Designing a therapeutic hepatitis B vaccine to circumvent immune tolerance

An effective prophylactic hepatitis B virus (HBV) vaccine has long been available but is ineffective for chronic infection. The primary cause of chronic hepatitis B (CHB) and greatest impediment for a therapeutic vaccine is the direct and indirect effects of immune tolerance to HBV antigens. The resulting defective CD4+/CD8+ T cell response, poor cytokine production, insufficient neutralizing anti-body (nAb) and poor response to HBsAg vaccination characterize CHB infection. The objective of this study was to develop virus-like-particles (VLPs) that elicit nAb to prevent viral spread and prime CD4+/CD8+ T cells to eradicate intracellular HBV. Eight neutralizing B cell epitopes from the envelope PreS1 region were consolidated onto a species-variant of the HBV core protein, the woodchuck hepatitis core antigen (WHcAg). PreS1-specific B cell epitopes were chosen because of preferential expression on HBV virions. Because WHcAg and HBcAg are not crossreactive at the B cell level and only partially cross-reactive at the CD4+/CD8+ T cell level, CD4+ T cells specific for WHcAg-unique T cell sites can provide cognate T-B cell help for anti-PreS1 Ab production that is not curtailed by immune tolerance. Immunization of immune tolerant HBV transgenic (Tg) mice with PreS1-WHc VLPs elicited levels of high titer anti-PreS1 nAbs equivalent to wildtype mice. Passive transfer of PreS1 nAbs into human-liver chimeric mice prevented acute infection and cleared serum HBV from mice previously infected with HBV in a model of CHB. At the T cell level, PreS1-WHc VLPs and hybrid WHcAg/HBcAg DNA immunogens elicited HBcAg-specific CD4+ Th and CD8+ CTL responses

Mutant hepatitis B virus surface antigens (HBsAg) are immunogenic but may have a changed specificity

AbstractMutant hepatitis B virus with substitutions within the coding region for HBV surface antigen (HBsAg) has been found naturally in chronic carriers. It is therefore important to clarify whether the identified substitutions within the HBsAg have impact on the antigenicity and immunogenicity of HBsAg. A total of nine mutated HBV s-genes with single representative mutations were generated by site-directed mutagenesis and subcloned into an expression vector. The binding of polyclonal and monoclonal antibodies to these mutant HBsAg (mtHBsAg) was tested by immunofluorescence (IF) staining of cells transfected with the expression vectors. The amino acid (aa) substitutions like G145R, F134S, and C147W affected the binding of anti-HBs antibodies to corresponding mtHBsAg to different extents. The impact of aa substitutions G145R and F134S on the immunogenicity was accessed by genetic immunization of mice with vectors expressing middle HBsAg with the corresponding mutations. The immunized mice developed antibodies to recombinant HBsAg containing the HBV preS region and HBsAg-specific cytotoxic T-cell. However, the development of antibody response to wild-type small HBsAg was significantly impaired by the aa substitutions in HBsAg. Based on this fact, we further investigated whether the mtHBsAg with the aa substitution G145R is able to induce mutant-specific antibody responses. Strikingly, serum samples from mice immunized with mtHBsAg with G145R recognized plasma-derived mtHBsAg. Two mouse MAbs specific to mtHBsAg were generated. One MAb recognized mtHBsAg with G145R but not wild type and other mtHBsAg. We conclude that HBsAg with aa substitutions are immunogenic but may have a changed fine specificity

Association of Moderate Coffee Intake with Self-Reported Diabetes among Urban Brazilians

Coffee has been associated with reductions in the risk of non-communicable chronic diseases (NCCD), including diabetes mellitus. Because differences in food habits are recognizable modifying factors in the epidemiology of diabetes, we studied the association of coffee consumption with type-2 diabetes in a sample of the adult population of the Federal District, Brazil. This cross-sectional study was conducted by telephone interview (n = 1,440). A multivariate analysis was run controlling for socio-behavioural variables, obesity and family antecedents of NCCD. A hierarchical linear regression model and a Poisson regression were used to verify association of type-2 diabetes and coffee intake. The independent variables which remained in the final model, following the hierarchical inclusion levels, were: first level—age and marital status; second level—diabetes and dyslipidaemias in antecedents; third level—cigarette smoking, supplement intake, body mass index; and fourth level—coffee intake (≤100 mL/d, 101 to 400 mL/day, and >400 mL/day). After adjusting hierarchically for the confounding variables, consumers of 100 to 400 mL of coffee/day had a 2.7% higher (p = 0.04) prevalence of not having diabetes than those who drank less than 100 mL of coffee/day. Compared to coffee intake of ≤100 mL/day, adults consuming >400 mL of coffee/day showed no statistically significant difference in the prevalence of diabetes. Thus, moderate coffee intake is favourably associated with self-reported type-2 diabetes in the studied population. This is the first study to show a relationship between coffee drinking and diabetes in a Brazilian population

