Association of Transcriptomic Signatures of Inflammatory Response with Viral Control after Dendritic Cell-Based Therapeutic Vaccination in HIV-1 Infected Individuals.

Systems vaccinology has seldomly been used in therapeutic HIV-1 vaccine research. Our aim was to identify early gene 'signatures' that predicted virus load control after analytical therapy interruption (ATI) in participants of a dendritic cell-based HIV-1 vaccine trial (DCV2). mRNA and miRNA were extracted from frozen post-vaccination PBMC samples; gene expression was determined by microarray method. In gene set enrichment analysis, responders showed an up-regulation of 14 gene sets (TNF-alpha/NFkB pathway, inflammatory response, the complement system, Il6 and Il2 JAK-STAT signaling, among others) and a down-regulation of 7 gene sets (such as E2F targets or interferon alpha response). The expression of genes regulated by three (miR-223-3p, miR-1183 and miR-8063) of the 9 differentially expressed miRNAs was significantly down-regulated in responders. The deregulation of certain gene sets related to inflammatory processes seems fundamental for viral control, and certain miRNAs may be important in fine-tuning these processes.

Using NS1 Flavivirus Protein Microarray to Infer Past Infecting Dengue Virus Serotype and Number of Past Dengue Virus Infections in Vietnamese Individuals.

BACKGROUND: In recent years, researchers have had an increased focus on multiplex microarray assays, in which antibodies are measured against multiple related antigens, for use in seroepidemiological studies to infer past transmission. METHODS: We assess the performance of a flavivirus microarray assay for determining past dengue virus (DENV) infection history in a dengue-endemic setting, Vietnam. We tested the microarray on samples from 1 and 6 months postinfection from DENV-infected patients (infecting serotype was determined using reverse-transcription polymerase chain reaction during acute, past primary, and secondary infection assessed using plaque reduction neutralization tests 6 months postinfection). RESULTS: Binomial models developed to discriminate past primary from secondary infection using the protein microarray (PMA) titers had high area under the curve (0.90-0.97) and accuracy (0.84-0.86). Multinomial models developed to identify most recent past infecting serotype using PMA titers performed well in those with past primary infection (average test set: κ = 0.85, accuracy of 0.92) but not those with past secondary infection (κ = 0.24, accuracy of 0.45). CONCLUSIONS: Our results suggest that the microarray will be useful in seroepidemiological studies aimed at classifying the past infection history of individuals (past primary vs secondary and serotype of past primary infections) and thus inferring past transmission intensity of DENV in dengue-endemic settings. Future work to validate these models should be undertaken in different transmission settings and with samples later after infection.

Alveolar barrier disruption in varicella pneumonia is associated with neutrophil extracellular trap formation.

Primary varicella-zoster virus (VZV) infection in adults is often complicated by severe pneumonia, which is difficult to treat and is associated with high morbidity and mortality. Here, the simian varicella virus (SVV) nonhuman primate (NHP) model was used to investigate the pathogenesis of varicella pneumonia. SVV infection resulted in transient fever, viremia, and robust virus replication in alveolar pneumocytes and bronchus-associated lymphoid tissue. Clearance of infectious virus from lungs coincided with robust innate immune responses, leading to recruitment of inflammatory cells, mainly neutrophils and lymphocytes, and finally severe acute lung injury. SVV infection caused neutrophil activation and formation of neutrophil extracellular traps (NETs) in vitro and in vivo. Notably, NETs were also detected in lung and blood specimens of varicella pneumonia patients. Lung pathology in the SVV NHP model was associated with dysregulated expression of alveolar epithelial cell tight junction proteins (claudin-2, claudin-10, and claudin-18) and alveolar endothelial adherens junction protein VE-cadherin. Importantly, factors released by activated neutrophils, including NETs, were sufficient to reduce claudin-18 and VE-cadherin expression in NHP lung slice cultures. Collectively, the data indicate that alveolar barrier disruption in varicella pneumonia is associated with NET formation.

Analysis of Virus and Host Proteomes During Productive HSV-1 and VZV Infection in Human Epithelial Cells.

Herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV) are two closely related human alphaherpesviruses that persistently infect most adults worldwide and cause a variety of clinically important diseases. Herpesviruses are extremely well adapted to their hosts and interact broadly with cellular proteins to regulate virus replication and spread. However, it is incompletely understood how HSV-1 and VZV interact with the host proteome during productive infection. This study determined the temporal changes in virus and host protein expression during productive HSV-1 and VZV infection in the same cell type. Results demonstrated the temporally coordinated expression of HSV-1 and VZV proteins in infected cells. Analysis of the host proteomes showed that both viruses affected extracellular matrix composition, transcription, RNA processing and cell division. Moreover, the prominent role of epidermal growth factor receptor (EGFR) signaling during productive HSV-1 and VZV infection was identified. Stimulation and inhibition of EGFR leads to increased and decreased virus replication, respectively. Collectively, the comparative temporal analysis of viral and host proteomes in productively HSV-1 and VZV-infected cells provides a valuable resource for future studies aimed to identify target(s) for antiviral therapy development.

Proteomic Profiling of Mouse Helper T Cell Differentiation.

Helper T cell differentiation is a key process in the regulation of adaptive immune responses. Here, mouse Th1 and Th2 cells are profiled using high-throughput proteomics to increase the understanding of the molecular biology of Th differentiation to support the design of prophylactic and therapeutic intervention strategies for (infectious) diseases. Protein profiling of Th1/Th2 differentiated cells results in the quantification of almost 6000 proteins of which 41 are differentially expressed at FDR < 0.1, and 19 at the FDR < 0.05 level, respectively. Differential protein expression analysis identifies a number of the expected canonical Th differentiation markers, and gene set analysis using the REACTOME database and a hypergeometric test (FDR < 0.05) confirms that helper T cell pathways are the top sets that are differentially expressed. Additionally, by network analysis, many differentially expressed proteins are associated with the Th1 and Th2 pathways. Data are available via PRIDE database with identifier PXD004532.

Studies into the mechanism of measles-associated immune suppression during a measles outbreak in the Netherlands.

Measles causes a transient immune suppression, leading to increased susceptibility to opportunistic infections. In experimentally infected non-human primates (NHPs) measles virus (MV) infects and depletes pre-existing memory lymphocytes, causing immune amnesia. A measles outbreak in the Dutch Orthodox Protestant community provided a unique opportunity to study the pathogenesis of measles immune suppression in unvaccinated children. In peripheral blood mononuclear cells (PBMC) of prodromal measles patients, we detected MV-infected memory CD4+ and CD8+ T cells and naive and memory B cells at similar levels as those observed in NHPs. In paired PBMC collected before and after measles we found reduced frequencies of circulating memory B cells and increased frequencies of regulatory T cells and transitional B cells after measles. These data support our immune amnesia hypothesis and offer an explanation for the previously observed long-term effects of measles on host resistance. This study emphasises the importance of maintaining high measles vaccination coverage.

Phase I clinical trial of an intranodally administered mRNA-based therapeutic vaccine against HIV-1 infection.

OBJECTIVE: The efficacy of therapeutic vaccines against HIV-1 infection has been modest. New inerts to redirect responses to vulnerable sites are urgently needed to improve these results. DESIGN: We performed the first-in-human clinical trial with naked mRNA (iHIVARNA) combining a dendritic cell activation strategy (TriMix:CD40L+CD70+caTLR4 RNA) with a novel HIV immunogen sequences (HTI immunogen). METHODS: A dose escalation, phase I clinical trial was performed in 21 chronic HIV-1-infected patients under ART who received three intranodal doses of mRNA (weeks 0, 2 and 4) as follow: TriMix-100 g, TriMix-300 g, TriMix-300 g with HTI-300 g, TriMix-300 g with HTI-600 g, TriMix-300 g with HTI-900 g. Primary end-point was safety and secondary-exploratory end-points were immunogenicity, changes in viral reservoir and transcriptome. RESULTS: Overall, the vaccine was secure and well tolerated. There were 31 grade 1/2 and 1 grade 3 adverse events, mostly unrelated to the vaccination. Patients who received the highest dose showed a moderate increase in T-cell responses spanning HTI sequence at week 8. In addition, the proportion of responders receiving any dose of HTI increased from 31% at w0 to 80% postvaccination. The intervention had no impact on caHIV-DNA levels, however, caHIV-RNA expression and usVL were transiently increased at weeks 5 and 6 in the highest dose of iHIVARNA, and these changes were positively correlated with HIV-1-specific-induced immune responses. CONCLUSION: This phase I dose-escalating trial showed that iHIVARNA administration was safe and well tolerated, induced moderate HIV-specific T-cell responses and transiently increased different viral replication readouts. These data support further exploration of iHIVARNA in a phase II study. CLINICALTRIALS. GOV IDENTIFIER: NCT02413645.

