Cell-mediated immune responses to influenza vaccination in ... - Nature

Bone Marrow Transplantation (2005) 36, 411–415 & 2005 Nature Publishing Group All rights reserved 0268-3369/05 $30.00

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Cell-mediated immune responses to influenza vaccination in healthy volunteers and allogeneic stem cell transplant recipients G Avetisyan1,2, E Ragnavo¨lgyi3, GT Toth4, M Hassan1,2 and P Ljungman1,2 1 Department of Medicine, Division of Hematology, Karolinska Institutet, Stockholm, Sweden; 2Department of Hematology, Karolinska University Hospital/Huddinge, Stockholm, Sweden; 3Institute of Immunology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary; and 4Department of Medical Chemistry, A. Szentgyorgyi University, Szeged, Hungary

Summary: Influenza is one of the most common respiratory diseases in humans. The response to vaccination is frequently poor in immunosuppressed individuals. The aim of the present study was to develop an enzyme-linked immunospot (ELISPOT) assay for measuring of the specific T-cell response to influenza vaccination. In all, 18 healthy subjects and six stem cell transplantation (SCT) patients tested before and 4 weeks after influenza vaccination were included in the present study. Peripheral blood lymphocytes were stimulated with four influenza peptides; three based on sequences from the hemagglutinin and one from the M1 protein. The ELISPOT assay and the measurement of intracellular IFN-c production were used to determine the cell-mediated responses after stimulation with the peptides. Influenza vaccination elicited strong cell-mediated immune responses in the healthy controls to all four peptides with 3.2–6.9-fold increases in the number of IFN-c producing spots/106 cells. By intracellular staining, it was suggested that CD4 þ cells mediated the responses to the hemagglutinin peptides. In contrast, there was no increase in the number of IFN-c producing cells response after vaccination in the six SCT patients. In conclusion, our results suggest that the ELISPOT assay might be used as a complement to serology for monitoring of future influenza vaccine studies in SCT patients. Bone Marrow Transplantation (2005) 36, 411–415. doi:10.1038/sj.bmt.1705064; published online 27 June 2005 Keywords: influenza; vaccination; cell-mediated immunity; elispot; stem cell transplantation

Influenza is one of the most widely spread human infectious diseases and causes significant morbidity and mortality every year. About 20% of children and 5% of adults worldwide develop symptomatic influenza A or B infection each year.1 The risk of influenza varies between seasons. In

Correspondence: Professor P Ljungman, Department of Hematology, Karolinska University Hospital/Huddinge, Stockholm, Sweden; E-mail: [email protected] Received 11 February 2005; accepted 3 May 2005; published online 27 June 2005

some groups of individuals (eg young children, elderly people and immunocompromised stem cell and organ recipients) the risk to develop life-threatening complications increases. Vaccination with an inactivated, attenuated vaccine can reduce serious morbidity and mortality associated with influenza infection. National authorities of many countries recommend annual influenza vaccination for those 465 years of age; residents of nursing homes; adults and children with chronic medical conditions; for patients with suppressed immunity.2–4 Recovery from influenza infection involves both humoral and cell-mediated immune mechanisms. Specific antibodies are essential for protection against primary influenza infections but due to antigenic drift and shift previous antibodies might not be protective against new strains. Whereas influenza vaccination of healthy individuals can induce humoral immune responses in 60–90%,4 most studies in immunocompromised patients show lower rates of efficacy varying between 10 and 50% using serological end points for controlling responses to vaccination.5–10 Although antibodies play critical roles in protection, activation of T-lymphocytes, in particular, the CD4 þ helper subpopulation is essential for supporting virusspecific effector cell functions.11 Few studies have addressed the possibility to investigate cell-mediated immune response as a marker of vaccine responsiveness.12 The aim of the present study was, therefore, to develop an enzyme-linked immunospot (ELISPOT) technique for measuring influenza-specific T-cell response for use in future prospective studies aiming to improve existent vaccination schemes in immunosuppressed recipients.