Kupffer Cells Hasten Resolution of Liver Immunopathology in Mouse Models of Viral Hepatitis

Kupffer cells (KCs) are widely considered important contributors to liver injury during viral hepatitis due to their pro-inflammatory activity. Herein we utilized hepatitis B virus (HBV)-replication competent transgenic mice and wild-type mice infected with a hepatotropic adenovirus to demonstrate that KCs do not directly induce hepatocellular injury nor do they affect the pathogenic potential of virus-specific CD8 T cells. Instead, KCs limit the severity of liver immunopathology. Mechanistically, our results are most compatible with the hypothesis that KCs contain liver immunopathology by removing apoptotic hepatocytes in a manner largely dependent on scavenger receptors. Apoptotic hepatocytes not readily removed by KCs become secondarily necrotic and release high-mobility group box 1 (HMGB-1) protein, promoting organ infiltration by inflammatory cells, particularly neutrophils. Overall, these results indicate that KCs resolve rather than worsen liver immunopathology

Activation of Toll-Like Receptor 3 Impairs the Dengue Virus Serotype 2 Replication through Induction of IFN-β in Cultured Hepatoma Cells

Toll-like receptors (TLRs) play an important role in innate immunity against invading pathogens. Although TLR signaling has been indicated to protect cells from infection of several viruses, the role of TLRs in Dengue virus (DENV) replication is still unclear. In the present study, we examined the replication of DENV serotype 2 (DENV2) by challenging hepatoma cells HepG2 with different TLR ligands. Activation of TLR3 showed an antiviral effect, while pretreatment of other TLR ligands (including TLR1/2, TLR2/6, TLR4, TLR5 or TLR7/8) did not show a significant effect. TLR3 ligand poly(I∶C) treatment prior to viral infection or simultaneously, but not post-treatment, significantly down-regulated virus replication. Pretreatment with poly(I∶C) reduced viral mRNA expression and viral staining positive cells, accompanying an induction of the type I interferon (IFN-β) and type III IFN (IL-28A/B). Intriguingly, neutralization of IFN-β alone successfully restored the poly(I∶C)-inhibited replication of DENV2. The poly(I∶C)-mediated effects, including IFN induction and DENV2 suppression, were significantly reversed by IKK inhibitor, further suggesting that IFN-β is the dominant factor involved in the poly(I∶C) mediated antiviral effect. Our study presented the first evidence to show that activation of TLR3 is effective in blocking DENV2 replication via IFN-β, providing an experimental clue that poly(I∶C) may be a promising immunomodulatory agent against DENV infection and might be applicable for clinical prevention

Single Nucleotide Polymorphisms of Toll-Like Receptor 4 Decrease the Risk of Development of Hepatocellular Carcinoma

BACKGROUND: Toll-like receptor 4 (TLR4) is a key innate immunity receptor that initiates an inflammatory response. Growing evidence suggests that mutation of TLR4 gene may play a role in the development of cancers. This study aimed to investigate the temporal relationship of single nucleotide polymorphisms of TLR4 and the risk of hepatocellular carcinoma, a single center-based case-control study was conducted. METHODS: A systematic genetic analysis of sequence variants of TLR4 by evaluating ten single-nucleotide polymorphisms was performed from 216 hepatocellular carcinoma cases and 228 controls. RESULTS: Six single nucleotide polymorphisms of the TLR4 in the 5'-untranslated region and intron were associated with risk of hepatocellular carcinoma. Individuals carrying the heterozygous genotypes for the rs10759930, rs2737190, rs10116253, rs1927914, rs12377632 and rs1927911 had significantly decreased risk of hepatocellular carcinoma (adjusted odds ratio [OR], from 0.527 to 0.578, P<0.01) comparing with those carrying wild-type homozygous genotypes. In haplotype analysis, one haplotype (GCCCTTAG) of TLR4 was associated significantly with decrease of the occurrence of hepatocellular carcinoma (OR, 0.556, 95% confidence interval [CI], 0.407-0.758, P = 0.000). CONCLUSIONS: Collectively, these results suggested that the risk of hepatocellular carcinoma was associated with TLR4 sequence variation. TLR4 single nucleotide polymorphisms may play an important protective role in the development of hepatocellular carcinoma

Identification of a Putative Network of Actin-Associated Cytoskeletal Proteins in Glomerular Podocytes Defined by Co-Purified mRNAs