Profiling of 3696 Nuclear Receptor-Coregulator Interactions: A Resource for Biological and Clinical Discovery.

Nuclear receptors (NRs) are ligand-inducible transcription factors that play critical roles in metazoan development, reproduction, and physiology and therefore are implicated in a broad range of pathologies. The transcriptional activity of NRs critically depends on their interaction(s) with transcriptional coregulator proteins, including coactivators and corepressors. Short leucine-rich peptide motifs in these proteins (LxxLL in coactivators and LxxxIxxxL in corepressors) are essential and sufficient for NR binding. With 350 different coregulator proteins identified to date and with many coregulators containing multiple interaction motifs, an enormous combinatorial potential is present for selective NR-mediated gene regulation. However, NR-coregulator interactions have often been determined experimentally on a one-to-one basis across diverse experimental conditions. In addition, NR-coregulator interactions are difficult to predict because the molecular determinants that govern specificity are not well established. Therefore, many biologically and clinically relevant NR-coregulator interactions may remain to be discovered. Here, we present a comprehensive overview of 3696 NR-coregulator interactions by systematically characterizing the binding of 24 nuclear receptors with 154 coregulator peptides. We identified unique ligand-dependent NR-coregulator interaction profiles for each NR, confirming many well-established NR-coregulator interactions. Hierarchical clustering based on the NR-coregulator interaction profiles largely recapitulates the classification of NR subfamilies based on the primary amino acid sequences of the ligand-binding domains, indicating that amino acid sequence is an important, although not the only, molecular determinant in directing and fine-tuning NR-coregulator interactions. This NR-coregulator peptide interactome provides an open data resource for future biological and clinical discovery as well as NR-based drug design.

Analysis of Mouse Brain Transcriptome After Experimental Duvenhage Virus Infection Shows Activation of Innate Immune Response and Pyroptotic Cell Death Pathway.

Rabies is an important neglected disease, characterized by invariably fatal encephalitis. Several studies focus on understanding the pathogenic mechanisms of the prototype lyssavirus rabies virus (RABV) infection, and little is known about the pathogenesis of rabies caused by other lyssaviruses. We sought to characterize the host response to Duvenhage virus infection and compare it with responses observed during RABV infection by gene expression profiling of brains of mice with the respective infections. We found in both infections differentially expressed genes leading to increased expression of type I interferons (IFNs), chemokines, and proinflammatory cytokines. In addition several genes of the IFN signaling pathway are up-regulated, indicating a strong antiviral response and activation of the negative feedback mechanism to limit type I IFN responses. Furthermore we provide evidence that in the absence of significant neuronal apoptotic death, cell death of neurons is mediated via the pyroptotic pathway in both infections. Taken together, we have identified several genes and/or pathways for both infections that could be used to explore novel approaches for intervention strategies against rabies.

Dendritic cell immunotherapy followed by cART interruption during HIV-1 infection induces plasma protein markers of cellular immunity and neutrophil recruitment.

