Major HBV splice variant encoding a novel protein important for infection.

BACKGROUND & AIMS: HBV expresses more than 10 spliced RNAs from the viral pregenomic RNA, but their functions remain elusive and controversial. To address the function of HBV spliced RNAs, we generated splicing-deficient HBV mutants and conducted experiments to assess the impact of these mutants on HBV infection. METHODS: HepG2-NTCP cells, human hepatocyte chimeric FRG mice (hu-FRG mice), and serum from patients with chronic hepatitis B were used for experiments on HBV infection. Additionally, SHifter assays and cryo-electron microscopy were performed. RESULTS: We found the infectivity of splicing-deficient HBV was decreased 100-1,000-fold compared with that of wild-type HBV in hu-FRG mice. Another mutant, A487C, which loses the most abundant spliced RNA (SP1), also exhibits severely impaired infectivity. SP1 hypothetically encodes a novel protein HBcSP1 (HBc-Cys) that lacks the C-terminal cysteine from full-length HBc. In the SHifter assay, HBcSP1 was detected in wild-type viral particles at a ratio of about 20-100% vs. conventional HBc, as well as in the serum of patients with chronic hepatitis B, but not in A487C particles. When infection was conducted with a shorter incubation time of 4-8 h at lower PEG concentrations in HepG2-NTCP cells, the entry of the A487C mutant was significantly slower. SP1 cDNA complementation of the A487C mutant succeeded in rescuing its infectivity in hu-FRG mice and HepG2-NTCP cells. Moreover, cryo-electron microscopy revealed a disulfide bond between HBc cysteine 183 and 48 in the HBc intradimer of the A487C capsid, leading to a locked conformation that disfavored viral entry in contrast to the wild-type capsid. CONCLUSIONS: Prior studies unveiled the potential integration of the HBc-Cys protein into the HBV capsid. We confirmed the proposal and validated its identity and function during infection. IMPACT AND IMPLICATIONS: HBV SP1 RNA encodes a novel HBc protein (HBcSP1) that lacks the C-terminal cysteine from conventional HBc (HBc-Cys). HBcSP1 was detected in cell culture-derived HBV and confirmed in patients with chronic infection by both immunological and chemical modification assays at 10-50% of capsid. The splicing-deficient mutant HBV (A487C) impaired infectivity in human hepatocyte chimeric mice and viral entry in the HepG2-NTCP cell line. Furthermore, these deficiencies of the splicing-deficient mutant could be rescued by complementation with the SP1-encoded protein HBcSP1. We confirmed and validated the identity and function of HBcSP1 during infection, building on the current model of HBV particles.

Mapping the conformational epitope of a therapeutic monoclonal antibody against HBsAg by in vivo selection of HBV escape variants.

BACKGROUND AND AIMS: Hepatitis B immunoglobulin (HBIG) has been routinely applied in the liver transplantation setting to block HBV reinfection of grafts. However, new monoclonal anti-HBV surface antibodies have been developed to replace HBIG. The epitopes of such monoclonal antibodies may affect the emergence of escape variants and deserve study. APPROACH AND RESULTS: The conformational epitope of sLenvervimab, a surrogate form of Lenvervimab, which is a monoclonal anti-HBsAg antibody currently under phase 3 trial, was investigated by selecting escape mutants from a human liver chimeric mouse. HBV-infected chimeric mice treated with sLenvervimab monotherapy showed an initial decline in circulating HBsAg levels, followed by a quick rebound in 1 month. Sequencing of circulating or liver HBV DNA revealed emerging variants, with replacement of amino acid E164 or T140, two residues widely separated in HBsAg. E164 HBV variants strongly resisted sLenvervimab neutralization in cell culture infection, and the T140 variant moderately resisted sLenvervimab neutralization. Natural HBV variants with amino-acid replacements adjacent to E164 were constructed and examined for sLenvervimab neutralization effects. Variants with K160 replacement also resisted neutralization. These data revealed the conformational epitope of sLenvervimab. CONCLUSIONS: Selection of antibody-escape HBV variants in human chimeric mice works efficiently. Analysis of such emerging variants helps to identify anchor amino-acid residues of the conformational epitope that are difficult to discover by conventional approaches.

Redox sensor NPGPx restrains ZAP70 activity and modulates T cell homeostasis.

