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Sample records for human neutral ceramidase

  1. An Arabidopsis neutral ceramidase mutant ncer1 accumulates hydroxyceramides and is sensitive to oxidative stress

    PubMed Central

    Li, Jian; Bi, Fang-Cheng; Yin, Jian; Wu, Jian-Xin; Rong, Chan; Wu, Jia-Li; Yao, Nan

    2015-01-01

    Ceramidases hydrolyze ceramide into sphingosine and fatty acids and, although ceramidases function as key regulators of sphingolipid homeostasis in mammals, their roles in plants remain largely unknown. Here, we characterized the Arabidopsis thaliana ceramidase AtNCER1, a homolog of human neutral ceramidase. AtNCER1 localizes predominantly on the endoplasmic reticulum. The ncer1 T-DNA insertion mutants had no visible phenotype, but accumulated hydroxyceramides, and showed increased sensitivity to oxidative stress induced by methyl viologen. Plants over-expressing AtNCER1 showed increased tolerance to oxidative stress. These data indicate that the Arabidopsis neutral ceramidase affects sphingolipid homeostasis and oxidative stress responses. PMID:26150824

  2. Identification of a novel amidase motif in neutral ceramidase

    PubMed Central

    2005-01-01

    Neutral CDases (ceramidases) are newly identified enzymes with important roles in cell regulation, but little is known about their catalytic mechanisms. In the present study the full-length human neutral CDase was cloned and expressed in the yeast double-knockout strain Δypc1Δydc1, which lacks the yeast CDases YPC1p and YDC1p. Biochemical characterization of the human neutral CDase showed that the enzyme exhibited classical Michaelis–Menten kinetics, with an optimum activity at pH 7.5. Activity was enhanced by Na+ and Ca2+. Mg2+ and Mn2+ were somewhat stimulatory, but Zn2+, Cu2+ and Fe2+ inhibited the enzyme. Dithiothreitol and 2-mercaptoethanol dose-dependently inhibited neutral CDase. In order to identify which amino acids were involved in the catalytic action of neutral CDase, the purified enzyme was subjected to chemical modifications. It was observed that the serine residue modifier di-isopropyl fluorophosphate dose-dependently inhibited activity, implicating a serine residue in the catalytic action. From an alignment of the sequences of the neutral CDases from different species, all conserved serine residues were selected for site-directed mutagenesis. Of the six aligned serine residues that were mutated to alanine, only the S354A mutant lost its activity totally. Ser354 falls within a very highly conserved hexapeptide sequence GDVSPN, which itself was in the middle of a larger conserved sequence, namely NXGDVSPNXXGP/XXC. Moreover, mutations of Asp352 and Cys362 in the consensus sequence to alanine resulted in loss of activity of neutral CDase. Hence the present study identified a novel amidase sequence containing a critical serine residue that may function as a nucleophile in the hydrolytic attack on the amide bond present in ceramide. PMID:16229686

  3. Identification of a novel amidase motif in neutral ceramidase.

    PubMed

    Galadari, Sehamuddin; Wu, Bill X; Mao, Cungui; Roddy, Patrick; El Bawab, Samer; Hannun, Yusuf A

    2006-02-01

    Neutral CDases (ceramidases) are newly identified enzymes with important roles in cell regulation, but little is known about their catalytic mechanisms. In the present study the full-length human neutral CDase was cloned and expressed in the yeast double-knockout strain Dypc1Dydc1, which lacks the yeast CDases YPC1p and YDC1p. Biochemical characterization of the human neutral CDase showed that the enzyme exhibited classical Michaelis-Menten kinetics, with an optimum activity at pH 7.5. Activity was enhanced by Na+ and Ca2+. Mg2+ and Mn2+ were somewhat stimulatory, but Zn2+, Cu2+ and Fe2+ inhibited the enzyme. Dithiothreitol and 2-mercaptoethanol dose-dependently inhibited neutral CDase. In order to identify which amino acids were involved in the catalytic action of neutral CDase, the purified enzyme was subjected to chemical modifications. It was observed that the serine residue modifier di-isopropyl fluorophosphate dose-dependently inhibited activity, implicating a serine residue in the catalytic action. From an alignment of the sequences of the neutral CDases from different species, all conserved serine residues were selected for site-directed mutagenesis. Of the six aligned serine residues that were mutated to alanine, only the S354A mutant lost its activity totally. Ser354 falls within a very highly conserved hexapeptide sequence GDVSPN, which itself was in the middle of a larger conserved sequence, namely NXGDVSPNXXGP/XXC. Moreover, mutations of Asp352 and Cys362 in the consensus sequence to alanine resulted in loss of activity of neutral CDase. Hence the present study identified a novel amidase sequence containing a critical serine residue that may function as a nucleophile in the hydrolytic attack on the amide bond present in ceramide. PMID:16229686

  4. Identification and biochemical characterization of Laodelphax striatellus neutral ceramidase

    PubMed Central

    Zhou, Y.; Lin, X-W; Zhang, Y-R; Huang, Y-J; Zhang, Ch-H; Yang, Q; Li, H-Y; Yuan, J-Q; Cheng, J-A; Xu, R.; Mao, C.; Zhu, Z-R

