Huajing Wang

A Broadly Flavivirus Cross-Neutralizing Monoclonal Antibody that Recognizes a Novel Epitope within the Fusion Loop of E Protein

Flaviviruses are a group of human pathogenic, enveloped RNA viruses that includes dengue (DENV), yellow fever (YFV), West Nile (WNV), and Japanese encephalitis (JEV) viruses. Cross-reactive antibodies against Flavivirus have been described, but most of them are generally weakly neutralizing. In this study, a novel monoclonal antibody, designated mAb 2A10G6, was determined to have broad cross-reactivity with DENV 1–4, YFV, WNV, JEV, and TBEV. Phage-display biopanning and structure modeling mapped 2A10G6 to a new epitope within the highly conserved flavivirus fusion loop peptide, the 98DRXW101 motif. Moreover, in vitro and in vivo experiments demonstrated that 2A10G6 potently neutralizes DENV 1–4, YFV, and WNV and confers protection from lethal challenge with DENV 1–4 and WNV in murine model. Furthermore, functional studies revealed that 2A10G6 blocks infection at a step after viral attachment. These results define a novel broadly flavivirus cross-reactive mAb with highly neutralizing activity that can be further developed as a therapeutic agent against severe flavivirus infections in humans.

A novel functional motif of osteopontin for human lymphocyte migration and survival

Osteopontin (OPN) is an extracellular matrix protein of pleiotropic properties and plays an important role in regulating lymphocyte adhesion and cytokine production associated with inflammatory processes and autoimmune diseases. Here we developed and characterized a monoclonal antibody (mAbs) (23C3D3) specific for human OPN. This antibody could inhibit OPN-induced lymphocyte adhesion, migration and survival. Epitope mapping showed that 23C3D3 could specifically recognize the phage displayed peptide (43)WLNPDP(48). In addition, a synthesized mimetic peptide (mimotope) 23P ((40)VATWLNPDPSQK(51)) could block the binding of 23C3D3 to hOPN and significantly inhibit the hOPN-induced lymphocyte adhesion, migration and survival. Moreover, mutations on the WLNPDP motif of hOPN also markedly diminished its activity for lymphocyte activation. Interestingly, this novel epitope is located in the extremely retained domain in all species. The functioning assay indicates that this novel epitope is critically involved in the lymphocyte migration and survival through activating ERK and the transcription factor NF-kappaB pathway, which can be inhibited by the motif (43)WLNPDP(48) blocking antibody, 23C3D3. These results suggest that this novel epitope of OPN may provide a potential therapeutic target for the treatment of T cell-mediated immune diseases.

Enhanced myeloid differentiation factor 88 promotes tumor metastasis via induction of epithelial-mesenchymal transition in human hepatocellular carcinoma.

Metastasis is the leading cause of death in patients with hepatocellular carcinoma (HCC) after curative resection. Therefore, it is critical to understand the mechanisms underlying tumor metastasis in HCC. We have previously shown that elevated expression of myeloid differentiation factor 88 (MyD88) may promote tumor growth and metastasis in HCC. In this study, we reported that enhanced expression of MyD88 promoted epithelial–mesenchymal transition (EMT) properties and tumor-initiating capabilities in HCC cells. MyD88 was found to be able to interact with p85, a regulatory subunit of phosphoinositide 3-kinase (PI3-K), independent of TLR/IL-1R-mediated response and caused PI3-K/v-akt murine thymoma viral oncogene homolog (Akt) activation, which resulted in subsequent phosphorylation of glycogen synthase kinase-3β and stabilization of Snail, a critical EMT mediator. Consistently, we observed a significant correlation between MyD88 expression and p-Akt levels in a cohort of HCC patients, and found that the combination of these two parameters have better prognostic value for HCC patients. Taken together, these results suggest that elevated MyD88 may facilitate HCC metastasis by promoting EMT properties and tumor-initiating capabilities via PI3–K/Akt pathway.

Osteopontin induces autophagy to promote chemo-resistance in human hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is a major health burden worldwide for its high incidence and mortality. Osteopontin (OPN) is a chemokine-like, matricellular phosphoglycoprotein whose expression is elevated in various types of cancer including HCC. OPN has been shown to be involved in tumorigenesis, chemo-resistance, metastasis and sustaining stem-like properties of cancer cells. Autophagy is a cellular process by which cytoplasmic components are degraded and recycled for maintaining cellular homeostasis. There is increasing evidence supports that autophagy plays a critical role for stem-like properties and chemo-resistance of cancer cells. However, the relationship between OPN and autophagy in maintaining cancer stem-like properties and chemo-resistance is yet to be clarified. Herein, we found that secreted OPN induced autophagy via binding with its receptor integrin αvβ3 and sustaining FoxO3a stability. OPN-elicited autophagy could promote cancer cell survival and resistance to chemotherapy drugs, as well as stem-like properties. Our findings indicated that OPN was capable of promoting chemo-resistance of HCCs via autophagy, which might provide a new strategy for the treatment of HCC.

