Development of AAV-delivered broadly neutralizing anti-human ACE2 antibodies against SARS-CoV-2 variants.

The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in the emergence of new variants that are resistant to existing vaccines and therapeutic antibodies, has raised the need for novel strategies to combat the persistent global COVID-19 epidemic. In this study, a monoclonal anti-human angiotensin-converting enzyme 2 (hACE2) antibody, ch2H2, was isolated and humanized to block the viral receptor-binding domain (RBD) binding to hACE2, the major entry receptor of SARS-CoV-2. This antibody targets the RBD-binding site on the N terminus of hACE2 and has a high binding affinity to outcompete the RBD. In vitro, ch2H2 antibody showed potent inhibitory activity against multiple SARS-CoV-2 variants, including the most antigenically drifted and immune-evading variant Omicron. In vivo, adeno-associated virus (AAV)-mediated delivery enabled a sustained expression of monoclonal antibody (mAb) ch2H2, generating a high concentration of antibodies in mice. A single administration of AAV-delivered mAb ch2H2 significantly reduced viral RNA load and infectious virions and mitigated pulmonary pathological changes in mice challenged with SARS-CoV-2 Omicron BA.5 subvariant. Collectively, the results suggest that AAV-delivered hACE2-blocking antibody provides a promising approach for developing broad-spectrum antivirals against SARS-CoV-2 and potentially other hACE2-dependent pathogens that may emerge in the future.

Epithelial cell adhesion molecule (EpCAM) regulates HGFR signaling to promote colon cancer progression and metastasis.

BACKGROUND: Epithelial cell adhesion molecule (EpCAM) is known to highly expression and promotes cancer progression in many cancer types, including colorectal cancer. While metastasis is one of the main causes of cancer treatment failure, the involvement of EpCAM signaling in metastatic processes is unclear. We propose the potential crosstalk of EpCAM signaling with the HGFR signaling in order to govern metastatic activity in colorectal cancer. METHODS: Immunoprecipitation (IP), enzyme-linked immunosorbent assay (ELISA), and fluorescence resonance energy transfer (FRET) was conducted to explore the extracellular domain of EpCAM (EpEX) and HGFR interaction. Western blotting was taken to determine the expression of proteins in colorectal cancer (CRC) cell lines. The functions of EpEX in CRC were investigated by proliferation, migration, and invasion analysis. The combined therapy was validated via a tail vein injection method for the metastasis and orthotopic colon cancer models. RESULTS: This study demonstrates that the EpEX binds to HGFR and induces downstream signaling in colon cancer cells. Moreover, EpEX and HGF cooperatively mediate HGFR signaling. Furthermore, EpEX enhances the epithelial-to-mesenchymal transition and metastatic potential of colon cancer cells by activating ERK and FAK-AKT signaling pathways, and it further stabilizes active ß-catenin and Snail proteins by decreasing GSK3ß activity. Finally, we show that the combined treatment of an anti-EpCAM neutralizing antibody (EpAb2-6) and an HGFR inhibitor (crizotinib) significantly inhibits tumor progression and prolongs survival in metastatic and orthotopic animal models of colon cancer. CONCLUSION: Our findings illuminate the molecular mechanisms underlying EpCAM signaling promotion of colon cancer metastasis, further suggesting that the combination of EpAb2-6 and crizotinib may be an effective strategy for treating cancer patients with high EpCAM expression.

Collagen-binding peptides for the enhanced imaging, lubrication and regeneration of osteoarthritic articular cartilage.

