SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects, materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

With emergence of MHC class I tetramers loaded with CD8+ T-cell viral epitopes, it is possible to study virus-specific CD8 cells in humans during infection and after vaccination. MHC class I tetramers was used to detect the frequency of haemagglutinin (HA)-specific T cells in 26 healthy influenza-vaccinated humans. Peripheral blood was collected before, and 7, 14 and 28 days after vaccination. Four-colour flow cytometry was used for monitoring of vaccine induced T-cell response. In 15 donors, two- to fivefold increase in frequency of HA-specific T cells was observed 7 days after vaccination. In addition, in 12 of these donors, this increase was accompanied with fourfold increase of H1N1 antibody titre. The increase in frequency of HA-specific CD8+/IFN-γ+ cells was low and peaked 28 days after vaccination in three of the six donors tested. Frequencies of HA-specific CD8+ T cells and antibody titre returned to prevaccination values 1 year after vaccination. Subunit influenza vaccines have the ability to induce HA-specific CD8+ cells. As the immune response to this vaccine decreased significantly after 1 year, our results confirm the importance of annual immunization for adequate protection.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects, materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Influenza A viruses cause annual epidemics of acute respiratory infection that often result in significant morbidity and mortality in human population. The virus appears to evade the immune system by changing its surface haemagglutinin (HA) and neuraminidase (NA) proteins every few years. As the virus mutates continuously, vaccination must be repeated annually before each influenza season with an updated vaccine to assure a good match with the circulating influenza strains. Antibody titre against HA proteins provides a correlate of protection, especially after immunization with inactivated influenza virus vaccine but it does not reflect necessarily other important immunological responses to vaccination such as the generation of virus-specific CD8+ cytotoxic T lymphocytes (CTL) and helper CD4+ T cells [1]. Virus-specific CTL have been implicated as necessary for the clearance of the influenza virus during infection [2, 3] and consequently are valuable population to induce by influenza vaccination. Published data for humans are inconsistent regarding whether inactivated influenza subunit vaccines have the ability to induce sustained cytotoxic T-cell response [4]. These observations prompted us to examine whether immunization with this type of influenza vaccine stimulates CTL response. In addition, since the same vaccine has been recommended for the time of the study conduct, we also investigated the necessity for repeated immunization.

In the present study, MHC class I tetramers [5] was used with an attempt to quantify ex vivo low frequency of influenza-specific T cells directed against HLA-A*0201-restricted influenza A antigens. Haemagglutinin inhibition (HI) assay was used for detection of specific antiviral antibodies titre in sera of naturally infected and influenza-vaccinated donors [6]. The repertoire of human CTL response to influenza A viruses targets multiple viral proteins including relatively conserved internal viral proteins, such as the nucleoprotein (NP), matrix protein (M), non-structural protein (NS), polymerases and the outer, more variable glycoproteins HA and NA [7, 8]. Previous data showed that memory CTL specific for outer viral proteins (HA and NA) like those specific for internal proteins may be either subtype cross-reactive or subtype specific when a different subtype of influenza A virus infects [9]. In the present work, we monitored the frequency of influenza A haemagglutinin antigens (A/New Caledonia/H1N1, HA344-353, HA541-549)-specific CTL in peripheral blood mononuclear cells (PBMC) of 26 HLA-A*0201+ influenza immunized healthy donors before, 7, 14 and 28 days after vaccination. In addition, virus-specific T cells were also followed 1 year after vaccination in six donors. Four-colour flow cytometry enabled us to define subpopulations of antigen-specific T cells on the basis of expression of surface molecules. In addition, we also determined activation status of tetramer+ CTL after vaccination by means of measuring the expression level of activation marker CD38 [10, 11]. As IFN-γ+ influenza-specific memory T cells are important for a rapid response to influenza reinfection [12], we also analysed the capability of influenza-specific CD8+ T cells to produce IFN-γ following vaccination.

