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Background: Dengue virus (DENV) is a significant public health threat in tropical and subtropical regions of the world. A therapeutic antibody against the viral envelope (E) protein represents a promising immunotherapy for disease control. Methodology/Principal Findings: We generated seventeen novel mouse monoclonal antibodies (mAbs) with high reactivity against E protein of dengue virus type 2 (DENV-2). The mAbs were further dissected using recombinant E protein domain I-II (E-DI-II) and III (E-DIII) of DENV-2. Using plaque reduction neutralization test (PRNT) and mouse protection assay with lethal doses of DENV-2, we identified four serotype-specific mAbs that had high neutralizing activity against DENV-2 infection. Of the four, E-DIII targeting mAb DB32-6 was the strongest neutralizing mAb against diverse DENV-2 strains. Using phage display and virus-like particles (VLPs) we found that residue K310 in the E-DIII A-strand was key to mAb DB32-6 binding E-DIII. We successfully converted DB32-6 to a humanized version that retained potency for the neutralization of DENV-2 and did not enhance the viral infection. The DB32-6 showed therapeutic efficacy against mortality induced by different strains of DENV-2 in two mouse models even in post-exposure trials. Conclusions/Significance: We used novel epitope mapping strategies, by combining phage display with VLPs, to identify the important A-strand epitopes with strong neutralizing activity. This study introduced potential therapeutic antibodies that might be capable of providing broad protection against diverse DENV-2 infections without enhancing activity in humans. [ABSTRACT FROM AUTHOR]

Background: The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs) against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored. Methodology/Principal Findings: We developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three recognized a novel epitope involving residues (Q211, D215, P217) at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol recognized multiple residues in A strand or residues in C strand/CC9 loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer. Conclusions/Significance: Our analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level. [ABSTRACT FROM AUTHOR]

Lung cancer is the leading cause of cancer-related mortality worldwide. The lack of tumor specificity remains a major drawback for effective chemotherapies and results in dose-limiting toxicities. However, a ligand-mediated drug delivery system should be able to render chemotherapy more specific to tumor cells and less toxic to normal tissues. In this study, we isolated a novel peptide ligand from a phage-displayed peptide library that bound to non-small cell lung cancer (NSCLC) cell lines. The targeting phage bound to several NSCLC cell lines but not to normal cells. Both the targeting phage and the synthetic peptide recognized the surgical specimens of NSCLC with a positive rate of 75% (27 of 36 specimens). In severe combined immunodeficiency (SCID) mice bearing NSCLC xenografts, the targeting phage specifically bound to tumor masses. The tumor homing ability of the targeting phage was inhibited by the cognate synthetic peptide, but not by a control or a WTY-mutated peptide. When the targeting peptide was coupled to liposomes carrying doxorubicin or vinorelbine, the therapeutic index of the chemotherapeutic agents and the survival rates of mice with human lung cancer xenografts markedly increased. Furthermore, the targeting liposomes increased drug accumulation in tumor tissues by 5.7- fold compared with free drugs and enhanced cancer cell apoptosis resulting from a higher concentration of bioavailable doxorubicin. The current study suggests that this tumor-specific peptide may be used to create chemotherapies specifically targeting tumor cells in the treatment of NSCLC and to design targeted gene transfer vectors or it may be used one in the diagnosis of this malignancy. [ABSTRACT FROM AUTHOR]

This document is a correction notice for an article titled "Development of a Humanized Antibody with High Therapeutic Potential against Dengue Virus Type 2." The correction states that there was an error in the use of panels in Figure 1C to represent the results in Figure 1D. An updated version of Figure 1 is provided to correct this mistake. The original data supporting all the results in the article and supporting information files are available from the corresponding author, except for the original western blot images underlying the DB19-4 panel in Figure 1B, which are no longer available. The authors apologize for the error in the published article. [Extracted from the article]

