Design of TLR2-ligand-synthetic long peptide conjugates for therapeutic vaccination of chronic HBV patients.

Synthetic long peptide (SLP) vaccination is a promising new treatment strategy for patients with a chronic hepatitis B virus (HBV) infection. We have previously shown that a prototype HBV-core protein derived SLP was capable of boosting CD4+ and CD8+ T cell responses in the presence of a TLR2-ligand in chronic HBV patients ex vivo. For optimal efficacy of a therapeutic vaccine in vivo, adjuvants can be conjugated to the SLP to ensure delivery of both the antigen and the co-stimulatory signal to the same antigen-presenting cell (APC). Dendritic cells (DCs) express the receptor for the adjuvant and are optimally equipped to efficiently process and present the SLP-contained epitopes to T cells. Here, we investigated TLR2-ligand conjugation of the prototype HBV-core SLP. Results indicated that TLR2-ligand conjugation reduced cross-presentation efficiency of the SLP-contained epitope by both monocyte-derived and naturally occurring DC subsets. Importantly, cross-presentation was improved after optimization of the conjugate by either shortening the SLP or by placing a valine-citrulline linker between the TLR2-ligand and the long SLP, to facilitate endosomal dissociation of SLP and TLR2-ligand after uptake. HBV-core SLP conjugates also triggered functional patient T cell responses ex vivo. These results provide an import step forward in the design of a therapeutic SLP-based vaccine to cure chronic HBV.

Elevated serum levels of soluble CD14 in HBeAg-positive chronic HBV patients upon Peginterferon treatment are associated with treatment response.

Pegylated IFNα (PEG-IFN) is one of the treatment options for chronic HBV (CHB) patients. However, the high patient treatment burden and limited response rate together clearly ask for biomarkers to predict PEG-IFN response. Soluble CD14 (sCD14) is considered a marker for immune activation and has been shown to predict clinical outcome of HIV infection. However, studies on sCD14 in CHB infection are inconclusive, and its relationship with clinical outcome is largely unknown. Here, we measured sCD14 levels in CHB patients and investigated whether changes in sCD14 level related to PEG-IFN response. Serum sCD14 levels were determined in 15 healthy controls, 15 acute self-limited HBV, 60 CHB patients in different disease phases and 94 HBeAg+ CHB patients at week 0 and week 12 of a 52-week PEG-IFN treatment. Response to PEG-IFN treatment was defined as HBeAg seroconversion or HBeAg loss at 26 weeks post-treatment. The mean sCD14 level in acute HBV patients (3.0 µg/mL) was significantly higher than in CHB patients (2.4 µg/mL) and healthy controls (2.4 µg/mL). In CHB patients receiving PEG-IFN, a significant increase in sCD14 was found after 12-week treatment (median week 0:2.1 µg/mL; week 12:3.7 µg/mL). After 12-week treatment, the fold change (FC = w12/w0) in sCD14 was significantly higher in responders compared to nonresponders (HBeAg seroconversion: median FCresponder  = 2.1 vs FCnonresponder  = 1.6; HBeAg loss: median FCresponder  = 2.2 vs FCnonresponder  = 1.5). Receiver operating characteristic curves demonstrated that FC-sCD14wk12/wk0 levels can be of significant value as a stopping rule to select patients at week 12 who are not likely to benefit from further PEG-IFN treatment.

HBV-Derived Synthetic Long Peptide Can Boost CD4+ and CD8+ T-Cell Responses in Chronic HBV Patients Ex Vivo.