Subjects, materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects, materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Subjects and samples.  In total, 94 individuals (average age 32, range 22–59) were screened to obtain HLA-A*0201+ group of adult healthy volunteers. HLA-A*02+ status was tested using the microlymphocytotoxicity (MLCT) serological technique [13] and/or fluorescein isothiocyanate anti-HLA-A*02 antibody (Proimmune, Oxford, UK). HLA-A*0201 typing was performed using polymerase chain reaction with sequence-specific primers (PCR-SSP) as described previously [14]. HLA-A*0201+ status was confirmed in 39 donors. Individuals were immunized with influenza vaccine Agrippal (Chiron, Italy) during the winter seasons of 2001–2002 or 2002–2003. This vaccine was produced from inactivated influenza A (A/New Caledonia/H1N1-like, A/Moscow/H3N2-like) and B (B/Hong Kong/-like, B/Shangdong-like) virus surface proteins according to WHO recommendations [15]. Two donors have been previously vaccinated, one in season 2000–2001 and the other more than 10 years ago. Subjects did not have known allergy to eggs, egg products or chicken protein and they did not suffer from influenza-like disease within previous 12 months. In addition, samples from six HLA-A*0201 donors who were immunized during winter season of 2001–2002 were also obtained 1 year after vaccination. The studies were approved by the ethical review board at Croatian National Institute of Public Health, and all participants provided informed consent. Finally, PMBC and sera from 26 donors were successfully collected before, 7, 14 and 28 days after vaccination.

Peripheral blood mononuclear cells were isolated from peripheral venous blood by Ficoll–Paque (Pharmacia Biotech, Uppsala, Sweden) following the manufacturer’s instructions. The cells were stored in liquid nitrogen and used within 6 months.

HLA-A*0201 tetramers.  Phycoerythrin (PE)-labelled HLA class I tetramers (Proimmune, Oxford, UK) were loaded with HLA-A*0201-restricted matrix protein M158-66 peptide (GILGFVFTL) [16] and two New Caledonia virus haemagglutinin peptides HA344-353 (GLFGAIAGFI) and HA541-549 (VLLVSLGAI) [8].

Immunofluorescence staining.  The following conjugated murine anti-human monoclonal antibodies (mAb) were used: CD3-APC, CD38-CyChrome (BD Biosciences, San Jose, CA, USA), CD8-PE-Cy5 (Dako, Glostrup, Denmark) and CD8-FITC (Serotec, Oxford, UK). For direct staining, cryopreserved PBMC (2 × 106) were washed twice with 0.1% bovine serum albumin (BSA) in PBS (Wash buffer), and first stained with HLA-tetramers at 4 °C for 20 min in the dark and then for additional 25 min with anti-CD3, anti-CD8 and anti-CD38 surface antibodies or with isotype- and fluorochrome-matched control antibodies. After incubation, cells were washed two times with Wash buffer, resuspended in 0.5 ml of PBS with 1% FCS (foetal calf serum) and 2,5% paraformaldehyde (Fix solution) and analysed immediately on a flow cytometer (FACSCalibur; BD Biosciences).

Controls.  Peripheral blood mononuclear cells from HLA-A*0201 donors (n = 2) isolated before, 7, 14 and 28 days after vaccination and PBMC from influenza-infected HLA-A*0201 adult patients (n = 2) were used as a negative control. As a positive control we used either PBMC from influenza-infected HLA-A*0201+ adult patients (n = 4) or influenza-specific T-cell lines, which were generated from PBMC of HLA-A*0201 influenza-vaccinated donors. The samples from influenza-infected patients were obtained in acute phase of illness (1–3 days after disease onset) and than in convalescence, 14 days after the onset of illness when patients were symptom-free.

Influenza infection was confirmed using rapid indirect immunofluorescence test in nasopharyngeal aspirate and/or direct fluorescent antibody (DFA) assay [17]. Influenza virus strain types were determined by HI assay.

For the generation of influenza-specific T-cell lines, freshly isolated PBMC were incubated in 12-well plates with 1 μm of three different HLA-A*0201-restricted influenza-derived peptides: (1) matrix protein M158-66 peptide (GILGFVFTL); (2) New Caledonia virus haemagglutinin peptides HA344-353 (GLFGAIAGFI); or (3) HA541-549 (VLLVSLGAI) in RPMI 1640 supplemented with 10% human AB serum (RPMI-AB) and antibiotics at density of 1 × 106 per well. Incubation was performed at 37 °C and 5% CO2. After 1-h incubation, 2 × 106 cells per well from the same donor were added. RPMI-AB supplemented with 10 U/ml of human recombinant IL-2 (R&D Systems, Minneapolis, MN, USA) was changed on third and seventh day of cell culture. After 10-day incubation, cells were harvested and four-colour immunofluorescence staining was performed as described before.

