Bone Marrow Transplantation (2015) 50, 858–864 © 2015 Macmillan Publishers Limited All rights reserved 0268-3369/15 www.nature.com/bmt
ORIGINAL ARTICLE
Evaluation of pretransplant influenza vaccination in hematopoietic SCT: a randomized prospective study A Ambati1,2, LSV Boas3, P Ljungman1,4, L Testa5, JF de Oliveira6, M Aoun7, V Colturato5, M Maeurer2,7 and CM Machado3,5 Pretransplant influenza vaccination of the donor or allogeneic hematopoietic SCT (HSCT) candidate was evaluated in a randomized study. One hundred and twenty-two HSCT recipients and their donors were assigned to three randomization groups: no pretransplant vaccination (n = 38), donor pretransplant vaccination (n = 44) or recipient pretransplant vaccination (n = 40). Specific IgG was assessed by both hemagglutinin inhibition (HI) and, in 57 patients, by an indirect influenza-specific ELISA at specified times after HSCT. Vaccinated donors had seroprotective HI titers for Ags H1 and H3 (Po 0.001) compared with the other groups at the time of donation. The titers against H1 (P = 0.028) and H3 (P o 0.001) were highest in the pretransplant recipient vaccination group until day 180 after transplantation. A significant difference was found in the specific Ig levels against pandemic H1N1 at 6 months after SCT (P = 0.02). The mean IgG levels against pandemic H1N1 and generic H1N1 and H3N2 were highest in the pretransplant recipient vaccination group. We conclude that pretransplant recipient vaccination improved the influenza-specific seroprotection rates. Bone Marrow Transplantation (2015) 50, 858–864; doi:10.1038/bmt.2015.47; published online 23 March 2015
INTRODUCTION Influenza can be severe in hematopoietic SCT (HSCT) recipients with up to 30% mortality rates in untreated patients.1 Among 286 HSCT patients, 43.7% were hospitalized, 32.5% developed lower respiratory tract disease, 11.5% required mechanical ventilation and 6.3% died from the recent pandemic outbreak.2 Preventive strategies including vaccination, prompt diagnosis and the use of neuraminidase inhibitors have lowered the risk of complications and fatal outcome.1,3,4 Vaccination is the primary mode of prevention, although seroresponse rates in HSCT patients are poor. They are recommended to start vaccination after 6 months or even earlier from transplantation during the influenza season, possibly followed by a second dose.4,5 Studies have shown that the persistence of pre-existing donor and recipient immunity in HSCT recipients is of importance, but either have a finite duration.6–9 Pretransplant vaccination of the donor, the recipient or both has been evaluated previously and was found to positively influence Ag-specific IgG levels in the first year after transplant.10–13 Accordingly, we hypothesized that pretransplant vaccination of the donor or HSCT candidate would elicit a certain degree of protection against influenza in the first months after transplantation via two mechanisms: prompting a rise in the levels of circulating influenza antibodies in the vaccinated HSCT candidate; or by transferring primed memory cells in the case of donor vaccination. We conducted a randomized prospective study to evaluate alternative schedules of pretransplant influenza vaccination in an HSCT setting.
MATERIALS AND METHODS Study design and subjects An open randomized prospective study was conducted from October 2007 to January 2010 in two Brazilian HSCT centers: Amaral Carvalho Foundation (center A) and Federal University of Minas Gerais (center B). The ethics committees of the participating institutions approved the study; all consecutive allogeneic HSCT recipients with a related donor were invited to participate in the study. D/R pairs with one or more of the following criteria were excluded from the study: (1) HSCT expected to occur less than a week from the date of recruitment; (2) egg, chicken protein, neomycin or thimerosal allergy history; (3) past history of severe reaction following influenza vaccine; (4) influenza vaccine received o6 months before HSCT date. The patients in this study did not receive any prophylactic Igs. Written informed consent was obtained from donors and recipients. The primary objective was to evaluate if pretransplant vaccination of the donor or HSCT candidates would improve influenza seroprotection rates early after HSCT. Secondary objectives were to evaluate the impact of pretransplant vaccination in seroresponse rates following influenza vaccination on day 180 (d180), and the occurrence of documented influenza infections during this period (pretransplant vaccine efficacy (VE)). The subjects were assigned to three randomization groups: no pretransplant vaccination (group 1), donor pretransplant vaccination (group 2) and recipient pretransplant vaccination (group 3). D/R pairs were randomized by the method of randomly permuted blocks. A list of random numbers with their respective randomization group was generated through the website of the University of Tuffs (available at http://www.tufts.edu/ ~ gdallal/randomize.htm). Allocation group cards were prepared, placed in opaque envelopes, sequentially numbered and sealed. Sets of 20 envelopes were distributed alternately between the two participating HSCT centers during the recruitment period. After signature of the informed consent, the envelopes were opened in numerical order. The study was not blinded. On d180,
1 Division of Hematology, Department of Medicine Huddinge, Karolinska Institutet, Stockholm, Sweden; 2Centre for Allogeneic Stem Cell Transplantation (CAST), Karolinska University Hospital, Stockholm, Sweden; 3Virology Laboratory (LIM 52-HCFMUSP), Institute of Tropical Medicine, University of São Paulo, São Paulo, Brazil; 4Department of Hematology, Karolinska University Hospital, Stockholm, Sweden; 5Hematopoietic Stem Cell Transplant Program, Amaral Carvalho Foundation, Jahu, Brazil; 6Clinics Hospital – School of Medicine, Federal University of Minas Gerais, Belo Horizonte, Brazil and 7Division of Therapeutic Immunology (TIM), Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden. Correspondence: Dr CM Machado, Virology Laboratory (LIM 52-HCFMUSP), Institute of Tropical Medicine, University of São Paulo, Avenida Dr Enéas de Carvalho Aguiar, 470, São Paulo 05403-000, Brazil, E-mail: [email protected] Received 7 October 2014; revised 12 January 2015; accepted 15 January 2015; published online 23 March 2015
Pretransplant influenza vaccination A Ambati et al
859 all groups received one dose of influenza vaccine, as currently recommended5,14 (Figure 1).
performed by direct fluorescent assay and real-time PCR as described previously.3,4
Influenza vaccination
Hemagglutination inhibition assay
Adults and children were included, and the age distribution of the entire cohort is shown in Supplementary Table 1. Donors and recipients from 6 months to o3 years received 0.25 mL, and those from 3 to 8 years old received 0.5 mL of the vaccine. During the study period, donors and/or recipients were vaccinated from April to January, according to the randomization group. Donor vaccination was administered ~ 15 days before donation. Recipient vaccination was administered at two time points: pretransplant, to HSCT candidates assigned to randomization group 3 only; and on d180, to all participants as mentioned above. The vaccines recommended for the Southern hemisphere during the study were: A/New Caledonia/20/99 (H1N1), A/Wisconsin/67/2005 (H3N2), B/Malaysia/2506/2004 in 2007; A/Solomon Islands/3/2006 (H1N1), A/Brisbane/12/2007 (H3N2), B/Florida/4/2006 in 2008; and A/Brisbane/59/2007 (H1N1), A/Brisbane/10/2007 (H3N2), B/Florida/4/2006 in 2009.
Anti-hemagglutinin inhibition (HI) Ab levels against each strain of influenza (H1, H3 and B) corresponding to the administered vaccines were determined in duplicate and measured as described previously.3,4,15 Reciprocal Ab titers were analyzed after logarithmic transformation.
Definitions Seroprotection was defined by the presence of HI Ab ⩾ 1:40. Seroresponse was defined by ⩾ 4-fold rise in HI Ab titers after vaccination. Donor seroresponse was evaluated around HSCT day. Recipient seroresponse to influenza vaccine was evaluated at d30 and after d180 vaccination. Severe aplastic anemia with o15 blood transfusions, acute leukemia in first remission and CML in chronic phase were defined as low risk. All the rest were considered as high risk.
