Annual influenza epidemics result in 3–5 million cases of illness and infection.1 Children, a natural reservoir for influenza, shed more virus and for a longer period, thus driving transmission more widely in the population. This causes a significant economic burden through direct medical costs and loss of earnings due to illness.1 During the recent 2009 H1N1 pandemic, there were high hospitalization rates among children younger than 5 years of age, which is in accordance with the high hospitalization rates experienced by children due to influenza-associated illness, during seasonal epidemics.2–4 Thus, young children are a recognized “risk group” for developing severe or complicated disease after influenza infection and were identified as a priority vaccination group during the 2009 H1N1 pandemic.
During this outbreak, several pandemic influenza vaccines were used to combat the spread of infection worldwide. Approximately one-third of the licensed pandemic vaccines used an adjuvant, a common method of potentiating adaptive immune responses after vaccination.5 A number of studies compared adjuvanted and nonadjuvanted pandemic vaccines to optimize health-care strategies.6–9
A randomized, multicenter UK-based clinical trial was conducted in 2009 to assess the safety and immunogenicity of a nonadjuvanted whole virion H1N1 influenza vaccine (Celvapan; Baxter Vaccines, Vienna, Austria) compared with an AS03B-adjuvanted split virion pandemic vaccine (Pandemrix; GlaxoSmithKline Vaccines, Rixensart, Belgium). The adjuvanted vaccine, while more reactogenic, was more immunogenic and, importantly, achieved high seroconversion rates in children younger than 3 years of age.10 A follow-up study, conducted 1 year later, demonstrated that while hemagglutinin inhibition (HI) titers had waned, for the recipients of the AS03B-adjuvanted split virion vaccine, the HI titers generally remained above 1:32. In this follow-on trial, children were vaccinated with the nonadjuvanted 2010/2011 seasonal trivalent influenza vaccine (TIV) and the recipients of the original AS03B-adjuvanted split virion pandemic vaccine continued to show the highest HI titers.11
TIV in addition to inducing humoral immunity has been shown to boost influenza-specific cellular responses in a small percentage of adult recipients.13,14 Cellular responses are thought to play an important protective role during influenza infection, and more importantly T-cell responses against internal influenza antigens can provide heterosubtypic immunity.15,16 However, little is known about the influenza-specific T-cell response in children or the ability to boost such responses after TIV vaccination. This study was undertaken to evaluate the T-cell responses to internal influenza antigens in children, <3 or between 3 and 13 years of age, who had previously been vaccinated with a nonadjuvanted whole virion H1N1 influenza vaccine or an adjuvanted split virion pandemic vaccine and to assess if these responses were boosted after TIV immunization.
MATERIALS AND METHODS
Between 26 September and 11 December 2009, during the second wave of the influenza A (H1N1) pandemic in the United Kingdom, an open-label, randomized, parallel group, phase II study was conducted at 5 UK sites (Oxford, Bristol, Southampton, Exeter and London) in children aged 6 months to 12 years to compare the safety, reactogenicity and immunogenicity of 2 novel H1N1 vaccines in a 2-dose regimen, a nonadjuvanted whole virion H1N1 influenza vaccine (Celvapan) and AS03B-adjuvanted split virion H1N1 influenza vaccine (Pandemrix).7,10 Each dose of AS03B-adjuvanted split virion H1N1 influenza vaccine (0.25 mL, half the adult dose) contained 1.875 µg of hemagglutinin (HA) antigen, while each dose (0.5 mL) of the nonadjuvanted whole virion vaccine contained 7.5 µg of HA from influenza A/California/07/2009 (H1N1).
Participants in this trial were invited back in December 2010 to assess the persistence of microneutralization antibody titers against H1N1 virus and optionally to receive a single dose of the 2010–2011 TIV (Fluarix; GlaxoSmithKline) a split viron, nonadjuvanted vaccine containing 15 µg of HA from each of A/California/7/2009(H1N1), A/Perth/16/2009(H3N2) and B/Brisbane/60/2008.
