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Previously, Jackson et al.

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In contrast, we here performed an in-depth analysis of infected DCs. The present study clearly shows that we were able to generate a phenotype in DC by selectively deletion or reinsertion of specific genes from the viral backbone. As described by Jackson et al. Unfortunately, the species specificity of the B19R protein might interfere with in vivo immunogenicity analysis in mice, such as performed by Jackson et al.

By restoring replication competence we were able to increase the expression of the transgene, which is important for the ability to induce robust T-cell responses in vivo. That enhanced transgene expression leads to enhanced cross-presentation to HIV- and vaccinia-specific T cells is expected from the observations of others [50] — [52] that level and stability of antigen expression are the two most important factors in the efficiency of cross-presentation and cross-priming.

The Leiden University Medical Center, the University of Washington, and the Institute for Research in Biomedicine obtained written, informed consent from every blood donor in order to collect PBMC samples and approved the use of the material for this study. The study was approved by the institutional review board and by the ethics committee from the Centre Hospitalier Universitaire Vaudois and all patients gave written informed consent to use their material to make cell lines.

Monocyte derived dendritic cells moDCs were obtained from cryopreserved or freshly isolated peripheral blood mononuclear cells PBMCs from buffy coats of healthy blood donors. Specificity was confirmed after 4 weeks of culture.


Although these CD8 T cells were not cloned from a limiting dilution, Cells were restimulated every two weeks. Cells were left untreated for at least two weeks before use in antigen presentation assay. Clone CM. Two other vaccinia-specific CD8 T cell clones were used and have shown similar results. Virological and pathogenic characterization of these vectors in cultured cells and in mice is described Kibler et al.

Cultures were harvested immediately after infection or at 3, 12 and 24 hours post infection. Virus was released from cells by multiple rounds of freezing and thawing and titered on permissive BHK cells or BSC40 cells by plaque staining assays. The expression of Gag protein was measured in moDCs and HeLa cells at 6 and 24 hours after infection. To this end, cells were infected for one hour at MOI 1 and 5 and subsequently washed thoroughly. After 6 and 24 hours incubation, cells were harvested and Gag expression was determined by intracellular staining with an anti-Gag specific antibody KC57, Beckman Coulter.

Immunostimulation through bacterial nucleic acids

After one hour of incubation, the cells were washed extensively and plated into well plates. Supernatant from infected DCs was harvested 48 hours post infection.

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Cells were extensively washed to remove residual virus. After 18 hours, intracellular cytokine staining ICS was performed as described [56]. Then, the reaction was quenched with one volume of FCS and cells were washed twice. Quantification and quality control of extracted RNA was obtained as previously described [58].

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Illumina probe data were exported from BeadStudio as raw data and were screened for quality; samples failing chip visual inspection and control examination were removed. Probeset from the two Illumina platforms were mapped to a common probeset Id using a mapping file provided by Illumina. A dataset containing probeset common to both platforms was then used for subsequent steps. Gene expression data was preprocessed and analyzed using Bioconductor www. Bioconductor's genefilter package was used to filter out genes with low expression and insufficient variation in expression across all samples tested.

Expression values retained after this filtering process presented intensities greater than units in at least 2 samples and a log base 2 scale of at least 0. The resulting matrix showing filtered probeset as rows and samples as columns was used as input for subsequent statistical analysis. P values from the resulting comparison were adjusted for multiple testing according to the method of Benjamini and Hochberg [61].

This method controls the false discovery rate, which was set to 0. To determine whether our expression data sets obtained from gene expression profiling of dendritic cells infected with different poxviruses are enriched in known biological pathways, we used Gene Set Enrichnment Analysis GSEA , a non-parametric annotation-driven statistical analysis method. The statistical significance of a gene set's ES is estimated by an empirical genes-based permutation test procedure.

To account for multiple hypotheses testing, GSEA normalizes the ES for each gene set to account for variation in set sizes and calculates a false discovery rate FDR corresponding to each normalized ES. Each column in the heatmap represents a replicate between 2 and The genes indicated in the right vertical line represent some of the genes that are involved in the indicated pathways. Each column in the heatmap represents a replicate between 12 and List of genes involved in the antigen processing and presentation pathway and B-cell function pathway. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field.

Abstract Attenuated poxviruses are safe and capable of expressing foreign antigens. Introduction Development of an effective HIV-1 vaccine inducing both broadly neutralizing antibodies and virus-specific T cells has the best chance to inhibit HIV-1 replication, infection and acquisition. Download: PPT. Figure 1. Table 1. Restored replication competence of NYVAC in human cells We also generated a virus mutant with reintroduced genes restoring virus replication competence Table 1.

Figure 2. Figure 3. Combination of the B19R deletion and replication competence resulted in expression of pathways targeted by both single mutants We performed gene set enrichment analysis GSEA [42] to identify the pathways that are differentially expressed in cDCs and pDCs infected with different NYVAC mutants.


Figure 4. Table 2. Gene set enrichment analysis revealed the induction of distinct signaling pathways in response to recombinant NYVAC The above-described results focused on pathways expected to be targeted by the B19R deletion and restoration of replication competence. Figure 5. Improved cross-presentation of replication-competent NYVAC In addition to the effects of these virus mutants on DC maturation, we studied the functional differences between the different recombinant virus vectors in vitro.

Figure 7. Figure 8. Discussion To explore potential improvements of the immunogenicity of the NYVAC vector, we have used two strategies: deletion of a poxvirus gene known to encode a protein that may affect the immune response and development of attenuated replication-competent in human cells NYVAC. Materials and Methods Ethics statement The Leiden University Medical Center, the University of Washington, and the Institute for Research in Biomedicine obtained written, informed consent from every blood donor in order to collect PBMC samples and approved the use of the material for this study.

Cells Monocyte derived dendritic cells moDCs were obtained from cryopreserved or freshly isolated peripheral blood mononuclear cells PBMCs from buffy coats of healthy blood donors. Supporting Information. Figure S1. Figure S2. Table S1. Table S2. Table S3. Table S4. Table S5. Table S6. Table S7. Table S8. Acknowledgments We thank all blood donors for their contribution.

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Several immunostimulating drugs which have attracted interest contain a purine as the effective component. This is not surprising in view of the fact that many genetically determined immunodeficiencies can be traced to defects of enzymes which play a crucial role in purine biosynthesis. Finally, the potential role of lymphokines as stimulators of the immunosystem is briefly described.

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Some of these glycoproteins have recently become available for clinical trials. Others will be made available through genetic engineering. The therapeutic utility of these compounds is not yet clear; they will, however, be of great value as probes for the study of immune functions and for the development of immunopharmacology. Unable to display preview. Download preview PDF.

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