Indirect IgG ELISA Blood and nasal wash sampling Donor serum samples were taken at study admission (baseline) and around HSCT day. Recipient serum samples were taken at baseline and on days 0 (d0), +30 (d30), +60 (d60), +100 (d100), +180 (d180) and after d180 vaccination. Nasal washes were taken from symptomatic HSCT recipients as described previously.3,4 Respiratory symptom surveillance was performed two times a week during the first 3 months of HSCT, and thereafter by phone calls. In the presence of symptoms, influenza diagnosis was
Randomization N =122
Group 1 No vaccine N =38
Group 2 Donor vaccination N =44
Group 3 Recipient vaccination N = 40
Serum IgG against influenza Ags (Baxter GmbH, Wien, Austria), such as A/H1N1/California/7/09 NYMC X-179A, A/Solomon Islands/3/2006 (H1N1), A/Uruguay/716/2007 (H3N2) and B/Brisbane/60/2008, were determined by indirect ELISA. Briefly, Ag-coated (1 μg/mL) 96-well ELISA plate (Nunc, Roskilde, Denmark) was washed three times with PBS+0.05% Tween-20 (washing buffer) (Sigma-Aldrich, St Louis, MO, USA) followed by blocking with 2% BSA in PBS at room temperature for 1 h. The plate was washed thrice and diluted (1:100) serum was added and left at room temperature for 2 h. After another five washes, the secondary anti-human IgG (1:1000) ALP-conjugated streptavidin (Mabtech, Stockholm, Sweden) was added to the plate and left at room temperature for 1 h, and then pNPP substrate (Thermo Fisher, Waltham, MA, USA) was added. OD was measured at 405 nm (Molecular Devices, Sunnyvale, CA, USA) after 30 min. In each assay, a standard serum and IgG standard (Sigma-Aldrich) prepared through 1:2 serial dilutions (1500–11.7 μg/mL) were used to report the relative Ag-specific IgG.
Statistical analysis
Post-transplant follow-up: serological and clinical evaluation
Respiratory symptom surveillance
Blood sampling
Nasal wash sampling if symptoms
Days 0, 30, 60, 100, 180 2-4 wk after d180 vaccination
Figure 1. Study algorithm of the open randomized prospective study in 122 transplant recipients and related donors conducted from October 2007 to January 2010 in two Brazilian HSCT centers: Amaral Carvalho Foundation and Federal University of Minas Gerais.
Table 1.
Patients’ demographic characteristics were summarized and compared among randomization groups using χ2 or Fisher’s exact test for categorical variables (as appropriate). Wilcoxon's rank-sum or Kruskal–Wallis test was used for continuous variables. The geometric mean titer was calculated according to the time of sampling, for each influenza Ag, to demonstrate the dynamics of HI antibodies up to d180. The analysis of variance and the T-test were used to compare the differences between randomization groups. Cox proportional hazards models were used to evaluate the variables associated with recipient seroprotection up to d60 and with recipient seroresponse after d180 vaccination, for each influenza Ag. Variables with P ⩽ 0.1 in the univariable models were candidates for multivariable models. Two-sided P-values o0.05 were considered statistically significant. The incidence of documented influenza was estimated by cumulative incidence curves. Pretransplant VE was measured by calculating the incidence rate of influenza among vaccinated
Patient characteristics according to the randomization group
Variable Age Gender UD risk SC source MoAb conditioning Acute GVHD Chronic GVHDa Pre-HSCT SP-H1b Pre-HSCT SP-H3 Pre-HSCT SP-B
Category 425 years Male High BM Yes Grades II–IV Yes Yes Yes Yes
Group 1 (%), N = 38 20 22 18 21 13 08 10 08 09 28
(54.1) (57.9) (47.4) (55.3) (34.2) (23.5) (32.3) (21.1) (23.7) (73.7)
Group 2 (%), N = 44 23 36 21 25 11 13 12 06 19 31
(52.3) (81.8) (47.7) (56.8) (25) (29.5) (35.3) (14) (44.2) (72.1)
Group 3 (%), N = 40 18 26 19 20 09 13 10 08 12 18
(45) (65) (47.5) (50) (23.1) (33.3) (32.3) (21.1) (31.6) (47.4)
P-value 0.69 0.054 0.99 0.60 0.50 0.65 0.95 0.63 0.14 0.025
Abbreviations: SC = stem cell source; UD = underlying disease risk. aAmong 96 HSCT recipients alive after day 100. bPre-HSCT SP-H1 = pretransplant seroprotective rates to Ag H1.
© 2015 Macmillan Publishers Limited
Bone Marrow Transplantation (2015) 858 – 864
Pretransplant influenza vaccination A Ambati et al
Geometric mean titer
860 H1 antigen
35 30 25 20
*
15 10 5 0
Group 1: No vaccination Group 2: Donor vaccination Group 3: Recipient vaccination
Geometric mean titer
Pre-HSCT
0
30
Percentage
H1 H3 B No.
180
Days after HSCT
60
100
180
Days after HSCT
B antigen
120 100 80 60 40 20 0 0
30
Group 1
90 80 70 60 50 40 30 20 10 0
100
**
Pre-HSCT
Figure 2.
60 H3 antigen
35 30 25 20 15 10 5 0 Pre-HSCT
Geometric mean titer
30
0
60 Group 2
100
180
Days after HSCT
Group 3
Dynamics of HI IgG titers up to day 180 according to randomization and different influenza Ags.
H1 H3 B
Pre HSCT 18 32.8 63.3
0-30 28.2 45 77.1
60 20 30.8 57.5
100 14.2 30.2 50.9
180 12.6 27.6 46
128
131
120
106
87
Days after HSCT
Figure 3. Proportion of HSCT recipients with HI seroprotective titers ⩾ 1:40 during the follow-up. and unvaccinated individuals. The VE was evaluated by the formula VE = ((r0 − r1):r0), where r0 is the rate of influenza in unvaccinated and r1 is the rate in vaccinated. All analysis was carried out with 'R' version 3.0.2 and SPSS version 18.
RESULTS One hundred and twenty-two donor–recipient pairs were included in the study, 78 from center A (64%) and 44 from center B (36%). Thirty-eight pairs were included in group 1 (no vaccination); 44 in group 2 (donor vaccination); and 40 in group 3 (recipient vaccination) (Figure 1 and Table 1). The majority of the patients were vaccinated during the winter (39.3%) and spring (25%). However, no significant difference was seen in season distribution among randomization groups (P = 0.51). Donor baseline seroprotection rates Serum samples from 117 of the 122 donors were taken at a median of 12 (58–6) days before HSCT. At baseline, seroprotection rates for Ags H1, H3 and B were 17.9%, 24.8% and 71.8%, respectively. Bone Marrow Transplantation (2015) 858 – 864
Donor seroresponse following pretransplant vaccination Donors randomized to group 2 received influenza vaccine immediately after baseline blood sampling. Forty-four donors were vaccinated at a median time of 11.5 (31–6) days before HSCT date. Donor seroresponse rates to Ags H1, H3 and B were 61.4% (27/44), 75% (33/44) and 54.5% (24/44), respectively. Donor seroprotection rates at stem cell donation HI assay was performed in serum samples from 119 of the 122 donors. Forty-nine donors (41.2%) showed HI Ab titers ⩾ 1:40 against the H1 Ag, 55 (46.2%) against the H3 Ag and 91 (76.5%) against the B Ag. In comparison with baseline rates, a 23.3% rise in seroprotection rate was observed against the H1 Ag (P o0.001), 21.4% against the H3 Ag (P o0.001) and 4.7% against the B Ag (P o 0.001). Donors belonging to the randomization group 2 were more likely to have seroprotective HI Ab titers against H1 and H3 Ags compared with donors belonging to groups 1 and 3 (P o 0.001). For the B Ag, similar seroprotective rates were observed among the randomization groups (P = 0.74). Recipient baseline seroprotection rates Determination of HI Ab was performed in 119 of the 122 HSCT candidates. The seroprotection rates at baseline for H1, H3 and B Ags were 18.5% (22/119 patients), 33.6% (40/119 patients) and 64.7% (77/119 patients), respectively. At baseline, the seroprotection rates did not differ significantly in donors as compared with recipients (data not shown). Recipient seroresponse rates up to d30 In the pretransplant period, 40 HSCT candidates assigned to group 3 received pretransplant vaccinations at a median of 10.5 (50–6) days before transplantation. The seroresponse rate was evaluated in 39 serum samples of the 40 recipients. In one patient, matched serum was unavailable. Seroresponse rates were 17.9% (7/39) for Ag H1, 25.6% (10/39) for Ag H3 and 30.8% (10/39) for Ag B. © 2015 Macmillan Publishers Limited