Cell surface and intracellular IFN-γ staining.  Peripheral blood mononuclear cells (2 × 106) were washed in Wash buffer and incubated with 10 μg/ml final concentration of New Caledonia virus haemagglutinin peptide HA344-353 in the presence of 1 μg/ml anti-CD28/anti-CD49d mAb (BD Biosciences) and Brefeldin A (BFA, 10 μg/ml) (Sigma Chemical Co., St Louis, MO, USA). PBMC were cultured in RPMI 1640 (G) supplemented with 10% human AB serum at 37 °C and 5% CO2 for 5 h.

Cells incubated in presence of anti-CD28/anti-CD49d mAb and BFA were used as a negative control. As a positive control we used cells incubated with 1 μg/ml Staphylococcal enterotoxin B, SEB (Sigma Chemical Co.) in presence of 1 μg/ml anti-CD28/anti-CD49d mAb and BFA (10 μg/ml).

After 5-h culture, cells were washed with Wash buffer and fixed with 4% formaldehyde and 0.5% FCS in PBS for 20 min in the dark at 4 °C. Cells were then washed in PBS containing 0.1% saponin and 0.5% sodium azide (Sigma Chemical Co.) in PBS (Permeabilizing buffer). Cell pellets were subsequently incubated with anti-IFN-γ-FITC, CD69-PE, anti-CD3 APC and anti-CD8 PerCP or with isotype- and fluorochrome-matched control antibodies (all from BD Biosciences) for 30 min at 4 °C. The stained cells were again washed in Permeabilizing buffer, resuspended in 2% formaldehyde in PBS, and analysed immediately on a flow cytometer.

Flow cytometry analysis.  Four-colour FACS analyses were performed using dual-laser, four-colour FACSCalibur flow cytometer and CellQuest software. Lymphocytes (gated from scatter graph based on ‘live cell’ population, size and density of the population) double positive for CD3 and CD8 were designated cytotoxic T cells. For each sample analysed for the co-expression of tetramer- and CD38-staining (the later was used as a marker of lymphocyte activation), at least 50,000 CD3+CD8+ events were collected. For the analysis of influenza-specific IFN-γ-producing CD8+ T cells, we collected 30,000 CD3+CD8+ events. Flow cytometry experiments for all the samples of one donor were performed in the same day.

Antibody response.  Inhibition of haemagglutination (IH) assay was used for detection of specific antiviral antibodies titre [6]. To monitor antibody responses of the immunized to the vaccine, all obtained sera were titrated simultaneously. Sera were prepared and then tested as described in [6].

Statistical analysis.  Kinetics of T-cell response and antibody titre following influenza vaccination were analysed by Friedman anova followed by post hoc tests. All analyses were performed with statistica v 6 (StatSoft, Inc., Tulsa, OK, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects, materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Antibody titre dynamics following vaccination

The influenza-specific humoral response to A/New Caledonia/H1N1-like virus strain was monitored by HI assay in sera obtained before, and 7, 14 or 28 days after vaccination. A HI titre ≥40 is considered the level of antibody response to influenza vaccination that is predictive of protection [18]. Therefore, individuals defined as having an intact humoral response and fulfilling the definition of so called responders should produce (1) a fourfold rise in HI titre to any one of strains in vaccine pre- to post-vaccination and (2) a post-vaccination HI titre ≥40 to at least one of strains in vaccine [19]. Three of 26 individuals had A/New Caledonia/H1N1 HI antibody titre ≥40 before vaccination. One of these donors was immunized in year 2000 with influenza subunit vaccine that contained instead of New Caledonia-like virus, an A/Beijing/262/95 (H1N1)-like virus. Other presumably had prior infection with H1N1-like influenza virus, although no medical record was available to confirm this assumption. In 16 donors vaccination induced fourfold increase of H1N1 antibody titre, which peaked 14 and 28 days after vaccination (Fig. 1).

image

Figure 1.  Antibody titre dynamics following vaccination. A/New Caledonia/H1N1 influenza strain antibody titre elicited by vaccination in HLA-A*0201+ donors before, and 7, 14 and 28 days after vaccination; Friedman anova followed by post hoc tests, statistica v 6 (StatSoft, Inc., Tulsa, OK, USA), boxes show medians and 25th and 75th percentiles, and whiskers show 10th and 90th percentiles.