    2013-01-01

    Ceramidases are a group of enzymes that catalyze hydrolysis of ceramides to generate fatty acid and sphingosine. In this study, we report the cloning and characterization of the rice small brown planthopper Laodelphax striatellus neutral ceramidase (nCDase), LsnCer. LsnCer was identified by sequencing the transcriptome of Laodelphax striatellus. LsnCer is a protein of 717 amino acids with a predicted molecular weight of 79.3 kDa. Similar to other known nCDases, LsnCer has a pH optimum at 8.0 and a temperature optimum at 37 °C for its in vitro activity. LsnCer activity is inhibited by Zn2+ significantly and Fe2+ slightly. LsnCer has broad substrate specificity with preference for ceramides with a medium acyl-chain or a mono unsaturated long acyl-chain. Infection with the rice strip virus (RSV) or treatment with insecticides significantly increased LsnCer mRNA expression and its enzymatic activity in L. striatellus. These results suggest that LsnCer is a bona fide nCDase that may have a role in adaption of L. striatellus to environmental stresses. PMID:23601004

  5. Loss of neutral ceramidase increases inflammation in a mouse model of inflammatory bowel disease

    PubMed Central

    Snider, Ashley J.; Wu, Bill X.; Jenkins, Russell W.; Sticca, Jonathan A.; Kawamori, Toshihiko; Hannun, Yusuf A.; Obeid, Lina M.

    2012-01-01

    Sphingolipids are emerging as important mediators of immune and inflammatory responses. We have previously demonstrated that sphingosine-1-phosphate (S1P) and its synthetic enzyme sphingosine kinase-1 (SK1) play an important role in inflammatory bowel disease. S1P generation is dependent on SK phosphorylation of sphingosine. Generation of sphingosine results only from the breakdown of ceramide by ceramidases (CDase). In this study, we set out to determine the role of neutral CDase (nCDase) in S1P generation and inflammatory bowel disease. To this end, we established nCDase expression is increased in patients with ulcerative colitis. Using the dextran sulfate sodium (DSS)-induced colitis model, we determined nCDase activity increased in colon epithelium, but not submucosa, in wild-type (WT) mice. Following DSS, ceramide levels were elevated in colon epithelium from WT and nCDase−/− mice, while S1P levels were significantly elevated only in the epithelium of nCDase−/− mice. Similarly, cyclooxygenase-2 (Cox-2) levels were significantly elevated only in the epithelium of nCDase−/− mice. Neutral CDase−/− mice also exhibited higher endotoxin levels in circulation, as well as higher circulating levels of S1P. This increase in S1P in nCDase−/− mice was accompanied by a marked leukocytosis, most notably circulating neutrophils and lymphocytes. Taken together these data demonstrate that loss of nCDase results in an unexpected increase in S1P generation in inflammation, and suggests that nCDase may actually protect against inflammation. PMID:22940715

  6. Ceramidases: regulators of cellular responses mediated by ceramide, sphingosine, and sphingosine-1-phosphate

    PubMed Central

    Mao, Cungui; Obeid, Lina M.

    2008-01-01

    Summary Ceramidases catalyze hydrolysis of ceramides to generate sphingosine (SPH), which is phosphorylated to form sphingosine-1-phosphate (S1P). Ceramide, SPH, and S1P are bioactive lipids that mediate cell proliferation, differentiation, apoptosis, adhesion, and migration, likely by controlling hydrolysis of ceramides and generation of SPH and S1P. Presently, 5 human ceramidases encoded by 5 distinct genes have been cloned: acid ceramidase (AC), neutral ceramidase (NC), alkaline ceramidase 1 (ACER1), alkaline ceramidase 2 (ACER2), and alkaline ceramidase 3 (ACER3). Each human ceramidase has a mouse counterpart. AC, NC, and ACER1–3 have maximal activities in acidic, neutral, and alkaline environments, respectively. ACER1–3 have similar protein sequences but no homology to AC and NC. AC and NC also have distinct protein sequences. The human AC (hAC) was implicated in Farber disease, and hAC may be important for cell survival. The mouse AC (mAC) is needed for early embryo survival. NC is protective against inflammatory cytokines, and the mouse NC (mNC) is required for the catabolism of ceramides in the digestive tract. ACER1 is critical in mediating cell differentiation by controlling the generation of SPH and S1P and that ACER2’s role in cell proliferation and survival depends on its expression or the cell type in which it is found. Here, we discuss the role of each ceramidase in regulating cellular responses mediated by ceramides, SPH, and S1P. PMID:18619555

  7. Molecular cloning and characterization of a human cDNA and gene encoding a novel acid ceramidase-like protein.

    PubMed

    Hong, S B; Li, C M; Rhee, H J; Park, J H; He, X; Levy, B; Yoo, O J; Schuchman, E H