Pretransplant influenza vaccination A Ambati et al
861 Table 2.
Variables associated with seroprotection on day 60 according to influenza Ag
Ag
P-value
Univariate
P-value
Multivariate
Variable
Category
Seroprotection, N/total (%)
H1
HI titer ⩾ 1:40 pre-HSCT HI titer ⩾ 1:40 at d0 Seroresponse up to d30 UD risk Acute GVHD
Yes; no Yes; no Yes; no High; low Yes; no
12/19 14/23 07/17 06/52 03/42
(63.2); 10/90 (11.1) (60.9); 08/88 (9.1) (41.2); 15/93 (16.1) (11.5); 16/60 (26.7) (7.1); 17/66 (26.8)
o0.0001 o0.0001 0.018 0.04 0.015
20.5 1.4 8.7 0.5 0.17
2.7 0.2 1.5 0.12 0.03
153.4 8.7 50.6 1.9 0.85
0.003 0.68 0.016 0.32 0.031
H3
HI titer ⩾ 1:40 pre-HSCT HI titer ⩾ 1:40 at d0 Seroresponse up to d30 Randomization group Acute GVHD
Yes; no Yes; no Yes; no Group 1; other Yes; no
22/37 29/45 12/15 06/34 10/42
(59.5); 13/72 (18.1 (64.4); 07/66 (10.6) (80); 24/95 (25.3) (17.6); 30/78 (38.5) (23.8); 24/66 (36.4)
o0.0001 o0.0001 o0.0001 0.030 0.17
6.7 4.1 34.0 1.02 0.5
0.77 0.57 5.3 0.45 0.15
57.6 29.6 218.3 2.3 1.6
0.084 0.16 o0.0001 0.95 0.24
B
HI titer ⩾ 1:40 pre-HSCT HI titer ⩾ 1:40 at d0 Acute GVHD
Yes; no Yes; no Yes; no
50/68 (73.5); 16/41 (39) 58/81 (71.6); 09/30 (30) 23/42 (54.8); 44/66 (66.7)
o0.0001 o0.0001 0.21
1.9 4.17 0.6
0.6 1.3 0.2
5.6 13.3 1.4
0.23 0.016 0.27
Exp (B)
95% CI
Abbreviations: CI = confidence interval; HI = hemagglutinin inhibition; UD = underlying disease risk. Significant P values are in bold (Po0.05).
Geometric mean titers of HI antibodies during follow-up Analysis of variance showed a significant difference between randomization groups for H1 (P = 0.03) and H3 (P o0.001) Ags, but not for the B Ag (P = 0.07). Concerning the H1 Ag, group 3 was statistically different from group 1 (P = 0.01); for the H3 Ag, group 3 was statistically different from groups 1 (P o 0.01) and 2 (P o 0.01); and for the B Ag, group 3 was statistically different from group 2 (P = 0.04). The variation in geometric mean titer is shown in Figure 2.
Recipient seroprotection rates during follow-up The seroprotection rates varied during follow-up. Figure 3 shows the percentage of HSCT recipients with HI titers ⩾ 1:40 according to influenza Ag and time after HSCT.
Table 3. Seroresponse after day 180 vaccination according to randomization groups and Ags Seroresponse (%)
Variables associated with recipient seroresponse after d180 vaccination Up to d180, 30 of the 122 patients died, 12 relapsed and 9 had no follow-up sample. Twelve patients could not be vaccinated because of the lack of influenza vaccine. Thus, influenza vaccine was administered to 63 of the 122 recipients (51.6%), at a median of 193 (145–281) days after transplantation. Seroresponse was evaluated in 59 of the 63 (93.6%) vaccinated recipients, as matched serum samples were not available in four of them (Table 3). There was no association with response to vaccination by randomization group, underlying disease risk, conditioning with monoclonal antibodies, seroprotective HI titers on d60 or the occurrence of acute or chronic GVHD up to d180 (not shown). © 2015 Macmillan Publishers Limited
P-value
Group 1, N = 24
Group 2, N = 18
Group 3, N = 17
H1 No Yes
20 (42) 4 (36)
12 (25) 6 (54)
16 (33) 1 (9)
0.11
H3 No Yes
21 (43) 3 (30)
12 (24) 6 (60)
16 (33) 1 (10)
0.07
23 (43) 1 (20)
14 (26) 4 (80)
17 (31) 0 (0)
0.04
B No Yes
Variables associated with recipient seroprotection up to d60 The following variables were included in the analysis of the presence of HI titers ⩾ 1:40 on d60: randomization group (group 1 vs others), underlying disease risk, seroprotective HI titers before HSCT, seroresponse up to d30, seroprotective HI titers up to d30 and occurrence of GVHD up to d60. A multivariate analysis showed that a seroresponse upto d30 (pre-transplant vaccination) was significantly associated with seroprotective titers on day 60 for antigens H1 and H3 but not for B antigen (Table 2).
Pretransplant vaccination group, N = 59
Significant P values are in bold (Po0.05).
Indirect IgG ELISA We analyzed influenza-specific IgG using an indirect ELISA assay in a subgroup of 57 patients: 17 from the no pretransplant vaccination group, 20 from the donor pretransplant vaccination group and 20 from the recipient pretransplant vaccination group, respectively (Table 4). There was significant difference in the Ag-specific serum IgG level against A/H1N1/California/2009 (Figure 4) between the three study groups at 6 months after HSCT with the strongest increase seen in serum from HSCT recipients who received the vaccine before transplantation (P = 0.02). In the multivariate analysis, the only significant factor was the pretransplant vaccination group (P = 0.03), while the stem cell source showed a trend to influence the IgG levels (P = 0.07). There were no significant differences in the specific Ab levels against other influenza Ags, although in all cases the mean specific IgG levels were highest for the patient group vaccinated before transplantation. Documented influenza infections Eleven cases of influenza were diagnosed during the study period (4 in group 1, 4 in group 2 and 3 in group 3). One patient had influenza B and 10 had influenza A (six cases of seasonal H1N1 Bone Marrow Transplantation (2015) 858 – 864
Pretransplant influenza vaccination A Ambati et al
862 Table 4.
Characteristics of 57 patients included in the substudy evaluated by indirect Ag-specific IgG ELISA Group 1, N = 20
Group 2, N = 20
Group 3, N = 17
Patient age at transplant (years), median (range) Male
33 (13–57) 12 (60%)
25 (3–56) 17 (85%)
26 (3–39) 12 (70%)
Donor age at transplant (years), median (range) Male
33 (11–61) 13 (65%)
27 (1 -56) 10 (50%)
26 (5–48) 6 (35%)
5 (25%) 7 (35%)
4 (20%) 9 (45%)
7 (41.1%) 7 (41.1%)
7 (35%) 13 (65%)
8 (40%) 12 (60%)
6 (35%) 11 (64.7%)
GVHD Acute GVHD grade II–IV Chronic GVHD SC source Blood stem cells Marrow Abbreviation: SC = stem cell.
