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. 2015 Apr;89(7):3819-32.
doi: 10.1128/JVI.03061-14. Epub 2015 Jan 21.

Interleukin-1- and type I interferon-dependent enhanced immunogenicity of an NYVAC-HIV-1 Env-Gag-Pol-Nef vaccine vector with dual deletions of type I and type II interferon-binding proteins

Julie Delaloye  1 , Abdelali Filali-Mouhim  2 , Mark J Cameron  2 , Elias K Haddad  2 , Alexandre Harari  3 , Jean-Pierre Goulet  2 , Carmen E Gomez  4 , Beatriz Perdiguero  4 , Mariano Esteban  4 , Giuseppe Pantaleo  3 , Thierry Roger  1 , Rafick-Pierre Sékaly  2 , Thierry Calandra  5
Affiliations

Interleukin-1- and type I interferon-dependent enhanced immunogenicity of an NYVAC-HIV-1 Env-Gag-Pol-Nef vaccine vector with dual deletions of type I and type II interferon-binding proteins

Julie Delaloye et al. J Virol. 2015 Apr.

Abstract

NYVAC, a highly attenuated, replication-restricted poxvirus, is a safe and immunogenic vaccine vector. Deletion of immune evasion genes from the poxvirus genome is an attractive strategy for improving the immunogenic properties of poxviruses. Using systems biology approaches, we describe herein the enhanced immunological profile of NYVAC vectors expressing the HIV-1 clade C env, gag, pol, and nef genes (NYVAC-C) with single or double deletions of genes encoding type I (ΔB19R) or type II (ΔB8R) interferon (IFN)-binding proteins. Transcriptomic analyses of human monocytes infected with NYVAC-C, NYVAC-C with the B19R deletion (NYVAC-C-ΔB19R), or NYVAC-C with B8R and B19R deletions (NYVAC-C-ΔB8RB19R) revealed a concerted upregulation of innate immune pathways (IFN-stimulated genes [ISGs]) of increasing magnitude with NYVAC-C-ΔB19R and NYVAC-C-ΔB8RB19R than with NYVAC-C. Deletion of B8R and B19R resulted in an enhanced activation of IRF3, IRF7, and STAT1 and the robust production of type I IFNs and of ISGs, whose expression was inhibited by anti-type I IFN antibodies. Interestingly, NYVAC-C-ΔB8RB19R induced the production of much higher levels of proinflammatory cytokines (tumor necrosis factor [TNF], interleukin-6 [IL-6], and IL-8) than NYVAC-C or NYVAC-C-ΔB19R as well as a strong inflammasome response (caspase-1 and IL-1β) in infected monocytes. Top network analyses showed that this broad response mediated by the deletion of B8R and B19R was organized around two upregulated gene expression nodes (TNF and IRF7). Consistent with these findings, monocytes infected with NYVAC-C-ΔB8RB19R induced a stronger type I IFN-dependent and IL-1-dependent allogeneic CD4(+) T cell response than monocytes infected with NYVAC-C or NYVAC-C-ΔB19R. Dual deletion of type I and type II IFN immune evasion genes in NYVAC markedly enhanced its immunogenic properties via its induction of the increased expression of type I IFNs and IL-1β and make it an attractive candidate HIV vaccine vector.

Importance: NYVAC is a replication-deficient poxvirus developed as a vaccine vector against HIV. NYVAC expresses several genes known to impair the host immune defenses by interfering with innate immune receptors, cytokines, or interferons. Given the crucial role played by interferons against viruses, we postulated that targeting the type I and type II decoy receptors used by poxvirus to subvert the host innate immune response would be an attractive approach to improve the immunogenicity of NYVAC vectors. Using systems biology approaches, we report that deletion of type I and type II IFN immune evasion genes in NYVAC poxvirus resulted in the robust expression of type I IFNs and interferon-stimulated genes (ISGs), a strong activation of the inflammasome, and upregulated expression of IL-1β and proinflammatory cytokines. Dual deletion of type I and type II IFN immune evasion genes in NYVAC poxvirus improves its immunogenic profile and makes it an attractive candidate HIV vaccine vector.