Treatments for osteoarthritis would benefit from the enhanced visualization of injured articular cartilage and from the targeted delivery of disease-modifying drugs to it. Here, by using ex vivo human osteoarthritic cartilage and live rats and minipigs with induced osteoarthritis, we report the application of collagen-binding peptides, identified via phage display, that are home to osteoarthritic cartilage and that can be detected via magnetic resonance imaging when conjugated with a superparamagnetic iron oxide. Compared with the use of peptides with a scrambled sequence, hyaluronic acid conjugated with the collagen-binding peptides displayed enhanced retention in osteoarthritic cartilage and better lubricated human osteoarthritic tissue ex vivo. Mesenchymal stromal cells encapsulated in the modified hyaluronic acid and injected intra-articularly in rats showed enhanced homing to osteoarthritic tissue and improved its regeneration. Molecular docking revealed WXPXW as the consensus motif that binds to the α1 chain of collagen type XII. Peptides that specifically bind to osteoarthritic tissue may aid the diagnosis and treatment of osteoarthritic joints.

Structural basis of interleukin-17B receptor in complex with a neutralizing antibody for guiding humanization and affinity maturation.

Upregulation of interleukin-17 receptor B (IL-17RB) is known to be oncogenic, while other IL-17 receptors and ligands are generally involved in pro-inflammatory pathways. We identify a mouse neutralizing monoclonal antibody (mAb) D9, which blocks the IL-17RB/IL-17B pathway and inhibits pancreatic tumorigenesis in an orthotopic mouse model. The X-ray crystal structure of the IL-17RB ectodomain in complex with its neutralizing antibody D9 shows that D9 binds to a predicted ligand binding interface and engages with the A'-A loop of IL-17RB fibronectin III domain 1 in a unique conformational state. This structure also provides important paratope information to guide the design of antibody humanization and affinity maturation of D9, resulting in a humanized 1B12 antibody with marginal affinity loss and effective neutralization of IL-17B/IL-17RB signaling to impede tumorigenesis in a mouse xenograft model.

Reversal of pancreatic desmoplasia by a tumour stroma-targeted nitric oxide nanogel overcomes TRAIL resistance in pancreatic tumours.

OBJECTIVE: Stromal barriers, such as the abundant desmoplastic stroma that is characteristic of pancreatic ductal adenocarcinoma (PDAC), can block the delivery and decrease the tumour-penetrating ability of therapeutics such as tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), which can selectively induce cancer cell apoptosis. This study aimed to develop a TRAIL-based nanotherapy that not only eliminated the extracellular matrix barrier to increase TRAIL delivery into tumours but also blocked antiapoptotic mechanisms to overcome TRAIL resistance in PDAC. DESIGN: Nitric oxide (NO) plays a role in preventing tissue desmoplasia and could thus be delivered to disrupt the stromal barrier and improve TRAIL delivery in PDAC. We applied an in vitro-in vivo combinatorial phage display technique to identify novel peptide ligands to target the desmoplastic stroma in both murine and human orthotopic PDAC. We then constructed a stroma-targeted nanogel modified with phage display-identified tumour stroma-targeting peptides to co-deliver NO and TRAIL to PDAC and examined the anticancer effect in three-dimensional spheroid cultures in vitro and in orthotopic PDAC models in vivo. RESULTS: The delivery of NO to the PDAC tumour stroma resulted in reprogramming of activated pancreatic stellate cells, alleviation of tumour desmoplasia and downregulation of antiapoptotic BCL-2 protein expression, thereby facilitating tumour penetration by TRAIL and substantially enhancing the antitumour efficacy of TRAIL therapy. CONCLUSION: The co-delivery of TRAIL and NO by a stroma-targeted nanogel that remodels the fibrotic tumour microenvironment and suppresses tumour growth has the potential to be translated into a safe and promising treatment for PDAC.

Homogeneous antibody and CAR-T cells with improved effector functions targeting SSEA-4 glycan on pancreatic cancer.

Pancreatic cancer is usually asymptomatic in the early stages; the 5-y survival rate is around 9%; and there is a lack of effective treatment. Here we show that SSEA-4 is more expressed in all pancreatic cancer cell lines examined but not detectable in normal pancreatic cells; and high expression of SSEA-4 or the key enzymes B3GALT5 + ST3GAL2 associated with SSEA-4 biosynthesis significantly lowers the overall survival rate. To evaluate potential new treatments for pancreatic cancer, homogeneous antibodies with a well-defined Fc glycan for optimal effector functions and CAR-T cells with scFv construct designed to target SSEA-4 were shown highly effective against pancreatic cancer in vitro and in vivo. This was further supported by the finding that a subpopulation of natural killer (NK) cells isolated by the homogeneous antibody exhibited enhancement in cancer-cell killing activity compared to the unseparated NK cells. These results indicate that targeting SSEA-4 by homologous antibodies or CAR-T strategies can effectively inhibit cancer growth, suggesting SSEA-4 as a potential immunotherapy target for treating pancreatic disease.

