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Early and effective identification of severe coronavirus disease 2019 (COVID-19) may allow us to improve the outcomes of associated severe acute respiratory illness with fever and respiratory symptoms. This study analyzed plasma concentrations of heat shock protein gp96 in nonsevere (including mild and typical) and severe (including severe and critical) patients with COVID-19 to evaluate its potential as a predictive and prognostic biomarker for disease severity. Plasma gp96 levels that were positively correlated with interleukin-6 (IL-6) levels were significantly elevated in COVID-19 patients admitted to the hospital but not in non-COVID-19 patients with less severe respiratory impairment. Meanwhile, significantly higher gp96 levels were observed in severe than nonsevere patients. Moreover, the continuous decline of plasma gp96 levels predicted disease remission and recovery, whereas its persistently high levels indicated poor prognosis in COVID-19 patients during hospitalization. Finally, monocytes were identified as the major IL-6 producers under exogenous gp96 stimulation. Our results demonstrate that plasma gp96 may be a useful predictive and prognostic biomarker for disease severity and outcome of COVID-19. IMPORTANCE Early and effective identification of severe COVID-19 may allow us to improve the outcomes of associated severe acute respiratory illness with fever and respiratory symptoms. Some heat shock proteins (Hsps) are released during oxidative stress, cytotoxic injury, and viral infection and behave as danger-associated molecular patterns (DAMPs). This study analyzed plasma concentrations of Hsp gp96 in nonsevere and severe patients with COVID-19. Significantly higher plasma gp96 levels were observed in severe than those in nonsevere patients, and its persistently high levels indicated poor prognosis in COVID-19 patients. The results demonstrate that plasma gp96 may be a useful predictive and prognostic biomarker for disease severity and outcome of COVID-19.

Summary Upregulation and stabilization of Foxp3 expression in Tregs are essential for regulating Treg function and immune homeostasis. In this study, gp96 immunization showed obvious therapeutic effects in a Lyn–/– mouse model of systemic lupus erythematosus. Moreover, gp96 alleviated the initiation and progression of MOG-induced experimental autoimmune encephalomyelitis. Immunization of gp96 increased Treg frequency, expansion, and suppressive function. Gene expression profiling identified the NF-κB family member p65 and c-Rel as the key transcription factors for enhanced Foxp3 expression in Treg by gp96. Mutant gp96 within its Toll-like receptor (TLR) binding domain, TLR2 knockout mice, and mice with cell-specific deletion of MyD88, were used to demonstrate that gp96 activated Tregs and induced Foxp3 expression via a TLR2-MyD88-mediated NF-κB signaling pathway. Taken together, these results show that gp96 immunization restricted antibody-induced and Th-induced autoimmune diseases by integrating Treg expansion and activation, indicating its potential clinical usefulness against autoimmune diseases., Graphical abstract, Highlights • SLE symptoms in Lyn–/– mice are ameliorated by gp96 immunization • Tregs expanded by gp96 provide potential in suppressing Th-mediated EAE • Gp96 promotes Treg proliferation, stability, and suppressive function • Gp96 binds to and activates Treg in a TLR2-MyD88-NF-кB-Foxp3 pathway, Immune response, Genomics, Molecular biology

In hepatitis B virus (HBV) infection, the virus produces redundant hepatitis B surface antigen (HBsAg) that plays a key role in driving T-cell tolerance and viral persistence. However, currently available anti-HBV agents have no direct effect on HBsAg transcription and protein expression. In this study, we designed a heat shock protein gp96 inhibitor p37 with the cell penetrating peptide PTD (protein transduction domain of trans-activator of transcription), which mediated p37 internalization into hepatocytes. PTD-p37 effectively suppressed HBsAg expression and viral replication both in vitro and in vivo. We further provide evidence that PTD-p37 suppressed HBV enhancer/promoter activity via p53 upregulation. Moreover, PTD-p37 had antiviral activity against a lamivudine-resistant HBV strain. Considering that suppression of HBsAg expression is a major goal for treatment of HBV infection, our results provide a basis for developing a new therapeutic approaches targeting host factors against viral expression.

Currently, immunization with inactivated influenza virus vaccines is the most prevalent method to prevent infections. However, licensed influenza vaccines provide only strain-specific protection and need to be updated and administered yearly; thus, new vaccines that provide broad protection against multiple influenza virus subtypes are required. In this study, we demonstrated that intradermal immunization with gp96-adjuvanted seasonal influenza monovalent H1N1 split vaccine could induce cross-protection against both group 1 and group 2 influenza A viruses in BALB/c mouse models. Vaccination in the presence of gp96 induced an apparently stronger antigen-specific T cell response than split vaccine alone. Immunization with the gp96-adjuvanted vaccine also elicited an apparent cross-reactive CD8+ T cell response that targeted the conserved epitopes across different influenza virus strains. These cross-reactive CD8+ T cells might be recalled from a pool of memory cells established after vaccination and recruited from extrapulmonary sites to facilitate viral clearance. Of note, six highly conserved CD8+ T epitopes from the viral structural proteins hemagglutinin (HA), M1, nucleoprotein (NP), and PB1 were identified to play a synergistic role in gp96-mediated cross-protection. Comparative analysis showed that most of conservative epitope-specific cytotoxic T lymphocytes (CTLs) apparently induced by heterologous virus infection were also activated by gp96-adjuvanted vaccine, thus resulting in broader protective CD8+ T cell responses. Our results demonstrated the advantage of adding gp96 to an existing seasonal influenza vaccine to improve its ability to provide better cross-protection.IMPORTANCE Owing to continuous mutations in hemagglutinin (HA) or neuraminidase (NA) or recombination of the gene segments between different strains, influenza viruses can escape the immune responses developed by vaccination. Thus, new strategies aimed to efficiently activate immune response that targets to conserved regions among different influenza viruses are urgently needed in designing broad-spectrum influenza vaccine. Heat shock protein gp96 is currently the only natural T cell adjuvant with special ability to cross-present coupled antigen to major histocompatibility complex class I (MHC-I) molecule and activate the downstream antigen-specific CTL response. In this study, we demonstrated the advantages of adding gp96 to monovalent split influenza virus vaccine to improve its ability to provide cross-protection in the BALB/c mouse model and proved that a gp96-activated cross-reactive CTL response is indispensable in our vaccine strategy. Due to its unique adjuvant properties, gp96 might be a promising adjuvant for designing new broad-spectrum influenza vaccines.

Utilizing regulatory T cells (Treg) is a promising approach for treating autoimmune diseases, including systemic lupus erythematosus (SLE) and rheumatic diseases. Upregulation and stabilization of forkhead box P3 (Foxp3) expression in Tregs are essential for regulating Treg function and immune homeostasis. In this study, we examined the potential effect and mechanism of heat shock protein gp96 in regulating Foxp3 expression and Treg activation in vivo. High-dose gp96 immunization showed obvious therapeutic effects in a Lyn-/- mouse model of SLE, as evidenced by decreased auto-antibody titers, follicular helper T cells, and antibody-production plasma cells. Moreover, immunization of mice with gp96 alleviated the initiation and progression of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE). Immunization of gp96 increased Treg frequency, expansion, and suppressive function with no obvious effector T cell activation. Gene expression profiling was used to identify the NF-κB family member p65 and c-Rel as the key transcription factors for enhanced Foxp3 expression in Treg by gp96. Mutant gp96 within its Toll-like receptor (TLR) binding domain, TLR2 knockout (KO) mice, and mice with cell-specific deletion of MyD88, were used to demonstrate that gp96 activated Tregs and induced Foxp3 expression via a TLR2-MyD88-mediated NF-κB signaling pathway. Taken together, these results show that gp96 immunization restricted antibody- and Th-induced autoimmune diseases by integrating Treg expansion and activation, indicating its potential clinical usefulness against autoimmune diseases.

