Localized mucosal response to intranasal live attenuated influenza vaccine in adults

Abstract

Background: Influenza virus infection is a major public health burden worldwide. Available vaccines include the inactivated intramuscular trivalent vaccine and, more recently, an intranasal live attenuated influenza vaccine (LAIV). The measure of successful vaccination with the inactivated vaccine is a systemic rise in immunoglobulin G (IgG) level, but for the LAIV no such correlate has been established.

Methods: Seventy-nine subjects were given the LAIV FluMist. Blood was collected prior to vaccination and 3 days and 30 days after vaccination. Nasal wash was collected 3 days and 30 days after vaccination. Responses were measured systemically and in mucosal secretions for cytokines, cell activation profiles, and antibody responses.

Results: Only 9% of subjects who received LAIV seroconverted, while 33% of patients developed at least a 2-fold increase in influenza virus-specific immunoglobulin A (IgA) antibodies in nasal wash. LAIV induced a localized inflammation, as suggested by increased expression of interferon-response genes in mucosal RNA and increased granulocyte colony-stimulating factor (G-CSF) and IP-10 in nasal wash. Interestingly, patients who seroconverted had significantly lower serum levels of G-CSF before vaccination.

Conclusions: Protection by LAIV is likely provided through mucosal IgA and not by increases in systemic IgG. LAIV induces local inflammation. Seroconversion is achieved in a small fraction of subjects with a lower serum G-CSF level.

Figure 1.

Systemic immunity after receipt of live attenuated influenza vaccine (LAIV). A, Hemagglutinin inhibition (HAI) titer in serum on day 30 after LAIV vaccination relative to day 0 (before vaccination). Data are from 1 experiment with 79 subjects assayed in duplicate, using influenza A virus subtype H1N1. Continued line represents subjects with a ≥4-fold increase above baseline (seroconversion). Dashed line represents subjects with a 2-fold increase. B, Immunohistochemical analysis (IHC) to recognize serum antibodies against influenza A virus subtype H1N1. Data are the fold-increase on day 30 relative to day 0. C, Cytokine concentrations on days 0 and 3 after vaccination were analyzed by multiplex enzyme-linked immunosorbent assay (ELISA). The y-axis displays the transformed concentration of each cytokine, calculated as log₂(1 + x), where x is the ELISA-determined value. G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-monocyte colony-stimulating factor; IFN-α2, interferon α2; IFN-γ, interferon γ; IL-1b, interleukin, 1b; IL-6, interleukin 6; IL-12p70, interleukin 12p70; TNF-α, tumor necrosis factor α. D, Box plots of serum levels of G-CSF on day 0 (left), G-CSF on day 3 after vaccination (middle), and IFN-α2 on day 0 (right) between subjects who were positive (n = 9) or negative (n = 70) for seroconversion. The top and bottom of each rectangle give the 75th and 25th percentiles, respectively, while the bold line in the middle of the rectangle gives the 50th percentile. Outliers are indicated by open circles. *P < .05, by the paired Wilcoxon test.

Figure 2.

Analysis of gene expression induced by live attenuated influenza vaccine receipt in monocytes of peripheral blood, using quantitative reverse transcription polymerase chain reaction analysis of CD14⁺ monocytes on day 0 and 3 after vaccination. Fold-changes in expression are shown as the mean of the log₂ value between days 3 and 0 for 7 subjects who were negative for seroconversion (−) and 5 subjects who were positive for seroconversion (+). Error bars represent the standard error of the mean. *P < .05.

Figure 3.

Analysis of mucosal immunity induced by live attenuated influenza vaccine (LAIV) receipt. A, Nasal hemagglutinin-specific immunoglobulin A (IgA) antibody to influenza A virus subtype H1N1 are shown as fold-changes on day 30 after LAIV vaccination relative to day 3. Continued line represents the subjects with a ≥2-fold increase above the baseline value. Dashed line denotes a fold-change equal to 1 (ie, no change). B, Nasal cytokine concentrations on day 3 and 30 after vaccination were analyzed by multiplex enzyme-linked immunosorbent assay and are represented as a heat map. Each column represents 1 cytokine. The Basal column represents the concentration on day 30, and the Post column represents the concentration on day 3 after vaccination. C, Box plots of cytokine levels from nasal wash specimens on day 30 after vaccination (baseline) and day 3 after vaccination. The top and bottom of each rectangle give the 75th and 25th percentiles, respectively, while the bold line in the middle of the rectangle gives the 50th percentile. Outliers are indicated by open circles. *P < .0005, by the paired Wilcoxon test.

Figure 4.

Nasal mucosal gene signature profiles after live attenuated influenza vaccine receipt. RNA amplification and quantitative reverse transcription polymerase chain reaction of cell pellets of nasal wash specimens obtained on days 3 and 30 after vaccination were performed to analyze the expression of the interferon-regulated genes MX1, STAT1, BST2, IRF7, and RIG1. Error bars represent the standard error of the mean. *P < .05.