Only those children who completed the 2009/2010 pandemic influenza vaccine trial (described above) at the Oxford site were eligible for inclusion in the 2010/2011 trial. The seasonal 2010–2011 TIV from GlaxoSmithKline was administered by intramuscular injection (deltoid or anterior-lateral thigh depending on age and muscle bulk). Blood samples were collected immediately before vaccination (D0) and 21 days (plus 14 or minus 7 days) (D21) after vaccination for a total of 62 children which segregate into 4 groups (Fig. 1).
Ex Vivo IFN-γ ELISpot
Ex vivo interferon-gamma enzyme-linked immunosorbent spot (IFN-γ ELISpot) assays were performed using fresh peripheral blood mononuclear cells (PBMCs) washed and resuspended in R10 [Roswell Park Memorial Institute medium 1640 with 10% fetal bovine serum, 100 IU/mL penicillin, 1 mg/mL streptomycin (all Sigma) and 2 mM L-glutamine (GIBCO/Invitrogen)]. The ex vivo IFN-γ ELISpot was carried out as previously described.17 In brief, up to 3 mL of heparinized blood was layered over 3 mL of lymphoprep (Axis-Shield) in a 12-mL leucosep tube (Greiner) with PBMCs isolated by centrifugation at 1000g for 15 minutes before washing twice with R0 medium (R10 without fetal bovine serum) and final resuspension in R10. Fifteen-mer to 20-mer peptides, overlapping by 10 amino acid residues, spanning the whole of pandemic 2009 H1N1 nucleprotein (NP), matrix protein 1 (M1) and nonstructural protein 1 (NS1) proteins, were used to stimulate PBMCs at a final concentration of 10 µg/mL. These proteins were chosen as it has previously been demonstrated that the majority of adult memory influenza-specific T-cell responses are predominantly directed toward NP and M1,18 and these proteins along with NS1 are currently being evaluated as targets for universal influenza vaccines.17,19 The peptides were split into 4 pools of 20–26 peptides; pool 1 and 2 contained peptides from the NP sequence, pool 3 contained peptides from the M1 sequence and pool 4 contained peptides from the NS1 sequence. Fifty microliters of PBMCs (2 × 105 cells) and 50 µL of the peptides or positive control were added to duplicate wells on IPVH membranes coated with 10 µg/mL antihuman IFN-γ (GZ4). R10 was used as a negative control and phytohemagglutinin (PHA) at a final concentration of 10 µg/mL, and PHA and staphylococcal enterotoxin B (SEB) 5 µg/mL were used as positive controls. After an 18- to 20-hour incubation at 37°C, IFN-γ spot-forming cells were detected by staining membranes with 1 µg/mL antihuman IFN-γ (7-B6-1) followed by streptavidin-alkaline phosphatase (1 µg/mL) and development with ELISpot development buffer 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium chloride (Plus) Solution (Europa Bioproducts Ltd). IFN-γ spot-forming cells were counted with an AID ELISpot reader (AID Diagnostika) and the results are expressed as spot-forming units per million PBMCs calculated by subtracting the mean R10-negative control response from the mean peptide pool response. Plates were excluded if a response under 1000 spot-forming unit was read in the PHA wells.
Intracellular Cytokine Staining
Thawed PBMCs were restimulated for a total 18 hours in 96-well U-bottom plates (total volume of 200 µL) with 1 µg/mL antihuman CD49d (9F10), 1 µg/mL CD28 (CD28.2) and 10 µL of antihuman CD107a-PECy5 (eBIOH4A3) in addition to 4 µg/mL single pool of all NP+M1+NS1 peptides or dimethyl sulfoxide (unstimulated control). For the final 16 hours of stimulation, 1 µg/mL Golgi-Plug, containing Brefeldin A (BD), and 0.8 µg/mL Golgi-Stop, containing Monensin (BD), were added together. Cells were surface stained for CD4 eFluro605 (OKT4), CD14 eFluro 450 (61D3), CD20 eFluro 450 (2H7) and Live-Dead Aqua (Invitrogen). Cells were fixed with 100 µL of Fix/Perm Solution (BD) on ice for 10 minutes before staining intracellularly for tumor necrosis factor-α fluorescein isothiocyanate (MAb11), IL-2 PE (MQ1-17H12), IFN-γ PECy7 (4S.B3), CD3 Alexa700 (SP34-2) and CD8-APC Alexa780 (RPA-T8) all diluted in Perm-Wash (BD).
