The Split Virus Influenza Vaccine rapidly activates immune cells through Fcγ receptors

Abstract

Seasonal influenza vaccination is one of the most common medical procedures and yet the extent to which it activates the immune system beyond inducing antibody production is not well understood. In the United States, the most prevalent formulations of the vaccine consist of degraded or "split" viral particles distributed without any adjuvants. Based on previous reports we sought to determine whether the split influenza vaccine activates innate immune receptors-specifically Toll-like receptors. High-dimensional proteomic profiling of human whole-blood using Cytometry by Time-of-Flight (CyTOF) was used to compare signaling pathway activation and cytokine production between the split influenza vaccine and a prototypical TLR response ex vivo. This analysis revealed that the split vaccine rapidly and potently activates multiple immune cell types but yields a proteomic signature quite distinct from TLR activation. Importantly, vaccine induced activity was dependent upon the presence of human sera indicating that a serum factor was necessary for vaccine-dependent immune activation. We found this serum factor to be human antibodies specific for influenza proteins and therefore immediate immune activation by the split vaccine is immune-complex dependent. These studies demonstrate that influenza virus "splitting" inactivates any potential adjuvants endogenous to influenza, such as RNA, but in previously exposed individuals can elicit a potent immune response by facilitating the rapid formation of immune complexes.

Keywords: Fcγ receptors; Immunology; Influenza; Mass cytometry; Vaccine.

Conflict of interest statement

Conflict of interest statement:

No conflicts to declare.

Copyright © 2014 Elsevier Ltd. All rights reserved.

Figures

Figure 1

Comparison of signaling network activation induced by either SV or a TLR7/8 agonist. (A and B) Freshly isolated human whole-blood was stimulated with PBS, SV (15ug/ml), or R848 (10ug/ml) for 30mins prior to RBC lysis and fixation. Cells were then stained with isotope labeled mAbs against surface proteins and signaling proteins and prepared for 31-parameter mass cytometric analysis. Cell populations were identified as CD11c+ CD33+ HLADR+ CD14hi Monocytes, CD11c+ CD33+ HLADR+ CD16hi Monocytes, CD66+ Granulocytes, HLADR+ CD123+ pDCs, HLADR+ CD1c+ cDCs, CD3− CD7+ NK cells, CD19+ CD20+ B cells, and CD3+ T cells. See Figure S1 for detailed gating strategy. Signaling induction was calculated as the difference of arcsinh median intensity compared with PBS control. A representative experiment is shown from 4 independent mass cytometry experiments conducted on 7 adult donors.

Figure 2

Dose response dynamics of SV versus TLR7/8 stimulation. Whole-blood was stimulated at varying concentrations of either SV or R848 and monitored for S6 phosphorylation using 10-parameter flow cytometry. Cell populations were defined as CD33+ HLADR+ CD14hi Monocytes, CD33+ HLADR+ CD16hi Monocytes, CD66+ Granulocytes, CD33+ HLADR+ CD14− CD16− cDCs, CD56+ NK cells, CD20+ B cells, and CD3+ T cells. Signaling induction was calculated as the % of cells showing greater than basal S6 phosphorylation. Mean data points of replicates from 1 donor are shown.

Figure 3

Comparison of cytokine production between SV and a TLR7/8 agonist. (A) Freshly isolated human whole-blood was stimulated with PBS, 15ug/ml SV, or 5ug/ml R848 for 6hrs prior to RBC lysis and fixation. Secretion inhibitors were added for either the entirety of stimulation (SV) or after 2hrs (R848). Fixed cells were then prepared for 34-parameter mass cytometric intracellular cytokine staining (ICS) analysis. Cytokine positive monocytes and pDCs were defined as cells showing signal greater than 102 counts. Cytokine positive NK cells were defined as cells showing signal greater than the 99th percentile of unstimulated cells. SV also did not induce IFNα in pDCs when secretion inhibitors were added after 2hrs. One representative experiment is shown. (B) Quantitation of the variance in cytokine production based on 5 independent experiments conducted on 3 adult donors. Bar graphs show mean ±SD.

Figure 4

SV signaling activity is induced by influenza protein derived immune complexes. (A) Whole-blood and PBMCs prepared via Ficoll density centrifugation were stimulated with either PBS, SV, or R848 at 10ug/ml for 30mins prior to fixation and flow cytometric analysis as in Figure 2. Representative data depicting CD16hi monocytes from 3 experiments (2 donors) are shown. (B) Intact whole-blood or plasma depleted whole-blood was stimulated with either SV, SV pre-incubated with autologous plasma, SV pre-incubated with autologous purified polyclonal human IgG, or recombinant H1N1 hemagglutinin (5ug/ml) for 30mins prior to fixation and flow cytometric analysis. See methods section for detailed information on pre-incubation. Representative data from 7 experiments are shown.

Figure 5

SV activation is Fc and FcγR dependent. (A) Plasma depleted whole-blood was stimulated with PBS, SV preincubated with polyclonal F(ab′)2, or SV preincubated with intact IgG. Representative data from 2 experiments are shown. (B) PBMCs treated with blocking mABs against CD16 and CD32 or isotype control mAbs were stimulated with .75–3ug/ml of SV + IgG complexes for 30mins prior to fixation and S6 phosphorylation analysis. Representative data from 3 experiments conducted on 2 donors are shown.

Figure 6

Comparison of different vaccine formulations. (A) Whole-blood or PBMCs were stimulated with 15ug/ml SV or WIV for 6hrs prior to centrifugation and plasma or supernatant isolation. MFI fold change was calculated as in Fig. S2B. Representative data from 4 independent experiments are shown; bar graphs show mean ±SD from 3 donors. (B) Whole-blood was stimulated (.3μg/ml HA) with either split or live attenuated viral particles for 40mins and assayed for signaling pathway activation as in Figure 1. Mean arcsinh difference ±SD from 3 donors is shown.