The glomerular podocyte is a highly specialized and polarized kidney cell type that contains major processes and foot processes that extend from the cell body. Foot processes from adjacent podocytes form interdigitations with those of adjacent cells, thereby creating an essential intercellular junctional domain of the renal filtration barrier known as the slit diaphragm. Interesting parallels have been drawn between the slit diaphragm and other sites of cell-cell contact by polarized cells. Notably mutations in several genes encoding proteins localized to the foot processes can lead to proteinuria and kidney failure. Mutations in the Wilm's tumor gene (WT1) can also lead to kidney disease and one isoform of WT1, WT1(+KTS), has been proposed to regulate gene expression post-transcriptionally. We originally sought to identify mRNAs associated with WT1(+KTS) through an RNA immunoprecipitation and microarray approach, hypothesizing that the proteins encoded by these mRNAs might be important for podocyte morphology and function. We identified a subset of mRNAs that were remarkably enriched for transcripts encoding actin-binding proteins and other cytoskeletal proteins including several that are localized at or near the slit diaphragm. Interestingly, these mRNAs included those of α-actinin-4 and non-muscle myosin IIA that are mutated in genetic forms of kidney disease. However, isolation of the mRNAs occurred independently of the expression of WT1, suggesting that the identified mRNAs were serendipitously co-purified on the basis of co-association in a common subcellular fraction. Mass spectroscopy revealed that other components of the actin cytoskeleton co-purified with these mRNAs, namely actin, tubulin, and elongation factor 1α. We propose that these mRNAs encode a number of proteins that comprise a highly specialized protein interactome underlying the slit diaphragm. Collectively, these gene products and their interactions may prove to be important for the structural integrity of the actin cytoskeleton in podocytes as well as other polarized cell types

Liver Is Able to Activate Naïve CD8+ T Cells with Dysfunctional Anti-Viral Activity in the Murine System

The liver possesses distinct tolerogenic properties because of continuous exposure to bacterial constituents and nonpathogenic food antigen. The central immune mediators required for the generation of effective immune responses in the liver environment have not been fully elucidated. In this report, we demonstrate that the liver can indeed support effector CD8+ T cells during adenovirus infection when the T cells are primed in secondary lymphoid tissues. In contrast, when viral antigen is delivered predominantly to the liver via intravenous (IV) adenovirus infection, intrahepatic CD8+ T cells are significantly impaired in their ability to produce inflammatory cytokines and lyse target cells. Additionally, intrahepatic CD8+ T cells generated during IV adenovirus infection express elevated levels of PD-1. Notably, lower doses of adenovirus infection do not rescue the impaired effector function of intrahepatic CD8+ T cell responses. Instead, intrahepatic antigen recognition limits the generation of potent anti-viral responses at both priming and effector stages of the CD8+ T cell response and accounts for the dysfunctional CD8+ T cell response observed during IV adenovirus infection. These results also implicate that manipulation of antigen delivery will facilitate the design of improved vaccination strategies to persistent viral infection

Plasma biomarkers of depressive symptoms in older adults

The pathophysiology of negative affect states in older adults is complex, and a host of central nervous system and peripheral systemic mechanisms may play primary or contributing roles. We conducted an unbiased analysis of 146 plasma analytes in a multiplex biochemical biomarker study in relation to number of depressive symptoms endorsed by 566 participants in the Alzheimer's Disease Neuroimaging Initiative (ADNI) at their baseline and 1-year assessments. Analytes that were most highly associated with depressive symptoms included hepatocyte growth factor, insulin polypeptides, pregnancy-associated plasma protein-A and vascular endothelial growth factor. Separate regression models assessed contributions of past history of psychiatric illness, antidepressant or other psychotropic medicine, apolipoprotein E genotype, body mass index, serum glucose and cerebrospinal fluid (CSF) τ and amyloid levels, and none of these values significantly attenuated the main effects of the candidate analyte levels for depressive symptoms score. Ensemble machine learning with Random Forests found good accuracy (∼80%) in classifying groups with and without depressive symptoms. These data begin to identify biochemical biomarkers of depressive symptoms in older adults that may be useful in investigations of pathophysiological mechanisms of depression in aging and neurodegenerative dementias and as targets of novel treatment approaches

Toll-like receptor 4 signaling in liver injury and hepatic fibrogenesis

Toll-like receptors (TLRs) are a family of transmembrane pattern recognition receptors (PRR) that play a key role in innate and adaptive immunity by recognizing structural components unique to bacteria, fungi and viruses. TLR4 is the most studied of the TLRs, and its primary exogenous ligand is lipopolysaccharide, a component of Gram-negative bacterial walls. In the absence of exogenous microbes, endogenous ligands including damage-associated molecular pattern molecules from damaged matrix and injured cells can also activate TLR4 signaling. In humans, single nucleotide polymorphisms of the TLR4 gene have an effect on its signal transduction and on associated risks of specific diseases, including cirrhosis. In liver, TLR4 is expressed by all parenchymal and non-parenchymal cell types, and contributes to tissue damage caused by a variety of etiologies. Intact TLR4 signaling was identified in hepatic stellate cells (HSCs), the major fibrogenic cell type in injured liver, and mediates key responses including an inflammatory phenotype, fibrogenesis and anti-apoptotic properties. Further clarification of the function and endogenous ligands of TLR4 signaling in HSCs and other liver cells could uncover novel mechanisms of fibrogenesis and facilitate the development of therapeutic strategies