients had protective antibody levels recipient populations. Thus, even the early group patients, against polio prior to BMT. The geometric mean titres regardless of their lower general immunologic recovery, (GMTs) to PV1, PV2 and PV3 were higher among the were able to respond to the vaccinations. present donors and patients prior to BMT than those among There is no previous information on the influence of the patients pretransplant in the study of Engelhard et al3 acute GVHD on the ability to retain antibodies to poliovirus tested by a comparable methodology. However, similar pro- after BMT. In our study the poliovirus antibody titres in portions of the present donors and patients had protective patients with acute GVHD declined earlier compared to antibody titres prior to BMT to the three poliovirus types patients without acute GVHD. Vaccination responses were as the BMT recipients in Sweden4,15 and Israel.3 similar despite the preceding acute GVHD. Chronic GVHD All patients in the early group except one showed protec- did not influence vaccination responses. However, approxi- tive antibody titres to all three types of polio prior to vacci- mately 2 years after the third polio vaccine dose patients nation at 6 months after BMT, while this was seen only in with chronic GVHD had lower antibody titres than patients two-thirds of the patients in the late group at 18 months without. In previous studies with relatively small patient groups, recipients without chronic GVHD either had higher after BMT. In our previous study on poliovirus immunity 3 using the same methodology, as few as two out of seven antibody titres to polio both prior to and after vaccinations or there was no clear influence by chronic GVHD.4 Chronic BMT recipients were immune to all three poliovirus sero- GVHD has considerable variability in the mode of onset, types at 12 months after BMT.6 In the study by Ljungman the organ systems involved and the rate of progression.11 et al4 two-thirds of the patients were seropositive one year In different studies the severity of chronic GVHD and time post-transplantation. interval to vaccinations may be different and explain the In the present study the GMTs of the antibodies to PV1, inconsistent results. PV2 and PV3 were similar in the two vaccination groups We conclude that polio vaccination should not be post- after each vaccination given at 6, 8 and 14 months or at poned for more than 6 months after BMT. Patients with 18, 20 and 26 months after BMT. The prevaccination chronic GVHD should be vaccinated with the same GMTs were lower in the late than in the early group, as schedule. expected. After the first vaccine dose the fold responses in the antibody titre were greater in the late than in the early group leading to similar post-vaccination GMTs. In spite Acknowledgements of the differences in fold responses, the antibody titres after completing the vaccination schedule remained at highly We thank Elisa Lamminsalo for skilful technical assistance. This protective levels for at least 2 years in both vaccination study was supported by a grant from the Sigrid Juse´lius Foun- groups. However, the number of patients in the present dation, Helsinki, Finland. study is quite small, and the possible immunological inferi- ority of the early schedule compared with the late schedule might become apparent over time. The advantage of the References early regimen is that high antibody titres are maintained between 6 and 18 months after BMT. It has been shown 1 Lum LG. Immune recovery after bone marrow transplantation. earlier that a three-dose tetanus vaccination schedule start- Hematol Oncol Clin North Am 1990; 4: 659–675. ing at 6 months after BMT is immunogenic,16 and therefore 2 Storek J, Saxon A. Reconstitution of B cell immunity follow- it might be practical to give these two protein antigen vac- ing bone marrow transplantation. Bone Marrow Transplant cines at the same time points. 1992; 9: 395–408. The response rates following one vaccine dose given at 3 Engelhard D, Handsher R, Naparstek E et al. Immune response to polio vaccination in bone marrow transplant 12 months after BMT have previously been either between recipients. Bone Marrow Transplant 1991; 8: 295–300. the responses to the first vaccine dose of our early and late 4 Ljungman P, Duraj V, Magnius L. Response to immunization group patients (PV1 and PV2) or lower (PV3) than those against polio after allogeneic marrow transplantation. 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