OBJECTIVES: To characterize the host response to dendritic cell-based immunotherapy and subsequent combined antiretroviral therapy (cART) interruption in HIV-1-infected individuals at the plasma protein level. DESIGN: An autologous dendritic cell (DC) therapeutic vaccine was administered to HIV-infected individuals, stable on cART. The effect of vaccination was evaluated at the plasma protein level during the period preceding cART interruption, during analytical therapy interruption and at viral reactivation. Healthy controls and post-exposure prophylactically treated healthy individuals were included as controls. METHODS: Plasma marker ('analyte') levels including cytokines, chemokines, growth factors, and hormones were measured in trial participants and control plasma samples using a multiplex immunoassay. Analyte levels were analysed using principle component analysis, cluster analysis and limma. Blood neutrophil counts were analysed using linear regression. RESULTS: Plasma analyte levels of HIV-infected individuals are markedly different from those of healthy controls and HIV-negative individuals receiving post-exposure prophylaxis. Viral reactivation following cART interruption also affects multiple analytes, but cART interruption itself only has only a minor effect. We find that Thyroxine-Binding Globulin (TBG) levels and late-stage neutrophil numbers correlate with the time off cART after DC vaccination. Furthermore, analysis shows that cART alters several regulators of blood glucose levels, including C-peptide, chromogranin-A and leptin. HIV reactivation is associated with the upregulation of CXCR3 ligands. CONCLUSIONS: Chronic HIV infection leads to a change in multiple plasma analyte levels, as does virus reactivation after cART interruption. Furthermore, we find evidence for the involvement of TBG and neutrophils in the response to DC-vaccination in the setting of HIV-infection.

1918 H1N1 Influenza Virus Replicates and Induces Proinflammatory Cytokine Responses in Extrarespiratory Tissues of Ferrets.

Background: The 1918 Spanish H1N1 influenza pandemic was the most severe recorded influenza pandemic with an estimated 20-50 million deaths worldwide. Even though it is known that influenza viruses can cause extrarespiratory tract complications-which are often severe or even fatal-the potential contribution of extrarespiratory tissues to the pathogenesis of 1918 H1N1 virus infection has not been studied comprehensively. Methods: Here, we performed a time-course study in ferrets inoculated intranasally with 1918 H1N1 influenza virus, with special emphasis on the involvement of extrarespiratory tissues. Respiratory and extrarespiratory tissues were collected after inoculation for virological, histological, and immunological analysis. Results: Infectious virus was detected at high titers in respiratory tissues and, at lower titers in most extrarespiratory tissues. Evidence for active virus replication, as indicated by the detection of nucleoprotein by immunohistochemistry, was observed in the respiratory tract, peripheral and central nervous system, and liver. Proinflammatory cytokines were up-regulated in respiratory tissues, olfactory bulb, spinal cord, liver, heart, and pancreas. Conclusions: 1918 H1N1 virus spread to and induced cytokine responses in tissues outside the respiratory tract, which likely contributed to the severity of infection. Moreover, our data support the suggested link between 1918 H1N1 infection and central nervous system disease.

Identification of differentially expressed peptides in high-throughput proteomics data.

With the advent of high-throughput proteomics, the type and amount of data pose a significant challenge to statistical approaches used to validate current quantitative analysis. Whereas many studies focus on the analysis at the protein level, the analysis of peptide-level data provides insight into changes at the sub-protein level, including splice variants, isoforms and a range of post-translational modifications. Statistical evaluation of liquid chromatography-mass spectrometry/mass spectrometry peptide-based label-free differential data is most commonly performed using a t-test or analysis of variance, often after the application of data imputation to reduce the number of missing values. In high-throughput proteomics, statistical analysis methods and imputation techniques are difficult to evaluate, given the lack of gold standard data sets. Here, we use experimental and resampled data to evaluate the performance of four statistical analysis methods and the added value of imputation, for different numbers of biological replicates. We find that three or four replicates are the minimum requirement for high-throughput data analysis and confident assignment of significant changes. Data imputation does increase sensitivity in some cases, but leads to a much higher actual false discovery rate. Additionally, we find that empirical Bayes method (limma) achieves the highest sensitivity, and we thus recommend its use for performing differential expression analysis at the peptide level.