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Figures

FIG 1
FIG 1
Venn diagrams and scatter plots of fold changes in expression of genes differentially induced by NYVAC-C, NYVAC-C-ΔB19R, and NYVAC-C-ΔB8RB19R in human monocytes. Venn diagrams (A, C, and E) depicting the number of genes differentially induced (fold change, >1.3; adjusted P value, <0.1) and scatter plots (B, D, and F) depicting fold changes in gene expression in human primary monocytes infected for 6 h with NYVAC-C, NYVAC-C-ΔB19R, or NYVAC-C-ΔB8RB19R (MOI, 5) relative to the levels of gene expression in control cells consisting of either mock-infected monocytes or monocytes infected with NYVAC-C. (A and B) Gene expression in NYVAC-C- versus NYVAC-C-ΔB19R-infected cells (MOI, 5) relative to that in mock-infected cell cultures; (C and D) gene expression in NYVAC-C-ΔB19R- versus NYVAC-C-ΔB8RB19R-infected cells (MOI, 5) relative to that in mock-infected cell cultures; (E and F) gene expression in NYVAC-C-ΔB19R- versus NYVAC-C-ΔB8RB19R-infected cells (MOI, 5) relative to that in NYVAC-C-infected cells. In the fold change scatter plots, the genes falling on the identity line (dark gray line) exhibited similar levels of expression in both systems. A loess curve (dashed blue line) was used to infer the local trend between the two systems. IFN-dependent genes differentially expressed in at least one comparison are highlighted in red. Solid lines indicate absolute 2-fold changes. The x and y axes in the scatter plots show the log2(FC) in the level of gene expression after 6 h of stimulation with NYVAC-C, NYVAC-C-ΔB19R, or NYVAC-C-ΔB8RB19R.
FIG 2
FIG 2
Gene expression values from the Illumina BeadArrays plotted against the OpenArray real-time PCR −ΔCT values for each target gene. Scatter plots comparing the fold change in gene expression between the Illumina microarrays and OpenArray real-time PCR (OA) in human primary monocytes infected for 6 h with NYVAC-C (A), NYVAC-C-ΔB19R (B), and NYVAC-C-ΔB8RB19R (C) relative to the levels of expression in mock-infected cells are shown. PCR targets were mapped to the BeadArray probes by matching the official gene symbols. The correlations indicated are the sample Pearson correlation coefficients.
FIG 3
FIG 3
Comparison of the patterns of pathway enrichment in monocytes infected with NYVAC-C-ΔB8RB19R and monocytes infected with NYVAC-C-ΔB19R. The pathways and genes induced in human primary monocytes infected for 6 h with NYVAC-C-ΔB8RB19R relative to those induced in human primary monocytes infected with NYVAC-C-ΔB19R are shown. Each row is an upregulated canonical innate immune pathway (Ingenuity software); each column represents an upregulated (red) or downregulated (blue) gene involved in one or more regulated pathways. The overrepresentation test was performed using Fisher's exact test, and the statistical significance, displayed on the right y axis, was achieved for P values of <0.05 [−log(P) > 1.3]. TNFR2, TNF receptor 2; DCs, dendritic cells; Com., communication; Inn., innate; Adap., adaptive; cAMP, cyclic AMP; G-alphaq signaling, G protein-alphaq signaling.
FIG 4
FIG 4
Heat map of IFN-filtered genes differentially expressed in human monocytes infected with NYVAC-C, NYVAC-C-ΔB19R, or NYVAC-C-ΔB8RB19R. Human primary monocytes were infected for 6 h with NYVAC-C, NYVAC-C-ΔB19R, or NYVAC-C-ΔB8RB19R (MOI, 5). The heat map depicts differential ISG expression, reported as the average fold change in gene expression relative to the levels of expression in mock-infected monocytes. The genes were immune filtered using three lists of IFN genes: IFN-α genes (A), IFN-γ genes (B), and genes common to both IFN-α and IFN-γ (C). The genes selected are the top genes significantly expressed in at least one contrast following an analysis of variance F test and selected to be differentially expressed on a fold change basis of up- or downregulation of 1.3-fold and an adjusted P value of <0.1. The color legend indicates fold changes over the levels of expression in mock-infected cells, and the fold changes are expressed on a log2 scale, where red and blue correspond to up- and downregulation, respectively.