    1999-12-01

    Computer-assisted database analysis of sequences homologous to human acid ceramidase (ASAH) revealed a 1233-bp cDNA (previously designated cPj-LTR) whose 266-amino-acid open reading frame had approximately 36% identity with the ASAH polypeptide. Based on this high degree of homology, we undertook further molecular characterization of cPj-LTR and now report the full-length cDNA sequence, complete gene structure (renamed human ASAHL since it is a human acid ceramidase-like sequence), chromosomal location, primer extension and promoter analysis, and transient expression results. The full-length human ASAHL cDNA was 1825 bp and contained an open-reading frame encoding a 359-amino-acid polypeptide that was 33% identical and 69% similar to the ASAH polypeptide over its entire length. Numerous short regions of complete identity were observed between these two sequences and two sequences obtained from the Caenorhabditis elegans genome database. The 30-kb human ASAHL genomic sequence contained 11 exons, which ranged in size from 26 to 671 bp, and 10 introns, which ranged from 150 bp to 6.4 kb. The gene was localized to the chromosomal region 4q21.1 by fluorescence in situ hybridization analysis. Northern blotting experiments revealed a major 2.0-kb ASAHL transcript that was expressed at high levels in the liver and kidney, but at relatively low levels in other tissues such as the lung, heart, and brain. Sequence analysis of the 5'-flanking region of the human ASAHL gene revealed a putative promoter region that lacked a TATA box and was GC rich, typical features of a housekeeping gene promoter, as well as several tissue-specific and/or hormone-induced transcription regulatory sites. 5'-Deletion analysis localized the promoter activity to a 1. 1-kb fragment within this region. A major transcription start site also was located 72 bp upstream from the ATG translation initiation site by primer extension analysis. Expression analysis of a green fluorescence protein/ASAHL fusion

  8. Acid Ceramidase Expression Modulates the Sensitivity of A375 Melanoma Cells to Dacarbazine*

    PubMed Central

    Bedia, Carmen; Casas, Josefina; Andrieu-Abadie, Nathalie; Fabriàs, Gemma; Levade, Thierry

    2011-01-01

    Dacarbazine (DTIC) is the treatment of choice for metastatic melanoma, but its response in patients remains very poor. Ceramide has been shown to be a death effector and to play an important role in regulating cancer cell growth upon chemotherapy. Among ceramidases, the enzymes that catabolize ceramide, acid ceramidase (aCDase) has been implicated in cancer progression. Here we show that DTIC elicits a time- and dose-dependent decrease of aCDase activity and an increase of intracellular ceramide levels in human A375 melanoma cells. The loss of enzyme activity occurred as a consequence of reactive oxygen species-dependent activation of cathepsin B-mediated degradation of aCDase. These events preceded autophagic features and loss of cell viability. Down-regulation of acid but not neutral or alkaline ceramidase 2 resulted in elevated levels of ceramide and sensitization to the toxic effects of DTIC. Conversely, inducible overexpression of acid but not neutral ceramidase reduced ceramide levels and conferred resistance to DTIC. In conclusion, we report that increased levels of ceramide, due to enhanced degradation of aCDase, are in part responsible for the cell death effects of DTIC. These results suggest that down-regulation of aCDase alone or in combination with DTIC may represent a useful tool in the treatment of metastatic melanoma. PMID:21700700

  9. The molecular medicine of acid ceramidase.

    PubMed

    Frohbergh, Michael; He, Xingxuan; Schuchman, Edward H

    2015-06-01

    Acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23; AC) is the lipid hydrolase responsible for the degradation of ceramide into sphingosine and free fatty acids within lysosomes. The enzymatic activity was first identified over four decades ago and is deficient in two rare inherited disorders, Farber lipogranulomatosis (Farber disease) and spinal muscular atrophy with myoclonic epilepsy (SMA-PME). Importantly, AC not only hydrolyzes ceramide into sphingosine within acidic compartments, but also can synthesize ceramide from sphingosine at neutral pH, suggesting that the enzyme may have diverse functions depending on its subcellular location and the local pH. Within cells, AC exists in a complex with other lipid hydrolases and requires a polypeptide cofactor (saposin D) for full hydrolytic activity. Recent studies also have shown that AC is overexpressed in several human cancers, and that inhibition of this enzyme may be a useful cancer drug target. Aberrant AC activity has also been described in several other common diseases. The cDNA and gene (ASAH1) encoding AC have been isolated, several mouse models of AC deficiency have been constructed, and the recombinant enzyme is currently being manufactured for the treatment of Farber disease and SMA-PME. Current information concerning the biology of this enzyme and its role in human disease is reviewed within. PMID:25938220

  10. The Arabidopsis ceramidase AtACER functions in disease resistance and salt tolerance.

    PubMed

    Wu, Jian-Xin; Li, Jian; Liu, Zhe; Yin, Jian; Chang, Zhen-Yi; Rong, Chan; Wu, Jia-Li; Bi, Fang-Cheng; Yao, Nan

    2015-03-01

    Ceramidases hydrolyze ceramide into sphingosine and fatty acids. In mammals, ceramidases function as key regulators of sphingolipid homeostasis, but little is known about their roles in plants. Here we characterize the Arabidopsis ceramidase AtACER, a homolog of human alkaline ceramidases. The acer-1 T-DNA insertion mutant has pleiotropic phenotypes, including reduction of leaf size, dwarfing and an irregular wax layer, compared with wild-type plants. Quantitative sphingolipid profiling showed that acer-1 mutants and the artificial microRNA-mediated silenced line amiR-ACER-1 have high ceramide levels and decreased long chain bases. AtACER localizes predominantly to the endoplasmic reticulum, and partially to the Golgi complex. Furthermore, we found that acer-1 mutants and AtACER RNAi lines showed increased sensitivity to salt stress, and lines overexpressing AtACER showed increased tolerance to salt stress. Reduction of AtACER also increased plant susceptibility to Pseudomonas syringae. Our data highlight the key biological functions of ceramidases in biotic and abiotic stresses in plants. PMID:25619405

  11. Acid ceramidase in prostate cancer radiation therapy resistance and relapse

    NASA Astrophysics Data System (ADS)

    Cheng, Joseph C.