A/California/H1N1/2009
A/Solomon/3/H1N1/2006 250
*
200
Relative IgG µg/ml
Relative IgG µg/ml
250
150 100 50
200 150 100 50 0
0 Recipient +
Control
Recipient +
Donor +
Donor +
A/Uruguay/H3N2/2008
500
500
400
400
Relative IgG µg/ml
Relative IgG µg/ml
B/Brisbane/60/2008
Control
300 200 100 0
300 200 100 0
Recipient +
Control
Donor +
Recipient +
Control
Donor +
Figure 4. Influenza-specific relative serum IgG assayed by an indirect ELISA in a subgroup (n = 57) at d180. Medians are indicated (Po0.05*). Control, no pretransplant influenza vaccination; donor+, influenza vaccination in donor pretransplant; recipient+, influenza vaccination in recipient pretransplant.
influenza and four cases of pandemic H1N1). The infections occurred at a median of 53 days after HSCT, ranging from d − 7 to d+187. Cumulative incidence of proven influenza infection was 7% up to d300. The cumulative incidence of influenza according to randomization group was 12% in group 1, 6% in group 2 and 2% in group 3 (P = 0.30). The four cases of pandemic influenza H1N1 were excluded in this analysis as the vaccine against pandemic H1N1 was not yet available at the time (Supplementary Figure 1). Four patients were treated, and in the remaining seven, the diagnosis was made retrospectively. Nine influenza cases had undetectable levels of HI antibodies when the diagnosis was made. Two patients acquired influenza despite having HI titers ⩾ 1:40. One patient from group 3 acquired seasonal influenza H1N1 despite seroprotective H1 Ab levels (1:80) and died of respiratory distress syndrome 5 days after influenza diagnosis. The other patient had H1 Ab titer of 1:40 but was infected with the pandemic influenza H1N1 with no complications. Four (36%) of the 11 cases of influenza A died (three seasonal H1N1 and one pandemic H1N1). Acute GVHD grade 4 may have contributed to Bone Marrow Transplantation (2015) 858 – 864
death in three of them. Mortality among untreated patients was 42.8%. Efficacy of pretransplant vaccination VE was evaluated only in the 86 patients from center A. Cases of pandemic A H1N1 were not considered in the analysis of VE as the patients did not receive vaccine against the pandemic virus. Three patients acquired seasonal influenza in group 1 (3/22; r0 = 13.6) and three in the other groups (3/64; r1 = 4.7). Thus, VE was 65.4% ((r0 − r1):r0). DISCUSSION Preventing influenza infection in HSCT patients is a complex task. The primary study objective was to improve recipient seroprotection during the first months after transplantation; a period in which influenza vaccine is frequently ineffective.16 Thus, we hypothesized that recipient vaccination before HSCT would boost © 2015 Macmillan Publishers Limited
Pretransplant influenza vaccination A Ambati et al
the levels of influenza antibodies and possibly protect the patient against influenza during the early period after HSCT. Another hypothesis tested by our study was whether donor vaccination might allow the transfer of influenza-specific memory cells, possibly favoring a rapid increase in Ab levels after recipient vaccination or exposure to influenza infection. As we included only donor–recipient pairs who had never received influenza vaccine o 6 months before HSCT, the baseline seroprotection rates were similar between donors and recipients, and reflected previous influenza infection. In fact, influenza B virus had circulated in 2007 most likely resulting in the higher Ab levels to the B Ag (460%) in comparison with the H1 Ag (~18%) or the H3 Ag (~30%) found in donors as well as in recipients. Although we did not see a significant effect of donor pretransplant vaccination, the pretransplant influenza vaccination of the HSCT candidate significantly improved the Ab titers to H1 and H3 Ags until d180. It is possible that an earlier, post-transplant vaccination either at 2 or 3 months after HSCT could have yielded better response in the vaccinated donor group. The benefit of early postHSCT vaccination of the recipient has been shown in studies assessing the response to other Ags such as tetanus, HIB (hemophilus influenzae type b), inactivated poliovirus and conjugated pneumococcal vaccine.17–20 This study shows that the immunogenicity of inactivated influenza vaccine when given before HSCT is poor, presumably reflecting the treatment of the underlying disease. Up to d30, recipient seroresponse rates varied between 22.9% (H1 Ag) and 25% (H3 and B Ag). This is significantly lower than the seroresponse rates of donors varying from 54.7% (B) to 75.4% (H3). In addition, HSCT patients respond poorly to influenza vaccination, with response rates around 20%, as seen previously.21–25 Although seroresponse rates following pretransplant vaccination were poor, we observed that patients who achieved protective Ab titers (⩾1:40) at HSCT day were more likely to maintain specific influenza immunity up to d60, supporting the strategy of pretransplant vaccination. Serum HI Ab titers of 1:40 are supposed to protective in the immunocompetent, but higher Ab levels might be required in subjects with impaired immunity.26 Inspite of having an HI ab titer of 1:80, a patient in the study population was diagnosed with influenza infection. In addition to the ‘classical’ HI assay, we determined specific IgG using an indirect ELISA with an aim to investigate if it would yield additional information. Limitations to this analysis are that the vaccine strains shifted between the two study years and that we only analyzed samples from a patient subpopulation. However, despite these limitations, we found that recipients vaccinated pretransplant had significantly higher IgG levels to A/H1N1 at 180 days after HSCT along with the trend to higher IgG levels against other influenza strains. After d180 vaccination, recipient seroresponse rates were also poor, varying from 8% (Ag B) to 20% (H1) comparable to pretransplant seroresponse rates. Previous studies have shown similar seroresponse rates varying from 20 to 35%.21–23,27 We found increased serum IgG to the H1 pandemic Ag by ELISA but not by HI at d180 in the recipient pretransplant vaccination group. This might be explained by that the ELISA measures IgG against epitopes from the hemagglutinin, the neuraminidase and trace amounts of the matrix 1 protein.28–31 Apart from the neutralization epitopes of HA, these other epitopes may be beneficial in Ab-dependent cell-mediated cytotoxicity and complement fixation. During follow-up, the proportion of patients with seroprotective titers was higher on d0 and d30 than at later time points, demonstrating that influenza immunity wanes over time. We found that the titer at baseline was significantly associated with protective titers to H1 and H3 influenza antigens on d60 in accordance with previous observations that if either donor or recipient is immunized pretransplant, the likelihood of transfer or © 2015 Macmillan Publishers Limited
persistence of immunity is increased.13,32–34 An early posttransplant vaccination is needed to retain immunity for prolonged periods. Despite the poor immunogenicity of the vaccine reflected by the low seroresponse and seroprotective rates during follow-up, the pretransplant VE was 465%. Previously, we observed a VE of 80% after influenza vaccination at d180.4 Unfortunately, the statistical analysis of other relevant risk factors, such as type of immunosuppressive therapy, the occurrence of acute or chronic GHVD and the presence of lymphopenia, was not possible because of the small number of patients with influenza. Severe influenza in otherwise healthy individuals is influenced by the viral pathogenicity and the host immune response, which is inherently different in HSCT recipients because of the immature Band T-cell reconstitution. A future vaccination strategy might be to vaccinate the recipient pretransplant and boost this response early within 3 months after transplantation in the recipient. Efforts should also therefore be made to limit the risk of influenza exposure in HSCT recipients. CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS We thank all the patient participants. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2008/00282-8), Vetenskaprådet (Swedish research council), Karolinska Instituet and Karolinska University Hospital.
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24 Dhédin N, Krivine A, Le Corre N, Mallet A, Lioure B, Bay J-O et al. Comparable humoral response after two doses of adjuvanted influenza A/H1N1pdm2009 vaccine or natural infection in allogeneic stem cell transplant recipients. Vaccine 2014; 32: 585–591. 25 Kunisaki KM, Janoff EN. Influenza in immunosuppressed populations: a review of infection frequency, morbidity, mortality, and vaccine responses. Lancet Infect Dis 2009; 9: 493–504. 26 Ring A, Marx G, Steer C, Harper P. Influenza vaccination and chemotherapy: a shot in the dark? Support Care Cancer 2002; 10: 462–465. 27 de Lavallade H, Garland P, Sekine T, Hoschler K, Marin D, Stringaris K et al. Repeated vaccination is required to optimize seroprotection against H1N1 in the immunocompromised host. Haematologica 2011; 96: 307–314. 28 Terajima M, Cruz J, Leporati AM, Orphin L, Babon JAB, Co MDT et al. Influenza A virus matrix protein 1-specific human CD8+ T-cell response induced in trivalent inactivated vaccine recipients. J Virol 2008; 82: 9283–9287. 29 Renfrey S, Watts A. Morphological and biochemical characterization of influenza vaccines commercially available in the United Kingdom. Vaccine 1994; 12: 747–752. 30 Garcia-Cañas V, Lorbetskie B, Girard M. Rapid and selective characterization of influenza virus constituents in monovalent and multivalent preparations using non-porous reversed-phase high performance liquid chromatography columns. J Chromatogr A 2006; 1123: 225–232. 31 Garcia-Cañas V, Lorbetskie B, Cyr TD, Hefford MA, Smith S, Girard M. Approach to the profiling and characterization of influenza vaccine constituents by the combined use of size-exclusion chromatography, gel electrophoresis and mass spectrometry. Biologicals 2010; 38: 294–302. 32 Wimperis JZ, Brenner MK, Prentice HG, Reittie JE, Karayiannis P, Griffiths PD et al. Transfer of a functioning humoral immune system in transplantation of T-lymphocyte-depleted bone marrow. Lancet 1986; 1: 339–343. 33 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. 34 Molrine DC, Guinan EC, Antin JH, Parsons SK, Weinstein HJ, Wheeler C et al. Donor immunization with Haemophilus influenzae type b (HIB)-conjugate vaccine in allogeneic bone marrow transplantation. Blood 1996; 87: 3012–3018.