NOTCH1 signaling promotes protein stability of HER3 through the AKT pathway in squamous cell carcinoma of head and neck.

Epidermal growth factor receptor (EGFR) remains the sole druggable molecular target other than the PD1/PD-L1 pathway with meaningful clinical benefit in squamous cell carcinoma of head and neck (SCCHN). Human epidermal growth factor receptor 3 (HER3) confers the resistance to EGFR-targeted treatment in SCCHN. Thus, it is essential to determine the distribution and regulatory mechanisms of HER3 in SCCHN. We explored the prevalence of HER3 expression and its distribution within SCCHN by immunohistochemical staining and clinicopathological correlations were analyzed. The regulatory mechanism of HER3 expression was then dissected in vitro, using RT-PCR, Western blotting, and immunoprecipitation in a set of SCCHN cell lines. Subsequent in vivo validation in the murine model was also performed. We found that concomitant high expression of HER3 and its ligand NRG1 in SCCHN is associated with the increased presence of regional lymphatic metastasis and the majority of HER3 is located on the differentiated tumor cells. Further investigation revealed that HER3 is under positive control of NOTCH1 through transcriptional activation and inhibition of protein degradation through the polyubiquitination machinery via AKT pathway and USP8 deubiquitinating enzyme. In addition, loss of function of NOTCH1 suppresses HER3 expression through increased phosphorylation of serine 473 of AKT in SCCHN cells, and promotes the aggressiveness of the tumor cells. These data indicated that the level of HER3 is regulated by NOTCH1 in SCCHN both transcriptionally and post-translationally, and NOTCH1 is in a higher hierarchy in the regulatory system of the AKT pathway. Since NOTCH1 is inactivated in approximately 10% of SCCHN cases and this aberration strongly impacts the AKT pathway and diminishes HER3, exclusion of patients with NOTCH1-inactivated SCCHN may be beneficial for future clinical trials of HER3-targeting antibodies.

Combined Effect of Anti-SSEA4 and Anti-Globo H Antibodies on Breast Cancer Cells.

The globo-series glycosphingolipids (SSEA3, SSEA4, and Globo H) were shown to express in many cancers selectively, and a combination of anti-SSEA4 and anti-Globo H antibodies was able to suppress tumor growth in mice inoculated with breast cancer cell lines. To further understand the effect, we focused on the combined effect of the two antibodies in target binding and antibody-dependent cellular cytotoxicity (ADCC) in vitro. Here, we report that the binding of anti-Globo H antibody (VK9) to MDA-MB231 breast cancer cells was influenced by anti-SSEA4 antibody (MC813-70), and a combination of both antibodies induced a similar effect as did anti-SSEA4 antibodies alone in a reporter-based ADCC assay, indicating that SSEA4 is a major target in breast cancer due to its higher expression than Globo H. Furthermore, we showed that a homogeneous anti-SSEA4 antibody (chMC813-70-SCT) designed to maximize the ADCC activity can be used to isolate a subpopulation of natural killer (NK) cells that exhibit an ∼23% increase in killing the target cells as compared to the unseparated NK cells. These findings can be used to predict a therapy outcome based on the expression levels of antigens and evaluate therapeutic antibody development.

Rapid generation of mouse model for emerging infectious disease with the case of severe COVID-19.