Liver failure leads to the massive necrosis of hepatocytes, releasing large amounts of intracellular components including damage-associated molecular patterns (DAMPs). We found that extracellular gp96 levels in serum were elevated in patients with chronic hepatitis B infection (CHB) and acute-on-chronic liver failure (ACLF). Meanwhile, the gp96 level positively correlated with hepatic necroinflammation. We employed two mouse liver damage and liver failure models induced by lipopolysaccharide (LPS) plus d-galactosamine (d-Galn), and concanavalin A (ConA) to identify the function of extracellular gp96. As a result, the inhibition of extracellular gp96 by a specific peptide efficiently mitigated both LPS/d-Galn- and ConA-induced liver injury and immune hyperactivation, whereas exogenous gp96 aggravated the symptoms of hepatic injury in mice but not in Kupffer cells-ablated mice. The exposure of Kupffer cells to gp96 induced the secretion of pro-inflammatory cytokines. Collectively, our data demonstrate that gp96 released from necrotic hepatocytes aggravates immune hyperactivation and promotes liver damage and possibly the development of liver failure mainly by activating Kupffer cells.

Programmed death ligand 1 (PD-L1) has been recently shown to be a major obstacle to antiviral immunity by binding to its receptor programmed death 1 (PD-1) on specific IFN-γ producing T cells in chronic hepatitis B. Currently, IFN-α is widely used to treat hepatitis B virus (HBV) infection, but its antiviral effect vary greatly and the mechanism is not totally clear. We found that IFN-α/γ induced a marked increase of PD-L1 expression in hepatocytes. Signal and activators of transcription (Stat1) was then identified as a major transcription factor involved in IFN-α/γ-mediated PD-L1 elevation both in vitro and in mice. Blockage of the PD-L1/PD-1 interaction by a specific mAb greatly enhanced HBV-specific T cell activity by the gp96 adjuvanted therapeutic vaccine, and promoted HBV clearance in HBV transgenic mice. Our results demonstrate the IFN-α/γ-Stat1-PD-L1 axis plays an important role in mediating T cell hyporesponsiveness and inactivating liver-infiltrating T cells in the hepatic microenvironment. These data raise further potential interest in enhancing the anti-HBV efficacy of IFN-α and therapeutic vaccines.

The reciprocal interaction between influenza virus and host microRNAs (miRNAs) has been implicated in the regulation of viral replication and host tropism. However, the global roles of the cellular miRNA repertoire and the mechanisms of miRNA-mediated antiviral defense await further elucidation. In this study, we systematically screened 297 cellular miRNAs from human and mouse epithelial cells and identified five inhibitory miRNAs that efficiently inhibited influenza virus replication in vitro and in vivo. Among these miRNAs, hsa-mir-127-3p, hsa-mir-486-5p, hsa-mir-593-5p, and mmu-mir-487b-5p were found to target at least one viral gene segment of both the human seasonal influenza H3N2 and the attenuated PR8 (H1N1) virus, whereas hsa-miR-1-3p inhibited viral replication by targeting the supportive host factor ATP6V1A. Moreover, the number of miRNA binding sites in viral RNA segments was positively associated with the activity of host miRNA-induced antiviral defense. Treatment with a combination of the five miRNAs through agomir delivery pronouncedly suppressed viral replication and effectively improved protection against lethal challenge with PR8 in mice. These data suggest that the highly expressed miRNAs in respiratory epithelial cells elicit effective antiviral defenses against influenza A viruses and will be useful for designing miRNA-based therapies against viral infection.

Programmed death ligand 1 (PD-L1) has been recently shown to be a major obstacle to antiviral immunity by binding to its receptor programmed death 1 (PD-1) on specific IFN-γ producing T cells in chronic hepatitis B. Currently, IFN-α is widely used to treat hepatitis B virus(HBV) infection, but its antiviral effect vary greatly and the mechanism is not totally clear. We found that IFN-α/γ induced a marked increase of PD-L1 expression in hepatocytes. Signal and activators of transcription (Stat1) was then identified as a major transcription factor involved in IFN-α/γ-mediated PD-L1 elevation both in vitro and in mice. Blockage of the PD-L1/PD-1 interaction by a specific mAb greatly enhanced HBV-specific T cell activity by the gp96 adjuvanted therapeutic vaccine, and promoted HBV clearance in HBV transgenic mice. Our results demonstrate the IFN-α/γ-Stat1-PD-L1 axis plays an important role in mediating T cell hyporesponsiveness and inactivating liver-infiltrating T cells in the hepatic microenvironment. These data raise further potential interest in enhancing the anti-HBV efficacy of IFN-α and therapeutic vaccines.

The coordinated recognition of virus-derived T cell epitopes and MHC molecules by T cells plays a pivotal role in cellular immunity–mediated virus clearance. It has been demonstrated that the conformation of MHC class I (MHC I) molecules can be adjusted by the presented peptide, which impacts T cell activation. However, it is still largely unknown whether the conformational shift of MHC I influences the protective effect of virus-specific T cells. In this study, utilizing the Middle East respiratory syndrome coronavirus–infected mouse model, we observed that through the unusual secondary anchor Ile5, a CD8+ T cell epitope drove the conformational fit of Trp73 on the α1 helix of murine MHC I H-2Kd. In vitro renaturation and circular dichroism assays indicated that this shift of the structure did not influence the peptide/MHC I binding affinity. Nevertheless, the T cell recognition and the protective effect of the peptide diminished when we made an Ile to Ala mutation at position 5 of the original peptide. The molecular bases of the concordant recognition of T cell epitopes and host MHC-dependent protection were demonstrated through both crystal structure determination and tetramer staining using the peptide–MHC complex. Our results indicate a coordinated MHC I/peptide interaction mechanism and provide a beneficial reference for T cell–oriented vaccine development against emerging viruses such as Middle East respiratory syndrome coronavirus.

The commonly used Bacillus Calmette-Guérin (BCG) vaccine only induces moderate T cell responses and is less effective in protecting against pulmonary tuberculosis (TB) in adults and ageing populations. Thus, developing new TB vaccine candidates is an important strategy against the spread of Mycobacterium tuberculosis. Here, we demonstrated that immunization with heat-shock protein gp96 as an adjuvant led to a significantly increased CD4(+) and CD8(+) T cell response to a BCG vaccine. Secretion of the Th1-type cytokines was increased by splenocytes from gp96-immunized mice. In addition, adding gp96 as an adjuvant effectively improved the protection against intravenous challenge with Mycobacterium bovis BCG in mice. Our study reveals the novel property of gp96 in boosting the vaccine-specific T cell response and its potential use as an adjuvant for BCG vaccines against mycobacterial infection.