Structural Insights into the Neutralization Mechanism of Monoclonal Antibody 6C2 against Ricin

Background: The antibody 6C2 exhibited an unusually potent neutralizing ability against ricin. Results: We determined the crystal structure of 6C2 Fab in complex with RTA and mapped the epitope on RTA. Conclusion: The binding of 6C2 hinders the interaction between RTA and the ribosome, thus inhibiting the activities of RTA. Significance: Our findings further confirm the role of ribosomal elements in ricin activity and specificity. Ricin belongs to the type II ribosome-inactivating proteins that depurinate the universally conserved α-sarcin loop of rRNA. The RNA N-glycosidase activity of ricin also largely depends on the ribosomal proteins that play an important role during the process of rRNA depurination. Therefore, the study of the interaction between ricin and the ribosomal elements will be better to understand the catalysis mechanism of ricin. The antibody 6C2 is a mouse monoclonal antibody exhibiting unusually potent neutralizing ability against ricin, but the neutralization mechanism remains unknown. Here, we report the 2.8 Å crystal structure of 6C2 Fab in complex with the A-chain of ricin (RTA), which reveals an extensive antigen-antibody interface that contains both hydrogen bonds and van der Waals contacts. The complementarity-determining region loops H1, H2, H3, and L3 form a pocket to accommodate the epitope on the RTA (residues Asp96–Thr116). ELISA results show that Gln98, Glu99, Glu102, and Thr105 (RTA) are the key residues that play an important role in recognizing 6C2. With the perturbation of the 6C2 Fab-RTA interface, 6C2 loses its neutralization ability, measured based on the inhibition of protein synthesis in a cell-free system. Finally, we propose that the neutralization mechanism of 6C2 against ricin is that the binding of 6C2 hinders the interaction between RTA and the ribosome and the surface plasmon resonance and pulldown results confirm our hypothesis. In short, our data explain the neutralization mechanism of mAb 6C2 against ricin and provide a structural basis for the development of improved antibody drugs with better specificity and higher affinity.

A humanized anti-osteopontin antibody protects from Concanavalin A induced-liver injury in mice.

Osteopontin has been implicated in various inflammatory diseases including rheumatoid arthritis, multiple sclerosis, Crohns disease, and fulminant hepatitis. Increased expression of osteopontin has been detected in pathological foci of these diseases. RA and fulminant hepatitis have been successfully treated by administration of neutralizing anti-osteopontin antibody in mice. However, rodent antibodies are highly immunogenic in humans and therefore limited in their clinical application. Here, a murine monoclonal antibody 23C3 against human osteopontin, was humanized by complementarity-determining region grafting method based on computer-assisted molecular modeling. The humanized version of 23C3, denoted as Hu23C3, was shown to possess affinity comparable to that of its parental antibody. Hu23C3 could also inhibit monocyte migration in response to osteopontin in vitro. Furthermore, in vivo data showed that Hu23C3 significantly protects mice from Concanavalin A (Con A) induced-liver injury in association with the reduction of transaminase activities and improvement of liver injury. Mechanistic studies demonstrated that Hu23C3 inhibited T and NKT cell infiltration, and activation of nuclear factor κB (NF-κB) in the liver, resulting in reduction of TNF-α and IFN-γ production. Thus, our data strongly support that the humanized anti-osteopontin antibody, Hu23C3, may have a potential for the treatment of T cell mediated-hepatitis in human.

Contribution of TIP30 to chemoresistance in laryngeal carcinoma

Laryngeal squamous cell carcinoma (LSCC) is one of the most common carcinomas of the head and neck. Despite advances in diagnosis and treatment, the survival of patients with LSCC has not improved in the past two decades. TIP30, a newly identified tumour suppressor, appears to be involved in multiple processes during tumour development. Here, we investigated the involvement of TIP30 in chemoresistance of LSCC in vitro and in vivo. We showed that TIP30 expression decreased significantly in drug-selected cells (DSCs) of laryngeal carcinoma. Suppressing TIP30 enhanced resistance capability to multiple chemotherapy drugs, cell proliferation and self-renewal in Hep2 cells. Additionally, decreased self-renewal capacity and chemotherapeutic resistance were observed in DSCs overexpressing TIP30. Furthermore, TIP30 negatively regulated tumourigenesis and chemoresistance in LSCC cells subcutaneously transplanted into nude mice. Moreover, decreased TIP30 expression contributed to chemoresistance, self-renewal and proliferation of LSCC cells via nuclearlisation of β-catenin, a cell–cell adhesion and stem cell renewal regulator. Consistently, Kaplan–Meier and Cox proportional hazards regression modelling analyses showed that decreased TIP30 expression independently predicted poor survival in patients with LSCC. Taken together, our results reveal that TIP30 has a crucial role in chemoresistance of LSCC through the AKT/glycogen synthase kinase-3β/β-catenin signalling pathway and may be a promising candidate for improving LSCC chemotherapy.

