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Fusion genes were predicted by chimerascan + deFUSE, with BLAT validation.

Sarcomatoid metastatic renal cell carcinoma (mRCC) is associated with a poor prognosis, and the biology of the disease has been inadequately characterized. RNA sequencing (RNA-seq) was performed on adjacent benign, clear cell, and sarcomatoid components from clinical specimens with sarcomatoid mRCC. M phase and cell-cycle pathways were enriched in sarcomatoid versus adjacent clear cell components, suggesting greater cell proliferation. The expression of aurora kinase A (AURKA) was increased as part of these pathways, and its increased expression was validated by quantitative PCR (qPCR). Immunohistochemical (IHC) analysis revealed that AURKA levels were increased in sarcomatoid tissue compared with their benign or clear cell parts. The increase in AURKA correlated with increased mTOR pathway activity, as evidenced by increased expression of phosphorylated mTOR (S2448) and ribosomal protein S6K (T389). When AURKA was stably expressed in a RCC cell line (Renca), it resulted in increased expression and activity of mTOR, suggesting that overexpression of AURKA can activate the mTOR pathway. These results warrant the analysis of a larger clinical cohort and suggest that targeting AURKA and/or mTOR in patients with sarcomatoid mRCC should be explored.Implications: Comparative RNA-seq of adjacent sarcomatoid and clear cell histology of RCC indicates a proliferative phenotype and increased AURKA-dependent activation of mTOR signaling in sarcomatoid RCC, which could be targeted by available agents. Mol Cancer Res; 13(1); 130–7. ©2014 AACR.

RNA-seq QC data (C=clear cell, S=sarcomatoid, B=benign).

Forward and reverse primers.

Significantly different genes sarcomatoid vs. clear cell.

Introduction: Contact lenses coated with antibacterial agents may reduce the risk of microbial keratitis; however, to the best of our knowledge, such contact lenses are not available on the market. Methods: We determined the ability of zinc oxide nanoparticles (ZnO-NPs)-loaded soft contact lenses to prevent the adhesion of Staphylococcus aureus and Pseudomonas aeruginosa. Commercially acquired sterile silicone hydrogel contact lenses were soaked in ZnO-NPs (˂50 nm) suspensions of various concentrations, and the stability of the ZnO-NPs coating on contact lenses over 28 days was monitored using a UV-vis spectrophotometer. The cytotoxicity effects of ZnO-NPs on human corneal epithelial cells were evaluated using a lactate dehydrogenase (LDH) kit. Results and Discussion: The results showed that the ZnO-NPs coating on contact lenses was optimal from day seven onward. In the following assays, optimally, ZnO-NP-coated contact lenses were incubated with S. aureus and P. aeruginosa suspensions (1 x 105 colony forming unit) for 24 hr at 37°C, followed by enumeration using the plating method. Our data showed that 100 ppm of ZnO-NPs coating on contact lenses reduced the adhesion of 69.9% and 74.6% of S. aureus and P. aeruginosa significantly (p Conclusion: This study provides insights into the potential role of ZnO-NPs in developing contact lenses with antibacterial properties.

Human immunodeficiency virus type-1 (HIV-1) antigenic variation poses a great challenge for vaccine immunogen design to elicit broadly neutralizing antibodies (bNAbs). Over the last 10-15 years, great progress has been made to understand the conserved sites of sensitivity on HIV envelope glycoprotein spikes targeted by bNAbs. Plasma neutralization mapping and monoclonal antibody isolation efforts have revealed five major conserved epitope clusters. Most of this work has focused on subtype B and C-infected Caucasian or African donors. It is not clear if the same epitopes and epitope rank order preferences are also true in donors infected with different HIV-1 subtypes and with different racial backgrounds. To investigate this point, in this study we report the first attempt to profile the bNAb specificities of CRF01_AE-infected Malaysian plasmas. We first measured neutralization titers of 21 plasmas against a subtype A, B, and AE pseudovirus panel. This revealed that 14% (3 of 21) plasmas had cross-clade breadth. Focusing on the cross-neutralizing plasma P9, we used AE and JR-FL mutant pseudoviruses, gp120 monomer interference, and native polyacrylamide gel electrophoresis to better understand the neutralization specificity. P9 demonstrates CD4-binding-site specificity with trimer dependence and D368 independence.

Aim: Herein, we determined the anti-amoebic properties of propolis collected from different regions in Iran against Naegleria fowleri and Balamuthia mandrillaris. Background: Natural products remain a valuable source of compounds that are biologically active. Approximately one-third of well-known therapeutic drugs worldwide are derived from natural products. Objectives: Considering the use of propolis in folk medicine, here, for the first time, we evaluated anti-amoebic properties of propolis against free-living pathogenic amoebae, B. mandrillaris and N. fowleri. Methods: Propolis was collected from different regions of Iran and ethanolic extracts were prepared. Amoebicidal and cysticidal assays were accomplished to determine the effects of propolis against N. fowleri and B. mandrillaris. In vitro cytotoxicity assays were carried out to establish propolis effects on host cell damage. Finally, propolis metabolites were determined using spectrometric methods. Results: It was observed that propolis exhibited potent antiamoebic effects against trophozoites and cysts of both N. fowleri and B. mandrillaris at μg concentrations. When tested against cysts of both amoebae, only selected propolis exhibited cysticidal properties. The best activity was observed by propolis from the Kermanshah region, which depicted more than 95% amoebicidal activity and more than 60% cysticidal activity against both amoebae, respectively. Liquid chromatography- tandem mass spectrometry revealed that each extract is comprised of various components. Conclusion: For the first time, we showed that propolis has amoebicidal activity against both amoebae tested. Investigation into the individual activity of the components of propolis extracts and their mechanism of action against the aforementioned parasites might lead to the discovery of novel anti-amoebic compounds.

Acanthamoeba keratitis is a sight-endangering eye infection, and causative organism Acanthamoeba presents a significant concern to public health, given escalation of contact lens wearers. Contemporary therapy is burdensome, necessitating prompt diagnosis and aggressive treatment. None of the contact lens disinfectants (local and international) can eradicate Acanthamoeba effectively. Using a range of compounds targeting cellulose, ion channels, and biochemical pathways, we employed bioassay-guided testing to determine their anti-amoebic effects. The results indicated that acarbose, indaziflam, terbuthylazine, glimepiride, inositol, vildagliptin and repaglinide showed anti-amoebic effects. Compounds showed minimal toxicity on human cells. Therefore, effects of the evaluated compounds after conjugation with nanoparticles should certainly be the subject of future studies and will likely lead to promising leads for potential applications.