Emerging evidences implicate the contribution of ROS to T cell activation and signaling. The tyrosine kinase, ζ-chain-associated protein of 70 kDa (ZAP70), is essential for T cell development and activation. However, it remains elusive whether a direct redox regulation affects ZAP70 activity upon TCR stimulation. Here, we show that deficiency of non-selenocysteine containing phospholipid hydroperoxide glutathione peroxidase (NPGPx), a redox sensor, results in T cell hyperproliferation and elevated cytokine productions. T cell-specific NPGPx-knockout mice reveal enhanced T-dependent humoral responses and are susceptible to experimental autoimmune encephalomyelitis (EAE). Through proteomic approaches, ZAP70 is identified as the key interacting protein of NPGPx through disulfide bonding. NPGPx is activated by ROS generated from TCR stimulation, and modulates ZAP70 activity through redox switching to reduce ZAP70 recruitment to TCR/CD3 complex in membrane lipid raft, therefore subduing TCR responses. These results reveal a delicate redox mechanism that NPGPx serves as a modulator to curb ZAP70 functions in maintaining T cell homeostasis.

Differentiation of remitting neuromyelitis optica spectrum disorders from multiple sclerosis by integrating parameters from serum proteins and lymphocyte subsets.

Differential diagnosis for neuromyelitis optica spectrum disorder (NMOSD) and multiple sclerosis (MS) is always doubtful. To differentiate these diseases, we studied the immune status in the blood of patients with MS (n = 45) or NMOSD (n = 23) at remission phase. Remitting NMOSD patients had increased levels of CXCL13 and memory B cells, while remitting MS patients had elevated levels of galectin-9 and Th1 cells. A diagnostic model with these four variables is built to distinguish remitting NMOSD from MS with a sensitivity of 91.30%. Our diagnostic model may help to improve the differentiation of remitting NMOSD from MS.

Peripheral sensory neuron injury contributes to neuropathic pain in experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS)-induced neuropathic pain deteriorates quality of life in patients but is often refractory to treatment. In experimental autoimmune encephalomyelitis (EAE), a rodent model of MS, animals develop neuropathy and inflammation-induced tissue acidosis, which suggests the involvement of acid-sensing ion channels (ASICs). Also, peripheral neuropathy is reported in MS patients. However, the involvement of the peripheral nervous system (PNS) in MS neuropathic pain remains elusive. This study investigated the contribution of ASICs and peripheral neuropathy in MS-induced neuropathic pain. Elicited pain levels were as high in Asic1a-/-, Asic2-/- and Asic3-/- mice as wild-type mice even though only Asic1a-/- mice showed reduced EAE disease severity, which indicates that pain in EAE was independent of disease severity. We thus adopted an EAE model without pertussis toxin (EAEnp) to restrain activated immunity in the periphery and evaluate the PNS contribution to pain. Both EAE and EAEnp mice showed similar pain behaviors and peripheral neuropathy in nerve fibers and DRG neurons. Moreover, pregabalin significantly reduced neuropathic pain in both EAE and EAEnp mice. Our findings highlight the essential role of the PNS in neuropathic pain in EAE and pave the way for future development of analgesics without side effects in the CNS.

CXCR3 signaling in glial cells ameliorates experimental autoimmune encephalomyelitis by restraining the generation of a pro-Th17 cytokine milieu and reducing CNS-infiltrating Th17 cells.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) is a mouse model of multiple sclerosis (MS). It has been shown that Th17 cells are critical for EAE pathogenesis. Mice lacking CXCR3 develop aggravated EAE compared with wild-type (WT) mice. This study investigated the effect of CXCR3 on Th17 expansion during EAE and further addressed the underlying mechanism. METHODS: Both active EAE and adoptive-transfer EAE experiments were employed for studying EAE pathogenesis in WT and CXCR3(-/-) mice. Demyelination and leukocyte infiltration in the spinal cord of mice were analyzed by luxol fast blue staining and flow cytometry analysis, respectively. Glial cells expressing CXCR3 in the spinal cord were analyzed by immunofluorescence staining. Cytokine and chemokine levels in the spinal cord were analyzed using quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA). The glial cell line U87MG was employed for studying the CXCR3 signaling-mediated mechanism regulating Th17 expansion. RESULTS: CXCR3(-/-) mice exhibited more severe EAE and had significantly increased central nervous system (CNS)-infiltrating Th17 cells compared with WT mice. Adoptive-transfer experiments showed that CXCR3(-/-) recipient mice that received Th17 cells polarized from splenocytes of myelin oligodendrocyte glycoprotein (MOG)-immunized CXCR3(-/-) mice or MOG-immunized WT mice always developed more severe EAE and had significantly increased CNS-infiltrating Th17 cells compared with WT recipient mice that received Th17 cells from the same origin. Furthermore, during EAE, the number of activated glial cells was increased in the CNS of MOG-immunized CXCR3(-/-) mice, and CXCR3-deficient glial cells expressed increased levels of cytokine genes required for Th17 expansion and recruitment. Finally, we found that extracellular signal-regulated kinase (ERK) activation elicited by CXCR3 signaling in U87MG cells attenuated the activation of NF-κB, a key transcription factor critical for the induction of IL-23 and CCL20, which are required for Th17 cell expansion and recruitment, respectively. CONCLUSIONS: This study demonstrates a previously unrecognized role of CXCR3 signaling in glial cells in negatively regulating Th17 cell expansion during EAE. Our results demonstrate that, in addition to its well-known role in the recruitment of immune cells, CXCR3 in CNS glial cells plays a critical role in restraining the pro-Th17 cytokine/chemokine milieu during EAE, thereby diminishing Th17 cell expansion in the CNS and suppressing disease development.

