Longevity of B-cell and T-cell responses after live attenuated influenza vaccination in children

Kristin G-I Mohn, Geir Bredholt, Karl A Brokstad, Rishi D Pathirana, Hans J Aarstad, Camilla Tøndel, Rebecca J Cox, Kristin G-I Mohn, Geir Bredholt, Karl A Brokstad, Rishi D Pathirana, Hans J Aarstad, Camilla Tøndel, Rebecca J Cox

Abstract

Background: The live attenuated influenza vaccine (LAIV) is the preferred vaccine for children, but the mechanisms behind protective immune responses are unclear, and the duration of immunity remains to be elucidated. This study reports on the longevity of B-cell and T-cell responses elicited by the LAIV.

Methods: Thirty-eight children (3-17 years old) were administered seasonal LAIV. Blood samples were collected before vaccination with sequential sampling up to 1 year after vaccination. Humoral responses were evaluated by a hemagglutination inhibition assay, and memory B-cell responses were evaluated by an enzyme-linked immunosorbent spot assay (ELISpot). T-cell responses were evaluated by interferon γ (IFN-γ) ELISpot analysis, and intracellular cytokine staining of CD4(+) T cells for detection of IFN-γ, interleukin 2, and tumor necrosis factor α was performed using flow cytometry.

Results: LAIV induced significant increases in B-cell and T-cell responses, which were sustained at least 1 year after vaccination. Strain variations were observed, in which the B strain elicited stronger responses. IFN-γ-expressing T cell counts increased significantly, and remained higher than prevaccination levels 1 year later. Expression of T-helper type 1 intracellular cytokines (interleukin 2, IFN-γ, and tumor necrosis factor α) increased after 1 dose and were boosted after the second dose. Hemagglutination inhibition titers were sustained for 1 year. Vaccine-induced memory B cell counts were significantly increased, and the response persisted for one year.

Conclusions: LAIV elicited B-cell and T-cell responses that persisted for at least 1 year in children. This is a novel finding that will aid future vaccine policy.

Keywords: IFN-γ; LAIV; T-cellular; humoral; influenza; longevity; pediatric.

© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.

Figures

Figure 1.
Figure 1.
Study design. Healthy children scheduled for elective tonsillectomy were recruited from the Ear, Nose, and Throat Department, Haukeland University Hospital (HUH), Bergen, Norway. Thirty-eight children received live attenuated influenza vaccine (LAIV), and 29 children (all

Figure 2.

Hemagglutination inhibition (HI) antibody titers…

Figure 2.

Hemagglutination inhibition (HI) antibody titers after vaccination. Children were intranasally vaccinated with 1…

Figure 2.
Hemagglutination inhibition (HI) antibody titers after vaccination. Children were intranasally vaccinated with 1 (for those aged ≥10 years) or 2 (for those aged A) and H3N2 (B) were measured at the following time points: day 0 (before vaccination), day 28 (after the first dose), day 56 (after the second dose), and days 180 and 360 after vaccination. Each symbol represents the HI response of 1 child, with bold horizontal lines and whiskers denoting geometric mean titers and 95% confidence intervals, respectively. The dotted line represents an HI titer of 40, considered the protective level [27]. The statistical significance of differences from prevaccination levels was determined by analysis of variance, using the nonparametric Kruskal–Wallis test. **P < .01, ***P < .001, and ****P < .0001.

Figure 3.

Long-term interferon γ (IFN-γ) immune…

Figure 3.

Long-term interferon γ (IFN-γ) immune response in blood after live attenuated influenza vaccination…

Figure 3.
Long-term interferon γ (IFN-γ) immune response in blood after live attenuated influenza vaccination (LAIV). The long-term immune response was evaluated by measuring the number of IFN-γ–producing T cells, measured as spot-forming cells (SFCs)/106 peripheral blood mononuclear cells (PBMCs) after LAIV, using the IFN-γ enzyme-linked immunosorbent spot assay. Children were intranasally vaccinated with 1 (for those aged <10 years) or 2 (for those aged ≥10 years; doses were administered at a 28-day interval) doses of LAIV. Blood samples were collected at 0, 28, 56, 180, and 360 days after vaccination. Each symbol represents the influenza virus–specific SFCs/106 PBMCs for each child for each influenza strain in the vaccine (A, B, C), with bold horizontal lines and whiskers denoting mean values and standard errors of the mean, respectively. The dotted line represents 100 SFCs/106 PBMCs, considered the protective level [15]. The statistical significance of differences from prevaccination levels was determined by analysis of variance, using the nonparametric Kruskal–Wallis test. *P < .05, **P < .01, ***P < .001, and ****P < .0001.

Figure 4.

The CD4 + T cell…

Figure 4.

The CD4 + T cell cytokine (T-helper type 1 [Th1]) response before and…

Figure 4.
The CD4+ T cell cytokine (T-helper type 1 [Th1]) response before and after vaccination. Peripheral blood mononuclear cells obtained before vaccination (day 0) and 28 and 56 days after vaccination were simulated overnight with split-virus antigen from a mixture of the 3 virus strains in the vaccine (H1N1, H3N2, and B). The percentage of CD4+ T cells secreting either single (A) or multiple (B) Th1 cytokines was measured by multiparametric flow cytometry. *P < .05, by the Student t test, compared with the CD4+ T-cell response before vaccination (day 0). Abbreviations: IFN-γ, interferon γ; IL-2, interleukin 2; TNF-α, tumor necrosis factor α.