Under the immune pressure of cytotoxic T cells (CTLs), hepatitis B virus (HBV) evolves to accumulate mutations more likely within epitopes to evade immune detection. However, little is known about the specific patterns of the immune pressure-associated HBV mutation of T-cell epitopes and their link to disease progression. Here, we observed a correlation of the accumulated variants on HBV core protein (HBc) with the disease severity of HBV infection. Further analysis indicated that these substitutions were mostly located within CD8+ T-cell epitopes of HBc protein, which were systematically screened and identified in an unbiased manner in our study. From individual peptide level to the human leukocyte antigen I (HLA-I)-restricted population level, we elucidated that the mutations in these well-defined HLA-I-restricted T-cell epitopes significantly decreased antiviral activity-specific CTLs and were positively associated with clinical parameters and disease progression in HBV-infected patients. The molecular pattern for viral epitope variations based on the sequencing of 105 HBV virus genomes indicated that the C-terminal portion (Pc), especially the Pc-1 and Pc-2 positions, have the highest mutation rates. Further structural analysis of HLA-A*02 complexed to diverse CD8+ T-cell epitopes revealed that the highly variable C-terminal bulged peak of M-shaped HBc-derived epitopes are solvent exposed, and most of the CDR3βs of the T-cell receptor hover over them. These data shed light on the molecular and immunological mechanisms of T-cell immunity-associated viral evolution in hepatitis B progression, which is beneficial for designing immunotherapies and vaccines.IMPORTANCE The specific patterns of sequence polymorphisms of T-cell epitopes and the immune mechanisms of the HBV epitope mutation-linked disease progression are largely unclear. In this study, we systematically evaluated the contribution of CD8+ T cells to the disease progress-associated evolution of HBV. By evaluation of patient T-cell responses based on the peptide repertoire, we comprehensively characterized the association of clinical parameters in chronic hepatitis B with the antiviral T-cell response-associated mutations of the viruses from the single-epitope level to the overall HLA-I-restricted peptide levels. Furthermore, we investigated the molecular basis of the HLA-A2-restricted peptide immune escape and found that the solvent-exposed C-terminal portion of the epitopes is highly variable under CDR3β recognition. Our work may provide a comprehensive evaluation of viral mutations impacted by the host CTL response in HBV disease progression in the context of the full repertoire of HBc-derived epitopes.

Background HER2 gene amplification generates an enormous number of HER2 transcripts, but the global effects on endogenous miRNA targets including HER family members in breast cancer are unexplored. Methods We generated a HER2–3’UTR expressing vector to test the tumor-promoting properties in HER2 low expressing T47D and MCF7 cells. Through microarray analysis and real-time PCR analysis we identified genes that were regulated by HER2–3’UTR. Positive and negative manipulation of miRNA expression, response element mutational studies and transcript reporter assays were performed to explore the mechanism of competitive sequestration of miR125a/miRNA125b by HER2 3’UTR. To investigate if trastuzumab-induced upregulation of HER3 is also mediated through miRNA de-repression, we used the CRISPR/cas9 to mutate the endogenous HER2 mRNA in HER2 over-expressing Au565 cells. Finally, we looked at cohorts of breast cancer samples of our own and the TCGA to show if HER2 and HER3 mRNAs correlate with each other. Results The HER2 3’UTR pronouncedly promoted cell proliferation, colony formation, and breast tumor growth. High-throughput sequencing revealed a significant increase in HER3 mRNA and protein levels by the HER2 3’untranslated region (3’UTR). The HER2 3’UTR harboring a shared miR-125a/b response element induced miR-125a/b sequestration and thus resulted in HER3 mRNA derepression. Trastuzumab treatment upregulated HER3 via elevated HER2 mRNA expression, leading to trastuzumab resistance. Depletion of miR-125a/b enhanced the antitumor activity of trastuzumab. Microarray data from HER2-overexpressing primary breast cancer showed significant elevation of mRNAs for predicted miR-125a/b targets compared to non-targets. Conclusions These results suggest that HER2 3’UTR-mediated HER3 upregulation is involved in breast cell transformation, increased tumor growth, and resistance to anti-HER2 therapy. The combinatorial targeting of HER3 mRNA or miR-125a/b may offer an effective tool for breast cancer therapy. Electronic supplementary material The online version of this article (10.1186/s12943-018-0862-5) contains supplementary material, which is available to authorized users.

Persistent inflammation in chronic hepatitis plays a major role in the development of hepatocellular carcinoma (HCC). In this study, the major inflammatory cytokines expressed in chronic hepatitis, IL-6 and TNF-α, induced a marked decrease in microRNA-122 (miR-122) levels, and miR-122 expression was downregulated in the livers of chronic hepatitis B (CHB) patients. The decrease of miR-122 caused upregulation of the proinflammatory chemokine CCL2. IL-6 and TNF-α suppressed miR-122 both by directly downregulating the transcription factor C/EBPα and indirectly upregulating c-myc, which blocks C/EBPα-mediated miR-122 transcription. In addition, IL-6 and TNF-α levels were elevated and miR-122 levels were decreased in mouse and rat models of diethylnitrosamine (DEN)-induced HCC. Restoration of miR-122 levels through delivery of agomir-122 suppressed DEN-induced hepatocarcinogenesis in mice. Our results show that inflammation-induced miR-122 downregulation in hepatitis contributes to carcinogenesis and suggest that increasing miR-122 may be an effective strategy for preventing HCC development in CHB patients.

The resistance to apoptosis displayed by liver cancer plays a key role in hepatocarcinogenesis, tumor progression, and resistance to chemo- or radio-therapy. In this study, we uncovered the potential role and mechanism of heat shock protein gp96 in regulating liver tumor cell growth and apoptosis. P53 protein was identified as a gp96 client protein by profiling apoptosis-related proteins in gp96-knockdown liver cancer cells. Overexpression and knockdown studies both demonstrated that gp96 decreases p53 protein levels, and gp96 regulated cell apoptosis in a p53-dependent manner. We further provide evidence that gp96 interacts with both p53 and Mdm2 to enhance Mdm2-mediated p53 ubiquitination and degradation. Moreover, targeting gp96 with siRNA induced cell apoptosis and led to the suppression of liver tumor growth in vivo. In conclusion, we elucidated an underlying mechanism by which gp96 promotes p53 degradation via increasing Mdm2 E3 ligase activity and provided a new therapeutic strategy to target the gp96-mediated anti-apoptotic characteristic of hepatocellular carcinoma.

HER2 receptor dimerization is a critical step in the HER2 activation process. Here, we demonstrated that heat shock protein gp96 on cell membrane interacts with HER2, facilitates HER2 dimerization and promotes cell proliferation. Cell membrane gp96 levels were observed to correlate with HER2 phosphorylation in primary breast tumors. Finally, we provide evidence that targeting gp96 with a specific monoclonal antibody led to decreased cell growth and increased apoptosis in vitro, and suppression of tumor growth in vivo. Our work represents a new therapeutic strategy for inhibiting HER2 signaling in cancer.

During virus infection, viral RNAs and mRNAs function as blueprints for viral protein synthesis and possibly as pathogen-associated molecular patterns (PAMPs) in innate immunity. Here, considering recent research progress in microRNAs (miRNAs) and competitive endogenous RNAs (ceRNAs), we speculate that viral RNAs act as sponges and can sequester endogenous miRNAs within infected cells, thus cross-regulating the stability and translational efficiency of host mRNAs with shared miRNA response elements. This cross-talk and these reciprocal interactions between viral RNAs and host mRNAs are termed “competitive viral and host RNAs” (cvhRNAs). We further provide recent experimental evidence for the existence of cvhRNAs networks in hepatitis B virus (HBV), as well as Herpesvirus saimiri (HVS), lytic murine cytomegalovirus (MCMV) and human cytomegalovirus (HCMV) infections. In addition, the cvhRNA hypothesis also predicts possible cross-regulation between host and other viruses, such as hepatitis C virus (HCV), HIV, influenza virus, human papillomaviruses (HPV). Since the interaction between miRNAs and viral RNAs also inevitably leads to repression of viral RNA function, we speculate that virus may evolve either to employ cvhRNA networks or to avoid miRNA targeting for optimal fitness within the host. CvhRNA networks may therefore play a fundamental role in the regulation of viral replication, infection establishment, and viral pathogenesis.