Supplementary Information accompanies this paper on Bone Marrow Transplantation website (http://www.nature.com/bmt)
Bone Marrow Transplantation (2015) 858 – 864
© 2015 Macmillan Publishers Limited
ORIGINAL ARTICLE
Evaluation of pretransplant influenza vaccination in hematopoietic SCT: a randomized prospective study A Ambati1,2, LSV Boas3, P Ljungman1,4, L Testa5, JF de Oliveira6, M Aoun7, V Colturato5, M Maeurer2,7 and CM Machado3,5 Pretransplant influenza vaccination of the donor or allogeneic hematopoietic SCT (HSCT) candidate was evaluated in a randomized study. One hundred and twenty-two HSCT recipients and their donors were assigned to three randomization groups: no pretransplant vaccination (n = 38), donor pretransplant vaccination (n = 44) or recipient pretransplant vaccination (n = 40). Specific IgG was assessed by both hemagglutinin inhibition (HI) and, in 57 patients, by an indirect influenza-specific ELISA at specified times after HSCT. Vaccinated donors had seroprotective HI titers for Ags H1 and H3 (Po 0.001) compared with the other groups at the time of donation. The titers against H1 (P = 0.028) and H3 (P o 0.001) were highest in the pretransplant recipient vaccination group until day 180 after transplantation. A significant difference was found in the specific Ig levels against pandemic H1N1 at 6 months after SCT (P = 0.02). The mean IgG levels against pandemic H1N1 and generic H1N1 and H3N2 were highest in the pretransplant recipient vaccination group. We conclude that pretransplant recipient vaccination improved the influenza-specific seroprotection rates. Bone Marrow Transplantation (2015) 50, 858–864; doi:10.1038/bmt.2015.47; published online 23 March 2015
INTRODUCTION Influenza can be severe in hematopoietic SCT (HSCT) recipients with up to 30% mortality rates in untreated patients.1 Among 286 HSCT patients, 43.7% were hospitalized, 32.5% developed lower respiratory tract disease, 11.5% required mechanical ventilation and 6.3% died from the recent pandemic outbreak.2 Preventive strategies including vaccination, prompt diagnosis and the use of neuraminidase inhibitors have lowered the risk of complications and fatal outcome.1,3,4 Vaccination is the primary mode of prevention, although seroresponse rates in HSCT patients are poor. They are recommended to start vaccination after 6 months or even earlier from transplantation during the influenza season, possibly followed by a second dose.4,5 Studies have shown that the persistence of pre-existing donor and recipient immunity in HSCT recipients is of importance, but either have a finite duration.6–9 Pretransplant vaccination of the donor, the recipient or both has been evaluated previously and was found to positively influence Ag-specific IgG levels in the first year after transplant.10–13 Accordingly, we hypothesized that pretransplant vaccination of the donor or HSCT candidate would elicit a certain degree of protection against influenza in the first months after transplantation via two mechanisms: prompting a rise in the levels of circulating influenza antibodies in the vaccinated HSCT candidate; or by transferring primed memory cells in the case of donor vaccination. We conducted a randomized prospective study to evaluate alternative schedules of pretransplant influenza vaccination in an HSCT setting.
MATERIALS AND METHODS Study design and subjects An open randomized prospective study was conducted from October 2007 to January 2010 in two Brazilian HSCT centers: Amaral Carvalho Foundation (center A) and Federal University of Minas Gerais (center B). The ethics committees of the participating institutions approved the study; all consecutive allogeneic HSCT recipients with a related donor were invited to participate in the study. D/R pairs with one or more of the following criteria were excluded from the study: (1) HSCT expected to occur less than a week from the date of recruitment; (2) egg, chicken protein, neomycin or thimerosal allergy history; (3) past history of severe reaction following influenza vaccine; (4) influenza vaccine received o6 months before HSCT date. The patients in this study did not receive any prophylactic Igs. Written informed consent was obtained from donors and recipients. The primary objective was to evaluate if pretransplant vaccination of the donor or HSCT candidates would improve influenza seroprotection rates early after HSCT. Secondary objectives were to evaluate the impact of pretransplant vaccination in seroresponse rates following influenza vaccination on day 180 (d180), and the occurrence of documented influenza infections during this period (pretransplant vaccine efficacy (VE)). The subjects were assigned to three randomization groups: no pretransplant vaccination (group 1), donor pretransplant vaccination (group 2) and recipient pretransplant vaccination (group 3). D/R pairs were randomized by the method of randomly permuted blocks. A list of random numbers with their respective randomization group was generated through the website of the University of Tuffs (available at http://www.tufts.edu/ ~ gdallal/randomize.htm). Allocation group cards were prepared, placed in opaque envelopes, sequentially numbered and sealed. Sets of 20 envelopes were distributed alternately between the two participating HSCT centers during the recruitment period. After signature of the informed consent, the envelopes were opened in numerical order. The study was not blinded. On d180,
1 Division of Hematology, Department of Medicine Huddinge, Karolinska Institutet, Stockholm, Sweden; 2Centre for Allogeneic Stem Cell Transplantation (CAST), Karolinska University Hospital, Stockholm, Sweden; 3Virology Laboratory (LIM 52-HCFMUSP), Institute of Tropical Medicine, University of São Paulo, São Paulo, Brazil; 4Department of Hematology, Karolinska University Hospital, Stockholm, Sweden; 5Hematopoietic Stem Cell Transplant Program, Amaral Carvalho Foundation, Jahu, Brazil; 6Clinics Hospital – School of Medicine, Federal University of Minas Gerais, Belo Horizonte, Brazil and 7Division of Therapeutic Immunology (TIM), Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden. Correspondence: Dr CM Machado, Virology Laboratory (LIM 52-HCFMUSP), Institute of Tropical Medicine, University of São Paulo, Avenida Dr Enéas de Carvalho Aguiar, 470, São Paulo 05403-000, Brazil, E-mail: [email protected] Received 7 October 2014; revised 12 January 2015; accepted 15 January 2015; published online 23 March 2015
Pretransplant influenza vaccination A Ambati et al
859 all groups received one dose of influenza vaccine, as currently recommended5,14 (Figure 1).
performed by direct fluorescent assay and real-time PCR as described previously.3,4
Influenza vaccination
Hemagglutination inhibition assay
Adults and children were included, and the age distribution of the entire cohort is shown in Supplementary Table 1. Donors and recipients from 6 months to o3 years received 0.25 mL, and those from 3 to 8 years old received 0.5 mL of the vaccine. During the study period, donors and/or recipients were vaccinated from April to January, according to the randomization group. Donor vaccination was administered ~ 15 days before donation. Recipient vaccination was administered at two time points: pretransplant, to HSCT candidates assigned to randomization group 3 only; and on d180, to all participants as mentioned above. The vaccines recommended for the Southern hemisphere during the study were: A/New Caledonia/20/99 (H1N1), A/Wisconsin/67/2005 (H3N2), B/Malaysia/2506/2004 in 2007; A/Solomon Islands/3/2006 (H1N1), A/Brisbane/12/2007 (H3N2), B/Florida/4/2006 in 2008; and A/Brisbane/59/2007 (H1N1), A/Brisbane/10/2007 (H3N2), B/Florida/4/2006 in 2009.