A functional motif QLYxxYP is essential for osteopontin induced T lymphocyte activation and migration

Osteopontin (OPN) plays an important role in regulating lymphocyte adhesion and cytokine production associated with inflammatory processes and autoimmune diseases. Here we developed and characterized a monoclonal antibody F8E11 specific for human OPN (hOPN). F8E11 could inhibit OPN-induced lymphocyte activation and migration. Epitope mapping showed that F8E11 could specifically recognize the peptide QLYxxYP. In addition, a synthesized mimetic peptide F8P (EEKQLYNKYPDA) could block the binding of F8E11 to hOPN and significantly inhibit the hOPN-induced lymphocyte migration. Moreover, mutations on the QLYxxYP motif of hOPN also markedly diminished its activity for lymphocyte activation and migration. The functioning assay indicated that this novel epitope is critically involved in the lymphocyte migration through activating MAPK/ERK/AP-1 pathway, which can be inhibited by the motif QLYxxYP blocking antibody, F8E11. These results suggest that this novel epitope of OPN may provide a potential therapeutic target for the treatment of T cell mediated-immune diseases.

Downregulation of ASPP2 improves hepatocellular carcinoma cells survival via promoting BECN1-dependent autophagy initiation

Autophagy is an important catabolic process, which sustains intracellular homeostasis and lengthens cell survival under stress. Here we identify the ankyrin-repeat-containing, SH3-domain-containing, and proline-rich region-containing protein 2 (ASPP2), a haploinsufficient tumor suppressor, as a molecular regulator of starvation-induced autophagy in hepatocellular carcinoma (HCC). ASPP2 expression is associated with an autophagic response upon nutrient deprivation and downregulation of ASPP2 facilitates autophagic flux, whereas overexpression of ASPP2 blocks this starvation-induced autophagy in HCC cells. Mechanistically, ASPP2 inhibits autophagy through regulating BECN1 transcription and formation of phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) complex. Firstly, ASPP2 inhibits p65/RelA-induced transcription of BECN1, directly by an ASPP2-p65/RelA-IκBα complex which inhibits phosphorylation of IκBα and the translocation of p65/RelA into the nucleus. Secondly, ASPP2 binds to BECN1, leading to decreased binding of PIK3C3 and UV radiation resistance-associated gene (UVRAG), and increased binding of Rubicon in PIK3C3 complex. Downregulation of ASPP2 enhances the pro-survival and chemoresistant property via autophagy in HCC cells in vitro and in vivo. Decreased ASPP2 expression was associated with increased BECN1 and poor survival in HCC patients. Therefore, ASPP2 is a key regulator of BECN1-dependent autophagy, and decreased ASPP2 may contribute to tumor progression and chemoresistance via promoting autophagy.

A bispecific antibody effectively neutralizes all four serotypes of dengue virus by simultaneous blocking virus attachment and fusion

ABSTRACT Although dengue virus (DENV) infection severely threatens the health of humans, no specific antiviral drugs are currently approved for clinical use against DENV infection. Attachment and fusion are 2 critical steps for the flavivirus infection, and the corresponding functional epitopes are located at E protein domain III (E-DIII) and domain II (E-DII), respectively. Here, we constructed a bispecific antibody (DVD-1A1D-2A10) based on the 2 well-characterized anti-DENV monoclonal antibodies 1A1D-2 (1A1D) and 2A10G6 (2A10). The 1A1D antibody binds E-DIII and can block the virus attaching to the cell surface, while the 2A10 antibody binds E-DII and is able to prevent the virus from fusing with the endosomal membrane. Our data showed that DVD-1A1D-2A10 retained the antigen-binding activity of both parental antibodies. Importantly, it was demonstrated to be significantly more effective at neutralizing DENV than its parental antibodies both in vitro and in vivo, even better than the combination of them. To eliminate the potential antibody-dependent enhancement (ADE) effect, this bispecific antibody was successfully engineered to prevent Fc-γ-R interaction. Overall, we generated a bispecific anti-DENV antibody targeting both attachment and fusion stages, and this bispecific antibody broadly neutralized all 4 serotypes of DENV without risk of ADE, suggesting that it has great potential as a novel antiviral strategy against DENV.