Figure 5.

Long-term memory B-cell (MBC) responses…

Figure 5.

Long-term memory B-cell (MBC) responses after live attenuated influenza vaccination (LAIV). The frequencies…

Figure 5.
Long-term memory B-cell (MBC) responses after live attenuated influenza vaccination (LAIV). The frequencies of influenza virus–specific immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM)–expressing MBCs before and after LAIV receipt. MBCs were stimulated to proliferate and differentiate into antibody-secreting cells by mitogens in vitro, and levels were subsequently measured by an enzyme-linked immunosorbent spot assay. The y-axis shows the percentage of influenza virus–specific MBCs. IgG+, IgM+, and IgA+ MBCs were measured against the 3 influenza virus strains in the vaccine. Data are represented as the percentage of antigen-specific IgG+, IgA+, and IgM+ MBCs among all IgG+, IgA+, and IgM+ MBCs, respectively. Each symbol represents 1 child. IgA+ and IgM+ MBC frequencies for day 180 were not determined because of laboratory constraints. The lines represent mean values ± standard errors of the mean. The statistical significance of differences from prevaccination levels was determined by analysis of variance, using the nonparametric Kruskal–Wallis test. *P < .05, **P < .01, ***P < .001, and ****P < .0001.
Figure 2.
Figure 2.
Hemagglutination inhibition (HI) antibody titers after vaccination. Children were intranasally vaccinated with 1 (for those aged ≥10 years) or 2 (for those aged A) and H3N2 (B) were measured at the following time points: day 0 (before vaccination), day 28 (after the first dose), day 56 (after the second dose), and days 180 and 360 after vaccination. Each symbol represents the HI response of 1 child, with bold horizontal lines and whiskers denoting geometric mean titers and 95% confidence intervals, respectively. The dotted line represents an HI titer of 40, considered the protective level [27]. The statistical significance of differences from prevaccination levels was determined by analysis of variance, using the nonparametric Kruskal–Wallis test. **P < .01, ***P < .001, and ****P < .0001.
Figure 3.
Figure 3.
Long-term interferon γ (IFN-γ) immune response in blood after live attenuated influenza vaccination (LAIV). The long-term immune response was evaluated by measuring the number of IFN-γ–producing T cells, measured as spot-forming cells (SFCs)/106 peripheral blood mononuclear cells (PBMCs) after LAIV, using the IFN-γ enzyme-linked immunosorbent spot assay. Children were intranasally vaccinated with 1 (for those aged <10 years) or 2 (for those aged ≥10 years; doses were administered at a 28-day interval) doses of LAIV. Blood samples were collected at 0, 28, 56, 180, and 360 days after vaccination. Each symbol represents the influenza virus–specific SFCs/106 PBMCs for each child for each influenza strain in the vaccine (A, B, C), with bold horizontal lines and whiskers denoting mean values and standard errors of the mean, respectively. The dotted line represents 100 SFCs/106 PBMCs, considered the protective level [15]. The statistical significance of differences from prevaccination levels was determined by analysis of variance, using the nonparametric Kruskal–Wallis test. *P < .05, **P < .01, ***P < .001, and ****P < .0001.
Figure 4.
Figure 4.
The CD4+ T cell cytokine (T-helper type 1 [Th1]) response before and after vaccination. Peripheral blood mononuclear cells obtained before vaccination (day 0) and 28 and 56 days after vaccination were simulated overnight with split-virus antigen from a mixture of the 3 virus strains in the vaccine (H1N1, H3N2, and B). The percentage of CD4+ T cells secreting either single (A) or multiple (B) Th1 cytokines was measured by multiparametric flow cytometry. *P < .05, by the Student t test, compared with the CD4+ T-cell response before vaccination (day 0). Abbreviations: IFN-γ, interferon γ; IL-2, interleukin 2; TNF-α, tumor necrosis factor α.
Figure 5.
Figure 5.
Long-term memory B-cell (MBC) responses after live attenuated influenza vaccination (LAIV). The frequencies of influenza virus–specific immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM)–expressing MBCs before and after LAIV receipt. MBCs were stimulated to proliferate and differentiate into antibody-secreting cells by mitogens in vitro, and levels were subsequently measured by an enzyme-linked immunosorbent spot assay. The y-axis shows the percentage of influenza virus–specific MBCs. IgG+, IgM+, and IgA+ MBCs were measured against the 3 influenza virus strains in the vaccine. Data are represented as the percentage of antigen-specific IgG+, IgA+, and IgM+ MBCs among all IgG+, IgA+, and IgM+ MBCs, respectively. Each symbol represents 1 child. IgA+ and IgM+ MBC frequencies for day 180 were not determined because of laboratory constraints. The lines represent mean values ± standard errors of the mean. The statistical significance of differences from prevaccination levels was determined by analysis of variance, using the nonparametric Kruskal–Wallis test. *P < .05, **P < .01, ***P < .001, and ****P < .0001.

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