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. 2008 Sep 15;26(39):5004-13.
doi: 10.1016/j.vaccine.2008.07.050. Epub 2008 Aug 6.

Gene profiling analysis of ALVAC infected human monocyte derived dendritic cells

Anke Harenberg  1 , Florine Guillaume, Elizabeth J Ryan, Nicolas Burdin, Franca Spada
Affiliations

Gene profiling analysis of ALVAC infected human monocyte derived dendritic cells

Anke Harenberg et al. Vaccine. .

Abstract

The recombinant canarypox virus ALVAC is being extensively studied as vaccine vector for the development of new vaccine strategies against chronic infectious diseases and cancer. However, the mechanisms by which ALVAC initiates the immune response have not been completely elucidated. In order to determine the type of innate immunity triggered by ALVAC, we characterized the gene expression profile of human monocyte derived dendritic cells (MDDCs) upon ALVAC infection. These cells are permissive to poxvirus infection and play a key role in the initiation of immune responses. The majority of the genes that were up-regulated by ALVAC belong to the type I interferon signaling pathway including IRF7, STAT1, RIG-1, and MDA-5. Genes involved in the NF-kappaB pathway were not up-regulated. The gene encoding for the chemokine CXCL10, a direct target of the transcription factor IRF3 was among those up-regulated and DC secretion of CXCL10 following exposure to ALVAC was confirmed by ELISA. Many downstream type I interferon activated genes with anti-viral activity (PKR, Mx, ISG15 and OAS among others) were also up-regulated in response to ALVAC. Among these, ISG15 expression in its unconjugated form by Western blot analysis was demonstrated. In view of these results we propose that ALVAC induces type I interferon anti-viral innate immunity via a cytosolic pattern-recognition-receptor (PRR) sensing double-stranded DNA, through activation of IRF3 and IRF7. These findings may aid in the design of more effective ALVAC-vectored vaccines.

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Figures

Fig. 1
Fig. 1
Maturation of human immature MDDCs upon exposure to ALVAC. (A) Human immature MDDCs (CD11c+ and CD14−), obtained from CD14 purified monocytes upon 6-day culture with GM-CSF and IL-4, were stimulated for 24 h with ALVAC (0.2 MOI), LPS (10 ng/ml) or left untreated (indicated by color code). (B) Plots show super-imposition of the surface expression of each maturation marker relative to the Ab isotype control under the three experimental conditions (ALVAC, LPS or medium). ALVAC infection increased the expression of DC maturation-associated markers CD40, CD80, CD86, CD83, HLA-1 (MHC-I), HLA-DR (MHC-II) on the surface of human MDDC. However, in contrast to LPS, ALVAC failed to up-regulate the CD25 marker.
Fig. 2
Fig. 2
Venn diagram showing the overlap between 1433 differentially up-regulated (A) and 1044 differentially down-regulated genes (B) in ALVAC infected MDDCs from four independent healthy donors. Each of the four circles represents the set of genes differentially expressed in each donor (fold increase >±1.5, p < 0.05). Numbers depicted in the intersections between the circles represent numbers of genes differentially up-regulated in two, three or four donors. (C) Ontology analysis of the genes differently regulated in response to ALVAC relative to untreated cells. In the bar chart, the Y-axis represents the significance threshold of a given gene involved in a particular function.
Fig. 3
Fig. 3
Proposed model of ALVAC induction of the type I IFN pathway in MDDCs (left side) and representation of type I IFN responsive gene expression levels from microarray analysis (right side). (Left side) We propose that ALVAC is detected by a cytoplasmic dsDNA sensor which leads to the phosphorylation of IRF3 via TBK1. Phosphorylated IRF3 homodimerizes or heterodimerizes with IRF7 and migrates to the nucleus where it directly induces the expression of IFN-α/β, CXCL10 and ISG15 among others. In a positive feedback loop type I IFNs induce gene expression of IRF7, MDA-5, RIG-1, etc. (Right side) Two-dimensional hierarchical clustering was performed using Rosetta Resolver System Software. Columns represent gene expression data for four distinct donors from an individual experiment. The intensity of the color red indicates the degree of up-regulation, genes which did not meet the required p-value of 0.05 are colored in grey.
Fig. 4
Fig. 4
Enhanced IFNβ gene transcription in ALVAC infected human MDDCs. MDDCs were incubated as indicated for 2, 6 or 16 h (Donor 1) or 16 h (Donor 5) with either medium alone or ALVAC at a MOI of 0.2 or Poly I:C at 10 μg/ml as a positive control as indicated. Amplification of the IFNβ and β-actin encoding genes was performed by RT-PCR as described in Section 2. A band of the expected size of 185 nt was detected in MDDCs upon ALVAC infection and Poly I:C stimulation. Under the current experimental conditions IFNβ gene transcription in MDDCs was evident after 6 h exposure to ALVAC (right figure, Donor 1).
Fig. 5
Fig. 5
Secretion of CXCL10 by ALVAC infected human MDDCs. MDDCs were incubated for 48 h in the presence or absence of ALVAC, recombinant IFN-α, neutralizing IFN antibody or control antibody as indicated in the figure. Supernatants were collected and levels of CXCL10 were quantified by ELISA. Results are expressed as mean concentration ± standard error and are representative of three independent experiments. Infection of MDDCs with the viral vector ALVAC induced secretion of CXCL10 that was partially inhibited by anti-type I IFN receptor antibody. As expected the non-neutralizing anti-type I IFN receptor antibody used as negative control had no effect on CXCL10 release.
Fig. 6
Fig. 6
Protein expression of ISG15 in MDDCs upon ALVAC exposure. MDDCs were stimulated with 0.2 MOI ALVAC or 1000 U/ml human leukocyte interferon (IFN-α and IFN-ω) for 48 h, harvested, lysed and subjected to SDS-PAGE. Expression of ISG15 was analyzed using anti-ISG antibody. Equal loading was confirmed by re-probing blots with an antibody against GAPDH. One representative result of three experiments is shown.
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