    Prostate tumor cell escape from ionizing radiation (IR)-induced killing can lead to disease progression and relapse. Sphingolipids such as ceramide and sphingosine 1-phosphate influence signal transduction pathways that regulate stress response in cancer cells. In particular, metabolism of apoptotic ceramide constitutes an important survival adaptation. Assessments of enzyme activity, mRNA, and protein demonstrated preferential upregulation of the ceramide deacylating enzyme acid ceramidase (AC) in irradiated cancer cells. Promoter-reporter and ChIP-qPCR assays revealed AC transcription by activator protein 1 (AP-1) is sensitive to pharmacological inhibition of de novo ceramide biosynthesis, identifying a protective feedback mechanism that mitigates the effects of IR-induced ceramide. Deregulation of c-Jun, in particular, induced marked radiosensitization in vitro and in vivo, which was rescued by ectopic AC over-expression. AC over-expression in prostate cancer clonogens surviving 80 Gray fractionated irradiation was associated with increased radioresistance and proliferation, suggesting a role in radiotherapy failure and relapse. Indeed, immunohistochemical analysis of human prostate cancer tissues revealed higher levels of AC after radiotherapy failure than therapy-naive adenocarcinoma, PIN, or benign tissues. By genetically downregulating AC with small interfering RNA (siRNA), we observed radiosensitization of cells using clonogenic and cytotoxicity assays. Finally, treatment with lysosomotropic small molecule inhibitors of AC, LCL385 or LCL521, induced prostate cancer xenograft radiosensitization and long-term suppression, suggesting AC is a tractable target for adjuvant radiotherapy.

  12. Acid ceramidase improves the quality of oocytes and embryos and the outcome of in vitro fertilization.

    PubMed

    Eliyahu, Efrat; Shtraizent, Nataly; Martinuzzi, Kurt; Barritt, Jason; He, Xingxuan; Wei, Hong; Chaubal, Sanjeev; Copperman, Alan B; Schuchman, Edward H

    2010-04-01

    A major challenge of assisted reproduction technologies (ARTs) is to mimic the natural environment required to sustain oocyte and embryo survival. Herein, we show that the ceramide-metabolizing enzyme, acid ceramidase (AC), is expressed in human cumulus cells and follicular fluid, essential components of this environment, and that the levels of this enzyme are positively correlated with the quality of human embryos formed in vitro. These observations led us to develop a new approach for oocyte and embryo culture that markedly improved the outcome of in vitro fertilization (IVF). The addition of recombinant AC (rAC) to human and mouse oocyte culture medium maintained their healthy morphology in vitro. Following fertilization, the number of mouse embryos formed in the presence of rAC also was improved (from approximately 40 to 88%), leading to approximately 5-fold more healthy births. To confirm these observations, immature bovine oocytes were matured in vitro and subjected to IVF in the presence of rAC. Significantly more high-grade blastocysts were formed, and the number of morphologically intact, hatched embryos was increased from approximately 24 to 70%. Overall, these data identify AC as an important component of the in vivo oocyte and embryo environment, and provide a novel technology for enhancing the outcome of assisted fertilization. Eliyahu, E., Shtraizent, N., Martinuzzi, K., Barritt, J., He, X., Wei, H., Chaubal, S., Copperman, A. B., Schuchman, E. H. Acid ceramidase improves the quality of oocytes and embryos and the outcome of in vitro fertilization. PMID:20007509

  13. Structural Basis of Human Parechovirus Neutralization by Human Monoclonal Antibodies

    PubMed Central

    Shakeel, Shabih; Westerhuis, Brenda M.; Ora, Ari; Koen, Gerrit; Bakker, Arjen Q.; Claassen, Yvonne; Wagner, Koen; Beaumont, Tim; Wolthers, Katja C.