Over 300,000 events were acquired on a Becton Dickinson LSRII flow cytometer using FACSDiva software (BD Biosciences) and analyzed using Flow Jo, Version 7.6 (Tree Star Inc). Unstained cells and single-stained antihuman compensation beads (BD Biosciences) were used as controls to automatically calculate compensation.
Participants with no detectable antigen-specific response on ELISpot assay had an arbitrary value of 1 assigned. ELISpot data were log10 transformed and geometric mean and 2-sided 95% confidence intervals (CIs) calculated. Regression analysis was carried out to assess whether vaccine group (adjuvanted or nonadjuvanted) or age at vaccination, in the original pandemic influenza vaccine study, influenced T-cell antigen-specific responses 1 year postvaccination (ie, D0 antigen response). The same approach was used to assess the antigen-specific response on D21 after TIV immunization while also adjusting for antigen-specific response at D0. Pearson partial correlation was used to analyze the correlation between the change in T-cell and B-cell response at D21 from D0, adjusting for age and vaccine group.
Of the 894 participants in the original study,10 323 took part in this follow-on study, 99 of whom were enrolled in Oxford. T-cell responses were assessed on a convenience sample of 62 children. Blood samples were obtained at D0 and D21 (Fig. 1A, time line). Of these 62 children, 14 were in group 1A; 12 were in group 2A; 15 were in group 1B and 21 were in group 2B (group details: Fig. 1B).
T-Cell Responses, in Children, to NP, M1 and NS1 1 Year After Pandemic Influenza Vaccination
All volunteers had detectable IFN-γ ELISpot responses to NP, M1 and NS1, on D0, except for 1 volunteer in group 1A and 1 in group 1B for whom responses were arbitrarily set to 1. The geometric mean summed antigen-specific response to NP, M1 and NS1 for group 1A on day 0 was 89 (95% CI 39–201); group 1B day 0: geometric mean 144 (95% CI 49–423); group 2A day 0: geometric mean 198 (95% CI 128–307); group 2B day 0: geometric mean 459 (95% CI 346–609) (Fig. 2A).
A scatter plot of D0 T-cell antigen-specific response plotted against age is shown in Figure 2B. Those who received AS03- adjuvanted vaccine during the pandemic influenza vaccination study had a significantly higher T-cell antigen-specific response 1 year postvaccination (P = 0.007). There was also a significantly higher T-cell antigen-specific response at D0 in those who were older in the original study (P < 0.0001), but no evidence of interaction effect between vaccine group and age was detected.
T-Cell Responses, to NP, M1 and NS1 After Seasonal TIV Vaccination, in Children Previously Vaccinated With Pandemic Influenza Vaccine
Summed T-cell ELISpot responses to NP, M1 and NS1, 21 days after vaccination with 2010/2011 seasonal TIV were as follows [group 1A day 21:, geometric mean 299 (95% CI 194–461); group 1B day 21: geometric mean 452 (95% CI 272–751); group 2A day 21: geometric mean 395 (95% CI 274–568); group 2B day 21: geometric mean 747 (95% CI 579–963)] (Fig. 3A).
Results from the multiple regression analysis suggested that vaccination with the adjuvanted vaccine produced a moderately higher post-TIV T-cell response (by 35% 95% CI: 0.5–80%; P = 0.05), while evidence of a significant effect of age was not observed (P = 0.69) (Fig. 3B).
Seasonal TIV Boosts T-Cell Responses, in Children, to NP, M1 and NS1
The strongest ELISpot response was observed in response to NP peptides, regardless of group; however, the highest fold increase in geometric mean between D0 and D21 was observed in M1 for groups 1A and 1B, NP in group 2B and NS1 in group 2A (Fig. 4).