Download figure to PowerPoint

In 12 of 26 donors fourfold increase of H1N1 antibody titre was accompanied with two- to fivefold increase of frequency of antigen-specific T cells 7 days after vaccination. Changes in either antibody titre or frequency of antigen-specific T cells were not observed in seven donors. In three donors, only frequency of antigen-specific T cells increased and four donors showed only increase in H1N1 antibody titre after vaccination. No correlation was found between HA-specific CD8 cells and antibody levels which is in consistence with previously published data [20].

Sensitivity of HLA-class I tetramers in detecting influenza-specific T cells

We used published sequences of immunodominant haemagglutinin epitopes [8] and identified two of these sequences (HA344-353 and HA541-549) in influenza A/New Caledonia/20/99(H1N1)-like virus that fulfilled HLA-A*0201-binding preferences using NCBI protein sequence viewer (accession no. DOI: CAC86622). We then proceeded to investigate whether these were immunogenic in vaccinated individuals. In addition, we have used a well-known immunodominant peptide M158-66 [16], which is conserved among different influenza A strains to compare the frequency of antigen-specific CD8+ T cells in naturally infected HLA-A*0201+ and HLA-A*0201 patients. We found virtually no CD8+tetramer+ cells in naturally infected HLA-A*0201 donors (Fig. 2A,B). In contrast, enriched population of M158-66-specific tetramer-binding cells were demonstrated among T cells obtained from influenza-infected HLA-A*0201 subjects (Fig. 2C,D) and to a higher extent among antigen-specific T-cell lines generated as described in Subjects, materials and methods (Fig. 2E,J). HLA-A*0201+ influenza infected patients (n = 4) had higher percentage of tetramer-binding T cells in convalescence phase than in acute phase.

image

Figure 2.  Representative dot plots of negative (A,B,F,J) and positive (C,D,E,G,H) controls used to detect the sensitivity of class I tetramer staining. PBMC obtained from influenza-infected HLA-A*0201 patients during acute infection (A) and than 2 weeks after in convalescence (B), as well as PBMC obtained from influenza-infected HLA-A*0201+ patients during acute infection (C) and 2 weeks after in convalescence (D) were labelled with M158-66 MHC tetramers (tetM1). PBMC obtained from HLA-A*0201 donors (F,G) and HLA-A*0201+ donors (H,I) after influenza vaccination were labelled with HA344-353 and HA541-549 MHC tetramers (tetHA344 and tetHA541) as described in Subjects, materials and methods. Live cells were gated by forward and side scattering and additionally for CD3 staining. A T-cell lines specific for M158-66 and HA344-353 were generated as described in Subjects, materials and methods and used through the study as a positive control for tetramer staining (E,J). X-axis represents the staining with tetramer and Y-axis represents staining with anti-CD8. Numbers above the boxes in dot plots are percentage of tetramer+CD8+ T cells.

Download figure to PowerPoint

A similar approach was used to determine the sensitivity for HA344-353 and HA541-549 tetramers. PBMC from influenza-vaccinated HLA-A*0201 (Fig. 2F,G) and HLA-A*0201+ (Fig. 2H,I) donors were used to determine the sensitivity of HA344-353 and HA541-549 MHC tetramer staining. We found populations of HA-specific CD8+CD3+ cells induced by vaccination in HLA-A*0201+-vaccinated donors in contrast to HLA-A*0201 donors.