Background Loss of monoubiquitination of histone H2B (H2Bub1) was found to be associated with poor differentiation, cancer stemness, and enhanced malignancy of non-small cell lung cancer (NSCLC). Herein, we investigated the biological significance and therapeutic implications of ubiquitin-specific protease 22 (USP22), an H2Bub1 deubiquitinase, in non-small cell lung cancer (NSCLC). Methods USP22 expression and its clinical relevance were assessed in NSCLC patients. The effects of USP22 knockout on sensitivity to cisplatin and irradiation, and growth, metastasis of NSCLC xenografts, and survival of cancer-bearing mice were investigated. The underlying mechanisms of targeting USP22 were explored. Results Overexpression of USP22 was observed in 49.0% (99/202) of NSCLC tissues; higher USP22 immunostaining was found to be associated with enhanced angiogenesis and recurrence of NSCLC. Notably, USP22 knockout dramatically suppressed in vitro proliferation, colony formation; and angiogenesis, growth, metastasis of A549 and H1299 in mouse xenograft model, and significantly prolonged survival of metastatic cancer-bearing mice. Furthermore, USP22 knockout significantly impaired non-homologous DNA damage repair capacity, enhanced cisplatin and irradiation-induced apoptosis in these cells. In terms of underlying mechanisms, RNA sequencing and gene ontology enrichment analysis demonstrated that USP22 knockout significantly suppressed angiogenesis, proliferation, EMT, RAS, c-Myc pathways, concurrently enhanced oxidative phosphorylation and tight junction pathways in A549 and H1299 NSCLC cells. Immunoblot analysis confirmed that USP22 knockout upregulated E-cadherin, p16; reduced ALDH1A3, Cyclin E1, c-Myc, and attenuated activation of AKT and ERK pathways in these cells. Conclusions Our findings suggest USP22 plays critical roles in the malignancy and progression of NSCLC and provide rationales for targeting USP22, which induces broad anti-cancer activities, as a novel therapeutic strategy for NSCLC patient.

Flap endonuclease 1 (FEN1) plays a crucial role in both DNA replication and damage repair. In this study, FEN1 expression and its clinical-pathologic significance in non–small-cell lung cancer (NSCLC) was investigated. Quantitative RT-PCR and immunohistochemistry analysis identified that both FEN1 mRNA and protein were highly overexpressed in about 36% of 136 cancer tissues compared to adjacent tissues, in which FEN1 was generally undetectable. Notably, patients with FEN1-overexpressed cancers were prone to have poor differentiation and poor prognosis. A strong positive correlation between the levels of FEN1 and Ki-67 staining was identified in these NSCLC tissues (r = 0.485), suggesting overexpressed FEN1 conferred a proliferative advantage to NSCLC. Furthermore, knockdown of FEN1 resulted in G1/S or G2/M phase cell cycle arrest and suppressed in vitro cellular proliferation in NSCLC cancer cells. Consistently, a selective FEN1 inhibitor was shown to effectively inhibit cellular proliferation of NSCLC cells in a dose-dependent manner. Additionally, knockdown of FEN1 significantly attenuated homologous DNA repair efficiency and enhanced cytotoxic effects of cisplatin in NSCLC cells. Taken together, these findings have indicated that overexpressed FEN1 represents a prognostic biomarker and potential therapeutic target for NSCLC treatment, which warrants further study.

Deregulated monoubiquitination of histone H2B (H2Bub1), mainly catalyzed by E3 ubiquitin-protein ligase RNF20/RNF40 complex, may play an important role in cancer. Here we investigate potential roles of H2Bub1 and the underlying mechanisms through which it contributes to cancer development and progression in lung adenocarcinoma. We show that downregulation of H2Bub1 through RNF20 knockdown dramatically decreases H3K79 and H3K4 trimethylation in both normal and malignant lung epithelial cell lines. Concurrently, global transcriptional profiling analysis reveals that multiple tumor-associated genes such as CCND3, E2F1/2, HOXA1, Bcl2 modifying factor (BMF), Met, and Myc; and signaling pathways of cellular dedifferentiation, proliferation, adhesion, survival including p53, cadherin, Myc, and anti-apoptotic pathways are differentially expressed or significantly altered in these lung epithelial cells upon downregulation of H2Bub1. Moreover, RNF20 knockdown dramatically suppresses terminal squamous differentiation of cultured bronchial epithelial cells, and significantly enhances proliferation, migration, invasion, and cisplatin resistance of lung cancer cells. Furthermore, immunohistochemistry analysis shows that H2Bub1 is extremely low or undetectable in > 70% of 170 lung adenocarcinoma samples. Notably, statistical analysis demonstrates that loss of H2Bub1 is significantly correlated with poor differentiation in lung adenocarcinoma (P = 0.0134). In addition, patients with H2Bub1-negative cancers had a trend towards shorter survival compared with patients with H2Bub1-positive cancers. Taken together, our findings suggest that loss of H2Bub1 may enhance malignancy and promote disease progression in lung adenocarcinoma probably through modulating multiple cancer signaling pathways. This article is protected by copyright. All rights reserved.

Previously, VLPs bearing JR-FL strain HIV-1 Envelope trimers elicited potent neutralizing antibodies (nAbs) in 2/8 rabbits (PLoS Pathog 11(5): e1004932) by taking advantage of a naturally absent glycan at position 197 that borders the CD4 binding site (CD4bs). In new immunizations, we attempted to improve nAb responses by removing the N362 glycan that also lines the CD4bs. All 4 rabbits developed nAbs. One targeted the N197 glycan hole like our previous sera. Two sera depended on the N463 glycan, again suggesting CD4bs overlap. Heterologous boosts appeared to reduce nAb clashes with the N362 glycan. The fourth serum targeted a N362 glycan-sensitive epitope. VLP manufacture challenges prevented us from immunizing larger rabbit numbers to empower a robust statistical analysis. Nevertheless, trends suggest that targeted glycan removal may improve nAb induction by exposing new epitopes and that it may be possible to modify nAb specificity using rational heterologous boosts.