    2015-01-01

    ABSTRACT Since it was first recognized in 2004 that human parechoviruses (HPeV) are a significant cause of central nervous system and neonatal sepsis, their clinical importance, primarily in children, has started to emerge. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases and has given moderate success. Direct inhibition of parechovirus infection using monoclonal antibodies is a potential treatment. We have developed two neutralizing monoclonal antibodies against HPeV1 and HPeV2, namely, AM18 and AM28, which also cross-neutralize other viruses. Here, we present the mapping of their epitopes using peptide scanning, surface plasmon resonance, fluorescence-based thermal shift assays, electron cryomicroscopy, and image reconstruction. We determined by peptide scanning and surface plasmon resonance that AM18 recognizes a linear epitope motif including the arginine-glycine-aspartic acid on the C terminus of capsid protein VP1. This epitope is normally used by the virus to attach to host cell surface integrins during entry and is found in 3 other viruses that AM18 neutralizes. Therefore, AM18 is likely to cause virus neutralization by aggregation and by blocking integrin binding to the capsid. Further, we show by electron cryomicroscopy, three-dimensional reconstruction, and pseudoatomic model fitting that ordered RNA interacts with HPeV1 VP1 and VP3. AM28 recognizes quaternary epitopes on the capsid composed of VP0 and VP3 loops from neighboring pentamers, thereby increasing the RNA accessibility temperature for the virus-AM28 complex compared to the virus alone. Thus, inhibition of RNA uncoating probably contributes to neutralization by AM28. IMPORTANCE Human parechoviruses can cause mild infections to severe diseases in young children, such as neonatal sepsis, encephalitis, and cardiomyopathy. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases. In order to develop more

  14. Elevation of the level and activity of acid ceramidase in Alzheimer's disease brain.

    PubMed

    Huang, Yu; Tanimukai, Hitoshi; Liu, Fei; Iqbal, Khalid; Grundke-Iqbal, Inge; Gong, Cheng-Xin

    2004-12-01

    Protein glycosylation modifies the processing of several key proteins involved in the molecular pathogenesis of Alzheimer's disease (AD). Aberrant glycosylation of tau and down-regulation of sialyltransferase in AD brain suggest a possible dysregulation of protein glycosylation that may play a role in AD. We therefore isolated major glycoproteins from AD brain by using lectin-affinity chromatographies and ion-exchange chromatography and further separated them using SDS-polyacylamide gel electrophoresis. Mass spectrometry analysis of 11 isolated glycoproteins led to their identification as: neuronal cell adhesion molecule, beta-globin, IgM heavy chain VH1 region precursor, contactin precursor, dipeptidylpeptidase VI, CD81 partner 3, prenylcysteine lyase, adipocyte plasma-associated protein, acid ceramidase and two novel proteins. We found that the level and activity of acid ceramidase (AC), one of the major identified human brain glycoproteins, were significantly elevated in AD brain. Immunohistochemical staining indicated that AC was located mainly in the cell bodies of neurons and colocalized with neurofibrillary tangles. Our findings suggest that AC might play a role in controlling neuronal apoptosis and that AC-mediated signalling pathways might be involved in the molecular mechanism of AD. PMID:15610181

  15. Acid ceramidase and the treatment of ceramide diseases: The expanding role of enzyme replacement therapy.

    PubMed

    Schuchman, Edward H

    2016-09-01

    Ceramides are a diverse group of sphingolipids that play important roles in many biological processes. Acid ceramidase (AC) is one key enzyme that regulates ceramide metabolism. Early research on AC focused on the fact that it is the enzyme deficient in the rare genetic disorder, Farber Lipogranulomatosis. Recent research has revealed that deficiency of the same enzyme is responsible for a rare form of spinal muscular atrophy associated with myoclonic epilepsy (SMA-PME). Due to their diverse role in biology, accumulation of ceramides also has been implicated in the pathobiology of many other common diseases, including infectious lung diseases, diabetes, cancers and others. This has revealed the potential of AC as a therapy for many of these diseases. This review will focus on the biology of AC and the potential role of this enzyme in the treatment of human disease. PMID:27155573

  16. Testing the Neutral Theory of Biodiversity with Human Microbiome Datasets.

    PubMed

    Li, Lianwei; Ma, Zhanshan Sam

    2016-01-01

    The human microbiome project (HMP) has made it possible to test important ecological theories for arguably the most important ecosystem to human health-the human microbiome. Existing limited number of studies have reported conflicting evidence in the case of the neutral theory; the present study aims to comprehensively test the neutral theory with extensive HMP datasets covering all five major body sites inhabited by the human microbiome. Utilizing 7437 datasets of bacterial community samples, we discovered that only 49 communities (less than 1%) satisfied the neutral theory, and concluded that human microbial communities are not neutral in general. The 49 positive cases, although only a tiny minority, do demonstrate the existence of neutral processes. We realize that the traditional doctrine of microbial biogeography "Everything is everywhere, but the environment selects" first proposed by Baas-Becking resolves the apparent contradiction. The first part of Baas-Becking doctrine states that microbes are not dispersal-limited and therefore are neutral prone, and the second part reiterates that the freely dispersed microbes must endure selection by the environment. Therefore, in most cases, it is the host environment that ultimately shapes the community assembly and tip the human microbiome to niche regime. PMID:27527985

  17. Testing the Neutral Theory of Biodiversity with Human Microbiome Datasets

    PubMed Central

    Li, Lianwei; Ma, Zhanshan (Sam)