Since the pandemic of COVID-19 has intensely struck human society, small animal model for this infectious disease is in urgent need for basic and pharmaceutical research. Although several COVID-19 animal models have been identified, many of them show either minimal or inadequate pathophysiology after SARS-CoV-2 challenge. Here, we describe a new and versatile strategy to rapidly establish a mouse model for emerging infectious diseases in one month by multi-route, multi-serotype transduction with recombinant adeno-associated virus (AAV) vectors expressing viral receptor. In this study, the proposed approach enables profound and enduring systemic expression of SARS-CoV-2-receptor hACE2 in wild-type mice and renders them vulnerable to SARS-CoV-2 infection. Upon virus challenge, generated AAV/hACE2 mice showed pathophysiology closely mimicking the patients with severe COVID-19. The efficacy of a novel therapeutic antibody cocktail RBD-chAbs for COVID-19 was tested and confirmed by using this AAV/hACE2 mouse model, further demonstrating its successful application in drug development.

ENO1 Promotes Lung Cancer Metastasis via HGFR and WNT Signaling-Driven Epithelial-to-Mesenchymal Transition.

ENO1 (α-enolase) expression is significantly correlated with reduced survival and poor prognosis in many cancer types, including lung cancer. However, the function of ENO1 in carcinogenesis remains elusive. In this study, we found that high expression of ENO1 is present in metastatic lung cancer cell lines and malignant tumors and is associated with poor overall survival of patients with lung cancer. Knockdown of ENO1 decreased cancer cell proliferation and invasiveness, whereas overexpression of ENO1 enhanced these processes. Moreover, ENO1 expression promoted tumor growth in orthotopic models and enhanced lung tumor metastasis in tail-vein injection models. These effects were mediated by upregulation of mesenchymal markers N-cadherin and vimentin and the epithelial-to-mesenchymal transition regulator SLUG, along with concurrent downregulation of E-cadherin. Mechanistically, ENO1 interacted with hepatocyte growth factor receptor (HGFR) and activated HGFR and Wnt signaling via increased phosphorylation of HGFR and the Wnt coreceptor LRP5/6. Activation of these signaling axes decreased GSK3ß activity via Src-PI3K-AKT signaling and inactivation of the ß-catenin destruction complex to ultimately upregulate SLUG and ß-catenin. In addition, we generated a chimeric anti-ENO1 mAb (chENO1-22) that can decrease cancer cell proliferation and invasion. chENO1-22 attenuated cancer cell invasion by inhibiting ENO1-mediated GSK3ß inactivation to promote SLUG protein ubiquitination and degradation. Moreover, chENO1-22 prevented lung tumor metastasis and prolonged survival in animal models. Taken together, these findings illuminate the molecular mechanisms underlying the function of ENO1 in lung cancer metastasis and support the therapeutic potential of a novel antibody targeting ENO1 for treating lung cancer. SIGNIFICANCE: This study shows that ENO1 promotes lung cancer metastasis via HGFR and WNT signaling and introduces a novel anti-ENO1 antibody for potential therapeutic use in lung cancer.

EpCAM Signaling Promotes Tumor Progression and Protein Stability of PD-L1 through the EGFR Pathway.

Although epithelial cell adhesion molecule (EpCAM) has previously been shown to promote tumor progression, the underlying mechanisms remain largely unknown. Here, we report that the EGF-like domain I within the extracellular domain of EpCAM (EpEX) binds EGFR, activating both AKT and MAPK signaling to inhibit forkhead transcription factor O3a (FOXO3a) function and stabilize PD-L1 protein, respectively. Treatment with the EpCAM neutralizing antibody, EpAb2-6, inhibited AKT and FOXO3a phosphorylation, increased FOXO3a nuclear translocation, and upregulated high temperature requirement A2 (HtrA2) expression to promote apoptosis while decreasing PD-L1 protein levels to enhance the cytotoxic activity of CD8+ T cells. In vivo, EpAb2-6 markedly extended survival in mouse metastasis and orthotopic models of human colorectal cancer. The combination of EpAb2-6 with atezolizumab, an anti-PD-L1 antibody, almost completely eliminated tumors. Moreover, the number of CD8+ T cells in combination-treated tumors was increased compared with atezolizumab alone. Our findings suggest a new combination strategy for cancer immunotherapy in patients with EpCAM-expressing tumors. SIGNIFICANCE: This study shows that treatment with an EpCAM neutralizing antibody promotes apoptosis while decreasing PD-L1 protein to enhance cytotoxic activity of CD8+ T cells.