Anti-hemagglutinin inhibition (HI) Ab levels against each strain of influenza (H1, H3 and B) corresponding to the administered vaccines were determined in duplicate and measured as described previously.3,4,15 Reciprocal Ab titers were analyzed after logarithmic transformation.
Definitions Seroprotection was defined by the presence of HI Ab ⩾ 1:40. Seroresponse was defined by ⩾ 4-fold rise in HI Ab titers after vaccination. Donor seroresponse was evaluated around HSCT day. Recipient seroresponse to influenza vaccine was evaluated at d30 and after d180 vaccination. Severe aplastic anemia with o15 blood transfusions, acute leukemia in first remission and CML in chronic phase were defined as low risk. All the rest were considered as high risk.
Indirect IgG ELISA Blood and nasal wash sampling Donor serum samples were taken at study admission (baseline) and around HSCT day. Recipient serum samples were taken at baseline and on days 0 (d0), +30 (d30), +60 (d60), +100 (d100), +180 (d180) and after d180 vaccination. Nasal washes were taken from symptomatic HSCT recipients as described previously.3,4 Respiratory symptom surveillance was performed two times a week during the first 3 months of HSCT, and thereafter by phone calls. In the presence of symptoms, influenza diagnosis was
Randomization N =122
Group 1 No vaccine N =38
Group 2 Donor vaccination N =44
Group 3 Recipient vaccination N = 40
Serum IgG against influenza Ags (Baxter GmbH, Wien, Austria), such as A/H1N1/California/7/09 NYMC X-179A, A/Solomon Islands/3/2006 (H1N1), A/Uruguay/716/2007 (H3N2) and B/Brisbane/60/2008, were determined by indirect ELISA. Briefly, Ag-coated (1 μg/mL) 96-well ELISA plate (Nunc, Roskilde, Denmark) was washed three times with PBS+0.05% Tween-20 (washing buffer) (Sigma-Aldrich, St Louis, MO, USA) followed by blocking with 2% BSA in PBS at room temperature for 1 h. The plate was washed thrice and diluted (1:100) serum was added and left at room temperature for 2 h. After another five washes, the secondary anti-human IgG (1:1000) ALP-conjugated streptavidin (Mabtech, Stockholm, Sweden) was added to the plate and left at room temperature for 1 h, and then pNPP substrate (Thermo Fisher, Waltham, MA, USA) was added. OD was measured at 405 nm (Molecular Devices, Sunnyvale, CA, USA) after 30 min. In each assay, a standard serum and IgG standard (Sigma-Aldrich) prepared through 1:2 serial dilutions (1500–11.7 μg/mL) were used to report the relative Ag-specific IgG.
Statistical analysis
Post-transplant follow-up: serological and clinical evaluation
Respiratory symptom surveillance
Blood sampling
Nasal wash sampling if symptoms
Days 0, 30, 60, 100, 180 2-4 wk after d180 vaccination
Figure 1. Study algorithm of the open randomized prospective study in 122 transplant recipients and related donors conducted from October 2007 to January 2010 in two Brazilian HSCT centers: Amaral Carvalho Foundation and Federal University of Minas Gerais.
Table 1.
Patients’ demographic characteristics were summarized and compared among randomization groups using χ2 or Fisher’s exact test for categorical variables (as appropriate). Wilcoxon's rank-sum or Kruskal–Wallis test was used for continuous variables. The geometric mean titer was calculated according to the time of sampling, for each influenza Ag, to demonstrate the dynamics of HI antibodies up to d180. The analysis of variance and the T-test were used to compare the differences between randomization groups. Cox proportional hazards models were used to evaluate the variables associated with recipient seroprotection up to d60 and with recipient seroresponse after d180 vaccination, for each influenza Ag. Variables with P ⩽ 0.1 in the univariable models were candidates for multivariable models. Two-sided P-values o0.05 were considered statistically significant. The incidence of documented influenza was estimated by cumulative incidence curves. Pretransplant VE was measured by calculating the incidence rate of influenza among vaccinated
Patient characteristics according to the randomization group
Variable Age Gender UD risk SC source MoAb conditioning Acute GVHD Chronic GVHDa Pre-HSCT SP-H1b Pre-HSCT SP-H3 Pre-HSCT SP-B
Category 425 years Male High BM Yes Grades II–IV Yes Yes Yes Yes
Group 1 (%), N = 38 20 22 18 21 13 08 10 08 09 28
(54.1) (57.9) (47.4) (55.3) (34.2) (23.5) (32.3) (21.1) (23.7) (73.7)
Group 2 (%), N = 44 23 36 21 25 11 13 12 06 19 31
(52.3) (81.8) (47.7) (56.8) (25) (29.5) (35.3) (14) (44.2) (72.1)
Group 3 (%), N = 40 18 26 19 20 09 13 10 08 12 18
(45) (65) (47.5) (50) (23.1) (33.3) (32.3) (21.1) (31.6) (47.4)
P-value 0.69 0.054 0.99 0.60 0.50 0.65 0.95 0.63 0.14 0.025
Abbreviations: SC = stem cell source; UD = underlying disease risk. aAmong 96 HSCT recipients alive after day 100. bPre-HSCT SP-H1 = pretransplant seroprotective rates to Ag H1.
© 2015 Macmillan Publishers Limited
Bone Marrow Transplantation (2015) 858 – 864
Pretransplant influenza vaccination A Ambati et al
Geometric mean titer
860 H1 antigen
35 30 25 20
*
15 10 5 0
Group 1: No vaccination Group 2: Donor vaccination Group 3: Recipient vaccination
Geometric mean titer
Pre-HSCT
0
30
Percentage
H1 H3 B No.
180
Days after HSCT
60
100
180
Days after HSCT
B antigen
120 100 80 60 40 20 0 0
30
Group 1
90 80 70 60 50 40 30 20 10 0
100
**
Pre-HSCT
Figure 2.
60 H3 antigen
35 30 25 20 15 10 5 0 Pre-HSCT
Geometric mean titer
30
0
60 Group 2
100
180
Days after HSCT
Group 3
Dynamics of HI IgG titers up to day 180 according to randomization and different influenza Ags.
H1 H3 B
Pre HSCT 18 32.8 63.3
0-30 28.2 45 77.1
60 20 30.8 57.5
100 14.2 30.2 50.9
180 12.6 27.6 46
128
131
120
106
87
Days after HSCT
Figure 3. Proportion of HSCT recipients with HI seroprotective titers ⩾ 1:40 during the follow-up. and unvaccinated individuals. The VE was evaluated by the formula VE = ((r0 − r1):r0), where r0 is the rate of influenza in unvaccinated and r1 is the rate in vaccinated. All analysis was carried out with 'R' version 3.0.2 and SPSS version 18.