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. Author summary: Upon the emergence of new infectious disease, animal model becomes a pivotal tool for study of disease mechanism and development of therapeutics. In this study, we propose a versatile approach that allows rapid generation of mouse model for novel infectious disease once the receptor of the pathogen is identified. We demonstrated this approach by generating a mouse model for COVID-19 in a month's time. These mice were capable of recapitulating severe COVID-19 in patients, and successfully applied in the development of a therapeutic antibody cocktail for the disease. This not only suggests the usefulness of this mouse model for the research on COVID-19, but also exhibit the utility of the proposed approach for establishing animal model for infectious disease. [ABSTRACT FROM AUTHOR]

Dengue is an increasing public health concern worldwide and requires efficient laboratory diagnostics. We evaluated three commercially available dengue rapid diagnostic tests—the Humasis Dengue Combo NS1 & IgG/IgM (Humasis, Korea), SD Bioline Dengue Duo NS1 Ag & IgG/IgM (SD Bioline, Korea), and CareUS Dengue Combo NS1 and IgM/IgG kits (WellsBio, Korea)—and compared them to reference immunoglobulin M (IgM) or immunoglobulin G (IgG) ELISAs and quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays. In total, 109 dengue-positive samples from children with acute symptomatic dengue and 63 dengue-negative samples from febrile and asymptomatic individuals were collected. For the nonstructural 1 protein (NS1) Ag test, the sensitivity and specificity were in the following order: CareUS (79.82 and 100%), Humasis (63.30 and 100%), and SD Bioline (48.62 and 100%). For IgM and IgG, CareUS had the highest sensitivities and specificities (89.91 and 100%; 82.57 and 100%, respectively), followed by SD Bioline (60.55 and 100%, 77.98 and 100%, respectively), and Humasis (51.38 and 98.21%, 72.48 and 95.24%, respectively). The IgM kits were more sensitive than the NS1 Ag or IgG kits; however, combining NS1 Ag and IgM reduced the number of missed cases. Therefore, the NS1 Ag plus IgM dengue kits increase the accuracy of the results. In our study, the CareUS Dengue Combo NS1 and IgM/IgG kit showed higher accuracy in performance with reference to qRT-PCR and ELISA results. [ABSTRACT FROM AUTHOR]

Despite the low case fatality, Zika virus (ZIKV) infection has been associated with microcephaly in infants and Guillain-Barré syndrome. Antiviral and vaccine developments against ZIKV are still ongoing; therefore, in the meantime, preventing the disease transmission is critical. Primarily transmitted by Aedes species mosquitoes, ZIKV also can be sexually transmitted. We used AG129 mice lacking interferon-α/β and -γ receptors to study the testicular pathogenesis and sexual transmission of ZIKV. Infection of ZIKV progressively damaged mouse testes, increased testicular oxidative stress as indicated by the levels of reactive oxygen species, nitric oxide, glutathione peroxidase 4, spermatogenesis-associated-18 homolog in sperm and pro-inflammatory cytokines including IL-1β, IL-6, and G-CSF. We then evaluated the potential role of the antioxidant ebselen (EBS) in alleviating the testicular pathology with ZIKV infection. EBS treatment significantly reduced ZIKV-induced testicular oxidative stress, leucocyte infiltration and production of pro-inflammatory response. Furthermore, it improved testicular pathology and prevented the sexual transmission of ZIKV in a male-to-female mouse sperm transfer model. EBS is currently in clinical trials for various diseases. ZIKV infection could be on the list for potential use of EBS, for alleviating the testicular pathogenesis with ZIKV infection and preventing its sexual transmission. [ABSTRACT FROM AUTHOR]