Ex vivo IFN-γ intracellular cytokine staining analysis was carried out to determine whether the IFN-γ detected in the ex vivo ELISpot was produced by CD31CD41 or CD31CD81 T cells. Intracellular cytokine staining was carried out on cryopreserved PBMCs from volunteers who had high ELISpot results and high numbers of cyropreserved cells. The mean percentage of CD31 T cells producing IFN-γ after antigen stimulation was 0.08% (95% CI 0.01–0.15). In the majority of cases, the percentage of antigen-specific IFN-γ1 CD41 cells (88% of IFN-γ1 T cells; 95% CI 79–97) was higher than the corresponding population of CD81 T cells.
Correlation Between T-Cell and B-Cell Responses
After adjusting for age and vaccine group, there was no evidence of a correlation between the change in the T-cell response (antigen-specific T-cell ELISpot) and the change in B-cell induced responses (HI and microneutralization titers) at D21 from D0 [partial correlation (95% CI) = −0.15 (−0.39, 0.10), P = 0.24] (Fig. 5).
In this study, we evaluated the T-cell response after TIV vaccination in pediatric recipients of the 2009 pandemic H1N1 vaccine. We demonstrated that, after 2010/2011 seasonal TIV vaccination, T-cell responses to internal influenza antigens were boosted, regardless of age or prior pandemic vaccination regime. Although antigen-specific T-cell IFN-γ responses 1 year after pandemic H1N1 vaccination were higher in the older age groups and in those receiving the adjuvanted split virion vaccine, fold increases after the TIV immunization were higher in those receiving the nonadjuvanted whole virion vaccine (albeit from a lower baseline). Indeed, when the change in response between D0 and D21 is adjusted for baseline response (D0) and for age, the boosting effect of TIV immunization is comparable across both treatment groups. The lower basal ELISpot responses and higher fold change, in young recipients of the nonadjuvanted whole virion pandemic vaccine, after TIV vaccination is probably reflective of the lower immunogenicity of the nonadjuvanted whole virion vaccine when compared with the adjuvanted vaccine and the limited number of exposures, for these young children, to seasonal influenza.
One of the key findings from this clinical study was the higher D0 T-cell responses to internal influenza antigens, when adjusted for age, in children who received an AS03B-adjuvanted split virion pandemic vaccine when compared with children vaccinated with nonadjuvanted whole virion pandemic vaccine. While the differences between the immunogenicity of the 2 pandemic vaccines may be partly attributed to vaccine type (ie, split virion versus whole virus), it is likely that the enhanced immunogenicity of the adjuvanted vaccine is largely due to the presence of the adjuvant: AS03B. The AS03B-adjuvanted pandemic influenza vaccine has previously been shown to be associated with increased humoral responses in children,7,10 and it is probable that the stronger T-cell response in recipients of the AS03B-pandemic vaccine reflects a generalized adjuvant-mediated augmentation of the adaptive immune response. Indeed, several oil-in-water adjuvants, including AS03B and MF59, have been shown to significantly enhance immunogenicity, in healthy adults and children, to inactivated influenza A/H5N1; both AS03B and MF59 were approved for clinical use to enhance immunogenicity of the recent pandemic H1N1 vaccines.20–22
Given the complex interplay of the various aspects of the immune system, it is not surprising that there was no evidence of a correlation between the change in the IFN-γ1 T-cell response and the change in B-cell response at D21 from D0. The induction of either the humoral or cellular immune response can confer protective immune responses toward influenza.15,23,24 However, the induction of both arms of the adaptive immune response inducing an efficacious and cross-clade heterosubtypic response is ideal.
