Innate responses induced by whole inactivated virus or subunit influenza vaccines in cultured dendritic cells correlate with immune responses in vivo

Abstract

Vaccine development involves time-consuming and expensive evaluation of candidate vaccines in animal models. As mediators of both innate and adaptive immune responses dendritic cells (DCs) are considered to be highly important for vaccine performance. Here we evaluated how far the response of DCs to a vaccine in vitro is in line with the immune response the vaccine evokes in vivo. To this end, we investigated the response of murine bone marrow-derived DCs to whole inactivated virus (WIV) and subunit (SU) influenza vaccine preparations. These vaccine preparations were chosen because they differ in the immune response they evoke in mice with WIV being superior to SU vaccine through induction of higher virus-neutralizing antibody titers and a more favorable Th1-skewed response phenotype. Stimulation of DCs with WIV, but not SU vaccine, resulted in a cytokine response that was comparable to that of DCs stimulated with live virus. Similarly, the gene expression profiles of DCs treated with WIV or live virus were similar and differed from that of SU vaccine-treated DCs. More specifically, exposure of DCs to WIV resulted in differential expression of genes in known antiviral pathways, whereas SU vaccine did not. The stronger antiviral and more Th1-related response of DCs to WIV as compared to SU vaccine correlates well with the superior immune response found in mice. These results indicate that in vitro stimulation of DCs with novel vaccine candidates combined with the assessment of multiple parameters, including gene signatures, may be a valuable tool for the selection of vaccine candidates.

Fig 1. Expression of surface activation markers on murine bone marrow-derived cDCs upon stimulation with virus or vaccine preparations.

Bone marrow cells were cultured for 9 days in the presence of GM-CSF. The resulting cDCs were then exposed to active virus (blue), WIV vaccine (green), SU vaccine (red) or medium (black) for 4, 12 or 24 hours. Unstained cells are indicated in grey. Maturation markers CD40, CD80, CD86 and MHCII were analyzed by flow cytometry. Histograms representative for two independent experiments are shown.

Fig 2. Cytokine production of cDCs upon stimulation with virus or vaccines.

Bone marrow cells were cultured for 9 days in the presence of GM-CSF. Cytokine production of these cDCs upon stimulation with active virus (black circles), WIV vaccine (inverted black triangles), SU vaccine (black squares) or medium (open diamonds) for 4, 12 or 24 hours is shown. Supernatants were analyzed for the presence of the indicated cytokines by Luminex technology. The results of two biological replicates for each condition are shown. Arrows indicate values under the detection limit.

Fig 3. Principal component analysis of stimulation of DCs with AV, WIV, SU or medium.

The numbers represent single arrays, with 2 biological replicates per condition tested. The lines have been added to link the samples that have been treated identically but have been harvested at different time points. The medium samples are indicated in black, SU in red, WIV in blue and AV in green. The first and second component explain 33% and 20% of the variation in the data, and correspond to the stimulus used in the experiment and the time after the start of stimulation.

Fig 4. Gene expression of cDCs upon stimulation with virus or vaccine preparations.

Murine cDCs were cultured and stimulated as described in the legend to Fig 1. RNA was isolated from the DCs and gene expression was measured by Affymetrix mouse 430 2.0 Genechip array. Two biological replicates per experimental condition were used. Genes were selected that were significantly (p<0.05) regulated compared to the time-matched medium control sample in at least one of the treatment groups at at least one of the sampling moments. This resulted in the inclusion of a total of 4812 genes into further analyses. A heatmap of all differentially regulated genes is shown, where upregulated genes are depicted in red and downregulated genes are depicted in green.

Fig 5. Gene Ontologies (GO) affected in murine bone marrow-derived cDCs upon stimulation with active virus or vaccine preparations.

Data sets obtained from the experiment described in the legend to Fig 3 were analyzed for significantly regulated GO using the annotation software package DAVID. The top ten GO identified for AV at t = 4h are listed in the figure. Data are presented in a heatmap. Higher intensity of the red color corresponds to a higher—log p value for the respective GO, thus to a higher probability that the respective GO was affected by the treatment.

Fig 6. Expression signatures of selected innate immune response-related genes affected by WIV- and SU vaccine.

Heatmap showing expression in cDCs stimulated with WIV- or SU vaccine for 4, 12 and 24 hours as compared to time-matched medium control cDCs. A selection of genes is shown that are known to be involved in transcription, the innate- and adaptive immune response. Upregulated genes are shown in red, whereas downregulated genes are shown in green. The asterix (*) indicates that there is a significant difference (p<0.05) in expression between WIV- and SU treated DCs (as measured in a direct Limma contrast between WIV and SU).