The demonstration that for infectious diseases vaccine-induced immunity is in principle only effective before rather than after infection occurs, provides valuable insights in understanding the nature of immune system and the challenges in cancer treatment. Besides the already known underlying counter-back mechanisms, the astronomical numbers of tumor cells in established tumors could overwhelm the limited amount of specific T cells induced by vaccination, which may account for the modest efficiency of immunotherapy against cancer. We speculate that the long window period for cancer development will allow immune-intervening strategies (e.g., the proper prophylactic vaccination) to promote adaptive mechanisms toward an enhanced immunosurveillance, which could effectively eradicate or at least control the few precancerous cells undergoing neoplastic transformation during early premalignant stages in cancer development, and protect the host from lethal tumor formation. It should be emphasized that the pre-cancer-associated antigens but not the tumor-associated antigens seem to be the suitable antigens for designing prophylactic cancer vaccines. In addition, an ideal prophylactic cancer vaccine may contain multiple pre-cancer-associated antigens, which will provide broad and effective immune protection in a heterogeneous human population. Finally, we demonstrated that placenta-derived gp96, which can be readily obtained in high amount for vaccination, has the ability to initiate antitumor T-cell immunity via association with multiple embryo-cancer antigens. Further understanding placental gp96 associated with carcinoembryonic antigen repertoires that orchestrate immune defense networks against cancer formation will allow to provide an effective prophylactic approach in cancer prevention.

Mutations in the core protein (HBc) of hepatitis B virus (HBV) are associated with aggressive hepatitis and advanced liver diseases in chronic hepatitis B (CHB). In this study, we identified the L60V variation in HBc that generates a new HLA-A2-restricted CD8 + T cell epitope by screening an overlapping 9-mer peptide pool covering HBc and its variants. The nonameric epitope V60 was determined by structural and immunogenic analysis. The HBc L60V variation is correlated with hepatic necroinflammation and higher viral levels, and it may be associated with a poor prognosis in CHB patients. Immunization with the defined HBV epitope V60 peptide elicited specific cytotoxic T lymphocyte (CTL)-induced liver injury in HLA-A2 + HBV transgenic mice. In addition, in vitro and in vivo experiments both demonstrated that the HBc L60V variation facilitates viral capsid assembly and increases HBV replication. These data suggest that the HBc L60V variation can impact both HBV replication and HBV-specific T cell responses. Therefore, our work provides further dissection of the impact of the HBc L60V variation, which orchestrates HBV replication, viral persistence, and immunopathogenesis during chronic viral infection.

The heat shock protein gp96 is an adjuvant that can elicit T cell responses against cancer and infectious diseases, via antigen presentation, in both rodent models and clinical trials. Its uptake and internalization into antigen presenting cells (APCs) is a critical step in gp96-mediated immune responses. This study examined strategies to improve the cell internalization and T cell activation of gp96. It was found that recombinant fusion with the cell-penetrating peptide TAT (trans-activator of transcription) slightly decreased the aggregation level of gp96 and significantly increased its internalization into macrophages. Furthermore, immunization with the TAT-gp96 fusion dramatically enhanced gp96-mediated hepatitis B virus (HBV)-specific T cell responses and its antiviral efficiency in HBV transgenic mice compared to rgp96. In addition, the inclusion of TAT significantly improved the antitumor T cell immune response to a gp96 vaccine in the B16 melanoma model. These results provide evidence that the efficient transduction of gp96 into APCs can significantly enhance the outcome of gp96-based immunotherapy, and therefore provide a basis for more efficient approaches to improving the immunoregulatory and adjuvant functions of this unique T cell adjuvant.

Hepatitis B virus (HBV) infection induces hepatocarcinogenesis and malignant progression, yet global effects of the redundant viral mRNAs produced during infection are unexplored. Here, microRNA (miRNA) target prediction and whole genome expression analysis revealed that HBV pre-C/C mRNA leads to upregulation of multiple let-7a targeted genes. A let-7a complementary region from nt 86 to 108 in the HBV genome was then identified in HBV pre-C/C, pre-S, and S mRNAs. The let-7a sequestration effect by HBV mRNAs was observed under transfection and virus infection, which is dependent on the let-7a response sequence. Moreover, we found reduced AGO2 binding, as well as functional mRNA and protein de-repression of let-7a targets (e.g., c-myc, K-RAS, and CCR7), upon viral mRNA expression. Let-7a levels in the liver were significantly decreased in hepatocellular carcinoma (HCC) patients with HBV infection and were negatively correlated with intrahepatic pre-S2 mRNA levels. Finally, both in vitro and in vivo studies demonstrated that let-7a inhibition by HBV mRNAs resulted in enhanced HCC cell colony formation and tumor growth, providing evidence of the oncogenic potential of HBV mRNAs.

The recognition between retinoic acid-inducible gene I-like receptors (RLRs) and viral RNA triggers an intracellular cascade of signaling to induce the expression of type I IFNs. Both positive and negative regulation of the RLR signaling pathway are important for the host antiviral immune response. Here, we demonstrate that the tetraspanin protein TSPAN6 inhibits RLR signaling by affecting the formation of the adaptor MAVS (mitochondrial antiviral signaling)-centered signalosome. We found that overexpression of TSPAN6 impaired RLR-mediated activation of IFN-stimulated response element, NF-κB, and IFN-β promoters, whereas knockdown of TSPAN6 enhanced the RLR-mediated signaling pathway. Interestingly, as the RLR pathway was activated, TSPAN6 underwent Lys-63-linked ubiquitination, which promoted its association with MAVS. The interaction of TSPAN6 and MAVS interfered with the recruitment of RLR downstream molecules TRAF3, MITA, and IRF3 to MAVS. Further study revealed that the first transmembrane domain of TSPAN6 is critical for its ubiquitination and association with MAVS as well as its inhibitory effect on RLR signaling. We concluded that TSPAN6 functions as a negative regulator of the RLR pathway by interacting with MAVS in a ubiquitination-dependent manner.

Gp96, a member of HSP90 family, is a versatile molecular chaperone with various newly-discovered functions, for example to serve as a low affinity, high capacity calcium binding protein, a natural adjuvant for therapeutic cancer vaccines, a tumor rejection antigen, an immune regulator to pathological cell death. Its multi-functional and structural characteristics make it also an interesting target to develop antibody-based therapeutics. However, its low immunogenicity to mice, because of its high-sequence similarity among different species, is an obstacle to obtain valuable monoclonal antibodies (MAbs). This is a common problem for any low immunogenic proteins, whose sequences share close identity between mice and other species. Here, a new strategy of priming was employed by swine endogenous full-length gp96 and then boosting by E. coli-system heterologously expressed gp96 N-terminal fragment (N-355) to generate MAbs. Twelve different highly-specific MAbs against swine/human endogenous gp96 were successfully obtained. The binding activities of these MAbs were confirmed by enzyme-linked immunosorbent assay (ELISA), Western blot (WB), immunofluorescence and flow cytometry analysis. This provides some important reagents for further research and potential therapeutics. The methods employed can be used for MAb production of any sequence-highly-conserved proteins between mice and swine/human (or any other species).