Novel monoclonal antibody against integrin α3 shows therapeutic potential for ovarian cancer.

Ovarian cancer has a high recurrence rate after platinum-based chemotherapy. To improve the treatment of ovarian cancer and identify ovarian cancer-specific antibodies, we immunized mice with the human ovarian carcinoma cell line, SKOV-3, and generated hybridoma clones. Several rounds of screening yielded 30 monoclonal antibodies (mAbs) with no cross-reactivity to normal cells. Among these mAbs, OV-Ab 30-7 was found to target integrin α3 and upregulate p53 and p21, while stimulating the apoptosis of cancer cells. We further found that binding of integrin α3 by OV-Ab 30-7 impaired laminin-induced focal adhesion kinase phosphorylation. The mAb alone or in combination with carboplatin and paclitaxel inhibited tumor progression and prolonged survival of tumor-bearing mice. Moreover, immunohistochemical staining of ovarian patient specimens revealed higher levels of integrin α3 in cancer cells compared with normal cells. By querying online clinical databases, we found that elevated ITGA3 expression in ovarian cancer is associated with poor prognosis. Taken together, our data suggest that the novel mAb, OV-Ab 30-7, may be considered as a potential therapeutic for ovarian cancer.

Imaging and biodistribution of radiolabeled SP90 peptide in BT-483 tumor bearing mice.

The objective of this study was to evaluate radiolabeled DOTA-SP90 as a radiotracer for breast cancer. The in vitro competition assay showed that radiolabeled DOTA-SP90 had significant binding affinity to BT-483 cancer cells. Biodistribution, nanoSPECT/CT and nanoPET/CT imaging results indicated that radiolabeled DOTA-SP90 can accumulate in tumors. In addition, radiolabeled DOTA-SP90 peptides can also detect metastatic tumors. Therefore, radiolabeled SP90 peptide may provide the potential capability as diagnostic agent for breast cancer patients.

Novel anti-EGFR scFv human antibody-conjugated immunoliposomes enhance chemotherapeutic efficacy in squamous cell carcinoma of head and neck.

BACKGROUND AND OBJECTIVES: Squamous cell carcinoma of head and neck (SCCHN) is the fifth most prevalent cancer worldwide. Because the anatomical complexity of this region, complete surgical resection is often not achievable and conventional chemotherapy would aid locoregional control and mitigate distant metastasis. Nonetheless, the nonspecific cytotoxicity and short in vivo half-life of conventional chemotherapeutic drugs limit their effects. Given the high frequency of overexpression of wild type epidermal growth factor receptor (EGFR), we exploit EGFR as a homing beacon for drug delivery system with cytotoxic payloads. MATERIALS AND METHODS: We generated fully human anti-EGFR single chain variable fragment (scFv)-conjugated immunoliposomes (IL) containing doxorubicin and vinorelbine and tested their anti-neoplastic efficacy in vitro and in vivo. RESULT: Our IL enhanced endocytosis and significantly reduced the half maximal inhibitory concentrations of the therapeutic payloads when compared to non-targeting liposomal counterparts in various cell lines of SCCHN. Furthermore, median survival time was significantly prolonged in subcutaneous and orthotopic SCCHN xenograft murine models treated with our IL formulations than those treated with non-targeting counterparts (94 days versus 60 days and 72 days versus 56 days, respectively) without evident increased systemic toxicity. CONCLUSION: The therapeutic index of the chemotherapeutic payloads was augmented by our EFGR-targeting IL formulation and they are warranted for further development and preclinical trial.

Development of therapeutic antibodies for the treatment of diseases.