The tumor microenvironment is composed of heterogeneous populations of cells, including cancer, immune, and stromal cells. Progression of tumor growth and initiation of metastasis is critically dependent on the reciprocal interactions between cancer cells and stroma. Through RNA-Seq and protein analyses, we found that cancer-associated fibroblasts derived from human breast cancer brain metastasis express significantly higher levels of chemokines CXCL12 and CXCL16 than fibroblasts from primary breast tumors or normal breast. To further understand the interplay between cancer cells and cancer-associated fibroblasts from each site, we developed three-dimensional organoids composed of patient-derived primary or brain metastasis cancer cells with matching cancer-associated fibroblasts. Three-dimensional CAF aggregates generated from brain metastasis promote migration of cancer cells more effectively than cancer-associated fibroblast aggregates derived from primary tumor or normal breast stromal cells. Treatment with a CXCR4 antagonist and/or CXCL16 neutralizing antibody, alone or in combination, significantly inhibited migration of cancer cells to brain metastatic cancer-associated fibroblast aggregates. These results demonstrate that human brain metastasis cancer-associated fibroblasts potently attract breast cancer cells via chemokines CXCL12 and CXCL16, and blocking CXCR6-CXCL16/CXCR4-CXCL12 receptor–ligand interactions may be an effective therapy for preventing breast cancer brain metastasis., Metastasis: brain metastases fuel further spread Breast cancer metastases to the brain secrete signaling molecules that promote additional cancer cells to migrate there. Peter P. Lee and colleagues from the City of Hope in Duarte, California, USA, analyzed protein and gene expression levels in brain metastases, and showed that it is the stromal cells (support cells such as fibroblasts), rather than the cancer cells themselves, that are the source of these homing signals. When compared against stromal cells derived from primary breast tumors or healthy breast tissue, they found that the stromal cells that had lodged themselves in the brain expressed the highest levels of CXCL12 and CXCL16, two chemokines involved in cell movement. Using three-dimensional aggregates, the researchers showed that these metastatic stromal cells promoted cancer cells migration more potently than stromal cells from primary tumors or normal breast tissues. Blocking the chemokine activity or that of its receptor impaired cancer cell movement, suggesting a possible therapeutic strategy for preventing brain metastasis in patients with breast cancer. These results highlight the importance of the tumor microenvironment and stromal cells in the metastasis process of breast cancer.

Background: Pathologic complete response (pCR) to HER2-targeting neoadjuvant therapy (NT) predicts for improved survival (Cortazar et al, Lancet, 2014). The addition of pertuzumab to trastuzumab and docetaxel increased pCR rates, and, as first line treatment for MBC led to longer overall survival ([OS] Swain et al, NEJM 2015). Avoidance of anthracyclines in the adjuvant setting for HER2+ BC reduced the risk of secondary hematologic malignancies without a detriment to OS (Slamon et al, NEJM, 20111). Finally, nab-paclitaxel (nab) might provide an advantage over other taxanes via decreased use of steroids and may lead to increased response rates (RR). We designed a study of pertuzumab (pert), trastuzumab (trast), and nab, testing the feasibility and efficacy of this regimen in the LABC and metastatic breast cancer settings. Materials and Methods: Pts with Stages II-III LABC received six cycles of NT with pert (day 1 q 21 days), trast, and nab 100 mg/m2 (both given IV, weekly). Pts with untreated MBC received the same regimen until progression, toxicities, or patient or physician preference led to stopping therapy. Primary endpoints included pCR (LABC) and RR and progression-free survival (PFS) in MBC. Forty pts with LABC and 25 pts with MBC were to be accrued. The study was designed to test whether the pCR rate of Neosphere (Gianni et al, Lancet Oncol, 2012, > 45.8%) and the PFS rate of CLEOPATRA (median of > 18.5 months) can be matched or exceeded. Procurement of serial samples for assessment of tumor gene expression, circulating tumor cells, miRNA, and serum DNA profiling for exploratory biomarker analysis was carried out. Results:Twenty-two of 28 already enrolled pts with LABC (clinical stage II:15, stage III: 7) completed NT. The median age was 53 (34-77). The pCR rate was 86% (6/7) for hormone receptor negative (HR-) and 40% (6/15) for HR+ pts, with an overall pCR of 55%. Three pts without pCR following NT had residual BC with a HER2 negative phenotype. Eighteen of 22 pts required nab dose modifications. The most frequent toxicities following NT included elevated liver function tests:27%, peripheral neuropathy:23%, hematological toxicities:17%, diarrhea:18%, infusion reactions:18%. In the MBC cohort there were 13 of 16 enrolled pts with > 2 months of follow-up. The median age was 47 (31-65), 62% had HR+ disease. A CR rate of 4/13 (31%) and confirmed RR of 77% were observed. The median number of cycles with pert, trast, nab was 9 (3+ to 41); 11 of 13 pts required dose modifications or delays (3 of the delays were due to primary breast surgery performed upon response to treatment). At a median follow-up of 19 months, PFS and OS estimates are 63% (95% CI 0.09-0.93), and 89% (95% CI 0.61-1.0). Conclusion: The non-anthracycline-containing regimen of pertuzumab, trastuzumab, and nab-paclitaxel induced a high pCR rate in HER2+ BC. PFS is encouraging in MBC. Outcome of the fully accrued cohorts inclusive of residual cancer burden scores in the LABC cohort, and correlative data with exploratory biomarker analysis will be presented. Citation Format: Somlo G, Frankel P, Yeon C, Yuan Y, Yim J, Kruper L, Taylor L, Mortimer J, Waisman J, Jones V, Vito C, Paz B, Huria A, Li D, Gaal C, Tong T, Tumyan L. Phase II trial of pertuzumab, trastuzumab, and nab-paclitaxel in patients (pts) with HER2 overexpressing (HER2+) locally advanced or inflammatory breast cancer (LABC) or untreated stage IV metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-21-35.

Malignant parotid tumors are heterogeneous and diverse. Accurate diagnosis requires a pathologist familiar with the various histologic subtypes, immunohistochemistry stains, and common translocations. Clinical course varies according to tumor subtype, ranging from indolent, slow-growing adenoid cystic carcinoma to rapidly progressive, possibly fatal, salivary ductal carcinoma. Histologic grade is important in prognosis and therapy. Surgery remains the mainstay of treatment when negative margins can be achieved. Radiation improves locoregional control of tumors with high-risk features. Chemotherapy for parotid tumors can be disappointing. Studies of new targeted therapies have not offered significant benefits.