RESULTS One hundred and twenty-two donor–recipient pairs were included in the study, 78 from center A (64%) and 44 from center B (36%). Thirty-eight pairs were included in group 1 (no vaccination); 44 in group 2 (donor vaccination); and 40 in group 3 (recipient vaccination) (Figure 1 and Table 1). The majority of the patients were vaccinated during the winter (39.3%) and spring (25%). However, no significant difference was seen in season distribution among randomization groups (P = 0.51). Donor baseline seroprotection rates Serum samples from 117 of the 122 donors were taken at a median of 12 (58–6) days before HSCT. At baseline, seroprotection rates for Ags H1, H3 and B were 17.9%, 24.8% and 71.8%, respectively. Bone Marrow Transplantation (2015) 858 – 864
Donor seroresponse following pretransplant vaccination Donors randomized to group 2 received influenza vaccine immediately after baseline blood sampling. Forty-four donors were vaccinated at a median time of 11.5 (31–6) days before HSCT date. Donor seroresponse rates to Ags H1, H3 and B were 61.4% (27/44), 75% (33/44) and 54.5% (24/44), respectively. Donor seroprotection rates at stem cell donation HI assay was performed in serum samples from 119 of the 122 donors. Forty-nine donors (41.2%) showed HI Ab titers ⩾ 1:40 against the H1 Ag, 55 (46.2%) against the H3 Ag and 91 (76.5%) against the B Ag. In comparison with baseline rates, a 23.3% rise in seroprotection rate was observed against the H1 Ag (P o0.001), 21.4% against the H3 Ag (P o0.001) and 4.7% against the B Ag (P o 0.001). Donors belonging to the randomization group 2 were more likely to have seroprotective HI Ab titers against H1 and H3 Ags compared with donors belonging to groups 1 and 3 (P o 0.001). For the B Ag, similar seroprotective rates were observed among the randomization groups (P = 0.74). Recipient baseline seroprotection rates Determination of HI Ab was performed in 119 of the 122 HSCT candidates. The seroprotection rates at baseline for H1, H3 and B Ags were 18.5% (22/119 patients), 33.6% (40/119 patients) and 64.7% (77/119 patients), respectively. At baseline, the seroprotection rates did not differ significantly in donors as compared with recipients (data not shown). Recipient seroresponse rates up to d30 In the pretransplant period, 40 HSCT candidates assigned to group 3 received pretransplant vaccinations at a median of 10.5 (50–6) days before transplantation. The seroresponse rate was evaluated in 39 serum samples of the 40 recipients. In one patient, matched serum was unavailable. Seroresponse rates were 17.9% (7/39) for Ag H1, 25.6% (10/39) for Ag H3 and 30.8% (10/39) for Ag B. © 2015 Macmillan Publishers Limited
Pretransplant influenza vaccination A Ambati et al
861 Table 2.
Variables associated with seroprotection on day 60 according to influenza Ag
Ag
P-value
Univariate
P-value
Multivariate
Variable
Category
Seroprotection, N/total (%)
H1
HI titer ⩾ 1:40 pre-HSCT HI titer ⩾ 1:40 at d0 Seroresponse up to d30 UD risk Acute GVHD
Yes; no Yes; no Yes; no High; low Yes; no
12/19 14/23 07/17 06/52 03/42
(63.2); 10/90 (11.1) (60.9); 08/88 (9.1) (41.2); 15/93 (16.1) (11.5); 16/60 (26.7) (7.1); 17/66 (26.8)
o0.0001 o0.0001 0.018 0.04 0.015
20.5 1.4 8.7 0.5 0.17
2.7 0.2 1.5 0.12 0.03
153.4 8.7 50.6 1.9 0.85
0.003 0.68 0.016 0.32 0.031
H3
HI titer ⩾ 1:40 pre-HSCT HI titer ⩾ 1:40 at d0 Seroresponse up to d30 Randomization group Acute GVHD
Yes; no Yes; no Yes; no Group 1; other Yes; no
22/37 29/45 12/15 06/34 10/42
(59.5); 13/72 (18.1 (64.4); 07/66 (10.6) (80); 24/95 (25.3) (17.6); 30/78 (38.5) (23.8); 24/66 (36.4)
o0.0001 o0.0001 o0.0001 0.030 0.17
6.7 4.1 34.0 1.02 0.5
0.77 0.57 5.3 0.45 0.15
57.6 29.6 218.3 2.3 1.6
0.084 0.16 o0.0001 0.95 0.24
B
HI titer ⩾ 1:40 pre-HSCT HI titer ⩾ 1:40 at d0 Acute GVHD
Yes; no Yes; no Yes; no
50/68 (73.5); 16/41 (39) 58/81 (71.6); 09/30 (30) 23/42 (54.8); 44/66 (66.7)
o0.0001 o0.0001 0.21
1.9 4.17 0.6
0.6 1.3 0.2
5.6 13.3 1.4
0.23 0.016 0.27
Exp (B)
95% CI
Abbreviations: CI = confidence interval; HI = hemagglutinin inhibition; UD = underlying disease risk. Significant P values are in bold (Po0.05).
Geometric mean titers of HI antibodies during follow-up Analysis of variance showed a significant difference between randomization groups for H1 (P = 0.03) and H3 (P o0.001) Ags, but not for the B Ag (P = 0.07). Concerning the H1 Ag, group 3 was statistically different from group 1 (P = 0.01); for the H3 Ag, group 3 was statistically different from groups 1 (P o 0.01) and 2 (P o 0.01); and for the B Ag, group 3 was statistically different from group 2 (P = 0.04). The variation in geometric mean titer is shown in Figure 2.
Recipient seroprotection rates during follow-up The seroprotection rates varied during follow-up. Figure 3 shows the percentage of HSCT recipients with HI titers ⩾ 1:40 according to influenza Ag and time after HSCT.
Table 3. Seroresponse after day 180 vaccination according to randomization groups and Ags Seroresponse (%)
Variables associated with recipient seroresponse after d180 vaccination Up to d180, 30 of the 122 patients died, 12 relapsed and 9 had no follow-up sample. Twelve patients could not be vaccinated because of the lack of influenza vaccine. Thus, influenza vaccine was administered to 63 of the 122 recipients (51.6%), at a median of 193 (145–281) days after transplantation. Seroresponse was evaluated in 59 of the 63 (93.6%) vaccinated recipients, as matched serum samples were not available in four of them (Table 3). There was no association with response to vaccination by randomization group, underlying disease risk, conditioning with monoclonal antibodies, seroprotective HI titers on d60 or the occurrence of acute or chronic GVHD up to d180 (not shown). © 2015 Macmillan Publishers Limited
P-value
Group 1, N = 24
Group 2, N = 18
Group 3, N = 17
H1 No Yes
20 (42) 4 (36)
12 (25) 6 (54)
16 (33) 1 (9)
0.11
H3 No Yes
21 (43) 3 (30)
12 (24) 6 (60)
16 (33) 1 (10)
0.07
23 (43) 1 (20)
14 (26) 4 (80)
17 (31) 0 (0)
0.04
B No Yes
Variables associated with recipient seroprotection up to d60 The following variables were included in the analysis of the presence of HI titers ⩾ 1:40 on d60: randomization group (group 1 vs others), underlying disease risk, seroprotective HI titers before HSCT, seroresponse up to d30, seroprotective HI titers up to d30 and occurrence of GVHD up to d60. A multivariate analysis showed that a seroresponse upto d30 (pre-transplant vaccination) was significantly associated with seroprotective titers on day 60 for antigens H1 and H3 but not for B antigen (Table 2).
Pretransplant vaccination group, N = 59
Significant P values are in bold (Po0.05).
Indirect IgG ELISA We analyzed influenza-specific IgG using an indirect ELISA assay in a subgroup of 57 patients: 17 from the no pretransplant vaccination group, 20 from the donor pretransplant vaccination group and 20 from the recipient pretransplant vaccination group, respectively (Table 4). There was significant difference in the Ag-specific serum IgG level against A/H1N1/California/2009 (Figure 4) between the three study groups at 6 months after HSCT with the strongest increase seen in serum from HSCT recipients who received the vaccine before transplantation (P = 0.02). In the multivariate analysis, the only significant factor was the pretransplant vaccination group (P = 0.03), while the stem cell source showed a trend to influence the IgG levels (P = 0.07). There were no significant differences in the specific Ab levels against other influenza Ags, although in all cases the mean specific IgG levels were highest for the patient group vaccinated before transplantation. Documented influenza infections Eleven cases of influenza were diagnosed during the study period (4 in group 1, 4 in group 2 and 3 in group 3). One patient had influenza B and 10 had influenza A (six cases of seasonal H1N1 Bone Marrow Transplantation (2015) 858 – 864
Pretransplant influenza vaccination A Ambati et al
862 Table 4.
Characteristics of 57 patients included in the substudy evaluated by indirect Ag-specific IgG ELISA Group 1, N = 20
Group 2, N = 20
Group 3, N = 17
Patient age at transplant (years), median (range) Male
33 (13–57) 12 (60%)
25 (3–56) 17 (85%)
26 (3–39) 12 (70%)
Donor age at transplant (years), median (range) Male
33 (11–61) 13 (65%)
27 (1 -56) 10 (50%)
26 (5–48) 6 (35%)
5 (25%) 7 (35%)
4 (20%) 9 (45%)
7 (41.1%) 7 (41.1%)
7 (35%) 13 (65%)
8 (40%) 12 (60%)
6 (35%) 11 (64.7%)
GVHD Acute GVHD grade II–IV Chronic GVHD SC source Blood stem cells Marrow Abbreviation: SC = stem cell.