It has been more than three decades since the first monoclonal antibody was approved by the United States Food and Drug Administration (US FDA) in 1986, and during this time, antibody engineering has dramatically evolved. Current antibody drugs have increasingly fewer adverse effects due to their high specificity. As a result, therapeutic antibodies have become the predominant class of new drugs developed in recent years. Over the past five years, antibodies have become the best-selling drugs in the pharmaceutical market, and in 2018, eight of the top ten bestselling drugs worldwide were biologics. The global therapeutic monoclonal antibody market was valued at approximately US$115.2 billion in 2018 and is expected to generate revenue of $150 billion by the end of 2019 and $300 billion by 2025. Thus, the market for therapeutic antibody drugs has experienced explosive growth as new drugs have been approved for treating various human diseases, including many cancers, autoimmune, metabolic and infectious diseases. As of December 2019, 79 therapeutic mAbs have been approved by the US FDA, but there is still significant growth potential. This review summarizes the latest market trends and outlines the preeminent antibody engineering technologies used in the development of therapeutic antibody drugs, such as humanization of monoclonal antibodies, phage display, the human antibody mouse, single B cell antibody technology, and affinity maturation. Finally, future applications and perspectives are also discussed.

Novel human Ab against vascular endothelial growth factor receptor 2 shows therapeutic potential for leukemia and prostate cancer.

Vascular endothelial growth factor receptor 2 (VEGFR2) is highly expressed in tumor-associated endothelial cells, where it modulates tumor-promoting angiogenesis, and it is also found on the surface of tumor cells. Currently, there are no Ab therapeutics targeting VEGFR2 approved for the treatment of prostate cancer or leukemia. Therefore, development of novel efficacious anti-VEGFR2 Abs will benefit cancer patients. We used the Institute of Cellular and Organismic Biology human Ab library and affinity maturation to develop a fully human Ab, anti-VEGFR2-AF, which shows excellent VEGFR2 binding activity. Anti-VEGFR2-AF bound Ig-like domain 3 of VEGFR2 extracellular region to disrupt the interaction between VEGF-A and VEGFR2, neutralizing downstream signaling of the receptor. Moreover, anti-VEGFR2-AF inhibited capillary structure formation and exerted Ab-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity in vitro. We found that VEGFR2 is expressed in PC-3 human prostate cancer cell line and associated with malignancy and metastasis of human prostate cancer. In a PC-3 xenograft mouse model, treatment with anti-VEGFR2-AF repressed tumor growth and angiogenesis as effectively and safely as US FDA-approved anti-VEGFR2 therapeutic, ramucirumab. We also report for the first time that addition of anti-VEGFR2 Ab can enhance the efficacy of docetaxel in the treatment of a prostate cancer mouse model. In HL-60 human leukemia-xenografted mice, anti-VEGFR2-AF showed better efficacy than ramucirumab with prolonged survival and reduced metastasis of leukemia cells to ovaries and lymph nodes. Our findings suggest that anti-VEGFR2-AF has strong potential as a cancer therapy that could directly target VEGFR2-expressing tumor cells in addition to its anti-angiogenic action.

PTPN3 suppresses lung cancer cell invasiveness by counteracting Src-mediated DAAM1 activation and actin polymerization.

Cancer cell migration plays a crucial role during the metastatic process. Reversible tyrosine phosphorylation by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) have been implicated in the regulation of cancer cell migration and invasion. However, the underlying mechanisms have not been fully elucidated. Here, we show that depletion of the FERM and PDZ domain-containing protein tyrosine phosphatase PTPN3 enhances lung cancer cell migration/invasion and metastasis by promoting actin filament assembly and focal adhesion dynamics. We further identified Src and DAAM1 (dishevelled associated activator of morphogenesis 1) as interactors of PTPN3. DAAM1 is a formin-like protein involved in the regulation of actin cytoskeletal remodeling. PTPN3 inhibits Src activity and Src-mediated phosphorylation of Tyr652 on DAAM1. The tyrosine phosphorylation of DAAM1 is essential for DAAM1 homodimer formation and actin polymerization. Ectopic expression of a DAAM1 phosphodeficient mutant inhibited F-actin assembly and suppressed lung cancer cell migration and invasion. Our findings reveal a novel mechanism by which reversible tyrosine phosphorylation of DAAM1 by Src and PTPN3 regulates actin dynamics and lung cancer invasiveness.