Cell lines generated from a chronic lymphocytic leukemia mouse model exhibit constitutive Btk and Akt signaling.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of mature CD5+ B cells in blood. Spontaneous apoptosis of CLL cells in vitro has hampered in-depth investigation of CLL pathogenesis. Here we describe the generation of three monoclonal mouse cell lines, EMC2, EMC4 and EMC6, from the IgH.TEµ CLL mouse model based on sporadic expression of SV40 large T antigen. The cell lines exhibit a stable CD5+CD43+IgM+CD19+ CLL phenotype in culture and can be adoptively transferred into Rag1-/- mice. RNA-seq analysis revealed only minor differences between the cell lines and their primary tumors and suggested that NF-κB and mTOR signaling pathways were involved in cell line outgrowth. In vitro survival and proliferation was dependent on constitutive phosphorylation of Bruton's tyrosine kinase (Btk) at Y551/Y223, and Akt(S473). Treatment of the cell lines with small molecule inhibitors specific for Btk (ibrutinib) or PI3K (idelalisib), which is upstream of Akt, resulted in reduced viability, proliferation and fibronectin-dependent cell adhesion. Treatment of cell line-engrafted Rag1-/- mice with ibrutinib was associated with transient lymphocytosis, reduced splenomegaly and increased overall survival. Thus, by generating stable cell lines we established a novel platform for in vitro and in vivo investigation of CLL signal transduction and treatment modalities.

Transcriptomic Analyses Reveal Differential Gene Expression of Immune and Cell Death Pathways in the Brains of Mice Infected with West Nile Virus and Chikungunya Virus.

West Nile virus (WNV) and chikungunya virus (CHIKV) are arboviruses that are constantly (re-)emerging and expanding their territory. Both viruses often cause a mild form of disease, but severe forms of the disease can consist of neurological symptoms, most often observed in the elderly and young children, respectively, for which the mechanisms are poorly understood. To further elucidate the mechanisms responsible for end-stage WNV and CHIKV neuroinvasive disease, we used transcriptomics to compare the induction of effector pathways in the brain during the early and late stage of disease in young mice. In addition to the more commonly described cell death pathways such as apoptosis and autophagy, we also found evidence for the differential expression of pyroptosis and necroptosis cell death markers during both WNV and CHIKV neuroinvasive disease. In contrast, no evidence of cell dysfunction was observed, indicating that cell death may be the most important mechanism of disease. Interestingly, there was overlap when comparing immune markers involved in neuroinvasive disease to those seen in neurodegenerative diseases. Nonetheless, further validation studies are needed to determine the activation and involvement of these effector pathways at the end stage of disease. Furthermore, evidence for a strong inflammatory response was found in mice infected with WNV and CHIKV. The transcriptomics profile measured in mice with WNV and CHIKV neuroinvasive disease in our study showed strong overlap with the mRNA profile described in the literature for other viral neuroinvasive diseases. More studies are warranted to decipher the role of cell inflammation and cell death in viral neuroinvasive disease and whether common mechanisms are active in both neurodegenerative and brain infectious diseases.

Cytokines and Chemokines Involved in Acute Retinal Necrosis.

Purpose: To investigate which cytokines and chemokines are involved in the immunopathogenesis of acute retinal necrosis (ARN), and whether cytokine profiles are associated with clinical manifestations, such as visual outcome. Methods: Serum and aqueous humor (AH) samples of 19 patients with ARN were analyzed by multiplex immunoassay. Infectious controls consisted of 18 patients with rubella virus-associated Fuchs' uveitis and 20 patients with ocular toxoplasmosis all confirmed by intraocular fluid analyses. The control group consisted of seven paired AH and serum samples from seven noninflammatory control patients with age-related cataract. In each sample, 4 anti-inflammatory, 12 proinflammatory, 2 vascular, and 4 other immune mediators were measured. In addition, various clinical characteristics were assessed. Results: In ARN, 10 of the 22 mediators, including most proinflammatory and vascular mediators such as IL-6, IL-8, IL-18, MIF, MCP-1, Eotaxin, IP-10, IL-15, sICAM-1, and sVCAM-1, were significantly elevated when compared to all controls. In addition, one anti-inflammatory mediator (IL-10) was significantly elevated in ARN as compared to the controls. No association was found between the time of sampling and the extent and levels of immune mediator expression. Conclusions: The pathogenesis of ARN is characterized by the presence of predominantly proinflammatory cytokines and chemokines with high expression levels as compared to other infectious causes of uveitis. There are no indications for an obvious Th-1 or Th-17 pathway. The combined data suggest that immune mediator expression is related to severity of disease, which is more fulminant in ARN, rather than to a specific uveitis entity.

