The four serotypes of dengue virus (DENV1-4) pose a serious threat to global health. Cross-reactive and non-neutralizing antibodies enhance viral infection, thereby exacerbating the disease via antibody-dependent enhancement (ADE). Studying the epitopes targeted by these enhancing antibodies would improve the immune responses against DENV infection. In order to investigate the roles of antibodies in the pathogenesis of dengue, we generated a panel of 16 new monoclonal antibodies (mAbs) against DENV4. Using plaque reduction neutralization test (PRNT), we examined the neutralizing activity of these mAbs. Furthermore, we used the in vitro and in vivo ADE assay to evaluate the enhancement of DENV infection by mAbs. The results indicate that the cross-reactive and poorly neutralizing mAbs, DD11-4 and DD18-5, strongly enhance DENV1-4 infection of K562 cells and increase mortality in AG129 mice. The epitope residues of these enhancing mAbs were identified using virus-like particle (VLP) mutants. W212 and E26 are the epitope residues of DD11-4 and DD18-5, respectively. In conclusion, we generated and characterized 16 new mAbs against DENV4. DD11-4 and D18-5 possessed non-neutralizing activities and enhanced viral infection. Moreover, we identified the epitope residues of enhancing mAbs on envelope protein. These results may provide useful information for development of safe dengue vaccine.
Vascular endothelial growth factor receptor 2 (VEGFR2) is highly expressed in tumorassociated 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 leukemiaxenografted 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.
Dengue virus (DENV), a global disease, is divided into four serotypes (DENV1-4). Cross-reactive and non-neutralizing antibodies against envelope (E) protein of DENV bind to the Fcγ receptors (FcγR) of cells, and thereby exacerbate viral infection by heterologous serotypes via antibody-dependent enhancement (ADE). Identification and modification of enhancing epitopes may mitigate enhancement of DENV infection. In this study, we characterized the cross-reactive DB21-6 and DB39-2 monoclonal antibodies (mAbs) against domain I-II of DENV; these antibodies poorly neutralized and potently enhanced DENV infection both in vitro and in vivo. In addition, two enhancing mAbs, DB21-6 and DB39-2, were observed to compete with sera antibodies from patients infected with dengue. The epitopes of these enhancing mAbs were identified using phage display, structural prediction, and mapping of virus-like particle (VLP) mutants. N8, R9, V12, and E13 are the reactive residues of DB21-6, while N8, R9, and E13 are the reactive residues of DB39-2. N8 substitution tends to maintain VLP secretion, and decreases the binding activity of DB21-6 and DB39-2. The immunized sera from N8 substitution (N8R) DNA vaccine exerted greater neutralizing and protective activity than wild-type (WT)-immunized sera, both in vitro and in vivo. Furthermore, treatment with N8R-immunized sera reduced the enhancement of mortality in AG129 mice. These results support identification and substitution of enhancing epitope as a novel strategy for developing safe dengue vaccines.
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