Dengue and Zika Virus Domain III-Flagellin Fusion and Glycan-Masking E Antigen for Prime-Boost Immunization.

The viral E proteins of dengue virus (DENV) and Zika virus (ZIKV) are the major viral proteins involved in receptor binding and fusion, and for the induction of protective antibodies against viral infections. DIII of the E proteins is an independent domain and stretches out on the virion surface that can elicit type-specific neutralizing antibodies. For recombinant DIII vaccine development, prime-boost immunizations can provide an advantage of eliciting more type-specific neutralizing antibodies by recalling DIII antigens after DIII booster to improve protection. Methods: The DIII of the E genes of DENV and ZIKV were fused with bacterial fliC gene for the expression of flagellin-DIII (FliC-DIII) fusion proteins. Prime-boost immunization strategies by the second-dose booster of four DENV serotype or ZIKV FliC-DIII fusion proteins were used to investigate the induction of neutralizing antibodies and protection against viral infections. Cross-reactive non-neutralizing antibodies in each group of antisera were also examined using in vitro antibody-dependent enhancement (ADE) assay. A series of glycan-masking E antigens were finally constructed for prime-boost immunizations to abolish the elicitation of cross-reactive non-neutralizing antibodies for ADE activity. Results: We showed that inclusion of a bivalent live-attenuated vaccine with a FliC-DIII booster is superior in eliciting neutralization titers and protection in vivo against all four-serotype DENVs. We also demonstrated that recombinant adenovirus vectors encoding four-serotype DENV prMEs with a FliC-DIII prime-boost scheme is capable of eliciting good antibody responses. In contract, recombinant adenovirus vector of ZIKV prME gene priming, followed by ZIKV FliC-DIII booster did not improve vaccine efficacy. The glycan-masking mutation on the ZIKV E protein ij loop (E-248NHT), but not on DENV2 E protein ij loop (E-242NHT), resulted in abolishing the elicitation of cross-reactive antibodies for DENV and ZIKV infection enhancements. Conclusions: Our findings can provide useful information for designing novel immunogens and vaccination strategies in an attempt to develop a safe and efficacious DENV or ZIKV vaccine.

The extracellular domain of epithelial cell adhesion molecule (EpCAM) enhances multipotency of mesenchymal stem cells through EGFR-LIN28-LET7 signaling.

Mesenchymal stem cells (MSCs) are widely considered to be an attractive cell source for regenerative therapies, but maintaining multipotency and self-renewal in cultured MSCs is especially challenging. Hence, the development and mechanistic description of strategies that help promote multipotency in MSCs will be vital to future clinical use. Here, using an array of techniques and approaches, including cell biology, RT-quantitative PCR, immunoblotting, immunofluorescence, flow cytometry, and ChIP assays, we show that the extracellular domain of epithelial cell adhesion molecule (EpCAM) (EpEX) significantly increases the levels of pluripotency factors through a signaling cascade that includes epidermal growth factor receptor (EGFR), signal transducer and activator of transcription 3 (STAT3), and Lin-28 homolog A (LIN28) and enhances the proliferation of human bone marrow MSCs. Moreover, we found that EpEX-induced LIN28 expression reduces the expression of the microRNA LET7 and up-regulates that of the transcription factor high-mobility group AT-hook 2 (HMGA2), which activates the transcription of pluripotency factors. Surprisingly, we found that EpEX treatment also enhances osteogenesis of MSCs under differentiation conditions, as evidenced by increases in osteogenic markers, including Runt-related transcription factor 2 (RUNX2). Taken together, our results indicate that EpEX stimulates EGFR signaling and thereby context-dependently controls MSC states and activities, promoting cell proliferation and multipotency under maintenance conditions and osteogenesis under differentiation conditions.