Different innate signatures induced in human monocyte-derived dendritic cells by wild-type dengue 3 virus, attenuated but reactogenic dengue 3 vaccine virus, or attenuated nonreactogenic dengue 1-4 vaccine virus strains.

DNA microarrays were used to assess the innate gene signature in human myeloid dendritic cells infected with chimeric dengue 1-4 vaccines, a wild-type dengue 3 virus, or a classically attenuated serotype 3 vaccine shown to be reactogenic in humans. We observed a very reproducible signature for each of the 4 chimeric dengue vaccines, involving stimulation of type I interferon and associated genes, together with genes encoding chemokines and other mediators involved in the initiation of adaptive responses. In contrast, wild-typeDEN3 virus induced a predominantly inflammatory profile, while the reactogenic attenuated serotype 3 vaccine appeared to induce a blunted response.

Cleavage of mitochondrial antiviral signaling protein in the liver of patients with chronic hepatitis C correlates with a reduced activation of the endogenous interferon system.

Hepatitis C virus (HCV) infection induces the endogenous interferon (IFN) system in the liver in some but not all patients with chronic hepatitis C (CHC). Patients with a pre-activated IFN system are less likely to respond to the current standard therapy with pegylated IFN-alpha. Mitochondrial antiviral signaling protein (MAVS) is an important adaptor molecule in a signal transduction pathway that senses viral infections and transcriptionally activates IFN-beta. The HCV NS3-4A protease can cleave and thereby inactivate MAVS in vitro, and, therefore, might be crucial in determining the activation status of the IFN system in the liver of infected patients. We analyzed liver biopsies from 129 patients with CHC to investigate whether MAVS is cleaved in vivo and whether cleavage prevents the induction of the endogenous IFN system. Cleavage of MAVS was detected in 62 of the 129 samples (48%) and was more extensive in patients with a high HCV viral load. MAVS was cleaved by all HCV genotypes (GTs), but more efficiently by GTs 2 and 3 than by GTs 1 and 4. The IFN-induced Janus kinase (Jak)-signal transducer and activator of transcription protein (STAT) pathway was less frequently activated in patients with cleaved MAVS, and there was a significant inverse correlation between cleavage of MAVS and the expression level of the IFN-stimulated genes IFI44L, Viperin, IFI27, USP18, and STAT1. We conclude that the pre-activation status of the endogenous IFN system in the liver of patients with CHC is in part regulated by cleavage of MAVS.

An amphipathic alpha-helix at the C terminus of hepatitis C virus nonstructural protein 4B mediates membrane association.

Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is an integral membrane protein that has been only poorly characterized to date. It is believed to comprise a cytosolic N-terminal part, a central part harboring four transmembrane passages, and a cytosolic C-terminal part. Here, we describe an amphipathic alpha-helix at the C terminus of NS4B (amino acid residues 229 to 253) that mediates membrane association and is involved in the formation of a functional HCV replication complex.

Innate immune responses in human dendritic cells upon infection by chimeric yellow-fever dengue vaccine serotypes 1-4.

Dengue infection is an important public health issue worldwide. The ChimeriVax-Dengue (CYD) vaccine uses yellow fever (YF) 17D vaccine as a live vector. Dendritic cells (DCs) play a key role in initiating immune responses and could be an important primary target of dengue infection. We investigated in vitro the consequences of CYD infection of DCs on their activation/maturation and cytokine production. In CYD-infected DCs, we observed an up-regulation of HLA-DR, CD80, CD86, and CD83. Cells exposed to CYD secreted type I interferons, monocyte chemoattractant protein 1 (MCP-1)/CC chemokine ligand 2 (CCL-2), interleukin-6 (IL-6), and low amounts of tumor necrosis factor-alpha (TNF-alpha), but no IL-10, IL-12, or IL-1alpha. Parental dengue viruses induced a similar array of cytokines, but more TNF-alpha, less IL-6, and less MCP-1/CCL-2 than induced by CYD. Chimeras thus induced DCs maturation and a controlled response accompanied by limited inflammatory cytokine production and consistent expression of anti-viral interferons, in agreement with clinical observations of safety and immunogenicity.

Do Th1 or Th2 sequence motifs exist in proteins? Identification of amphipatic immunomodulatory domains in Helicobacter pylori catalase.

The reasons why some proteins induce a particular type of T helper (Th) response are of fundamental importance but only partially understood. In the present study, amphipatic sequence motifs were identified in N- and C-terminal domains of Helicobacter pylori (Hp) catalase, which are linked to the induction of Th1 or Th2 immune responses, respectively. Alignment of these motifs with other proteins known to induce either Th1 or Th2 responses has lead to the identification of Th1 and Th2 consensus motifs, termed modulotopes. Their immunomodulatory potential was demonstrated by immunisation experiments using recombinant proteins comprising the C-terminal domain of catalase fused with one or several modulotopes and by co-immunisations of C- or N-terminal catalase domains with peptides containing these motifs. In addition to these in vivo data, in vitro assays using Limulus extracts suggested that modulotopes might interfere with responses triggered by danger signals such as LPS. Th1 and Th2 modulotopes are characterised by a specific hydrophobic/hydrophilic pattern, which might be the structural determinant for their activity. Our data suggest that Th1 and/or Th2 motifs may generally exist on proteins, thus offering the possibility of a rational modulation of the immune response.

HIV-1 gp41 and gp160 are hyperthermostable proteins in a mesophilic environment. Characterization of gp41 mutants.

HIV gp41(24-157) unfolds cooperatively over the pH range of 1.0-4.0 with T(m) values of > 100 degrees C. At pH 2.8, protein unfolding was 80% reversible and the DeltaH(vH)/DeltaH(cal) ratio of 3.7 is indicative of gp41 being trimeric. No evidence for a monomer-trimer equilibrium in the concentration range of 0.3-36 micro m was obtained by DSC and tryptophan fluorescence. Glycosylation of gp41 was found to have only a marginal impact on the thermal stability. Reduction of the disulfide bond or mutation of both cysteine residues had only a marginal impact on protein stability. There was no cooperative unfolding event in the DSC thermogram of gp160 in NaCl/P(i), pH 7.4, over a temperature range of 8-129 degrees C. When the pH was lowered to 5.5-3.4, a single unfolding event at around 120 degrees C was noted, and three unfolding events at 93.3, 106.4 and 111.8 degrees C were observed at pH 2.8. Differences between gp41 and gp160, and hyperthermostable proteins from thermophile organisms are discussed. A series of gp41 mutants containing single, double, triple or quadruple point mutations were analysed by DSC and CD. The impact of mutations on the protein structure, in the context of generating a gp41 based vaccine antigen that resembles a fusion intermediate state, is discussed. A gp41 mutant, in which three hydrophobic amino acids in the gp41 loop were replaced with charged residues, showed an increased solubility at neutral pH.