To distinguish recently activated tetramer-binding cells, analysis of CD38-expressing CD3+CD8+ cells was performed. This type of analysis demonstrated the increase of both recently activated as well resting tetramer+ cells 2 weeks following the onset of disease as described previously [21]. We also observed a small subset of tetramer staining cells among CD8 cells which we subsequently identified as CD19+ B cells (data not shown) and confirmed previous findings made by several authors of HLA unrestricted, non-specific binding of viral antigens [22].

Frequency of activated antigen-specific T cells before and after immunization

According to the vaccine manufacturer, the purified subunit vaccine we used to immunize healthy individuals consists mostly of HA and NA antigens. Due to the limitations of known immunodominant HA and NA epitopes presented in the HLA-A*0201 molecules at the time of the study design, we were able to analyse only the possible increase in HA-reactive CD8+ T cells. For this purpose, we used two peptides embedded in HLA-A*0201 molecules (HA344-353 and HA541-549). Co-staining of the tetramer+ cells with CD38 molecule was used to show whether these cells represents recently activated T cells (CD38+tet+) as an outcome of influenza vaccination. The frequencies of recently activated CD38 expressing HA-specific T cells were followed before, and 7, 14 days and 28 days after vaccination (Fig. 3). T-cell response was detectable for both tested tetramers and peaked on the 7 day post-vaccination; however, the frequency of HA344-353+ cells was higher in most of the immunized donors indicating a possible hierarchy in immunodominance among used peptide sequences (Fig. 4). Similar results were obtained if results were presented as number of tetramer-binding cells among 50,000 CD3+CD8+ T lymphocytes (data not shown). In 15 of 26 donors, two- to fivefold increase of frequency of both tetramer-binding populations of recently activated influenza-specific T cells was observed after vaccination.

image

Figure 3.  Kinetics of T-cell response to influenza vaccination in HLA-A*0201+ donors. Estimation of the frequency of recently activated HA344-353 (A), and HA541-549 (B) specific CD3+CD8+CD38+ T cells in peripheral blood in HLA-A*0201-vaccinated donors before, and 7, 14 and 28 days after vaccination. Frequencies are the percentages of tetramer-staining cells; Friedman anova followed by post hoc tests, statistica v 6 (StatSoft, Inc., Tulsa, OK, USA), boxes show medians and 25th and 75th percentiles, and whiskers show 10th and 90th percentiles.

Download figure to PowerPoint

image

Figure 4.  Representative dot plots of HA344-353 and HA541-549 tetramer staining of recently activated (CD38+, upper right quadrant) and resting (CD38, low right quadrant) tetramer+CD3+CD8+ cells in peripheral blood in HLA-A*0201 positive and negative donors before and 7 days after vaccination. X-axis represents the staining with tetramer and Y-axis represents staining with anti-CD38. Upper right quadrant shows staining of recently activated CD38+ cells, and low right quadrant shows resting CD38 cells.

Download figure to PowerPoint

Influenza-specific IFN-γ-secreting CD8 T cells upon ex vivo activation in vaccinated individuals

To determine whether the influenza-specific CD8+ T cells induced by subunit trivalent influenza vaccine are capable of producing the IFN-γ, we analysed CD8+ T cell IFN-γ secretion after influenza peptide activation in vitro. Attempts to stain influenza peptide stimulated cells with tetramers and IFN-γ in the same tube was unsuccessful because the frequency of IFN-γ producing cells was markedly reduced if the tetramer was added before the peptide in comparison to incubation with peptide only (data not shown). Limited number of cells obtained from vaccinated individuals and higher frequency of tetHA344-353+ cells prompted us to use HA344-353 peptide for PBMC stimulation. The frequencies of HA344-353-specific IFN-γ-secreting T cells expressing CD69 marker were measured before, and 7, 14 days and 28 days after vaccination in six vaccinated donors. Three donors showed 1.5- to fourfold increase in frequency of HA344-353-specific CD69+CD8+ T cells producing IFN-γ 7 days after vaccination and maintain it 28 days after vaccination (Fig. 5). Five donors reached 1.5- to twofold increase in frequency of HA344-353-specific CD69+CD8+ T cells producing IFN-γ 28 days after vaccination. There was no correlation between rise in frequency of tetHA344-353+CD38+CD8+ T cells and frequency of antigen-specific CD69+CD8+ T cells in vaccinated donors after vaccination.