Background: Pathologic complete response (pCR) and residual cancer burden (RCB scores of 0 [pCR] or 1[near CR]) after neoadjuvant chemotherapy (NCT) may predict for improved survival (Symmans et al. J Clin Oncol 25:4414-22, 2007). We set out to test the pCR rate with an anthracycline-free regimen of carboplatin (carb) and nab-paclitaxel (nab) in patients (pts) with triple negative breast cancer (TNBC). Materials and Methods: Forty-nine pts with stages II-III BC were to receive carb (AUC 6) on day 1 of a 28 day cycle, and nab 80 mg/m2 weekly, for a total of 4 cycles. Core biopsies were performed prior to NCT. Blood procurement for circulating tumor cell (CTC) analysis using the CellSearch platform was carried out pre-treatment, mid-treatment, and at surgery. We set out to assess the predictive value of Mammaprint (poor vs. good), BluePrint (basal, vs. luminal, vs. HER2) molecular subtype as well as microarray RNA and miRNA profiling, for pCR. Responses were also dichotomized as complete or near complete response (Symmans RCB scores of 0-1) vs. suboptimal response (RCB score > 1). Results: The median age was 53 (28-75). Pts presented with clinical stages II (63%) and III (37%). So far, 38 of the 49 pts accrued between 2/2012 and 6/2015, have undergone surgery, 68% of whom underwent modified radical mastectomy. The pCR rate (breast and lymph nodes in CR) was 53%, and RCB 0 and 1 were seen in 68% of pts. Toxicites included grade ¾ anemia (45%), thrombocytopenia (13%) and neutropenia (53%,1 pt with neutropenic fever). Dose adjustments were needed in over 80% of pts. Grades 2 or 3 peripheral neuropathy were seen in 8% each, and grades 3-4 fatigue (13%), hypokalemia (3%), and hyponatremia (3%) were observed. The median number of CTCs (pre-NCT) observed in 7 CTC positive pts of the first 27 pts who completed surgery was 1 (0-7), and 2 of the 7 pts continued to have CTCs at the time of surgery (1 CTC each), while 2 pts without CTCs pre-NCT had CTCs (1 each) detected at surgery. The final pt enrolled is expected to complete surgery by 10/2015. Results of sequential CTC assessments, MammaPrint/Blueprint and RNA/miRNA analysis of pre- and post-treatment specimens and their correlation with pCR will be presented. Conclusion: The non-anthracycline-containing regimen of carb and nab-paclitaxel induced a high pCR rate in TNBC, in preliminary analysis. Ongoing profiling may allow for future subset-specific modification of this regimen to increase pCR across all molecular subtypes of TNBC. Citation Format: Somlo G, Chung S, Frankel P, Hurria A, Koehler S, Kruper L, Mortimer JE, Paz B, Robinson K, Taylor L, Vito C, Waisman J, Yeon C, Yim J, Yuan Y, Tong T. Phase II trial of neoadjuvant chemotherapy with carboplatin and nab-paclitaxel in patients with triple negative locally advanced and inflammatory breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-14-10.

Background Dysregulated histone methyltransferase G9a may represent a potential cancer therapeutic target. The roles of G9a in tumorigenesis and therapeutics are not well understood in non-small cell lung cancer (NSCLC). Here we investigated the impact of G9a on tumor growth and signaling pathways in NSCLC. Methods Immunohistochemistry analyzed G9a expression in NSCLC tissues. Both siRNA and selective inhibitor were used to target G9a. The impact of targeting G9a on key genes, signaling pathways and growth were investigated in NSCLC cells by RNA sequencing analysis, rescue experiments, and xenograft models. Results Overexpression of G9a (≥ 5% of cancer cells showing positive staining) was found in 43.2% of 213 NSCLC tissues. Multiple tumor-associated genes including HP1α, APC2 are differentially expressed; and signaling pathways involved in cellular growth, adhesion, angiogenesis, hypoxia, apoptosis, and canonical Wnt signaling pathways are significantly altered in A549, H1299, and H1975 cells upon G9a knockdown. Additionally, targeting G9a by siRNA-mediated knockdown or by a selective G9a inhibitor UNC0638 significantly inhibited tumor growth, and dramatically suppressed Wnt signaling pathway in vitro and in vivo. Furthermore, we showed that treatment with UNC0638 restores the expression of APC2 expression in these cells through promoter demethylation. Restoring HP1α and silencing APC2 respectively attenuated the inhibitory effects on cell proliferation and Wnt signaling pathway in cancer cells in which G9a was silenced or suppressed. Conclusions These findings demonstrate that overexpressed G9a represents a promising therapeutic target, and targeting G9a potentially suppresses growth and Wnt signaling pathway partially through down-regulating HP1α and epigenetically restoring these tumor suppressors such as APC2 that are silenced in NSCLC. Electronic supplementary material The online version of this article (10.1186/s12943-018-0896-8) contains supplementary material, which is available to authorized users.

Although genetically compact, HIV-1 commandeers vast arrays of cellular machinery to sustain and protect it during cycles of viral outgrowth. Transposon-mediated saturation linker scanning mutagenesis was used to isolate fully replication-competent viruses harbouring a potent foreign epitope tag. Using these viral isolates, we performed differential isotopic labelling and affinity-capture mass spectrometric analyses on samples obtained from cultures of human lymphocytes to classify the vicinal interactomes of the viral Env and Vif proteins as they occur during natural infection. Importantly, interacting proteins were recovered without bias, regardless of their potential for positive, negative or neutral impact on viral replication. We identified specific host associations made with trimerized Env during its biosynthesis, at virological synapses, with innate immune effectors (such as HLA-E) and with certain cellular signalling pathways (for example, Notch1). We also defined Vif associations with host proteins involved in the control of nuclear transcription and nucleoside biosynthesis as well as those interacting stably or transiently with the cytoplasmic protein degradation apparatus. Our approach is broadly applicable to elucidating pathogen-host interactomes, providing high-certainty identification of interactors by their direct access during cycling infection. Understanding the pathophysiological consequences of these associations is likely to provide strategic targets for antiviral intervention.

In the coming age of Internet of Things, the underlying infrastructure that supports IoT applications will play a pivotal role. Bluetooth Low Energy Beacons, small radio frequency broadcasters that advertise their unique identification, have been highlighted for their possible usage in the IoT infrastructure, as a sensor network of BLE Beacons is capable of providing contextual and locational information to the users. However, as the size of the wireless sensor network has grown, finite battery capacity has proven to be a major challenge. Due to the limited battery capacity, Beacons require periodic maintenance and battery replacement, which results in increased beacon management cost and complexity. This paper attempts to remedy this problem through the integration of an energy harvesting mechanism with BLE Beacons, and explore the possibilities of using solar power to operate these devices. Experimental results for BLE Beacon power consumption and solar panel power output characteristics are presented, and therefore baseline parameters of the power requirements for sustainable BLE Beacons are established. Furthermore, a preliminary design of a solar-powered BLE Beacon is presented. It has been shown that a typical BLE Beacon with a transmission power of 0 dbm and advertising interval of 800 ms can be powered by a solar panel with surface area of 300 cm2, and a lithium ion rechargeable coin cell battery, LIR2450, with a nominal voltage of 3.6 V can be recharged by a solar panel with a surface area of 88 cm2.