FIG 5
FIG 5
Gene interaction networks differentially induced with NYVAC-C-ΔB19R and NYVAC-C-ΔB8RB19R in human monocytes. Human primary monocytes were infected for 6 h with NYVAC-C, NYVAC-C-ΔB19R, or NYVAC-C-ΔB8RB19R (MOI, 5). Gene interaction networks were built on the basis of genes differentially induced by NYVAC-C-ΔB19R (A, B) and NYVAC-C-ΔB8RB19R (C) relative to their levels of expression in cells infected with NYVAC-C or differentially induced by NYVAC-C-ΔB8RB19R relative to their levels of expression in cells infected with NYVAC-C-ΔB19R (D, E) by using the Ingenuity Pathways Knowledge Base (IPKB). Genes are highlighted in red (upregulation) or green (downregulation), and node properties are indicated by shape, as indicated in the legends. Interactions between the different nodes are given as solid (direct interaction) and dashed (indirect interaction) lines (edges), with various colors used for the different interaction types. The networks are the results of merging of the top two networks that received a high score by IPA.
FIG 6
FIG 6
Type I interferons, cytokines, and chemokines released by human monocytes infected with NYVAC-C, NYVAC-C-ΔB19R, and NYVAC-C-ΔB8RB19R. Monocytes from healthy volunteers were infected for 24 h with NYVAC-C, NYVAC-ΔB19R, or NYVAC-CΔB8RB19R, and the concentrations of IFN-α, IFN-β, CXCL10/IP-10, TNF, IL-1β, and IL-6 were measured in cell culture supernatants. Box plots of three separate experiments including a total of eight subjects are shown. Data points are means for duplicate or triplicate samples per subject. Bottom, median, and top lines, 25th, 50th, and 75th percentiles, respectively; vertical lines with whiskers, the range of values.
FIG 7
FIG 7
NYVAC-C-ΔB19R and NYVAC-C-ΔB8RB19R activate IRF3, IRF7, and STAT1 transcription factors. Cytosolic extracts were obtained from THP-1 cells infected for 6 h with NYVAC-C, NYVAC-C-ΔB19R, or NYVAC-C-ΔB8RB19R (MOI, 5). The expression of P-IRF3, IRF7, P-STAT1, STAT1, and tubulin was analyzed by Western blotting. Results are representative of those from three independent experiments.
FIG 8
FIG 8
Expression of chemokines and transcription factors induced by NYVAC-C-ΔB8RB19R in human monocytic cells is type I IFN dependent. Human THP-1 monocytic cells were preincubated for 1 h with control or anti-IFN-α/β antibody (10 μg/ml) and then stimulated for 3 h with NYVAC-C, NYVAC-C-ΔB19R, and NYVAC-C-ΔB8RB19R (MOI, 5). (A) Gene expression levels were analyzed by RT-PCR, and the results are expressed as the ratio of gene to HPRT mRNA levels. A.U., arbitrary units. (B) Cell-free supernatants were collected after 24 h to quantify the concentrations of IFN-β and CXCL10/IP-10. Data are means ± SDs for triplicate samples from one experiment and are representative of those from three independent experiments. *, P < 0.05 for anti-IFN-α/β versus control antibody.
FIG 9
FIG 9
Type 1 IFN- and IL-1-dependent induction of allogeneic CD4+ T cell response by primary monocytes infected with NYVAC-C-ΔB8RB19R. Primary human monocytes were infected for 18 h with NYVAC-C, NYVAC-C-ΔB19R, or NYVAC-C-ΔB8RB19R (MOI, 1) in the absence (A) or in the presence (B, C) of control, anti-IFN-α/β (B), or IL-1ra (C) antibody (10 μg/ml). Monocytes were collected, washed, and added to CD4+ T cells at monocyte/CD4+ T cell ratios ranging from 1/10 to 1/160 (A) or at a fixed 1/10 ratio (B, C). After 5 days, T cell proliferation was analyzed by measurement of the level of thymidine incorporation. Data are means ± SDs for triplicate samples from one experiment and are representative of those from two separate experiments. *, P < 0.05 for mutant versus wild-type NYVAC-C.
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