Background: Recently, the number of reports on focal adhesion kinase (FAK) as a vital therapeutic target in solid carcinomas has increased; however, the prognostic role of FAK status remains poorly understood. This study aims to evaluate the prognostic effect of FAK by means of a meta-analysis. Methods: We performed a systematic literature search in order to examine the correlation between expression of FAK and overall survival(OS). The hazard ratio (HR) of OS was used to measure survival. A random-effects model was used to pool study statistics. Sensitivity and publication bias analyses were also conducted. Results: Thirty eligible studies involving 4702 patients were included. The median expression rate of FAK was 54%. Meta-analysis of the HRs demonstrated that high FAK expression was associated with worse OS (average HR = 2.073, 95%confidence interval[CI]:1.712–2.510, p = 0.000). Regarding cancer type, FAK was associated with worse OS in gastric cancer (HR = 2.646,95% CI:1.743–4.017, p = 0.000), hepatocellular carcinoma (HR = 1.788,95% CI:1.228–2.602, p = 0.002), ovarian cancer (HR = 1.815, 95% CI: 1.193–2.762, p = 0.005), endometrial cancer (HR = 4.149, 95% CI:2.832–6.079, p = 0.000), gliomas (HR = 2.650, 95% CI: 1.205–5.829, p = 0.015), and squamous cell carcinoma (HR = 1,696, 95% CI: 1.030–2.793, p = 0.038). No association was found between HR and disease staging according to our meta-regression analysis. Conclusions: Our study shows that high expression of FAK is associated with a worse OS in patients with carcinomas, but the association between FAK and prognosis varies according to cancer type. The value of FAK status in clinical prognosis in cancer needs further research. [ABSTRACT FROM AUTHOR]

CT-guided percutaneous ablation for liver cancer treatment is a relevant technique for patients not eligible for surgery and with tumors that are inconspicuous on US imaging. The lack of real-time imaging and the use of a limited amount of CT contrast agent make targeting the tumor with the needle challenging. In this study, we evaluate a registration framework that allows the integration of diagnostic pre-operative contrast enhanced CT images and intra-operative non-contrast enhanced CT images to improve image guidance in the intervention. The liver and tumor are segmented in the pre-operative contrast enhanced CT images. Next, the contrast enhanced image is registered to the intra-operative CT images in a two-stage approach. First, the contrast-enhanced diagnostic image is non-rigidly registered to a non-contrast enhanced image that is conventionally acquired at the start of the intervention. In case the initial registration is not sufficiently accurate, a refinement step is applied using non-rigid registration method with a local rigidity term. In the second stage, the intra-operative CT-images that are used to check the needle position, which often consist of only a few slices, are registered rigidly to the intra-operative image that was acquired at the start of the intervention. Subsequently, the diagnostic image is registered to the current intra-operative image, using both transformations, this allows the visualization of the tumor region extracted from pre-operative data in the intra-operative CT images containing needle. The method is evaluated on imaging data of 19 patients at the Erasmus MC. Quantitative evaluation is performed using the Dice metric, mean surface distance of the liver border and corresponding landmarks in the diagnostic and the intra-operative images. The registration of the diagnostic CT image to the initial intra-operative CT image did not require a refinement step in 13 cases. For those cases, the resulting registration had a Dice coefficient for the livers of 91.4%, a mean surface distance of 4.4 mm and a mean distance between corresponding landmarks of 4.7 mm. For the three cases with a refinement step, the registration result significantly improved (p<0.05) compared to the result of the initial non rigid registration method (DICE of 90.3% vs 71.3% and mean surface distance of 5.1 mm vs 11.3 mm and mean distance between corresponding landmark of 6.4 mm vs 10.2 mm). The registration of the preoperative data with the needle image in 16 cases yielded a DICE of 90.1% and a mean surface distance of 5.2 mm. The remaining three cases with DICE smaller than 80% were classified as unsuccessful registration. The results show that this is promising tool for liver image registration in interventional radiology. [ABSTRACT FROM AUTHOR]

Here we presented a simple and effective membrane mimetic microfluidic device with antibody conjugated supported lipid bilayer (SLB) “smart coating” to capture viable circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) directly from whole blood of all stage clinical cancer patients. The non-covalently bound SLB was able to promote dynamic clustering of lipid-tethered antibodies to CTC antigens and minimized non-specific blood cells retention through its non-fouling nature. A gentle flow further flushed away loosely-bound blood cells to achieve high purity of CTCs, and a stream of air foam injected disintegrate the SLB assemblies to release intact and viable CTCs from the chip. Human blood spiked cancer cell line test showed the ~95% overall efficiency to recover both CTCs and CTMs. Live/dead assay showed that at least 86% of recovered cells maintain viability. By using 2 mL of peripheral blood, the CTCs and CTMs counts of 63 healthy and colorectal cancer donors were positively correlated with the cancer progression. In summary, a simple and effective strategy utilizing biomimetic principle was developed to retrieve viable CTCs for enumeration, molecular analysis, as well as ex vivo culture over weeks. Due to the high sensitivity and specificity, it is the first time to show the high detection rates and quantity of CTCs in non-metastatic cancer patients. This work offers the values in both early cancer detection and prognosis of CTC and provides an accurate non-invasive strategy for routine clinical investigation on CTCs. [ABSTRACT FROM AUTHOR]