More than 350 million people worldwide are chronically infected with hepatitis B virus (HBV). Broad repertoire and strong magnitude of HBV-specific T cell responses are thought to play key roles for virus control and clearance. Previous studies together with ours showed that heat shock protein gp96 as adjuvant induces antigen specific T cell responses, yet little is known for its anti-viral properties. Here, we investigated the role of gp96 mediated cellular and humoral immunity in antiviral effects in HBV transgenic mice. Immunization with HBV surface (HBsAg) and core (HBcAg) antigens combined formulation along with gp96 induced robust antiviral T-cell and antibody immunity against HBsAg and HBcAg. Compared with non-immunized control, immunization with gp96 adjuvant vaccine led to decrease of serum HBs level and HBc expression in hepatocyte by 45% and 90% at maximum, respectively, and decreased serum HBV-DNA level to below or close to the detection limit 4 weeks after the last immunization, suggesting the therapeutic effect. A significant enhancement in cellular responses towards HBcAg and increased infiltration of CD8+ T cells in liver of transgenic were observed under treatment with gp96 compared with no treatment (P < 0.05 or 0.01). Treatment with gp96 was capable of reducing Tregs by overall 30–40%. The superior immune responses induced with the aid of gp96 correlated with improved antiviral effect by vaccination with HBsAg and HBcAg. We conclude that gp96 may contribute to enhanced antiviral immunity in transgenic mice at least partly by Treg down-regulation. HBcAg may act as potent adjuvant for Th1 response. Our study reveals the novel property of gp96 in immune modulation and its potential use for breaking immunotolerance in immunotherapy of chronic HBV infection.

As the most abundant liver-specific microRNA (miRNA), miR-122 has been extensively studied for its role in the regulation of lipid metabolism, hepatocarcinogenesis and hepatitis C virus (HCV) replication, but little is known regarding its role in the replication of Hepatitis B virus (HBV), a highly prevalent hepatotropic virus that can cause life-threatening complications. In this study we examined the effects of antisense inhibition of miR-122 and transfection of a miR-122 mimic on HBV expression in hepatoma cells. The over-expression of miR-122 inhibited HBV expression, whereas the depletion of endogenous miR-122 resulted in increased production of HBV in transfected cells. We further found that the down-regulation of Heme oxygenase-1 (HO-1) by miR-122 plays a negative role in the miR-122-mediated inhibition of viral expression. Our study demonstrates the anti-HBV activity of miR-122, suggesting that therapies that increase miR-122 and HO-1 may be an effective strategy to limit HBV replication.

The heat shock protein gp96 elicits specific T cell responses to its chaperoned peptides against cancer and infectious diseases in both rodent models and clinical trials. Although gp96-induced innate immunity, via a subset of Toll like receptors (TLRs), and adaptive immunity, through antigen presentation, are both believed to be important for priming potent T cell responses, direct evidence for the role of gp96-mediated TLR activation related to its functional T cell activation is lacking. Here, we report that gp96 containing mutations in its TLR-binding domain failed to activate macrophages, but peptide presentation was unaffected. Moreover, we found that peptide-specific T cell responses, as well as antitumor T cell immunity induced by gp96, are severely impaired when the TLR-binding domain is mutated. These data demonstrate the essential role of the gp96-TLR interaction in priming T cell immunity and provide further molecular basis for the coupling of gp96-mediated innate with adaptive immunity.

Hepatitis B virus (HBV) chronically infects approximately 350 million people worldwide. The replication of HBV which genome is only 3.2 kb long relies heavily on host factors. Previous studies demonstrated that a highly expressed liver-specific microRNA (miRNA) miR-122 suppresses HBV expression and replication in multiple ways. In this study, we found that the miR-122 response elements in viral genome facilitate HBV expression and replication in miR-122 highly-expressed hepatocytes. Moreover, mutations in miR-122 response elements are correlated with viral loads and disease progression in HBV-infected patients. We next found that HBV mRNA with miR-122 response elements alone could lead to altered expression of multiple host genes by whole genome expression analysis. HBV mRNA-mediated miR-122 down-regulation plays a major role in HBV mRNA-induced differential gene expression. HBV mRNA could enhance viral replication via miR-122 degradation and the up-regulation of its target cyclin G1. Our study thereby reveals that under the unique condition of high abundance of miR-122 and viral mRNAs and much lower level of miR-122 target in HBV infection, HBV may have evolved to employ the miRNA-mediated virus and host mRNAs network for optimal fitness within hepatocytes.

In Hepatitis B virus (HBV) infection, the virus generates numerous viral mRNAs/proteins and viral loads, which plays a major role in driving T cell tolerance, viral persistence, and hepatocellular carcinoma. However, currently available anti-HBV agents have no direct effect on viral mRNA transcription and protein expression. In this study, we designed a recombinant fusion of p53 protein with the cell-penetrating peptide PTD (protein transduction domain of trans-activator of transcription), which mediated p53 internalization into hepatocytes. PTD-p53 effectively suppressed HBV transcription and antigen expression by interaction with viral enhancers. We further provide evidence that PTD-p53 counteracts the viral transcription feedback loop and effectively suppressed HBV production of viral mRNAs, as well as HBsAg, HBeAg, and HBcAg, both in vitro and in vivo. Our results thereby provide a basis for developing a new therapeutic approach against HBV infection.

ER (estrogen receptor)-α36, a variant of human ERα, activates non-genomic cell signaling pathways. ER-α36 on the cell membrane plays a role in breast cancer growth and development, and contributes to tamoxifen resistance. However, it is not understood how cell membrane expression of ER-α36 is regulated. In this study, we investigated the role of cell membrane glycoprotein 96 (mgp96) in the regulation of ER-α36 expression and signaling. We found that the C-terminal domain of mgp96 directly interacts with ER-α36 on the cell membrane of breast tumor cells. This interaction stabilizes the ER-α36 protein, thereby increasing its signaling, which, in turn, increases tumor cell growth and invasion. Moreover, targeting mgp96 with siRNA or monoclonal antibody (mAb) blocks the mgp96-ER-α36 interaction and inhibits breast cancer growth and invasion both in vitro and in vivo. These results provide insights into the modulation of cell membrane ER-α36 expression and suggest that mgp96 could be a potential therapeutic target for ER-α36-overexpressing breast cancer.

Cell membrane translocation of heat shock protein gp96 from the endoplasmic reticulum has been observed in multiple tumors and is associated with tumor malignancy. However, the cancer-intrinsic function and the related mechanism of cell membrane gp96 as a pro-oncogenic chaperone remain further elucidated. In this study, we found that inhibition of gp96 intramolecular conformational changes by a single α-helix peptide p37 dramatically increased its binding to HER2, whereas decreased HER2 dimerization, phosphorylation and downstream signaling. Targeting cell membrane gp96 promoted HER2 ubiquitination and subsequent lysosomal degradation, which led to decreased cell growth and increased apoptosis, and inhibited tumor growth in vivo. We also demonstrate that gp96 inhibitory peptide p37 synergized with trastuzumab to suppress cell growth and induce apoptosis. Our work demonstrates that blocking gp96 conformational changes directs HER2 for cellular degradation, and represents a new therapeutic strategy for inhibiting HER2 signaling in cancer.

Overexpression of urokinase plasminogen activator system or HER-2 (erbB-2) in breast cancer is associated with a poor prognosis. HER-2 overexpression is caused by HER-2 gene amplification. The anti-HER-2 antibody trastuzumab significantly improves clinical outcome for HER2-positive breast cancer. Drugs that target the uPA system are in early clinical trials. The aims of this study were to determine whether urokinase plasminogen activator receptor (uPAR) gene amplification occurs and whether analysis of individual tumor cells (TCs) in the blood or tissue can add information to conventional pathological analysis that could help in diagnosis and treatment. Analysis of individual TCs indicates that uPAR amplification occurs in a significant portion of primary breast cancers and also circulating tumor cells (CTCs) from patients with advanced disease. There was complete concordance between touch preps (TPs) and conventional pathological examination of HER-2 and uPAR gene status in primary tumors. There was also excellent concordance of HER-2 gene status between primary tumors and CTCs provided that acquisition of HER-2 gene amplification in CTCs was taken into account. Unexpectedly, gene amplification of HER-2 and uPAR occurred most frequently in the same TC and patient, suggesting a biological bias and potential advantage for coamplification. Expression of HER-2 and uPAR in primary tumors predicted gene status in 100 and 92% of patients, respectively.