image

Figure 5.  An increase of the frequency of HA344-353 tetramer+CD8+ T cells (A) and HA344-353-specific CD69+CD8+ T cells producing IFN-γ (B) in successfully influenza-vaccinated individual. Representative dot plots of (A) recently activated (CD38+, upper right quadrant) and resting (CD38, low right quadrant) HA344-353 tetramer+CD8+ T cells and (B) HA344-353-specific CD69+CD8+ T cells producing IFN-γ in peripheral blood of HLA-A*0201+ subject that showed immune response to influenza vaccine before, 7, 14, and 28 days after vaccination. Antibody titres for H1N1 influenza virus subtype are given in brackets. (C) Frequency HA344-353-specific CD69+CD8+ T cells producing IFN-γ in peripheral blood of six HLA-A*0201+-vaccinated donors before, 7, 14 and 28 days after vaccination.

Download figure to PowerPoint

Follow-up of tetramer-specific cells in immunized individuals

Previous data showed that humoral response to influenza vaccination return to baseline levels prior immunization after 1 year so annual revaccination is recommended especially for elderly population [19]. As the same vaccine was recommended for both seasons at which our study was conducted, this enabled us to investigate the necessity for repeated immunization. HI test was performed in samples from six HLA-A*0201 donors obtained before, and 7, 14, 28 days and 12 months after vaccination. Only two of six tested individuals retained post-vaccination HI titre ≥40 for A/New Caledonia/H1N1 influenza virus subtype 12 months after vaccination (Fig. 6C). In addition to the humoral response, we also determined the presence of tetramer+ cells in those individuals 12 months after vaccination. The frequency of tetramer+ cells was determined for both recently activated (CD38+) and resting cells (CD38). In all tested individuals, tetramer binding among CD38+ cells showed significant downregulation 12 months after vaccination (Fig. 6A,B).

image

Figure 6.  Follow-up of tetramer-specific cells and antibody titre in immunized individuals. Number of recently activated CD38+ T cells specific for A/New Caledonia/H1N1 antigens HA344-353 (A), and HA541-549 (B) in 50,000 CD3+CD8+ T cells in PBMC of HLA-A*0201-vaccinated donors before, and 7, 14 28 days and 12 months after vaccination. Antibody titre for A/New Caledonia/H1N1 influenza virus subtype in sera of HLA-A*0201-vaccinated donors before, 7, 14, 28 days and 12 months after vaccination (C). Friedman anova followed by post hoc tests, statistica v 6 (StatSoft, Inc., Tulsa, OK, USA) showed significant downregulation of tetramer+ cells 12 months of immunization.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects, materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

In this study, we have measured serum antibody titre and frequency of CD8+ influenza-specific T cells in immunized humans. HLA class I/peptide tetramers enabled us both detection and characterization of CD8+ T cells directed against influenza A viral antigens. PBMC from influenza-infected HLA-A*0201 and HLA-A*0201+ patients were used as a negative and positive controls respectively.

Although the currently recommended subunit trivalent influenza vaccines primarily stimulate humoral response, we were able to demonstrate a significant increase clearly in a frequency of A/New Caledonia/H1N1-like-specific CD8 cells that recognize two HA epitopes of this virus in HLA-A*0201+-vaccinated subjects. In three donors, the increase of frequency of antigen-specific T cells was not accompanied with increased H1N1 antibody titres. The underlying mechanism for these findings has yet to be investigated.

In a previous study which compared T-cell response to influenza vaccination between frail elderly subjects and healthy subjects, only M158-66-specific T cells were enumerated [23]. Here, we have analysed two dominant CD8 T-cell epitopes (HA344-353 and HA541-549), which are present in HA peptide of A/New Caledonia/H1N1-like viruses and also in the used vaccine. In most subjects, higher percentage of HA344-353-specific CD8 T cells was observed. Additional labelling of cells with CD38 enabled us to distinguish activated T cells (CD38+) that reflected recent immune activation on exposure to antigen. At present, we cannot confirm whether the observed increase represented an expansion of pre-existing HA344-353 memory T cells or de novo expansion of naïve HA344-353 T cells. It is likely that in addition to the two epitopes examined, other epitopes in particular those presented by HLA-B molecules [7] contribute to the overall pool of specific CD8 T cells which contribute to viral clearance and recovery from infection.