A/California/H1N1/2009
A/Solomon/3/H1N1/2006 250
*
200
Relative IgG µg/ml
Relative IgG µg/ml
250
150 100 50
200 150 100 50 0
0 Recipient +
Control
Recipient +
Donor +
Donor +
A/Uruguay/H3N2/2008
500
500
400
400
Relative IgG µg/ml
Relative IgG µg/ml
B/Brisbane/60/2008
Control
300 200 100 0
300 200 100 0
Recipient +
Control
Donor +
Recipient +
Control
Donor +
Figure 4. Influenza-specific relative serum IgG assayed by an indirect ELISA in a subgroup (n = 57) at d180. Medians are indicated (Po0.05*). Control, no pretransplant influenza vaccination; donor+, influenza vaccination in donor pretransplant; recipient+, influenza vaccination in recipient pretransplant.
influenza and four cases of pandemic H1N1). The infections occurred at a median of 53 days after HSCT, ranging from d − 7 to d+187. Cumulative incidence of proven influenza infection was 7% up to d300. The cumulative incidence of influenza according to randomization group was 12% in group 1, 6% in group 2 and 2% in group 3 (P = 0.30). The four cases of pandemic influenza H1N1 were excluded in this analysis as the vaccine against pandemic H1N1 was not yet available at the time (Supplementary Figure 1). Four patients were treated, and in the remaining seven, the diagnosis was made retrospectively. Nine influenza cases had undetectable levels of HI antibodies when the diagnosis was made. Two patients acquired influenza despite having HI titers ⩾ 1:40. One patient from group 3 acquired seasonal influenza H1N1 despite seroprotective H1 Ab levels (1:80) and died of respiratory distress syndrome 5 days after influenza diagnosis. The other patient had H1 Ab titer of 1:40 but was infected with the pandemic influenza H1N1 with no complications. Four (36%) of the 11 cases of influenza A died (three seasonal H1N1 and one pandemic H1N1). Acute GVHD grade 4 may have contributed to Bone Marrow Transplantation (2015) 858 – 864
death in three of them. Mortality among untreated patients was 42.8%. Efficacy of pretransplant vaccination VE was evaluated only in the 86 patients from center A. Cases of pandemic A H1N1 were not considered in the analysis of VE as the patients did not receive vaccine against the pandemic virus. Three patients acquired seasonal influenza in group 1 (3/22; r0 = 13.6) and three in the other groups (3/64; r1 = 4.7). Thus, VE was 65.4% ((r0 − r1):r0). DISCUSSION Preventing influenza infection in HSCT patients is a complex task. The primary study objective was to improve recipient seroprotection during the first months after transplantation; a period in which influenza vaccine is frequently ineffective.16 Thus, we hypothesized that recipient vaccination before HSCT would boost © 2015 Macmillan Publishers Limited
Pretransplant influenza vaccination A Ambati et al
the levels of influenza antibodies and possibly protect the patient against influenza during the early period after HSCT. Another hypothesis tested by our study was whether donor vaccination might allow the transfer of influenza-specific memory cells, possibly favoring a rapid increase in Ab levels after recipient vaccination or exposure to influenza infection. As we included only donor–recipient pairs who had never received influenza vaccine o 6 months before HSCT, the baseline seroprotection rates were similar between donors and recipients, and reflected previous influenza infection. In fact, influenza B virus had circulated in 2007 most likely resulting in the higher Ab levels to the B Ag (460%) in comparison with the H1 Ag (~18%) or the H3 Ag (~30%) found in donors as well as in recipients. Although we did not see a significant effect of donor pretransplant vaccination, the pretransplant influenza vaccination of the HSCT candidate significantly improved the Ab titers to H1 and H3 Ags until d180. It is possible that an earlier, post-transplant vaccination either at 2 or 3 months after HSCT could have yielded better response in the vaccinated donor group. The benefit of early postHSCT vaccination of the recipient has been shown in studies assessing the response to other Ags such as tetanus, HIB (hemophilus influenzae type b), inactivated poliovirus and conjugated pneumococcal vaccine.17–20 This study shows that the immunogenicity of inactivated influenza vaccine when given before HSCT is poor, presumably reflecting the treatment of the underlying disease. Up to d30, recipient seroresponse rates varied between 22.9% (H1 Ag) and 25% (H3 and B Ag). This is significantly lower than the seroresponse rates of donors varying from 54.7% (B) to 75.4% (H3). In addition, HSCT patients respond poorly to influenza vaccination, with response rates around 20%, as seen previously.21–25 Although seroresponse rates following pretransplant vaccination were poor, we observed that patients who achieved protective Ab titers (⩾1:40) at HSCT day were more likely to maintain specific influenza immunity up to d60, supporting the strategy of pretransplant vaccination. Serum HI Ab titers of 1:40 are supposed to protective in the immunocompetent, but higher Ab levels might be required in subjects with impaired immunity.26 Inspite of having an HI ab titer of 1:80, a patient in the study population was diagnosed with influenza infection. In addition to the ‘classical’ HI assay, we determined specific IgG using an indirect ELISA with an aim to investigate if it would yield additional information. Limitations to this analysis are that the vaccine strains shifted between the two study years and that we only analyzed samples from a patient subpopulation. However, despite these limitations, we found that recipients vaccinated pretransplant had significantly higher IgG levels to A/H1N1 at 180 days after HSCT along with the trend to higher IgG levels against other influenza strains. After d180 vaccination, recipient seroresponse rates were also poor, varying from 8% (Ag B) to 20% (H1) comparable to pretransplant seroresponse rates. Previous studies have shown similar seroresponse rates varying from 20 to 35%.21–23,27 We found increased serum IgG to the H1 pandemic Ag by ELISA but not by HI at d180 in the recipient pretransplant vaccination group. This might be explained by that the ELISA measures IgG against epitopes from the hemagglutinin, the neuraminidase and trace amounts of the matrix 1 protein.28–31 Apart from the neutralization epitopes of HA, these other epitopes may be beneficial in Ab-dependent cell-mediated cytotoxicity and complement fixation. During follow-up, the proportion of patients with seroprotective titers was higher on d0 and d30 than at later time points, demonstrating that influenza immunity wanes over time. We found that the titer at baseline was significantly associated with protective titers to H1 and H3 influenza antigens on d60 in accordance with previous observations that if either donor or recipient is immunized pretransplant, the likelihood of transfer or © 2015 Macmillan Publishers Limited
persistence of immunity is increased.13,32–34 An early posttransplant vaccination is needed to retain immunity for prolonged periods. Despite the poor immunogenicity of the vaccine reflected by the low seroresponse and seroprotective rates during follow-up, the pretransplant VE was 465%. Previously, we observed a VE of 80% after influenza vaccination at d180.4 Unfortunately, the statistical analysis of other relevant risk factors, such as type of immunosuppressive therapy, the occurrence of acute or chronic GHVD and the presence of lymphopenia, was not possible because of the small number of patients with influenza. Severe influenza in otherwise healthy individuals is influenced by the viral pathogenicity and the host immune response, which is inherently different in HSCT recipients because of the immature Band T-cell reconstitution. A future vaccination strategy might be to vaccinate the recipient pretransplant and boost this response early within 3 months after transplantation in the recipient. Efforts should also therefore be made to limit the risk of influenza exposure in HSCT recipients. CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS We thank all the patient participants. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2008/00282-8), Vetenskaprådet (Swedish research council), Karolinska Instituet and Karolinska University Hospital.
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