The ability of Tetra-O-methyl nordihydroguaiaretic acid (M4N) to induce rapid cell death in combination with Etoposide, Rapamycin, or UCN-01 was examined in LNCaP cells, both in cell culture and animal experiments. Mice treated with M4N drug combinations with either Etoposide or Rapamycin showed no evidence of tumor and had a 100% survival rate 100 days after tumor implantation. By comparison all other vehicles or single drug treated mice failed to survive longer than 30 days after implantation. This synergistic improvement of anticancer effect was also confirmed in more than 20 cancer cell lines. In LNCaP cells, M4N was found to reduce cellular ATP content, and suppress NDUFS1 expression while inducing hyperpolarization of mitochondrial membrane potential. M4N-treated cells lacked autophagy with reduced expression of BNIP3 and ATG5. To understand the mechanisms of this anticancer activity of M4N, the effect of this drug on three cancer cell lines (LNCaP, AsPC-1, and L428 cells) was further examined via transcriptome and metabolomics analyses. Metabolomic results showed that there were reductions of 26 metabolites essential for energy generation and/or production of cellular components in common with these three cell lines following 8 hours of M4N treatment. Deep RNA sequencing analysis demonstrated that there were sixteen genes whose expressions were found to be modulated following 6 hours of M4N treatment similarly in these three cell lines. Six out of these 16 genes were functionally related to the 26 metabolites described above. One of these up-regulated genes encodes for CHAC1, a key enzyme affecting the stress pathways through its degradation of glutathione. In fact M4N was found to suppress glutathione content and induce reactive oxygen species production. The data overall indicate that M4N has profound specific negative impacts on a wide range of cancer metabolisms supporting the use of M4N combination for cancer treatments. [ABSTRACT FROM AUTHOR]

Lipocalin-type prostaglandin D synthase (L-PGDS) is a member of the lipocalin superfamily, which is composed of secretory transporter proteins, and binds a wide variety of small hydrophobic molecules. Using this function, we have reported the feasibility of using L-PGDS as a novel drug delivery vehicle for poorly water-soluble drugs. In this study, we show the development of a drug delivery system using L-PGDS, one that enables the direct clinical use of 7-ethyl-10-hydroxy-camptothecin (SN-38), a poorly water-soluble anti-cancer drug. In the presence of 2 mM L-PGDS, the concentration of SN-38 in PBS increased 1,130-fold as compared with that in PBS. Calorimetric experiments revealed that L-PGDS bound SN-38 at a molecular ratio of 1:3 with a dissociation constant value of 60 μM. The results of an in vitro growth inhibition assay revealed that the SN-38/L-PGDS complexes showed high anti-tumor activity against 3 human cancer cell lines, i.e., Colo201, MDA-MB-231, and PC-3 with a potency similar to that of SN-38 used alone. The intravenous administration of SN-38/L-PGDS complexes to mice bearing Colo201 tumors showed a pronounced anti-tumor effect. Intestinal mucositis, which is one of the side effects of this drug, was not observed in mice administered SN-38/L-PGDS complexes. Taken together, L-PGDS enables the direct usage of SN-38 with reduced side effects. [ABSTRACT FROM AUTHOR]