Subjects and methods Human subjects In all, 18 adult healthy volunteers (10 males and eight females) were included in the study. They were recruited from the staff of the Departments of Medicine and Clinical Science at the Karolinska University Hospital. The median age was 36.5 (range 26–53) years. In addition, six patients undergoing allogeneic unrelated stem cell transplantation (SCT) were included in the study. The patients’ median age was 42 years (range 19–57). The study was approved by the ethical committee of Karolinska Institute.

Influenza vaccination in healthy subjects and SCT recipients G Avetisyan et al

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All subjects were immunized in the deltoid muscle with one dose of trivalent inactivated influenza subunit vaccine containing 15 mg hemagglutinin. The patients were immunized at 3 months after SCT and followed under one influenza season. The study including the healthy volunteers was performed during two subsequent influenza seasons making the compositions of the vaccines different. The H1N1 strain was the same during both seasons (A/New Caledonia/20/99) while the H3N2 (2003/2004 A/Moscow/10/99; 2004/2005 A/Fujian/411/2002) and B (2003/2004 Hong Kong/330/2001; 2004/2005 Shanghai/ 361/2002) differed between the seasons.

Preparation of peripheral blood mononuclear cells Blood samples were obtained prior to and 4 weeks after vaccination. Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation of heparinized blood on a Lymphoprep gradient (Nycomed, Oslo, Norway). Cells were washed twice in tissue culture medium RPMI (RPMI1640-Hepes with Glutamax-I, GIBCO BRL, NY, USA) and resuspended to a concentration of 2 106/ml in RPMI 1640 supplemented with 10% heat inactivated fetal bovine serum (FBS) (GIBCO BRL, NY, USA).

Peptides Initially five peptides synthesized at the A. Szentgyorgyi University, Szeged, Hungary, covering the 306–341 region of uncleaved human influenza A virus hemagglutinin (HA0)13 were available for the study. Titration experiments to choose the optimal concentrations were performed in three of the healthy subjects. Peptides were dissolved in dimethylsulfoxide (DMSO) (Sigma, Steinheim, Germany) to the concentrations of 1 mg/ml. Four different dilutions were tested by ELISPOT assay (1, 2, 5, 10 ng/well). Three peptides comprising H1 (HA306–318), H2 (HA317–341), and H3 (HA316–341) subtype sequences were selected all in the concentration of 5 ng/well. In addition the HLA-A0201 restricted M1 peptide (GILGFVFTL, INNOVAGEN AB, Lund, Sweden) was included.

washing, cells were developed by adding 100 ml/well of colorimetric substrate (BCIP/NBT-plus, Mabtech AB) and then counted in an ELISPOT reader. Each spot was produced by a single IFN-g secreting T-cell–spot-forming cell (SFC). All tests were performed in duplicate and the mean values were calculated. A response was considered to be positive when the number of spots in the wells with peptide-stimulated cells, after subtraction of the background (wells without peptide stimulation), was at least two-fold greater than the number of background spots14 and more than 20/106 SFC.

Detection of intracellular cytokine production by flow cytometry The intracellular staining method was used for identifying the phenotype of INF-g producing T cells activated by peptide stimulation. The staining was performed using the protocol described by Rauser et al.15 PBMC were stimulated for 6 h with 1 mg/ml influenza peptides. Brefeldin A (10 mg/ml, Sigma-Aldrich, St Louis, MO) was added for the last 4 h of the incubation. A positive control was obtained by stimulating the cells with 0.5 mg/ml PMA (Phorbol 12-myrestate 13-acetate) and 1 mg/ml Ionomycin (both from Sigma-Aldrich, St Louis, MO, USA). The cells were permeabilized and stained with fluorochrome-labeled anti-CD3, anti-CD4 or anti-CD8 and anti-IFN-g antibodies (Becton Dickinson, San Jose, CA, USA). Samples were analyzed by flow cytometry using FACSCalibur (Becton Dickinson). The positive limit for this test was 0.05% of IFN producing T cells.

Statistical analysis Responses before and after vaccination were compared by Wilcoxon’s signed rank test. Comparisons of groups were performed with the Mann–Whitney U-test (two-sided).