Background: Vaccination with synthetic long peptides (SLP) is a promising new treatment strategy for chronic hepatitis B virus (CHB). SLP can induce broad T-cell responses for all HLA types. Here we investigated the ability of a prototype HBV-core (HBc)-sequence-derived SLP to boost HBV-specific T cells in CHB patients ex vivo. Methods: HBc-SLP was used to assess cross-presentation by monocyte-derived dendritic cells (moDC) and BDCA1+ blood myeloid DC (mDC) to engineered HBV-specific CD8+ T cells. Autologous SLP-loaded and toll-like receptor (TLR)-stimulated DC were used to activate patient HBc-specific CD8+ and CD4+ T cells. Results: HBV-SLP was cross-presented by moDC, which was further enhanced by adjuvants. Patient-derived SLP-loaded moDC significantly increased autologous HBcAg18-27-specific CD8+ T cells and CD4+ T cells ex vivo. HBV-specific T cells were functional as they synthesized tumor necrosis factor-alpha and interferon-gamma. In 6/7 of patients blockade of PD-L1 further increased SLP effects. Also, importantly, patient-derived BDCA1+ mDC cross-presented and activated autologous T-cell responses ex vivo. Conclusions: As a proof of concept, we showed a prototype HBc-SLP can boost T-cell responses in patients ex vivo. These results pave the way for the development of a therapeutic SLP-based vaccine to induce effective HBV-specific adaptive immune responses in CHB patients.

Hepatitis B Virus Surface Antigen Activates Myeloid Dendritic Cells via a Soluble CD14-Dependent Mechanism.

UNLABELLED: Hepatitis B virus (HBV) infection can cause chronic liver disease, which is associated with increased risk of liver cirrhosis, liver failure, and liver cancer. Clearance of HBV infection requires effective HBV-specific immunity; however, the immunological mechanisms that determine the development of effective HBV-specific immunity are poorly understood. Dendritic cells (DC) play a pivotal role in the regulation of antiviral immunity. Here, we investigated the interaction between HBV surface antigen (HBsAg), the main envelope glycoprotein of HBV, and BDCA1(+) myeloid dendritic cells (mDC). Exposure of peripheral blood-derived BDCA1(+) mDC to HBsAg resulted in strong DC maturation, cytokine production, and enhanced capacity to activate antigen-specific cytotoxic T cells (CTLs). By using neutralizing antibodies, crucial roles for CD14 and Toll-like receptor 4 (TLR4) in HBsAg-mediated BDCA1(+) mDC maturation were identified. Concordantly, HBsAg-mediated DC maturation required fetal calf serum (FCS) or human plasma, naturally containing soluble CD14 (sCD14). Intriguingly, HBsAg-induced DC maturation was significantly reduced in umbilical cord blood plasma, which contained less sCD14 than adult plasma, indicating that sCD14 is an important host factor for recognition of HBsAg by DC and subsequent DC activation. A direct interaction between sCD14 and HBsAg was demonstrated by using enzyme-linked immunosorbent assay (ELISA). Moreover, sCD14-HBsAg complexes were detected both in vitro and in sera of HBV-infected patients. The abundance of sCD14-HBsAg complexes varied between chronic HBV disease stages and correlated with activation of BDCA1(+) mDC in vivo We conclude that HBsAg activates BDCA1(+) DC via an sCD14-dependent mechanism. These findings provide important novel insights into the initiation of HBV-specific immunity and facilitate development of effective immunotherapeutic interventions for HBV. IMPORTANCE: Hepatitis B virus (HBV) infection is a significant health problem, as it causes progressive liver injury and liver cancer in patients with chronic HBV infection, which affects approximately 250 million individuals worldwide. Some of the infected adults and the majority of neonates fail to mount an effective immune response and consequently develop chronic infection. The viral and host factors involved in the initiation of effective HBV-specific immune responses remain poorly understood. Here we identified CD14 and TLR4 as receptors for HBsAg, the main HBV envelope antigen. HBsAg induced strong maturation of dendritic cells (DC), which have a central role in regulation of virus-specific immunity. These results provide essential novel insights into the mechanisms underlying the initiation of HBV-specific immunity. Intriguingly, since neonates have naturally low sCD14, the finding that serum-derived sCD14 is a crucial host factor for recognition of HBsAg by DC may have implications for immunity of neonates to HBV infection.

PI3K-PKB hyperactivation augments human plasmacytoid dendritic cell development and function.