    2016-01-01

    The human microbiome project (HMP) has made it possible to test important ecological theories for arguably the most important ecosystem to human health—the human microbiome. Existing limited number of studies have reported conflicting evidence in the case of the neutral theory; the present study aims to comprehensively test the neutral theory with extensive HMP datasets covering all five major body sites inhabited by the human microbiome. Utilizing 7437 datasets of bacterial community samples, we discovered that only 49 communities (less than 1%) satisfied the neutral theory, and concluded that human microbial communities are not neutral in general. The 49 positive cases, although only a tiny minority, do demonstrate the existence of neutral processes. We realize that the traditional doctrine of microbial biogeography “Everything is everywhere, but the environment selects” first proposed by Baas-Becking resolves the apparent contradiction. The first part of Baas-Becking doctrine states that microbes are not dispersal-limited and therefore are neutral prone, and the second part reiterates that the freely dispersed microbes must endure selection by the environment. Therefore, in most cases, it is the host environment that ultimately shapes the community assembly and tip the human microbiome to niche regime. PMID:27527985

  18. Cross-Neutralization between Human and African Bat Mumps Viruses

    PubMed Central

    Katoh, Hiroshi; Kubota, Toru; Ihara, Toshiaki; Maeda, Ken; Kidokoro, Minoru

    2016-01-01

    Recently, a new paramyxovirus closely related to human mumps virus (MuV) was detected in bats. We generated recombinant MuVs carrying either or both of the fusion and hemagglutinin-neuraminidase bat virus glycoproteins. These viruses showed replication kinetics similar to human MuV in cultured cells and were neutralized efficiently by serum from healthy humans. PMID:26982800

  19. Cross-Neutralization between Human and African Bat Mumps Viruses.

    PubMed

    Katoh, Hiroshi; Kubota, Toru; Ihara, Toshiaki; Maeda, Ken; Takeda, Makoto; Kidokoro, Minoru

    2016-04-01

    Recently, a new paramyxovirus closely related to human mumps virus (MuV) was detected in bats. We generated recombinant MuVs carrying either or both of the fusion and hemagglutinin-neuraminidase bat virus glycoproteins. These viruses showed replication kinetics similar to human MuV in cultured cells and were neutralized efficiently by serum from healthy humans. PMID:26982800

  20. Alkaline ceramidase 2 regulates β1 integrin maturation and cell adhesion

    PubMed Central

    Sun, Wei; Hu, Wei; Xu, Ruijuan; Jin, Junfei; Szulc, Zdzislaw M.; Zhang, Guofeng; Galadari, Sehamuddin H.; Obeid, Lina M.; Mao, Cungui

    2009-01-01

    The polypeptide core of the integrin β1 subunit (β1) is glycosylated sequentially in the endoplasmic reticulum and the Golgi complex to form β1 precursor and mature β1, respectively. The β1 precursor to mature β1 conversion, termed β1 maturation, regulates the cell surface levels and function of β1-containing integrins, β1 integrins. Here we demonstrate that the human alkaline ceramidase 2 (ACER2), a Golgi enzyme, regulates β1 maturation by controlling the generation of sphingosine. ACER2 overexpression inhibited β1 maturation, thus leading to a decrease in the levels of mature β1 in T-REx HeLa cells, whereas RNA interference-mediated knockdown of ACER2 enhanced β1 maturation in MCF-7 cells. ACER2 overexpression decreased the cell surface levels of β1 integrins, thus inhibiting cell adhesion to fibronectin or collagen, whereas ACER2 knockdown has the opposite effects. Treatment with all-trans retinoic acid (ATRA) increased both the expression of ACER2 and the generation of sphingosine in HeLa cells and inhibited β1 maturation. ACER2 knockdown attenuated the inhibitory effects of ATRA on both β1 maturation and cell adhesion. In contrast, treatment with phorbol myristate acetate (PMA), a protein kinase C activator, decreased the expression of ACER2 and sphingosine in T-REx HeLa cells, thus enhancing β1 maturation. ACER2 overexpression inhibited the stimulatory effects of PMA on both β1 maturation and cell adhesion. These results suggest that the ACER2/sphingosine pathway plays an important role in regulating β1 maturation and cell adhesion mediated by β1 integrins.—Sun, W., Hu, W., Xu, R., Jin, J., Szulc, Z. M., Zhang, G., Galadari, S. H., Obeid, L. M, Mao, C. Alkaline ceramidase 2 regulates β1 integrin maturation and cell adhesion. PMID:18945876

  1. Regulation of alkaline ceramidase activity by the c-Src-mediated pathway.

    PubMed

    Sasaki, Hirotsune; Toyomura, Kaori; Matsuzaki, Wataru; Okamoto, Aya; Yamaguchi, Naoto; Nakamura, Hiroyuki; Murayama, Toshihiko