Amplification and overexpression of the HER-2 oncogene in breast cancer is felt to be stable over the course of disease and concordant between primary tumor and metastases. Therefore, patients with HER-2 -negative primary tumors rarely will receive anti-Her-2 antibody (trastuzumab, Herceptin) therapy. A very sensitive blood test was used to capture circulating tumor cells (CTCs) and evaluate their HER-2 gene status by fluorescence in situ hybridization. The HER-2 status of the primary tumor and corresponding CTCs in 31 patients showed 97% agreement, with no false positives. In 10 patients with HER-2 -positive tumors, the HER-2 /chromosome enumerator probe 17 ratio in each tumor was about twice that of the corresponding CTCs (mean 6.64 ± 2.72 vs. 2.8 ± 0.6). Hence, the ratio of the CTCs is a reliable surrogate marker for the expected high ratio in the primary tumor. Her-2 protein expression of 10 CTCs was sufficient to make a definitive diagnosis of the HER-2 gene status of the whole population of CTCs in 19 patients with recurrent breast cancer. Nine of 24 breast cancer patients whose primary tumor was HER-2 -negative each acquired HER-2 gene amplification in their CTCs during cancer progression, i.e., 37.5% (95% confidence interval of 18.8–59.4%). Four of the 9 patients were treated with Herceptin-containing therapy. One had a complete response and 2 had a partial response.

Targeted therapy is currently under intensive investigation due to the resistance of liver cancer to cytotoxic chemotherapies. Dissecting the molecular events that drive the progression of liver cancer and defining specific targets are urgently needed to develop efficient tailored therapies. Cell membrane gp96 (mgp96) has been implicated in tumor growth and malignancy. Here, we explored the functional and clinical relevance of mgp96 in liver cancer. We found that elevated mgp96 abundance was associated with tumor metastasis and recurrence in patients with primary liver tumors. Decreased KDELR1 levels in hepatoma cells contribute to cell membrane translocation of the normally ER-resident gp96. Urokinase-type plasminogen activator receptor (uPAR) was identified as a mgp96 client protein, and mgp96 stabilized uPAR protein. Our clinical results proved that elevated mgp96 abundance is positively correlated with uPAR expression levels in liver tumors. We further provided evidence that targeting mgp96 with siRNA or a specific mAb that blocked the mgp96-uPAR interaction led to inhibited cell growth, survival, and invasion in vitro, as well as the suppression of liver tumor growth and metastasis in vivo. mgp96 promotes liver cancer progression through increasing the protein stability and signaling of uPAR, and may be a new promising target for suppressing uPAR-mediated tumor growth and metastasis in liver cancer.

Cytotoxic T cells (CTLs) play a key role in the control of Hepatitis B virus (HBV) infection and viral clearance. However, most of identified CTL epitopes are derived from HBV of genotypes A and D, and few have been defined in virus of genotypes B and C which are more prevalent in Asia. As HBV core protein (HBc) is the most conservative and immunogenic component, in this study we used an overlapping 9-mer peptide pool covering HBc to screen and identify specific CTL epitopes. An unconventional HLA-A2-restricted epitope HBc141–149 was discovered and structurally characterized by crystallization analysis. The immunogenicity and anti-HBV activity were further determined in HBV and HLA-A2 transgenic mice. Finally, we show that mutations in HBc141–149 epitope are associated with viral parameters and disease progression in HBV infected patients. Our data therefore provide insights into the structure characteristics of this unconventional epitope binding to MHC-I molecules, as well as epitope specific CTL activity that orchestrate T cell response and immune evasion in HBV infected patients.

The commonly used inactivated or split influenza vaccines induce only induce minimal T cell responses and are less effective in preventing heterologous virus infection. Thus, developing cross-protective influenza vaccines against the spread of a new influenza virus is an important strategy against pandemic emergence. Here we demonstrated that immunization with heat shock protein gp96 as adjuvant led to a dramatic increased antigen-specific T cell response to a pandemic H1N1 split vaccine. Notably, gp96 elicited a cross-protective CD8(+) T cell response to the internal conserved viral protein NP. Although the split pH1N1vaccine alone has low cross-protective efficiency, adding gp96 as an adjuvant effectively improved the cross-protection against challenge with a heterologous virus in mice. Our study reveals the novel property of gp96 in boosting the T cell response against conserved epitopes of influenza virus and its potential use as an adjuvant for human pre-pandemic inactivated influenza vaccines against different viral subtypes.

More than 350 million people are chronically infected with hepatitis B virus, and dysfunctional T cell responses contribute to persistent viral infection and immunopathogenesis in chronic hepatitis B (CHB). However, the underlying mechanisms of T cell hyporesponsiveness remain largely undefined. Given the important role of microRNA-146a (miR-146a) in diverse aspects of lymphocyte function, we investigated the potential role and mechanism of miR-146a in regulating T cell immune responses in CHB. We found that miR-146a expression in T cells is significantly upregulated in CHB compared with healthy controls, and miR-146a levels were correlated with serum alanine aminotransaminase levels. Both inflammatory cytokines and viral factors led to miR-146a upregulation in T cells. Stat1 was identified as a miR-146a target that is involved in antiviral cytokine production and the cytotoxicity of CD4+ and CD8+ T cells. In vitro blockage of miR-146a in T cells in CHB greatly enhanced virus-specific T cell activity. Therefore, our work demonstrates that miR-146a upregulation in CHB causes impaired T cell function, which may contribute to immune defects and immunopathogenesis during chronic viral infection.

Up to 10% of cytosolic proteins are dependent on the mammalian heat shock protein 90 (HSP90) for folding. However, the interactors of its endoplasmic reticulum (ER) paralogue (gp96, Grp94 and HSP90b1) has not been systematically identified. By combining genetic and biochemical approaches, we have comprehensively mapped the interactome of gp96 in macrophages and B cells. A total of 511 proteins were reduced in gp96 knockdown cells, compared to levels observed in wild type cells. By immunoprecipitation, we found that 201 proteins associated with gp96. Gene Ontology analysis indicated that these proteins are involved in metabolism, transport, translation, protein folding, development, localization, response to stress and cellular component biogenesis. While known gp96 clients such as integrins, Toll-like receptors (TLRs) and Wnt co-receptor LRP6, were confirmed, cell surface HSP receptor CD91, TLR4 pathway protein CD180, WDR1, GANAB and CAPZB were identified as potentially novel substrates of gp96. Taken together, our study establishes gp96 as a critical chaperone to integrate innate immunity, Wnt signaling and organ development.