It was previously shown that CTL are detectable in blood after 6–14 days and reach peak level at day 14 than return to baseline 12 months after influenza vaccination [4] which is consistent with our results. We found that the frequency of two monitored populations of influenza-specific CTL (HA344-353, and HA541-549) significantly increased 7 days after vaccination.

As reported previously, specific antibody titre to A/New Caledonia/H1N1 strain significantly increased 14 and 28 days after vaccination in 16 donors which are in line with the ∼70% success rate in reaching protective antibody levels to A/New Caledonia/H1N1 in vaccinated individuals [6]. Antibody response is shown to peak 2–3 weeks after vaccination in primed individuals [24, 25] and wanes over time and it is generally twofold lower 6 months after vaccination [26]. As generation of anti- haemagglutinin antibodies by B lymphocytes is under control of CD4 cells, it is also interesting that the frequency of CD4+ influenza-specific T cells raised two- to fivefold 10 days after vaccination with trivalent-inactivated influenza vaccine [27]. We also followed immune response in six donors 1 year after vaccination and found that both antigen-specific CD8+ T cells and antibody levels returned to base line frequencies in all individuals tested. Although sustained antibody production and memory CD8 response seems to be regulated differently [23, 28], our results stress the importance of repeated immunization even in seasons when the same vaccine is recommended by WHO.

CD8+ T cells produce variable soluble factors such IFN-γ which plays an active role in immune response. Deng et al. [29] showed that majority of IFN-γ secreting CD8+ T cells following influenza vaccination express CD45R0 which corroborate with published data showing the capacity of memory T cells to exert effector function almost immediately. Although we added costimulatory antibodies anti-CD28 and anti-CD49d to maximize the detection of T cells with higher activation thresholds, we presume that ex vivo activation of PBMC was suboptimal since we used vaccine containing purified antigen. Responses to single immunodominant epitopes may be very low in some donors and are often not reflective of responses to the entire antigen [30]. It has been previously observed for viruses such as cytomegalovirus that using either peptide mix or virus lysate results in an increase in the detectable level of IFN-γ+ T cells [31].

Cellular activation to trigger cytokine production results in downregulation of the T-cell receptor [32] which required separate tetramer and intracellular staining. In contrast to tetramer technique, there are no HLA restrictions on performing intracellular cytokine analysis. In this study, vaccination with the subunit vaccine resulted in an increased IFN-γ-secreting influenza-specific T-cell response in four of six individuals tested but the average response for this group was minimal. These four volunteers showed the expected rise in antibody titre and in the frequency of HA344-353-specific CD8+ T cells. However, we did not found any correlation between frequencies of tet+CD38+CD8+ T cells and frequencies of antigen-specific CD69+CD8+ T cells after vaccination. The minimal cytokine response observed could indicate the lack of antigen processing and presentation of CD8+ T-cell restrictive antigens [33]. Therefore, antigen-specific CD8+ T cells capable of IFN-γ production may exist in vaccinated donors but may not be efficiently detected using our stimulation.

A limitation of the present study lies in the fact that the threshold of circulating antigen-specific T cells which will result in effective antiviral response following vaccination is unknown. Previous study reported that humans who demonstrated neither an antibody nor a cell mediated response had 25% risk of naturally influenza infection and approximate 15% risk of influenza infection had humans who develop either an antibody response, a cell mediated response, or both [19]. Neither of individuals included in the present study acquired influenza following vaccination. As neither of individuals included in the present study had influenza caused by New Caledonia virus, we cannot compare the levels of CTL response in these patients with the levels observed in vaccinated individuals. However, we speculate that the use of these assays may provide important insight into the quantity and quality of protective immune response to viral disease as well as to help in development of new strategies that enhance CD8 T-cell responses in existing and new antiviral vaccines [34].

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects, materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

This work was supported by Croatian Ministry of Science, Education and Sports grant (TP-01/0021-05) to A. G. We would like to thank A. Bezic-Redovnikovic, BA, for language editing.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects, materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References