Transcriptome assists prognosis of disease severity in respiratory syncytial virus infected infants.

Respiratory syncytial virus (RSV) causes infections that range from common cold to severe lower respiratory tract infection requiring high-level medical care. Prediction of the course of disease in individual patients remains challenging at the first visit to the pediatric wards and RSV infections may rapidly progress to severe disease. In this study we investigate whether there exists a genomic signature that can accurately predict the course of RSV. We used early blood microarray transcriptome profiles from 39 hospitalized infants that were followed until recovery and of which the level of disease severity was determined retrospectively. Applying support vector machine learning on age by sex standardized transcriptomic data, an 84 gene signature was identified that discriminated hospitalized infants with eventually less severe RSV infection from infants that suffered from most severe RSV disease. This signature yielded an area under the receiver operating characteristic curve (AUC) of 0.966 using leave-one-out cross-validation on the experimental data and an AUC of 0.858 on an independent validation cohort consisting of 53 infants. A combination of the gene signature with age and sex yielded an AUC of 0.971. Thus, the presented signature may serve as the basis to develop a prognostic test to support clinical management of RSV patients.

Changes in heterosubtypic antibody responses during the first year of the 2009 A(H1N1) influenza pandemic.

Seropositivity to avian influenza (AI) via low-level antibody titers has been reported in the general population and poultry-exposed individuals, raising the question whether these findings reflect true infection with AI or cross-reactivity. Here we investigated serological profiles against human and avian influenza viruses in the general population using a protein microarray platform. We hypothesized that higher antibody diversity across recent H1 and H3 influenza viruses would be associated with heterosubtypic reactivity to older pandemic- and AI viruses. We found significant heterogeneity in antibody profiles. Increased antibody diversity to seasonal influenza viruses was associated with low-level heterosubtypic antibodies to H9 and H7, but not to H5 AI virus. Individuals exposed to the recent 2009 A(H1N1) pandemic showed higher heterosubtypic reactivity. We show that there is a complex interplay between prior exposures to seasonal and recent pandemic influenza viruses and the development of heterosubtypic antibody reactivity to animal influenza viruses.

DC immunotherapy in HIV-1 infection induces a major blood transcriptome shift.

OBJECTIVE: This study aimed to evaluate the effect of dendritic cell (DC) vaccination against HIV-1 on host gene expression profiles. DESIGN: Longitudinal PBMC samples were collected from participants of the DC-TRN trial for immunotherapy against HIV. Microarray-assisted gene expression profiling was performed to evaluate the effects of vaccination and subsequent interruption of antiretroviral therapy on host genome expression. Data from the DC-TRN trial were compared with results from other vaccination trials. METHODS: We used Affymetrix GeneChips for microarray gene expression analysis. Data were analyzed by principal component analysis and differential gene expression was assessed using linear modeling. Gene ontology enrichment and gene set analysis were used to characterize differentially expressed genes. Transcriptome analysis included comparison with PBMCs obtained from DC-vaccinated melanoma patients and of healthy individuals who received seasonal influenza vaccination. RESULTS: DC-TRN immunotherapy in HIV-infected individuals resulted in a major shift in the transcriptome. Longitudinal analysis demonstrated that changes in the transcriptome sustained also during interruption of antiretroviral therapy. After DC-vaccination, the transcriptome was enriched for cellular immunity associated genes that were also induced in healthy adults who received live attenuated influenza virus vaccination. These beneficial responses were accompanied by detrimental signals of general immune activation. CONCLUSIONS: The DC-TRN induced changes in the transcriptome were profound, lasting, and consisted of both protective signals and signatures of inflammation and immune exhaustion, with a net result of decreased viral load, without clinical benefit. Thus transcriptome analysis provides useful information, dissecting both positive and negative effects, for the evaluation of safety and efficacy of immunotherapeutic strategies.