Introduction: Radiofrequency ablation (RFA) is used for the local treatment of liver cancer. RFA is effective for small (<3cm) tumors, but for tumors > 3 cm, there is a tendency to leave viable tumor cells in the margins or clefts of overlapping ablation zones. This increases the possibility of incomplete ablation or local recurrence. Lyso-Thermosensitive Liposomal Doxorubicin (LTLD), is a thermally sensitive liposomal doxorubicin formulation for intravenous administration, that rapidly releases its drug content when exposed to temperatures >40°C. When used with RFA, LTLD releases its doxorubicin in the vasculature around the zone of ablation-induced tumor cell necrosis, killing micrometastases in the ablation margin. This may reduce recurrence and be more effective than thermal ablation alone. Purpose: The purpose of this study was to optimize the RFA procedure used in combination with LTLD to maximize the local deposition of doxorubicin in a swine liver model. Pigs were anaesthetized and the liver was surgically exposed. Each pig received a single, 50 mg/m2 dose of the clinical LTLD formulation (ThermoDox®). Subsequently, ablations were performed with either 1, 3 or 6 sequential, overlapping needle insertions in the left medial lobe with total ablation time of 15, 45 or 90 minutes respectively. Two different RFA generators and probes were evaluated. After the final ablation, the ablation zone (plus 3 cm margin) was dissected out and examined for doxorubicin concentration by LC/MS and fluorescence. Conclusion: The mean Cmax of plasma total doxorubicin was 26.5 μg/ml at the end of the infusion. Overall, increased heat time from 15 to 45 to 90 minutes shows an increase in both the amount of doxorubicin deposited (up to ~100 μg/g) and the width of the ablation target margin to which doxorubicin is delivered as determined by tissue homogenization and LC/MS detection of doxorubicin and by fluorescent imaging of tissues. [ABSTRACT FROM AUTHOR]

Lung cancer ranks among the most common malignancies, and is the leading cause of cancer-related mortality worldwide. Chemotherapy for lung cancer can be made more specific to tumor cells, and less toxic to normal tissues, through the use of ligand-mediated drug delivery systems. In this study, we investigated the targeting mechanism of the ligand-mediated drug delivery system using a peptide, SP5-2, which specifically binds to non-small cell lung cancer (NSCLC) cells. Conjugation of SP5-2 to liposomes enhanced the amount of drug delivered directly into NSCLC cells, through receptor-mediated endocytosis. Functional SP5-2 improved the therapeutic index of Lipo-Dox by enhancing therapeutic efficacy, reducing side effects, and increasing the survival rate of tumor-bearing mice in syngenic, metastatic and orthotopic animal models. Accumulation of SP5-2-conjugated liposomal doxorubicin (SP5-2-LD) in tumor tissues was 11.2-fold higher than that of free doxorubicin, and the area under the concentration-time curve (AUC0–72 hours) was increased 159.2-fold. Furthermore, the experiment of bioavailability was assessed to confirm that SP5-2 elevates the uptake of the liposomal drugs by the tumor cells in vivo. In conclusion, the use of SP5-2-conjugated liposomes enhances pharmacokinetic properties, improves efficacy and safety profiles, and allows for controlled biodistribution and drug release. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) is the leading cause of arboviral diseases in humans worldwide. The envelope (E) protein of DENV is the major target of neutralizing antibodies (Abs). Previous studies have shown that a significant proportion of anti-E Abs in human serum after DENV infection recognize the highly conserved fusion loop (FL) of E protein. The role of anti-FL Abs in protection against subsequent DENV infection versus pathogenesis remains unclear. A human anti-E monoclonal Ab was used as a standard in a virion-capture ELISA to measure the concentration of anti-E Abs, [anti-E Abs], in dengue-immune sera from Nicaraguan patients collected 3, 6, 12 and 18 months post-infection. The proportion of anti-FL Abs was determined by capture ELISA using virus-like particles containing mutations in FL, and the concentration of anti-FL Abs, [anti-FL Abs], was calculated. Neutralization titers (NT50) were determined using a previously described flow cytometry-based assay. Analysis of sequential samples from 10 dengue patients revealed [anti-E Abs] and [anti-FL Abs] were higher in secondary than in primary DENV infections. While [anti-FL Abs] did not correlate with NT50 against the current infecting serotype, it correlated with NT50 against the serotypes to which patients had likely not yet been exposed ("non-exposed" serotypes) in 14 secondary DENV3 and 15 secondary DENV2 cases. These findings demonstrate the kinetics of anti-FL Abs and provide evidence that anti-FL Abs play a protective role against "non-exposed" serotypes after secondary DENV infection. Author Summary: The four serotypes of dengue virus (DENV) are the leading cause of mosquito-borne viral diseases in humans. Whereas infection with one DENV serotype is thought to confer protection against re-infection with that serotype, it can be either protective or enhance disease severity upon subsequent ("secondary") infection with a different serotype. The envelope (E) protein of DENV is the major target of neutralizing antibodies. Previously, we and others reported that a significant proportion of anti-E antibodies in human dengue-immune sera recognize the fusion loop (FL) of E protein. The role of anti-FL antibodies in protection against subsequent DENV infections versus pathogenesis remains unclear. In this study, we developed capture ELISAs to measure the concentrations of anti-E and anti-FL antibodies in sera of Nicaraguan dengue patients collected 3, 6, 12 and 18 months post-illness, and investigated the kinetics of these antibodies and their relationship to neutralization activity. While the concentrations of anti-FL antibodies did not correlate with 50% neutralization titers (NT50) against the current infecting serotype, it correlated with NT50 against serotypes to which patients had likely not yet been exposed ("non-exposed" serotypes) in secondary DENV infections. These findings provide evidence that anti-FL antibodies play a protective role against "non-exposed" serotypes after secondary DENV infection. [ABSTRACT FROM AUTHOR]