An HIV-1 vaccine remains elusive, in part because various factors limit the quantity and quality of the antibodies raised against the viral envelope glycoprotein complex (Env). We hypothesized that targeting Env vaccines directly to B cells, by fusing them to molecules that bind and activate these cells, would improve Env-specific antibody responses. Therefore, we fused trimeric Env gp140 to A PRoliferation-Inducing Ligand (APRIL), B-cell Activating Factor (BAFF), and CD40 Ligand (CD40L). The Env-APRIL, Env-BAFF, and Env-CD40L gp140 trimers all enhanced the expression of activation-induced cytidine deaminase (AID), the enzyme responsible for inducing somatic hypermutation, antibody affinity maturation, and antibody class switching. They also triggered IgM, IgG, and IgA secretion from human B cells in vitro . The Env-APRIL trimers induced higher anti-Env antibody responses in rabbits, including neutralizing antibodies against tier 1 viruses. The enhanced Env-specific responses were not associated with a general increase in total plasma antibody concentrations, indicating that the effect of APRIL was specific for Env. All the rabbit sera raised against gp140 trimers, irrespective of the presence of CD40L, BAFF, or APRIL, recognized trimeric Env efficiently, whereas sera raised against gp120 monomers did not. The levels of trimer-binding and virus-neutralizing antibodies were strongly correlated, suggesting that gp140 trimers are superior to gp120 monomers as immunogens. Targeting and activating B cells with a trimeric HIV-1 Env-APRIL fusion protein may therefore improve the induction of humoral immunity against HIV-1.

HIV-1 viruses and virus-like particles (VLPs) bear nonnative “junk” forms of envelope (Env) glycoprotein that may undermine the development of antibody responses against functional gp120/gp41 trimers, thereby blunting the ability of particles to elicit neutralizing antibodies. Here, we sought to better understand the nature of junk Env with a view to devising strategies for its removal. Initial studies revealed that native trimers were surprisingly stable in the face of harsh conditions, suggesting that junk Env is unlikely to arise by trimer dissociation or gp120 shedding. Furthermore, the limited gp120 shedding that occurs immediately after synthesis of primary HIV-1 isolate Envs is not caused by aberrant cleavage at the tandem gp120/gp41 cleavage sites, which were found to cleave in a codependent manner. A major VLP contaminant was found to consist of an early, monomeric form of gp160 that is glycosylated in the endoplasmic reticulum (gp160ER) and then bypasses protein maturation and traffics directly into particles. gp160ER was found to bind two copies of monoclonal antibody (MAb) 2G12, consistent with its exclusively high-mannose glycan profile. These findings prompted us to evaluate enzyme digests as a way to remove aberrant Env. Remarkably, sequential glycosidase-protease digests led to a complete or near-complete removal of junk Env from many viral strains, leaving trimers and viral infectivity largely intact. “Trimer VLPs” may be useful neutralizing antibody immunogens.

Eliciting broad tier 2 neutralizing antibodies (nAbs) is a major goal of HIV-1 vaccine research. Here we investigated the ability of native, membrane-expressed JR-FL Env trimers to elicit nAbs. Unusually potent nAb titers developed in 2 of 8 rabbits immunized with virus-like particles (VLPs) expressing trimers (trimer VLP sera) and in 1 of 20 rabbits immunized with DNA expressing native Env trimer, followed by a protein boost (DNA trimer sera). All 3 sera neutralized via quaternary epitopes and exploited natural gaps in the glycan defenses of the second conserved region of JR-FL gp120. Specifically, trimer VLP sera took advantage of the unusual absence of a glycan at residue 197 (present in 98.7% of Envs). Intriguingly, removing the N197 glycan (with no loss of tier 2 phenotype) rendered 50% or 16.7% (n = 18) of clade B tier 2 isolates sensitive to the two trimer VLP sera, showing broad neutralization via the surface masked by the N197 glycan. Neutralizing sera targeted epitopes that overlap with the CD4 binding site, consistent with the role of the N197 glycan in a putative “glycan fence” that limits access to this region. A bioinformatics analysis suggested shared features of one of the trimer VLP sera and monoclonal antibody PG9, consistent with its trimer-dependency. The neutralizing DNA trimer serum took advantage of the absence of a glycan at residue 230, also proximal to the CD4 binding site and suggesting an epitope similar to that of monoclonal antibody 8ANC195, albeit lacking tier 2 breadth. Taken together, our data show for the first time that strain-specific holes in the glycan fence can allow the development of tier 2 neutralizing antibodies to native spikes. Moreover, cross-neutralization can occur in the absence of protecting glycan. Overall, our observations provide new insights that may inform the future development of a neutralizing antibody vaccine., Author Summary Here we show that native HIV-1 Env spikes expressed in a natural membrane context can induce potent tier 2 nAbs in rabbits. These antibodies reacted exclusively with epitopes present on these trimers and not with isolated Env subunits. Intriguingly, the neutralizing sera were found to take advantage of natural gaps in the carbohydrate defenses of Env spikes of the vaccine strain. Some sera were able to neutralize heterologous isolates, provided that a key, regulating glycan was removed. Overall, these findings suggest that native, membrane-expressed trimers hold promise for further development as vaccine candidates. In the future, by adapting our current findings, we might be able to encourage nAb development to key conserved sites by introducing additional, targeted gaps in the trimer's glycan shell. We suggest that the rare ability to predictably induce potent autologous neutralizing antibodies to field isolates, as we report here, provides a foundation for exploring new strategies aimed at inducing neutralization breadth which is widely expected to be essential for vaccine-induced protection.

Elevated ubiquitin-specific protease 22 (USP22) is associated with enhanced malignancy, therapeutic resistance, and poor prognosis of various human cancers. In this study, the clinicopathological significance of USP22, therapeutic implication of targeting USP22, and underlying molecules of USP22 knockdown were investigated in human non-small cell lung cancer (NSCLC). Immunohistochemistry analysis revealed that USP22 protein was dramatically elevated in ~ 67% of 197 NSCLC tissues compared with adjacent normal lung tissues. Importantly, statistical analysis demonstrated that elevated USP22 was positively associated with advanced cancer stage and a trend to poor prognosis of NSCLC patients. Moreover, siRNA-mediated knockdown of USP22 induced a cell cycle arrest in G1/S phase and significantly suppressed cell proliferation and soft agar colony formations in NSCLC cell lines. In addition, knockdown of USP22 significantly enhanced cisplatin-induced genotoxic effects in NSCLC cell lines. Furthermore, USP22 silencing substantially down-regulated SIRT1 and upregulated p53 tumor suppressor in NSCLC cells, indicating downregulation of USP22 may boost p53-mediated tumor suppression and DNA damage response in NSCLC cells. Knockdown of USP22 also increased global histone H2B monoubiquitylation, H3K4 and H3K79 trimethylation leading to global gene expression profile changes in these NSCLC cells. These results suggest that targeting USP22 will have broad anti-cancer effects via altering multiple cancer signaling pathways. Taken together, data of this study further indicate that the elevated USP22 may potentially represent a prognostic factor and therapeutic target for NSCLC patients. Note: This abstract was not presented at the meeting. Citation Format: Keqiang Zhang, Tommy R Tong, Xinwei Yun, Rebecca Nelson, Nardi Isaac, Ravi Salgia, Dan Raz. Elevated USP22 is a potential therapeutic target for human non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4451. doi:10.1158/1538-7445.AM2017-4451