    2014-05-15

    Ceramidase hydrolyzes ceramide to fatty acids and sphingosine, and sphingosine is then converted to sphingosine-1-phosphate. Ceramide and sphingosine-1-phosphate act as signaling molecules. Although stimuli coupling to protein kinases-dependent systems have been shown to regulate ceramidase activity, the exact role of c-Src-mediated signal has not been elucidated. We examined the effects of the downregulation of c-Src activity and c-Src overexpression on ceramidase activity in cells. In A549, CHO, and HeLa cells labeled with a fluorescent ceramide, 4-nitrobenzo-2-oxa-1,3-diazole-labeled C6-ceramide (NBD-ceramide), the downregulation of c-Src by c-Src-shRNA and pharmacological inhibitors including SU6656 decreased levels of NBD-caproic acid. The overexpression of c-Src increased NBD-caproic acid levels in CHO and HeLa cells. Similar results were obtained in Na3VO4-treated cells having higher NBD-caproic acid levels. The downregulation and overexpression of c-Src decreased and increased ceramidase activity, respectively, in the lysates of A549 cells at pH 8.8. The ceramidase sensitivity to substrates, pH, and Ca(2+) suggest that the c-Src- and SU6656-sensitive ceramidase is alkaline ceramidase (ACER), possibly Ca(2+)-activated ACER2. Serum starvation increased both ceramidase activity at pH 8.8 and expression of ACER2. Our data suggest that c-Src-mediated signal positively regulates ACER activity in a Ca(2+)-independent manner. PMID:24708996

  2. Human-like antibodies neutralizing Western equine encephalitis virus

    PubMed Central

    Hülseweh, Birgit; Rülker, Torsten; Pelat, Thibaut; Langermann, Claudia; Frenzel, Andrè; Schirrmann, Thomas; Dübel, Stefan; Thullier, Philippe; Hust, Michael

    2014-01-01

    This study describes the development of the first neutralizing antibodies against Western equine encephalitis virus (WEEV), a member of the genus Alphavirus. WEEV is transmitted by mosquitoes and can spread to the human central nervous system, causing symptoms ranging from mild febrile reactions to life-threatening encephalitis. WEEV has been classified as a biological warfare agent by the US Centers for Disease Control and Prevention. No anti-WEEV drugs are currently commercially available. Neutralizing antibodies are useful for the pre- and post-exposure treatment of WEEV infections. In this study, two immune antibody gene libraries were constructed from two macaques immunized with inactivated WEEV. Four antibodies were selected from these libraries and recloned as scFv-Fc, with a human Fc part. These antibodies bound WEEV specifically in ELISA with little or no cross-reaction with other alphaviruses. They were further analyzed by immunohistochemistry. All binders were suitable for the intracellular detection of WEEV particles. Neutralizing activity was determined in vitro. Three of the four antibodies were found to be neutralizing; about 1 ng/mL of the best antibody (ToR69–3A2) neutralized 50% of 5x104 TCID50/mL. Due to its human-like nature with a germinality index of 89% (VH) and 91% (VL), the ToR69–3A2 antibody is a promising candidate for future passive vaccine development. PMID:24518197

  3. Human recombinant neutralizing antibodies against hantaan virus G2 protein.

    PubMed

    Koch, Joachim; Liang, Mifang; Queitsch, Iris; Kraus, Annette A; Bautz, Ekkehard K F

    2003-03-30

    Old world hantaviruses, causing hemorrhagic fever with renal syndrome (HFRS), still present a public health problem in Asia and Eastern Europe. The majority of cases has been recorded in China. The aim of our study was to generate human recombinant neutralizing antibodies to a hantavirus by phage display technology. To preserve the structural identity of viral protein, the panning procedure was performed on native Hantaan (HTN) (76-118) virus propagated in Vero-E6 cells. In total, five complete human recombinant IgG antibodies were produced in a baculovirus expression system. All of them were able to completely neutralize HTN, and Seoul (SEO) virus in a plaque reduction neutralization test (PRNT). Three of these antibodies could also completely neutralize Dobrava (DOB) virus but not Puumala (PUU) virus. All antibodies bind to Hantaan virus G2 protein localized in the virus envelope. The sequence areas within the HTN (76-118)-G2 protein detected by five selected antibodies were mapped using peptide scans. Two partial epitopes, 916-KVMATIDSF-924 and 954-LVTKDIDFD-963, were recognized, which presumably are of paramount importance for docking of the virus to host cell receptors. A consensus motif 916-KVXATIXSF-924 could be identified by mutational analysis. The neutralizing antibodies to the most widely distributed hantaviruses causing HFRS might be promising candidates for the development of an agent for prevention and treatment of HFRS in patients. PMID:12706090

  4. Acid Ceramidase in Melanoma: EXPRESSION, LOCALIZATION, AND EFFECTS OF PHARMACOLOGICAL INHIBITION.

    PubMed

    Realini, Natalia; Palese, Francesca; Pizzirani, Daniela; Pontis, Silvia; Basit, Abdul; Bach, Anders; Ganesan, Anand; Piomelli, Daniele

    2016-01-29

    Acid ceramidase (AC) is a lysosomal cysteine amidase that controls sphingolipid signaling by lowering the levels of ceramides and concomitantly increasing those of sphingosine and its bioactive metabolite, sphingosine 1-phosphate. In the present study, we evaluated the role of AC-regulated sphingolipid signaling in melanoma. We found that AC expression is markedly elevated in normal human melanocytes and proliferative melanoma cell lines, compared with other skin cells (keratinocytes and fibroblasts) and non-melanoma cancer cells. High AC expression was also observed in biopsies from human subjects with Stage II melanoma. Immunofluorescence studies revealed that the subcellular localization of AC differs between melanocytes (where it is found in both cytosol and nucleus) and melanoma cells (where it is primarily localized to cytosol). In addition to having high AC levels, melanoma cells generate lower amounts of ceramides than normal melanocytes do. This down-regulation in ceramide production appears to result from suppression of the de novo biosynthesis pathway. To test whether AC might contribute to melanoma cell proliferation, we blocked AC activity using a new potent (IC50 = 12 nM) and stable inhibitor. AC inhibition increased cellular ceramide levels, decreased sphingosine 1-phosphate levels, and acted synergistically with several, albeit not all, antitumoral agents. The results suggest that AC-controlled sphingolipid metabolism may play an important role in the control of melanoma proliferation. PMID:26553872