Heterosubtypic protection toward different or drifted influenza isolates is principally thought to be generated through cross-reactive memory T cells and anti-HA and NA stalk-specific antibodies.15,16,25–27 Numerous preclinical and clinical studies have demonstrated that cross-reactive T-cell responses directed toward internal influenza proteins can be protective, and most importantly, cross-clade protection is achieved.15,16,26,28–30 The majority of adult memory T-cell responses are predominantly directed toward NP and M1,18 and these proteins are currently being evaluated as antigenic targets for universal influenza vaccines.17 Our findings demonstrate that TIV can induce a small but significant boost in T-cell reactivity toward internal influenza antigens, in the majority of children, which is in contrast to adult studies where boosting was detected in only 10–12% of participants after TIV administration.13,14 In this study, we demonstrate that the predominant pediatric T-cell IFN-γ response to internal influenza antigens, after vaccination, was CD4+ T-cell derived which is in contrast to the predominantly CD81 T-cell response detected in adults resulting from natural exposure to influenza.17 An age-dependent increase of virus-specific CD81 T-cell responses has been observed in unvaccinated healthy children,31 and further studies will be required to delineate the role of the vaccination-induced IFN-γ1 CD41 T cells observed in this study after pediatric vaccination.
We thank the volunteers and clinical teams involved in the trials and Katherine Collins for discussions. The recruitment to this study was supported by the NIHR Oxford Comprehensive Biomedical Research Centre programme (including salary support for MDS), and the Thames Valley Comprehensive Local Research Network. T.L. and A.J.S. are James Martin fellows. A.J.P. and A.V.S.H. are Jenner Institute Investigators and James Martin Senior Fellows. S.C.G. is a Jenner Institute Investigator.
1. Molinari NA, Ortega-Sanchez IR, Messonnier ML, et al. The annual impact of seasonal influenza in the US: measuring disease burden and costs. Vaccine. 2007;25:5086–5096
2. Bautista E, Chotpitayasunondh T, Gao Z, et al. Clinical aspects of pandemic 2009 influenza A (H1N1) virus infection. N Engl J Med. 2010;362:1708–1719
3. Nguyen-Van-Tam JS, Openshaw PJ, Hashim A, et al. Risk factors for hospitalisation and poor outcome with pandemic A/H1N1 influenza: United Kingdom first wave (May-September 2009). Thorax. 2010;65:645–651
4. Poehling KA, Edwards KM, Weinberg GA, et al. The underrecognized burden of influenza in young children. N Engl J Med. 2006;355:31–40
5. Scheifele DW, Ward BJ, Dionne M, et al. Evaluation of adjuvanted pandemic H1N1(2009) influenza vaccine after one and two doses in young children. Pediatr Infect Dis J. 2011;30:402–407
6. Nicholson KG, Abrams KR, Batham S, et al. A randomised, partially observer blind, multicentre, head-to-head comparison of a two-dose regimen of Baxter and GlaxoSmithKline H1N1 pandemic vaccines, administered 21 days apart. Health Technol Assess. 2010;14:193–334
7. Waddington C, Andrews N, Hoschler K, et al. Open-label, randomised, parallel-group, multicentre study to evaluate the safety, tolerability and immunogenicity of an AS03(B)/oil-in-water emulsion-adjuvanted (AS03(B)) split-virion versus non-adjuvanted whole-virion H1N1 influenza vaccine in UK children 6 months to 12 years of age. Health Technol Assess. 2010;14:1–130
8. Roman F, Vaman T, Gerlach B, et al. Immunogenicity and safety in adults of one dose of influenza A H1N1v 2009 vaccine formulated with and without AS03A-adjuvant: preliminary report of an observer-blind, randomised trial. Vaccine. 2010;28:1740–1745