Most HIV-1-specific neutralizing antibodies isolated to date exhibit unusual characteristics that complicate their elicitation. Neutralizing antibodies that target the V1V2 apex of the HIV-1 envelope (Env) trimer feature unusually long protruding loops, which enable them to penetrate the HIV-1 glycan shield. As antibodies with loops of requisite length are created through uncommon recombination events, an alternative mode of apex binding has been sought. Here, we isolated a lineage of Env apex-directed neutralizing antibodies, N90-VRC38.01-11, by using virus-like particles and conformationally stabilized Env trimers as B cell probes. A crystal structure of N90-VRC38.01 with a scaffolded V1V2 revealed a binding mode involving side-chain-to-side-chain interactions that reduced the distance the antibody loop must traverse the glycan shield, thereby facilitating V1V2 binding via a non-protruding loop. The N90-VRC38 lineage thus identifies a solution for V1V2-apex binding that provides a more conventional B cell pathway for vaccine design.

Purpose Increasing evidence has demonstrated that men taking the anticoagulant warfarin have a lower risk of developing prostate cancer. This phenomenon is not observed in other cancers. We sought to determine if the target of warfarin, vitamin K epoxide reductase (VKOR), is expressed in benign and cancerous prostate tissues and if a functional single nucleotide polymorphism (SNP) in the VKOR gene is associated with prostate cancer risk. Materials and methods The expression of VKOR was quantified by immunohistochemistry in an institutional series of 54 radical prostatectomy samples and metastatic biopsies, as well as in 40 other cancers and matched benign tissues on a tissue microarray. Genotyping of SNP rs2359612 was performed in a prospective series of 57 patients. Results VKOR is highly expressed in benign human prostate epithelial cells but is not expressed or expressed at very low levels in cancerous cells. This expression pattern is unique to prostate cancer. Additionally, the proportion of the carrier C allele of rs2359612 in the patients with prostate cancer was significantly higher than in the population, suggesting an association between this allele and the risk of having a diagnosis of prostate cancer. Conclusions The expression of VKOR in benign prostate epithelial cells, along with the association between a functional VKOR SNP and prostate cancer risk, suggests a possible role for VKOR in mediating the effect of warfarin on prostate cancer risk. Larger multi-institutional cohort studies are warranted, as are molecular studies on the role of VKOR in prostate cancer development.

Sarcomatoid metastatic renal cell carcinoma (mRCC) is associated with a poor prognosis, and the biology of the disease has been inadequately characterized. RNA sequencing (RNA-seq) was performed on adjacent benign, clear cell, and sarcomatoid components from clinical specimens with sarcomatoid mRCC. M phase and cell-cycle pathways were enriched in sarcomatoid versus adjacent clear cell components, suggesting greater cell proliferation. The expression of aurora kinase A (AURKA) was increased as part of these pathways, and its increased expression was validated by quantitative PCR (qPCR). Immunohistochemical (IHC) analysis revealed that AURKA levels were increased in sarcomatoid tissue compared with their benign or clear cell parts. The increase in AURKA correlated with increased mTOR pathway activity, as evidenced by increased expression of phosphorylated mTOR (S2448) and ribosomal protein S6K (T389). When AURKA was stably expressed in a RCC cell line (Renca), it resulted in increased expression and activity of mTOR, suggesting that overexpression of AURKA can activate the mTOR pathway. These results warrant the analysis of a larger clinical cohort and suggest that targeting AURKA and/or mTOR in patients with sarcomatoid mRCC should be explored. Implications: Comparative RNA-seq of adjacent sarcomatoid and clear cell histology of RCC indicates a proliferative phenotype and increased AURKA-dependent activation of mTOR signaling in sarcomatoid RCC, which could be targeted by available agents. Mol Cancer Res; 13(1); 130–7. ©2014 AACR.

Molecular and structural characterization is reported for a new broad and potent monoclonal antibody against HIV that binds to an epitope bridging the gp41 and gp120 subunits — the antibody affects a step in virus entry after binding to CD4 and before engagement of CCR5. This paper describes a broadly neutralizing HIV-specific monoclonal antibody that binds with high potency to a novel HIV-1 envelope glycoprotein epitope. Molecular and structural characterization of the new antibody, named 35O22, show that it is specific for a new site of vulnerability made up of amino acids and glycans bridging the gp41 and gp120 subunits. The antibody affects a step in virus entry after binding to CD4 and before engagement of CCR5. Serologic analysis indicates that antibodies to this newly recognized site of vulnerability are commonly elicited by natural infection, raising the prospect that in may be a promising potential vaccine target. The isolation of human monoclonal antibodies is providing important insights into the specificities that underlie broad neutralization of HIV-1 (reviewed in ref. 1). Here we report a broad and extremely potent HIV-specific monoclonal antibody, termed 35O22, which binds a novel HIV-1 envelope glycoprotein (Env) epitope. 35O22 neutralized 62% of 181 pseudoviruses with a half-maximum inhibitory concentration (IC50)

In natural infection, antibodies interact with HIV-1 primarily through nonfunctional forms of envelope glycoproteins (Env), including uncleaved (UNC) gp160 and gp41 stumps. These antigens are important to fully characterize, as they may be decoys that promote nonneutralizing responses and may also be targets for nonneutralizing effector responses. In this study, we compared the antigenic properties of Env expressed in situ on pseudovirion virus-like particle (VLP) surfaces and soluble gp120 using harmonized enzyme-linked immunosorbent assays (ELISAs) and a panel of 51 monoclonal antibodies (MAbs). Only 32 of 46 soluble gp120-reactive MAbs recognized the primary UNC gp160 antigen of VLPs. Indeed, many epitopes were poorly exposed (C1, V2, C1-C4, C4, C4-V3, CD4 induced [CD4i], and PGT group 3) or obscured (C2, C5, and C1-C5) on VLPs. In further studies, VLP Env exhibited an increased degree of inter-MAb competition, the epicenter of which was the base of the V3 loop, where PGT, 2G12, V3, and CD4 binding site specificities competed. UNC gp160 also underwent more drastic soluble CD4 (sCD4)-induced conformational changes than soluble gp120, exposing CD4i, C1-C4, and V2 epitopes. A greater propensity of UNC gp160 to undergo conformational changes was also suggested by the induction of CD4i MAb binding to VLPs by a V3 MAb as well as by soluble CD4. The same effect was not observed for soluble gp120. Taken together, our data suggest that membrane-expressed UNC gp160 exists in a less “triggered” conformational state than soluble gp120 and that MAb binding to UNC gp160 tends to have greater conformational consequences.