Background: The envelope (E) protein of dengue virus (DENV) is the major immunogen for dengue vaccine development. At the C-terminus are two α-helices (EH1 and EH2) and two transmembrane domains (ET1 and ET2). After synthesis, E protein forms a heterodimer with the precursor membrane (prM) protein, which has been shown as a chaperone for E protein and could prevent premature fusion of E protein during maturation. Recent reports of enhancement of DENV infectivity by anti-prM monoclonal antibodies (mAbs) suggest the presence of prM protein in dengue vaccine is potentially harmful. A better understanding of prM-E interaction and its effect on recognition of E and prM proteins by different antibodies would provide important information for future design of safe and effective subunit dengue vaccines. Methodology/Principal Findings: In this study, we examined a series of C-terminal truncation constructs of DENV4 prME, E and prM. In the absence of E protein, prM protein expressed poorly. In the presence of E protein, the expression of prM protein increased in a dose-dependent manner. Radioimmunoprecipitation, sucrose gradient sedimentation and pulsechase experiments revealed ET1 and EH2 were involved in prM-E interaction and EH2 in maintaining the stability of prM protein. Dot blot assay revealed E protein affected the recognition of prM protein by an anti-prM mAb; truncation of EH2 or EH1 affected the recognition of E protein by several anti-E mAbs, which was further verified by capture ELISA. The E protein ectodomain alone can be recognized well by all anti-E mAbs tested. Conclusions/Significance: A C-terminal domain (EH2) of DENV E protein can affect the expression and stability of its chaperone prM protein. These findings not only add to our understanding of the interaction between prM and E proteins, but also suggest the ectodomain of E protein alone could be a potential subunit immunogen without inducing anti-prM response. [ABSTRACT FROM AUTHOR]

Background: The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs) against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored. Methodology/Principal Findings: We developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three recognized a novel epitope involving residues (Q211, D215, P217) at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol recognized multiple residues in A strand or residues in C strand/CC′ loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer. Conclusions/Significance: Our analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level. Author Summary: Dengue virus is the leading cause of arboviral diseases worldwide. The envelope protein is the major target of neutralizing antibodies and vaccine development. While previous studies have reported several epitopes on envelope protein, the possibility of interdomain epitopes and the relationship of epitopes to neutralizing potency remain unexplored. We developed a high throughput dot blot assay by using 67 alanine mutants of surface-exposed envelope residues as a systematic approach to identify epitopes recognized by mouse monoclonal antibodies and polyclonal human sera. Our results suggested the presence of interdomain epitopes more frequent than previously appreciated. Compared with monoclonal antibodies generated by traditional protocol, the potent neutralizing monoclonal antibodies generated by a new protocol showed several unique features of their epitopes. Moreover, the predominant epitopes of antibodies against envelope protein in polyclonal sera can be identified by this assay. These findings have implications for future development of epitope-specific diagnostics and epitope-based dengue vaccine, and add to our understanding of humoral immune responses to dengue virus at the epitope level. [ABSTRACT FROM AUTHOR]