Peptide-based immunotherapy of experimental autoimmune encephalomyelitis without anaphylaxis.

Administration of peptide antigens in tolerogenic form holds promise as a specific treatment for autoimmune and allergic disorders. However, experiments in rodent autoimmune models have highlighted the risk of anaphylaxis in response to systemic peptide application once the aberrant immune response is underway. Thus, mice with clinical signs of experimental autoimmune encephalomyelitis (EAE) or diabetes have been reported to suffer fatal anaphylaxis upon administration of native autoantigenic peptides. Clearly, this might represent a significant barrier to the use of synthetic peptides in the treatment of ongoing human autoimmune conditions. Here we describe the development of an altered peptide ligand (APL) engineered to prevent anaphylaxis (no antibody binding) whilst retaining the ability to silence pathogenic myelin-reactive T lymphocytes. Administration of the APL to mice with an ongoing anti-myelin immune response did not cause anaphylaxis, but led to complete protection from the subsequent induction of EAE and, when given during ongoing EAE, led to a rapid remission of clinical signs. The approach of removing antibody recognition whilst maintaining the desired functional effect (in this case T cell tolerance) may be of value in other situations in which there is a risk of triggering anaphylaxis with peptide-based drugs.

Correction of malignant behavior of tumor cells by traditional Chinese herb medicine through a restoration of p53.

We have previously demonstrated that a UVC-induced tumorigenic HeLa x skin fibroblast cell line could be induced to form a more normal phenotypic state ('reversion'), including loss of IAP expression. We have now used the loss of IAP expression to monitor the enhancement of this reversion in the cervical cancer cell line, HeLa, by a traditional Chinese herb medicine (TCM), Yigan Kang (YGK). IAP level decreased, and the reversion frequency increased, in a dose-dependent manner at concentrations of YGK of more than 10 mg. YGK significantly repressed E6/E7 oncogenes at the transcriptional level, with subsequent reactivation of p53 and p21 expression (P<0.01). YGK had little effect on the cell cycle of HeLa cells and slightly increased the apoptotic cell death between 20 and 40 mg. In vivo, tumorigenicity studies were performed using six different animal experimental protocols, which demonstrated that YGK was effective at inducing reversion of the tumorigenic phenotype, with YGK-treated HeLa cells showing much less aggressive tumor growth than untreated cells. YGK may raise the possibility of the continuing management of some cancers as a chronic condition in which the malignant behavior of the tumor cells is constrained.

IL-15Ralpha is a negative regulator of TCR-activated proliferation in CD4+ T cells.

Although IL-15 is known to be a T cell growth factor, the function in T cells of IL-15Ralpha, its high affinity receptor, remains unclear. We found that murine IL-15Ralpha(-/-) CD4(+) T cells hyperproliferated in response to TCR stimulation, in vitro and in vivo, and displayed a lower TCR activation threshold than wild-type CD4(+) T cells. TCR-induced activation of Zap70 and of the phospholipase C-gamma1-NFATp, Ras-ERK-c-Fos, and Rac-JNK-c-Jun pathways was all augmented in IL-15Ralpha(-/-) CD4(+) T cells. This in turn led to earlier IL-2Ralpha induction and higher IL-2 production, which most likely contribute to the hyperproliferation of IL-15Ralpha(-/-) CD4(+) T cells. Exogenous IL-15 reduced levels of TCR-activated signals, transcription factors, IL-2, and IL-2Ralpha, and division in wild-type CD4(+) T cells. These results reveal IL-15Ralpha to be a negative regulator for CD4(+) T cell activation and demonstrate a novel layer of regulation of TCR signaling by a cytokine system.