Background HIV-1 infected cells can establish new infections by crossing the vaginal epithelia and subsequently producing virus in a milieu that avoids the high microbicide concentrations of the vaginal lumen. Findings To address this problem, here, we report that pretreatment of HIV-infected peripheral blood mononuclear cells (PBMCs) with a 27 amino acid CD4-mimetic, M48U1, causes dramatic and prolonged reduction of infectious virus output, due to its induction of gp120 shedding. Conclusions M48U1 may, therefore, be valuable for prophylaxis of mucosal HIV-1 transmission.

Despite almost 30 years of research, no effective vaccine has yet been developed against HIV-1. Probably such a vaccine would need to induce both an effective T cell and antibody response. Any vaccine component focused on inducing humoral immunity requires the HIV-1 envelope (Env) glycoprotein complex as it is the only viral protein exposed on the virion surface. HIV-1 has evolved several mechanisms to evade broadly reactive neutralizing antibodies. One such a mechanism involves variable loop domains, which are highly flexible structures that shield the underlying conserved epitopes. We hypothesized that removal of such loops would increase the exposure and immunogenicity of these conserved regions. Env variable loop deletion however often leads to protein misfolding and aggregation because hydrophobic patches becoming solvent accessible. We have therefore previously used virus evolution to acquire functional Env proteins lacking the V1V2 loop. We then expressed them in soluble (uncleaved) gp140 forms. Three mutants were found to perform optimally in terms of protein expression, stability, trimerization and folding. In this study, we characterized the immune responses to these antigens in rabbits. The V1V2 deletion mutant Delta V1V2.9.VK induced a prominent response directed to epitopes that are not fully available on the other Env proteins tested but that effectively bound and neutralized the Delta V1V2 Env virus. This Env variant also induced more efficient neutralization of the tier 1 virus SF162. The immune refocusing effect was lost after booster immunization with a full-length gp140 protein with intact V1V2 loops. Collectively, this result suggests that deletion of variable domains could alter the specificity of the humoral immune response, but did not result in broad neutralization of neutralization-resistant virus isolates

Monoubiquitination of histone H2B (H2Bub1) is an important chromatin modification that plays multiple roles in transcription, mRNA processing, DNA repair, cell differentiation, and maintenance of stemness. Recent studies have indicated that dysregulation of H2Bub1 and the resulting alterations of global gene expression are key contributors in cellular transformation and metastasis. However, the importance of H2B ubiquitination in lung cancer is unknown. Here, by using immunohistochemical analysis (IHC), we have demonstrated that the protein level of H2Bub1 is frequently decreased in a cohort of human lung cancer tissues in comparison to paired adjacent normal tissues. We also found that decrease in H2Bub1 is associated with increased cellular proliferation, as measured by Ki67, and is associated with decreased overall survival. Furthermore, using RNA-seq and transcriptional profiling analysis, we identified that reduction of H2Bub1 via attenuating the expression of histone H2B E3 ubiquitin ligase ring finger protein 20 (RNF20) leads to significant changes of global gene expression, modulating multiple malignancy-promoting genes, including c-Met, E2F2, and BMF. Signaling pathways including p53, DNA replication, DNA repair, and cellular proliferation, and regulation of apoptosis in lung cancer cells are also altered. Additionally, we have further shown that decrease of H2Bub1 by RNF20 knockdown significantly promotes growth, migration/invasion, and strongly enhances resistance to cytotoxic chemotherapy-induced apoptosis in lung cancer cells. Our findings suggest that dysregulation of H2Bub1 signaling may play an important role in growth and drug resistance of lung cancers, and may potentially serve as a prognostic biomarker and therapeutic target for lung cancer patients. Citation Format: Keqiang Zhang, Jinhui Wang, Xiwei Wu, Theresa A. Reno, Tommy R. Tong, Jae Kim, Dan Raz. Decreased H2B monoubiquitination is associated with enhanced proliferation and resistance to apoptosis in human lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4523.

e635 Background: Multi-institution retrospective trials have shown that prolonged use of warfarin reduces the risk of prostate cancer (PC), an effect that is not observed for other cancers. The anti-coagulant warfarin prevents the gamma-carboxylation (gla) of target proteins by interfering with the vitamin K cycle through its inhibition of the vitamin K epoxide reductase (VKOR). Most known gla proteins are found in the clotting cascade, but here we investigate the role of gla of the androgen receptor (AR) and other warfarin-mediated effects in the prevention of PC by warfarin. Methods: We treated mice with warfarin and performed RNA-seq on prostate tissues. We used immunoprecipitation (IP) followed by mass spec sequencing to identify gla sites on AR. We used cell culture models to investigate the role of AR-gla and other pathways identified by RNA-seq data in the mechanism of action of warfarin. Finally, we sequenced a SNP in VKOR and performed IHC to detect VKOR expression in prostatectomy tissues in an institutional series. Results: RNA-seq data show alteration of several pathways in warfarin-treated mouse prostates, including AR signaling, hormone and lipid synthesis, and genes known to affect PC (e.g. SPINK1). Mass spec shows that AR is gla’d at amino acid 2E, which is known to be mutated in partial androgen insensitivity syndrome patients. Warfarin inhibits AR conformation change and transcriptional activity in benign (non-cancerous) prostate cells. VKOR knock-down recapitulates effects of warfarin treatment, suggesting an on target effect of the drug. Mutation of the AR-gla site has modest effects in benign prostate cells. In a small series of patients, we found that one allele of a VKOR SNP is found at a significantly higher rate in PC patients than expected. We also found that VKOR is highly expressed in normal human prostate epithelia but is lost in cancerous tissue, a pattern of expression that is unique to PC. Conclusions: Warfarin, likely through its ability to control AR-gla, inhibits AR transcriptional activity. The relative contribution of modest AR inhibition and the effect on other pathways must be investigated to determine how warfarin reduces the risk of PC and to identify optimal drug targets for development of chemopreventative therapeutics.