9. Cheong HJ, Song JY, Heo JY, et al. Immunogenicity and safety of the influenza A/H1N1 2009 inactivated split-virus vaccine in young and older adults: MF59-adjuvanted vaccine versus nonadjuvanted vaccine. Clin Vaccine Immunol. 2011;18:1358–1364
10. Waddington CS, Walker WT, Oeser C, et al. Safety and immunogenicity of AS03B adjuvanted split virion versus non-adjuvanted whole virion H1N1 influenza vaccine in UK children aged 6 months-12 years: open label, randomised, parallel group, multicentre study. BMJ. 2010;340:c2649
11. Walker WT, de Whalley P, Andrews N, et al. H1N1 antibody persistence 1 year after immunization with an adjuvanted or whole-virion pandemic vaccine and immunogenicity and reactogenicity of subsequent seasonal influenza vaccine: a multicenter follow-on study. Clin Infect Dis. 2012;54:661–669
12. Terajima M, Cruz J, Leporati AM, 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
13. Rastogi D, Wang C, Mao X, et al. Antigen-specific immune responses to influenza vaccine in utero. J Clin Invest. 2007;117:1637–1646
14. McMichael AJ, Gotch FM, Noble GR, et al. Cytotoxic T-cell immunity to influenza. N Engl J Med. 1983;309:13–17
15. Benton KA, Misplon JA, Lo CY, et al. Heterosubtypic immunity to influenza A virus in mice lacking IgA, all Ig, NKT cells, or gamma delta T cells. J Immunol. 2001;166:7437–7445
16. Berthoud TK, Hamill M, Lillie PJ, et al. Potent CD8+ T-cell immunogenicity in humans of a novel heterosubtypic influenza A vaccine, MVA-NP+M1. CID. 2011;52:1–7
17. Lee LY, Ha do LA, Simmons C, et al. Memory T cells established by seasonal human influenza A infection cross-react with avian influenza A (H5N1) in healthy individuals. J Clin Invest. 2008;118:3478–3490
18. Goodman AG, Heinen PP, Guerra S, et al. A human multi-epitope recombinant vaccinia virus as a universal T cell vaccine candidate against influenza virus. PLoS ONE. 2011;6:e25938
19. Atmar RL, Keitel WA. Adjuvants for pandemic influenza vaccines. Curr Top Microbiol Immunol. 2009;333:323–344
20. Khurana S, Verma N, Yewdell JW, et al. MF59 adjuvant enhances diversity and affinity of antibody-mediated immune response to pandemic influenza vaccines. Sci Transl Med. 2011;3:85ra48
21. Durando P, Iudici R, Alicino C, et al. Adjuvants and alternative routes of administration towards the development of the ideal influenza vaccine. Human Vaccines. 2011;7(Suppl):29–40
22. Jefferson T, Rivetti A, Harnden A, Di Pietrantonj C, Demicheli V. Vaccines for preventing influenza in healthy children. Cochrane Database Syst Rev. 2008 CD004879
23. Pulendran B, Ahmed R. Immunological mechanisms of vaccination. Nat Immunol. 2011;12:509–517
24. Wrammert J, Koutsonanos D, Li GM, et al. Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection. J Exp Med. 2011;208:181–193
25. Epstein SL, Kong WP, Misplon JA, et al. Protection against multiple influenza A subtypes by vaccination with highly conserved nucleoprotein. Vaccine. 2005;23:5404–5410
26. McElhaney JE, Xie D, Hager WD, et al. T cell responses are better correlates of vaccine protection in the elderly. J Immunol. 2006;176:6333–6339
27. Donnelly JJ, Friedman A, Ulmer JB, et al. Further protection against antigenic drift of influenza virus in a ferret model by DNA vaccination. Vaccine. 1997;15:865–868
28. Laddy DJ, Yan J, Khan AS, et al. Electroporation of synthetic DNA antigens offers protection in nonhuman primates challenged with highly pathogenic avian influenza virus. J Virol. 2009;83:4624–4630
29. Breathnach CC, Clark HJ, Clark RC, et al. Immunization with recombinant modified vaccinia Ankara (rMVA) constructs encoding the HA or NP gene protects ponies from equine influenza virus challenge. Vaccine. 2006;24:1180–1190
30. Remarque EJ, Nijhuis EW, Hinloopen B, et al. Correlation between the antibody response to influenza vaccine and helper T cell subsets in healthy aging. Vaccine. 1996;14:127–130
pediatric; trivalent influenza vaccine; T-cell responses; pandemic H1N1 vaccine