Alpha interferon (IFN-α)-based therapy can effectively treat chronic hepatitis B virus (HBV) infection, which causes life-threatening complications. Responses to IFN-α therapy vary greatly in chronic hepatitis B (CHB) patients, but underlying mechanisms are almost unknown. In this study, we found that IFN-α treatment induced a marked decrease of microRNA-122 (miR-122) expression in hepatocytes. We next showed that IFN-α-induced miR-122 downregulation was only partly due to transcriptional suppression. One IFN-stimulated gene (ISG), NT5C3, which was identified as a miR-122 target, efficiently inhibited miR-122 by binding and sequestering miR-122 with its mRNA 3′-untranslated region (3′-UTR), indicating that this ISG is involved in IFN-α-mediated miR-122 suppression. Notably, the inhibitory effect of IFN-α on miR-122 was completely abolished by blocking IFN-α-induced upregulation of NT5C3 mRNA expression by RNA interference (RNAi). Meanwhile, we observed that miR-122 dramatically inhibited HBV expression and replication. Finally, we showed that IFN-α-mediated HBV-inhibitory effects could be enhanced significantly by blocking IFN-α-induced downregulation of miR-122. We therefore concluded that IFN-α-induced inhibition of miR-122 may negatively affect the anti-HBV function of IFN-α. These data provide valuable insights for a better understanding of the antiviral mechanism of IFN-α and raise further potential interest in enhancing its anti-HBV efficacy.

As the most abundant liver-specific microRNA, miR-122 is involved in diverse aspects of hepatic function and neoplastic transformation. Our previous study showed that miR-122 levels are significantly decreased in hepatitis B virus (HBV)-infected patients, which may facilitate viral replication and persistence (S. Wang, L. Qiu, X. Yan, W. Jin, Y. Wang, L. Chen, E. Wu, X. Ye, G. F. Gao, F. Wang, Y. Chen, Z. Duan, and S. Meng, Hepatology 55: 730–741, 2012). Loss of miR-122 expression in patients with hepatitis B enhances hepatitis B virus replication through cyclin G1-modulated P53 activity.). In this study, we provide evidence that all HBV mRNAs harboring an miR-122 complementary site act as sponges to bind and sequester endogenous miR-122, indicating that the highly redundant HBV transcripts are involved in HBV-mediated miR-122 suppression. We next identified pituitary tumor-transforming gene 1 (PTTG1) binding factor (PBF) as a target of miR-122 and demonstrated that HBV replication causes an obvious increase in PBF levels. Furthermore, we observed that the miR-122 levels were decreased and PBF was upregulated in chronic hepatitis B (CHB) and hepatocellular carcinoma (HCC). Overexpression and knockdown studies both revealed that PBF enhances proliferation and invasion of HCC cells, and silencing PBF resulted in a dramatic reduction of HCC tumor growth in vivo . Mechanistic analysis demonstrated that PBF interacts with PTTG1 and facilitates PTTG1 nuclear translocation, subsequently increasing its transcriptional activities. Therefore, we identified a novel HBV mRNA-miR-122-PBF regulatory pathway that facilitates malignant hepatocyte growth and invasion in CHB which may contribute to CHB-induced HCC development and progression. Our work underscores the reciprocal interplay of host miRNA sequestration and depletion by viral mRNAs, which may contribute to chronic-infection-related cancer.

As the most abundant liver-specific microRNA, microRNA-122 (miR-122) is involved in various physiological processes in hepatic function as well as in liver pathology. There is now compelling evidence that miR-122, as a regulator of gene networks and pathways in hepatocytes, plays a central role in diverse aspects of hepatic function and in the progress of liver diseases. This liver-enriched transcription factors-regulated miRNA promotes differentiation of hepatocytes and regulates lipid metabolism. With regard to liver diseases, miR-122 was shown to stimulate hepatitis C virus (HCV) replication through a unique and unusual interaction with two binding sites in the 5′-UTR of HCV genome to mediate the stability of the viral RNA, whereas inhibit the expression and replication of hepatitis B virus (HBV) by a miR-122-cylin G1/p53-HBV enhancer regulatory pathway. In addition, miR-122 acts as a suppressor of cell proliferation and malignant transformation of hepatocytes with remarkable tumor inhibition activity. Notably, a clinical trial targeting miR-122 with the anti-miR-122 oligonucleotides miravirsen, the first miRNA targeted drug, has been initiated for treatment of HCV infection. With further understanding of the comprehensive roles of miR-122 in hepatic functions and the mechanisms involved in miR-122 down-regulation in chronic hepatitis or hepatocellular carcinoma, miR-122 appears to be a promising candidate for effective therapeutic approaches against tumor and infectious diseases.

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) has recently caused huge economic losses in the pig industry worldwide. Commercial vaccines, including inactivated vaccines and attenuated live vaccines, are available but fail to provide sustainable protection, especially against genetically heterologous strains. Thus several approaches have been used to develop more effective PRRSV vaccines and/or immune modulators to accelerate and magnify immune responses to PRRSV vaccines. Heat shock protein Gp96 is one such modulator that enhances both the innate and adaptive immune responses. In the present study, two B-cell epitopes and seven T-cell epitopes from PRRSV and a Pan DR T-helper cell epitope were synthesized and mixed with the N-terminal 22-355 aa of Gp96 (Gp96N) as an adjuvant, and immune responses were evaluated. Our results show that Gp96N activated PRRSV-specific humoral immune responses elicited by BCE-peptides and promoted the PRRSV-specific cellular immunity induced by TCE-peptides. Moreover, higher levels of IL-12 and TNF-α and lower levels of IL-4 and IL-10 were observed in the serum of Gp96N-vaccinated piglets compared to piglets immunized with no Gp96N, displaying a predominant Th1 type of immune response induced by Gp96N. Following challenge with the virulent HP-PRRSV isolate JXwn06, piglets vaccinated with the mixture of peptides and Gp96N presented with milder clinical symptoms, lower viremia, and less pathological lesions in their lungs, however, this vaccine could not provide lasting and effective protection against HP-PRRSV infection. These data provide important bases for the development of PRRSV epitope-based synthetic peptide vaccines combined with Gp96N as attractive immunomodulators in swine.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses in the pig industry worldwide. Currently available commercial vaccines provide limited protection due to delayed and weak cell-mediated immunity and neutralizing antibody production, thus the immunomodulators should be considered in order to improve the efficacy of PRRSV vaccines. Heat shock protein gp96 may be used as a modulator to enhance both innate and adaptive immune responses. In the present study, two multi-epitope subunit vaccines, named as Cp1 and Cp2, were designed based on the conserved B cell epitopes of viral proteins with the N-terminal 22–370 amino acids (aa) of porcine gp96 (Gp96N) chosen as the adjuvant. Immune responses elicited by the different combinations of Cp1/Cp2 and Gp96N were examined in mice and piglets. The results indicated that the group of Cp1/Cp2-Gp96N (CG) combination induced 3–4-fold higher titers of Cp1/Cp2-ELISA antibodies and neutralizing antibodies (NAs) in mice than the groups which received Cp1/Cp2 immunization alone or with Freund's adjuvant. Additionally, Gp96N significantly enhanced the levels of lymphocyte proliferative responses of splenocytes or peripheral blood mononuclear cells from vaccinated mice or piglets. The production of IFN-γ in mice splenocytes, TNF-α, IFN-γ, and IL-12 in sera of piglets were also remarkably increased with the treatment of Gp96N, while IL-4 was reduced by half and IL-10 was decreased to an undetectable level. These results suggest that the porcine Gp96N could effectively enhance the innate and adaptive immune responses of Cp1/Cp2 with a Th1-type bias. Therefore, the multi-epitope subunit vaccine Cp1/Cp2 co-administered with porcine Gp96N might potentially be a promising candidate vaccine for the prevention and control of PRRSV in pigs.