Plasmacytoid dendritic cells (pDCs) are considered potential tools or targets for immunotherapy. However, current knowledge concerning methodologies to manipulate their development or function remains limited. Here, we investigated the role of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB)-mammalian target of rapamycin (mTOR) axis in human pDC development, survival, and function. In vitro pDC generation from human cord blood-derived CD34(+) hematopoietic progenitors was reduced by pharmacologic inhibition of PI3K, PKB, or mTOR activity, and peripheral blood pDCs required PI3K-PKB-mTOR signaling to survive. Accordingly, activity of this pathway in circulating pDCs correlated with their abundance in peripheral blood. Importantly, introduction of constitutively active PKB or pharmacologic inhibition of negative regulator phosphatase and tensin homolog (PTEN) resulted in increased pDC numbers in vitro and in vivo. Furthermore, MHC class II and costimulatory molecule expression, and production of IFN-α and TNF-α, were augmented, which could be explained by enhanced IRF7 and NF-κB activation. Finally, the numerically and functionally impaired pDCs of chronic hepatitis B patients demonstrated reduced PI3K-PKB-mTOR activity. In conclusion, intact PI3K-PKB-mTOR signaling regulates development, survival, and function of human pDCs, and pDC development and functionality can be promoted by PI3K-PKB hyperactivation. Manipulation of this pathway or its downstream targets could be used to improve the generation and function of pDCs to augment immunity.

Tight control of STAT5 activity determines human CD34-derived interstitial dendritic cell and langerhans cell development.

Despite the crucial function of dendritic cells (DC) in immunity, the molecular mechanisms regulating human DC development remain poorly defined. STAT5 regulates various hematopoietic lineages and is activated by GM-CSF, a critical cytokine in DC development. In this study, we investigated the role of STAT5 during differentiation of human CD34(+) hematopoietic progenitors into precursor DC (pre-DC) and their subsequent differentiation toward interstitial DC and Langerhans cells. Inhibiting STAT5 activity by dominant-negative STAT5 promoted Langerhans cell commitment of hematopoietic progenitors but resulted in loss of pre-interstitial DC development, showing subset-specific regulation. Increasing the low endogenous STAT5 activity by ectopic STAT5 activation downregulated expression of the critical DC transcription factor PU.1 and abrogated commitment to either DC lineage. In contrast, high STAT5 activity was beneficial in already committed pre-DC: terminal DC differentiation was associated with increased endogenous STAT5 phosphorylation levels, JAK2-STAT5 inhibition reduced terminal DC differentiation, and conditional STAT5 activation in pre-DC improved development of BDCA-1(+), DC-SIGN(+), and Langerin(+) DC with normal maturation and T cell stimulation. These data show that STAT5 critically regulates human DC development, with specific requirements for the level of STAT5 activation at distinct differentiation stages. By regulating STAT5 activity, cytokines present at specific locations and under different pathophysiological conditions can determine the fate of DC precursors.

A nonredundant role for canonical NF-κB in human myeloid dendritic cell development and function.

The plastic role of dendritic cells (DCs) in the regulation of immune responses has made them interesting targets for immunotherapy, but also for pathogens or tumors to evade immunity. Functional alterations of DCs are often ascribed to manipulation of canonical NF-κB activity. However, though this pathway has been linked to murine myeloid DC biology, a detailed analysis of its importance in human myeloid DC differentiation, survival, maturation, and function is lacking. The myeloid DC subsets include interstitial DCs and Langerhans cells. In this study, we investigated the role of canonical NF-κB in human myeloid DCs generated from monocytes (monocyte-derived DCs [mo-DCs]) or CD34(+) progenitors (CD34-derived myeloid DCs [CD34-mDCs]). Inhibition of NF-κB activation during and after mo-DC, CD34-interstitial DC, or CD34-Langerhans cell differentiation resulted in apoptosis induction associated with caspase 3 activation and loss of mitochondrial transmembrane potential. Besides regulating survival, canonical NF-κB activity was required for the acquisition of a DC phenotype. Despite phenotypic differences, however, Ag uptake, costimulatory molecule and CCR7 expression, as well as T cell stimulatory capacity of cells generated under NF-κB inhibition were comparable to control DCs, indicating that canonical NF-κB activity during differentiation is redundant for the development of functional APCs. However, both mo-DC and CD34-mDC functionality were reduced by NF-κB inhibition during activation. In conclusion, canonical NF-κB activity is essential for the development and function of mo-DCs as well as CD34-mDCs. Insight into the role of this pathway may help in understanding how pathogens and tumors escape immunity and aid in developing novel treatment strategies aiming to interfere with human immune responses.