Results Controls

ELISPOT assay The 96-well PVDF (polyvinylidene difluoride, Millipore Corp., MA, USA) plates were prewet with 70% EtOH, then coated with 100 ml/well of anti-human IFN-g monoclonal antibody (mAb) 1-D1K (Mabtech AB, Stockholm, Sweden), diluted to 15 mg/ml in sterile filtered phosphatebuffered saline (PBS) (GIBCO BRL, NY, USA). The plates were kept overnight at 41C. A total of 100 000 PBMC plus 5 ml/well (1 mg/ml) peptides were added to each well in duplicate for each condition and incubated for 24 h at 371C in 5% CO2. After incubation the plates were washed with PBS then 100 ml of biotinylated mAb (7-B6-1-biotin, Mabtech AB), diluted in filtered PBS with 0.5% FBS to the concentration 1 mg/ml were added to each well. The plates were incubated 2 h at room temperature, washed with PBS and then incubated with 100 ml/well of streptavidine-alkaline phosphatase (Mabtech AB) diluted 1–1000 in PBS with 0.5% FBS for 1 h at room temperature. After Bone Marrow Transplantation

The ELISPOT results in the 18 healthy adults are summarized in Table 1. Vaccination was able to induce a cell-mediated immune response detectable by the ELISPOT method after stimulation with all three HA peptides. The

Table 1 Comparison of IFN-g production by T-cell stimulated with different peptides before and after influenza vaccination of healthy volunteers Peptide M1 H1 H2 H3

Median of SFC before vaccination 60 25 30 20

(30–100) (10–95) (10–75) (5–85)

Median of SFC after vaccination 192.5 102.5 100 137.5

(115–270) (65–225) (65–140) (55–320)

SFC ¼ spot-forming cell. In parenthesis – quartiles (lower-upper).

Fold increase

P-value

3.2 4.1 3.3 6.9

0.04 0.002 0.01 0.01


413 1000

N of SFC per 106 PBMC

900

Before After

800 700 600 500 400 300 200 100 0 C1 C2

C3 C4 C5 C6

C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 Healthy volunteers

Figure 1 IFN-g production after stimulation of PBLs from healthy volunteers with H3 influenza peptide.

Table 2 Comparison of IFN-g production by T-cell stimulated with different peptides before and after influenza vaccination of patients Peptide M1 H1 H2 H3

Median of SFC before vaccination 15 47.5 35 45

(10–20) (30–160) (20–140) (10–130)

Median of SFC after vaccination 20 22.5 30 47

(0–115) (10–190) (5–80) (0–135)

Fold increase

P-value

1.3 0.5 0.9 1.0

0.3 0.07 0.6 0.3

SFC ¼ spot-forming cell. In parenthesis – quartiles (lower-upper).

responses were significantly stronger after vaccination (Pp0.01 for all three peptides). There was no obvious difference in the immune response between controls vaccinated during the first or the second season. Individual results for the controls after stimulation with the H3 (HA316–341) peptide before and after vaccination are shown in Figure 1. Prior to vaccination, PBMC from 9/18, 10/18, and 9/18 individuals produced at least 20 IFN-g producing spots/106 cells after stimulation with H1, H2, and H3 peptides, respectively. The corresponding numbers 4 weeks after vaccination were 17/18, 17/18, and 16/18, respectively. In all, 13 out of 18 controls had at least doubled the number of IFN-g producing spots after compared to before vaccination. Intracellular staining and FACS analysis showed that cells responding to the HA peptides were CD4 þ cells (data not shown). Also the immune response to the M1 peptide became stronger after vaccination (Table 1). The responses were significantly stronger in HLA-A0201 positive individuals compared to nonA0201 individuals (Po0.01, data not shown).

Patients Six SCT patients were immunized at 3 months after SCT. The patients were studied immediately before and 4 weeks

after influenza vaccination. The number of IFN-g producing cells was significantly lower than in healthy controls and there was no significant increase in the number of IFNg producing cells in the SCT patients after vaccination (Table 2). IFN-g producing cells stimulated by the H3 peptide were detected before vaccination in 4 out of 6 patients (Figure 2). The corresponding numbers to H1 and H2 peptides were 5 out of 6 and 5 out of 6, respectively. One patient (no. 1) had a verified influenza A infection directly after vaccination and between the two ELISPOT assays. If this patient is excluded, only one patient doubled the number of IFN-g producing cells compared to before vaccination.