Time since onset of disease and individual clinical markers associate with transcriptional changes in uncomplicated dengue.

BACKGROUND: Dengue virus (DENV) infection causes viral haemorrhagic fever that is characterized by extensive activation of the immune system. The aim of this study is to investigate the kinetics of the transcriptome signature changes during the course of disease and the association of genes in these signatures with clinical parameters. METHODOLOGY/PRINCIPLE FINDINGS: Sequential whole blood samples from DENV infected patients in Jakarta were profiled using affymetrix microarrays, which were analysed using principal component analysis, limma, gene set analysis, and weighted gene co-expression network analysis. We show that time since onset of disease, but not diagnosis, has a large impact on the blood transcriptome of patients with non-severe dengue. Clinical diagnosis (according to the WHO classification) does not associate with differential gene expression. Network analysis however, indicated that the clinical markers platelet count, fibrinogen, albumin, IV fluid distributed per day and liver enzymes SGOT and SGPT strongly correlate with gene modules that are enriched for genes involved in the immune response. Overall, we see a shift in the transcriptome from immunity and inflammation to repair and recovery during the course of a DENV infection. CONCLUSIONS/SIGNIFICANCE: Time since onset of disease associates with the shift in transcriptome signatures from immunity and inflammation to cell cycle and repair mechanisms in patients with non-severe dengue. The strong association of time with blood transcriptome changes hampers both the discovery as well as the potential application of biomarkers in dengue. However, we identified gene expression modules that associate with key clinical parameters of dengue that reflect the systemic activity of disease during the course of infection. The expression level of these gene modules may support earlier detection of disease progression as well as clinical management of dengue.

Host proteome correlates of vaccine-mediated enhanced disease in a mouse model of respiratory syncytial virus infection.

UNLABELLED: Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants. Despite over 50 years of research, to date no safe and efficacious RSV vaccine has been licensed. Many experimental vaccination strategies failed to induce balanced T-helper (Th) responses and were associated with adverse effects such as hypersensitivity and immunopathology upon challenge. In this study, we explored the well-established recombinant vaccinia virus (rVV) RSV-F/RSV-G vaccination-challenge mouse model to study phenotypically distinct vaccine-mediated host immune responses at the proteome level. In this model, rVV-G priming and not rVV-F priming results in the induction of Th2 skewed host responses upon RSV challenge. Mass spectrometry-based spectral count comparisons enabled us to identify seven host proteins for which expression in lung tissue is associated with an aberrant Th2 skewed response characterized by the influx of eosinophils and neutrophils. These proteins are involved in processes related to the direct influx of eosinophils (eosinophil peroxidase [Epx]) and to chemotaxis and extravasation processes (Chil3 [chitinase-like-protein 3]) as well as to eosinophil and neutrophil homing signals to the lung (Itgam). In addition, the increased levels of Arg1 and Chil3 proteins point to a functional and regulatory role for alternatively activated macrophages and type 2 innate lymphoid cells in Th2 cytokine-driven RSV vaccine-mediated enhanced disease. IMPORTANCE: RSV alone is responsible for 80% of acute bronchiolitis cases in infants worldwide and causes substantial mortality in developing countries. Clinical trials performed with formalin-inactivated RSV vaccine preparations in the 1960s failed to induce protection upon natural RSV infection and even predisposed patients for enhanced disease. Despite the clinical need, to date no safe and efficacious RSV vaccine has been licensed. Since RSV vaccines have a tendency to prime for unbalanced responses associated with an exuberant influx of inflammatory cells and enhanced disease, detailed characterization of primed host responses has become a crucial element in RSV vaccine research. We investigated the lung proteome of mice challenged with RSV upon priming with vaccine preparations known to induce phenotypically distinct host responses. Seven host proteins whose expression levels are associated with vaccine-mediated enhanced disease have been identified. The identified protein biomarkers support the development as well as detailed evaluation of next-generation RSV vaccines.