UNLABELLED Hepatitis B virus (HBV) causes chronic infection in about 350 million people worldwide. Given the important role of the most abundant liver-specific microRNA, miR-122, in hepatic function and liver pathology, here we investigated the potential role and mechanism of miR-122 in regulating HBV replication. We found that miR-122 expression in liver was significantly down-regulated in patients with HBV infection compared with healthy controls, and the miR-122 levels were negatively correlated with intrahepatic viral load and hepatic necroinflammation. The depletion of endogenous miR-122 by its antisense inhibitor led to enhanced HBV replication, whereas overexpression of miR-122 by transfection of mimic or its expression vector inhibited viral production. We next identified cyclin G(1) as an miR-122 target from multiple candidate target genes that are involved in the regulation of HBV replication. Overexpression and knockdown studies both showed that cyclin G(1) regulated viral replication in HBV transfected cells. We also observed that cyclin G(1) expression was up-regulated in HBV-infected patients, and cyclin G(1) levels were inversely associated with miR-122 expression in liver tissues. Using coimmunoprecipitation, a luciferase reporter system, and electrophoretic mobility shift assay, we further demonstrated that cyclin G(1) specifically interacted with p53, and this interaction blocked the specific binding of p53 to HBV enhancer elements and simultaneously abrogated p53-mediated inhibition of HBV transcription. Finally, we show that miR-122 suppressed HBV replication in p53 wildtype cells but not in null isogenic cells. CONCLUSION miR-122 down-regulates its target cyclin G(1) , and thus interrupts the interaction between cyclin G(1) and p53 and abrogates p53-mediated inhibition of HBV replication. Our work shows that miR-122 down-regulation induced by HBV infection can impact HBV replication and possibly contribute to viral persistence and carcinogenesis.

Despite its potent immunostimulatory properties, vaccination with autologous tumor-derived gp96 has relatively modest antitumor effect in a range of clinical trials. Based on our previous study showing a gp96-mediated immune balance between CTL and Tregs, here we investigated possible synergy between gp96 vaccine and systemic Treg depletion on induction of antitumor T-cell immunity and the mechanisms accounting for synergistic efficacy. In gp96-peptide complex immunized BALB/c mice, anti-CD25 mAb treatment significantly increased IFN-γ-producing CD8(+) and CD4(+) T cells by about 1-2-fold in spleen and 40-50% in lymph node. A significantly higher number of peptide-specific CTL were observed under anti-CD25 mAb treatment compared with no treatment. Moreover, Treg depletion synergistically improved the anticancer activity of tumor-derived gp96 vaccine in the poorly immunogenic and highly tumorigenic B16 melanoma model in C57BL/6 J mice. While gp96 immunization alone led to the modest enhancement of CTL activities in spleen, the combination with Treg depletion dramatically increased tumor-specific CTL responses. In addition, the combination resulted in a significant increase of CD8(+) T-cell infiltration in tumor, which correlated with an enhanced inhibition of tumor growth. Our results provide evidence that targeting Tregs may provide a more efficient strategy to potentiate gp96-mediated T-cell responses and enhance the antitumor efficiency of gp96-based therapeutic vaccine.

Previous studies together with ours showed that heat shock protein gp96 as an adjuvant induces antigen specific T cell responses against cancer and infectious diseases. However, at present there is no efficient method to obtain high amount of full-length gp96 by in vitro expression. Here, we used the yeast Hansenula polymorpha as an efficient host for gp96 recombinant protein production. The transformant clones with highly expressed recombinant proteins were screened and selected by measuring the halo size which indicates enzymatic hydrolysis of starch in the medium. High-level production of gp96 (around 150 mg/mL) was achieved by using high-cell density fed-batch cultivations. We showed that peptide binding of the recombinant protein has similar specificity and intrinsic binding parameters as that of the native gp96. We next examined the self-assembly properties and high-order structures of the recombinant protein. Moreover, the H. polymorpha expressed recombinant gp96 can effectively induce HBV-specific CTL response in immunized mice while Escherichia coli-expressed gp96 cannot. Our results therefore may provide bases for structure and functional studies of gp96 and thereby potentially expedite the development of gp96-based vaccines for immunotherapy of cancer or infectious diseases.

Overexpression of urokinase plasminogen activator receptor (uPAR) or HER2 (erbB-2) in breast cancer is associated with a poor prognosis. We previously reported that gene amplification and overexpression of HER2 and uPAR occur in 70% of HER2-amplified tumor cells from blood or tissue of patients with breast cancer. In this study, we first examined whether depletion of HER2 and uPAR synergized in suppression of the growth of breast cancer cells that overexpress both HER2 and uPAR (SKBR3 and ZR 751). The results showed that depletion of either HER2 or uPAR by RNA interference suppressed cell growth and induced cell apoptosis, but that these effects were significantly enhanced in cells depleted of both HER2 and uPAR. Mechanistic analysis demonstrated that silencing of HER2 and uPAR caused suppression of MAPK signal pathways, resulting in decrease of ERK activity and prompting a high p38/ERK activity ratio. The level of the phosphorylated form of ERK was decreased in cells depleted of HER2, uPAR or both, and the effect in cells depleted of both is the most evident. Moreover, downregulation of uPAR synergized with trastuzumab to suppress the growth and induce apoptosis of SKBR3 and ZR 751 cells. uPAR RNAi significantly enhanced the effect of trastuzumab on inhibition of MAPK signal pathways. In conclusion, targeting HER2 and uPAR has a synergistic inhibitory effect on breast cancer cells. Our results provide evidence that simultaneous downregulation of HER2 and uPAR may offer an effective tool for breast cancer therapy.

HER2 analysis in circulating tumor cells (CTCs) may have clinical significance for HER2-targeted therapy as HER2-positive CTCs and disseminated tumor cells can be detected in patients with HER2-negative primary tumors who currently do not have access to HER2-targeted therapy. In this study, we performed quantitative analysis by confocal microscopy assay for evaluation of HER2 expression in individual tumor cells. HER2 testing by confocal microscopy assay exhibited high concordance with results of real-time PCR, Western blot analysis and FISH analysis. We found that there was a significant positive correlation between HER2 overexpression and gene amplification in individual CTCs, which provided validation of confocal microscopy assay for HER2 expression in CTCs. By using subsets of 10 consecutive cells (bins), we conclude that HER2 expression (3+) in CTCs predicts HER2 overexpression of tumor with high probability in breast cancer patients. These results may provide interpretive guidelines for HER2 status assay in CTCs and raise great opportunities for using CTCs as non-invasive and 'real-time' biopsy to examine and monitor the status of tumor markers.

Novel assay methods developed for the isolation and characterization of circulating tumor cells (CTC) of epithelial origin offer the potential of markers for the non-invasive gathering of clinical information relevant to the diagnosis, evolution and treatment of carcinoma. Of the numerous techniques currently used to analyze CTC, slide-based assays are perhaps the most common. While traditional combined immunocytochemical/brightfield microscopy systems continue to be the most frequently employed, fluorescence-based analysis is gaining in importance. This is partly because fluorescence microscopy analysis of slide-mounted CTC can provide simultaneously cytogenetic as well as morphologic and multiple phenotypic information. In particular, fluorescence microscopy analysis of slide-mounted CTC can accurately determine genetic changes at the chromosomal level in patients with recurrent disease. More importantly, by identifying genetic aberrations in CTC, it becomes possible to choose those patients most likely to benefit from a given treatment. The potential of this technique has already been demonstrated by employing fluorescence in situ hybridization (FISH) methods to measure expression of the HER2/ neu gene in tissue from patients with breast carcinoma for the specific purpose of identifying those patients most likely to respond to Trastuzumab targeted therapy. Here, we review the major methodologies used in the preparation and analysis of the slide-based assays.