612 Background: Clear cell renal cell carcinoma (ccRCC) metastasis is associated with a dismal prognosis. Early identification of patients at high or low risk of metastasis may help to guide appropriate treatment. There are currently no reliable clinico-pathologic and laboratory modalities for this purpose in a routine clinical setting. We previously performed a microarray study using frozen ccRCC specimens and identified dysregulation of 4 microRNAs (miRNAs), miR-10b, miR-139-5p, miR-130b and miR-199b-5p, to be highly associated ccRCC metastasis and prognosis. Methods: We established a training cohort including formalin-fixed paraffin-embedded (FFPE) specimens of localized (n = 13; pT1) and metastatic (n = 15; M1) ccRCCs. We measured the expression of the 4 miRNAs of the training cohort samples by quantitative RT-PCR, normalized with miR-24, one of the most stably expressed miRNAs in human renal tissue as we previously reported. Using the risk score method, we rebuilt a mathematical formula with cutoff points to predict a patient to be at high, low or equivocal risk of metastasis. We validated the signature in an FFPE test cohort of 265-case primary ccRCCs. We examined the signature performance and its correlation with the cancer-specific survival. Results: Multivariate logistic regression analysis showed the signature to be highly associated with ccRCC metastasis (OR 7.67, 95%Cl 2.80-21.09, p < 0.0001), with the overall sensitivity and specificity of 80% and 76%. The sensitivities and specificities were 72% and 78%, 70% and 67%, and 78% and 75% for stage I, II or III patients, respectively. The signature predicted metastasis of small ccRCCs ( ≤ 4.0 cm), with the sensitivity of 60% and specificity of 77%. The overall positive and negative predictive values (PPV and NPV) were 61% and 89%. The area under the curve of ROC was 0.8. The signature was also found to be well correlated with worse cancer-specific survival (HR 3.06, 95%Cl 1.49-6.93, p < 0.01). Conclusions: This optimized miRNA signature can reliably predict ccRCC metastasis and prognosis and may help to stratify patients for more appropriate therapies and suitable clinical trials.

Hypothetically, since native HIV-1 Env trimers are exclusively recognized by neutralizing antibodies, they might induce the neutralizing antibodies in a vaccine setting. This idea has not been evaluated due to the difficulty of separating trimers from nonfunctional Env (uncleaved gp160 and gp41 stumps). The latter are immunodominant and induce nonneutralizing antibodies. We previously showed that nonfunctional Env can be selectively cleared from virus-like particle (VLP) surfaces by enzyme digests (E. T. Crooks, T. Tong , K. Osawa, and J. M. Binley, J.Virol. 85:5825, 2011). Here, we investigated the effects of these digests on the antigenicity of VLPs and their sensitivity to neutralization. Before digestion, WT VLPs (bearing wild-type Env) and UNC VLPs (bearing uncleaved gp160) were recognized by various Env-specific monoclonal antibodies (MAbs), irrespective of their neutralizing activity, a result which is consistent with the presence of nonfunctional Env. After digestion, only neutralizing MAbs recognized WT VLPs, consistent with selective removal of nonfunctional Env (i.e., “trimer VLPs”). Digests eliminated the binding of all MAbs to UNC VLPs, again consistent with removal of nonfunctional Env. An exception was MAb 2F5, which weakly bound to digested UNC VLPs and bald VLPs (bearing no Env), perhaps due to lipid cross-reactivity. Trimer VLPs were infectious, and their neutralization sensitivity was largely comparable to that of undigested WT VLPs. However, they were ∼100-fold more sensitive to the MAbs 4E10 and Z13e1, suggesting increased exposure of the gp41 base. Importantly, a scatterplot analysis revealed a strong correlation between MAb binding and neutralization of trimer VLPs. This suggests that trimer VLPs bear essentially pure native trimer that should allow its unfettered evaluation in a vaccine setting.

A small proportion of HIV-infected individuals generate a neutralizing antibody (NAb) response of exceptional magnitude and breadth. A detailed analysis of the critical epitopes targeted by broadly neutralizing antibodies should help to define optimal targets for vaccine design. HIV-1-infected subjects with potent cross-reactive serum neutralizing antibodies were identified by assaying sera from 308 subjects against a multiclade panel of 12 “tier 2” viruses (4 each of subtypes A, B, and C). Various neutralizing epitope specificities were determined for the top 9 neutralizers, including clade A-, clade B-, clade C-, and clade A/C-infected donors, by using a comprehensive set of assays. In some subjects, neutralization breadth was mediated by two or more antibody specificities. Although antibodies to the gp41 membrane-proximal external region (MPER) were identified in some subjects, the subjects with the greatest neutralization breadth targeted gp120 epitopes, including the CD4 binding site, a glycan-containing quaternary epitope formed by the V2 and V3 loops, or an outer domain epitope containing a glycan at residue N332. The broadly reactive HIV-1 neutralization observed in some subjects is mediated by antibodies targeting several conserved regions on the HIV-1 envelope glycoprotein.

OA06.04 Background: Isolating new broadly neutralizing antibodies (bnAbs) provides fresh insights for rational HIV-1 vaccine design. Several sites of vulnerability to bnAbs are now well defined, and it is likely that additional targets exist. Virus-like particles (VLPs) that express Env trimers display all of the natural epitopes and are therefore attractive candidates for use as probes. However, VLP surfaces are also decorated with nonfunctional forms of HIV Env. Exposing VLPs to proteases clears nonfunctional Env, leaving native trimeric Env intact. The resulting “trimer-VLPs” preferentially bind to nAbs compared to non-neutralizing antibodies and therefore may have gained sufficient selective power for use as B cell probes to identify new bnAbs. Methods: PBMC from HIV-infected patients were stained with the enzyme-digested trimer-VLPs, and VLP-positive memory B cells were sorted by flow cytometry. Multiplex RT-PCR was performed on cell lysates to amplify kappa, lambda, and heavy chain genes. Cloned antibodies were assessed for neutralization breadth and potency against a panel of Env-pseudoviruses using the TZM-bl neutralization assay. Results: VLPs were used to sort B cells from the donor of the broad CD4bs antibody VRC13. The sort yielded relatives of VRC13, as well as unrelated sequences that exhibited high sequence divergence from germline VH genes and/or long CDR3 regions, which are characteristics of known bnAbs. Cloning and characterization of these Abs is ongoing. Conclusions: Unlike many previously used B cell probes, VLPs express multiple epitopes including those for trimer specific antibodies which allows for simultaneous sorting of B-cells of different specificities. Additionally, these probes are devoid of non-functional forms of Env and therefore preferentially bind to neutralizing Ab-producing B cells. VLPs therefore hold great promise for isolating novel HIV-specific bnAbs that can better inform vaccine design.

e15613 Background: The presence of sarc elements in pts with RCC portends a poor prognosis. Some studies have focused on epithelial-to-mesenchymal transition (EMT) as a driver of sarc ccRCC; howeve...