Discussion Influenza viruses are unique in their ability to cause recurrent illnesses, annual epidemics and more extended pandemics that spread rapidly and can affect all population groups.4 Patients with hematological disorders who undergo SCT experience immunological dysfunction for up to 1 year or longer.16 In a prospective study by EBMT approximately 20% of community respiratory virus infections in adult SCT recipients were due to influenza viruses.17 Death from influenza usually results from secondary bacterial pneumonia but can also be consequences of primary viral pneumonia in the absence of bacterial invasion.18 In published studies, mortality rates in SCT patients has varied between 17 and 57%.19–21 The immune defense against influenza virus infection involves both humoral and cellular immune mechanisms. Hemagglutinin is a major antigenic determinant of influenza viruses; 15 hemagglutinin subtypes (H1–H15) and nine neuroaminidase subtypes (N1–N9) have been identified for type A virus and only one subtype of hemagglutinin and one subtype of neuroaminidase are recognized for type B virus. Influenza specific CTL are able to lyse influenza virus-infected cells by recognition of viral internal nucleoprotein (NP). Bone Marrow Transplantation


414 1000

N of SFC per 106 PBMC

900 800 Before

700

After

600 500 400 300 200 100 0 P1

P2

P3

P4

P5

P6

Patients

Figure 2

IFN-g production after stimulation of PBLs from SCT patients with H3 influenza peptide.

Our study shows that PBMC, isolated from healthy subjects, contain CD4 þ T-lymphocytes able to secrete IFN-g upon in vitro stimulation with peptides comprising the conserved H1, H2, and H3 hemagglutitin sequences. We also demonstrate, that healthy immunocompetent individuals respond to influenza vaccination with increased numbers of IFN-g producing helper T-lymphocytes. The strongest responses were seen after stimulation with the H3 peptide. This is consistent with the fact, that this is the most frequent subtype circulating in the most recent epidemics. The GILGFVFTL M1 peptide has been used as a control peptide in studies of CMV specific immunity in HLA-0201 individuals. We found that 17 of 18 (94%) individuals had positive response to stimulation with the M1 peptide regardless of being HLA-A0201 positive although responses were stronger in the HLA-0201 positive controls. These results could be explained by the fact that influenza-specific CTL repertoires are broad (ie, in the same individual multiple epitopes are recognized) and multispecific (ie, includes recognition of several proteins).22 We also were able to assume that the responding cells to the M1 peptide were mainly CD8 þ cells possibly because the vaccine was prepared from inactivated split virus and contains M1 peptide. Most previous studies of the efficacy of influenza vaccination in SCT patients have used antibody titer increases as end points and it has been shown that the humoral immune responses were poor if influenza vaccine was given early after SCT.5,8 Recently a small study was published using lymphocyte proliferation to lysates of influenza H3N2 infected cells as stimulators showing that specific lymphocyte proliferation was increased after vaccination of a small group of children who had undergone SCT.12 Haining et al12 showed by a lymphocyte proliferation assay that existing memory cells was the cell type responding to vaccination and naı¨ ve cells were not recruited by vaccination. Furthermore, they found that CD4 responses were stronger than CD8 responses and that none of the patients developed specific IgG responses.12 Our study of a small group of adult allogeneic SCT patients Bone Marrow Transplantation

showed that low numbers of IFN-g producing cells were detectable also before vaccination. At the time these studies were performed, all patients were 100% donor chimeras suggesting that the cells responding were transferred from the stem cell donors. However, in most of the patients, the cells were unable to respond to vaccination in the same manner as in the healthy controls. It was interesting that one patient who developed influenza A infection was able to mount a strong immune response to all studied peptides. Whether this patient would have responded to vaccination is unknown since the infection developed very early after the vaccination. It has been shown previously that virusspecific donor T cells as well as donor B cells can be present early after SCT but in the absence of restimulation, they are lost with extended follow-up.23,24 Both the group studied by Haining et al and our group of SCT patients were very small and further studies are therefore needed. We conclude that ELISPOT can be used as a sensitive and specific assay for measuring cell-mediated immunity to influenza vaccine. This technique might be used in further studies to evaluate immune response to influenza and thereby assess novel vaccination strategies.

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