  5. Neutralization of Botulinum Neurotoxin Type E by a Humanized Antibody.

    PubMed

    Derman, Yağmur; Selby, Katja; Miethe, Sebastian; Frenzel, André; Liu, Yvonne; Rasetti-Escargueil, Christine; Avril, Arnaud; Pelat, Thibaut; Urbain, Remi; Fontayne, Alexandre; Thullier, Philippe; Sesardic, Dorothea; Lindström, Miia; Hust, Michael; Korkeala, Hannu

    2016-01-01

    Botulinum neurotoxins (BoNTs) cause botulism and are the deadliest naturally-occurring substances known to humans. BoNTs have been classified as one of the category A agents by the Centers for Disease Control and Prevention, indicating their potential use as bioweapons. To counter bio-threat and naturally-occurring botulism cases, well-tolerated antibodies by humans that neutralize BoNTs are relevant. In our previous work, we showed the neutralizing potential of macaque (Macaca fascicularis)-derived scFv-Fc (scFv-Fc ELC18) by in vitro endopeptidase immunoassay and ex vivo mouse phrenic nerve-hemidiaphragm assay by targeting the light chain of the botulinum neurotoxin type E (BoNT/E). In the present study, we germline-humanized scFv-Fc ELC18 into a full IgG hu8ELC18 to increase its immunotolerance by humans. We demonstrated the protection and prophylaxis capacity of hu8ELC18 against BoNT/E in a mouse model. A concentration of 2.5 ng/mouse of hu8ELC18 protected against 5 mouse lethal dose (MLD) in a mouse protection assay and complete neutralization of 1 LD50 of pure BoNT/E toxin was achieved with 8 ng of hu8ELC18 in mouse paralysis assay. Furthermore, hu8ELC18 protected mice from 5 MLD if injected up to 14 days prior to intraperitoneal BoNT/E administration. This newly-developed humanized IgG is expected to have high tolerance in humans. PMID:27626446

  6. Radiation-induced acid ceramidase confers prostate cancer resistance and tumor relapse

    PubMed Central

    Cheng, Joseph C.; Bai, Aiping; Beckham, Thomas H.; Marrison, S. Tucker; Yount, Caroline L.; Young, Katherine; Lu, Ping; Bartlett, Anne M.; Wu, Bill X.; Keane, Barry J.; Armeson, Kent E.; Marshall, David T.; Keane, Thomas E.; Smith, Michael T.; Jones, E. Ellen; Drake, Richard R.; Bielawska, Alicja; Norris, James S.; Liu, Xiang

    2013-01-01

    Escape of prostate cancer (PCa) cells from ionizing radiation–induced (IR-induced) killing leads to disease progression and cancer relapse. The influence of sphingolipids, such as ceramide and its metabolite sphingosine 1-phosphate, on signal transduction pathways under cell stress is important to survival adaptation responses. In this study, we demonstrate that ceramide-deacylating enzyme acid ceramidase (AC) was preferentially upregulated in irradiated PCa cells. Radiation-induced AC gene transactivation by activator protein 1 (AP-1) binding on the proximal promoter was sensitive to inhibition of de novo ceramide biosynthesis, as demonstrated by promoter reporter and ChIP-qPCR analyses. Our data indicate that a protective feedback mechanism mitigates the apoptotic effect of IR-induced ceramide generation. We found that deregulation of c-Jun induced marked radiosensitization in vivo and in vitro, which was rescued by ectopic AC overexpression. AC overexpression in PCa clonogens that survived a fractionated 80-Gy IR course was associated with increased radioresistance and proliferation, suggesting a role for AC in radiotherapy failure and relapse. Immunohistochemical analysis of human PCa tissues revealed higher levels of AC after radiotherapy failure than those in therapy-naive PCa, prostatic intraepithelial neoplasia, or benign tissues. Addition of an AC inhibitor to an animal model of xenograft irradiation produced radiosensitization and prevention of relapse. These data indicate that AC is a potentially tractable target for adjuvant radiotherapy. PMID:24091326

  7. Radiation-induced acid ceramidase confers prostate cancer resistance and tumor relapse.

    PubMed

    Cheng, Joseph C; Bai, Aiping; Beckham, Thomas H; Marrison, S Tucker; Yount, Caroline L; Young, Katherine; Lu, Ping; Bartlett, Anne M; Wu, Bill X; Keane, Barry J; Armeson, Kent E; Marshall, David T; Keane, Thomas E; Smith, Michael T; Jones, E Ellen; Drake, Richard R; Bielawska, Alicja; Norris, James S; Liu, Xiang

    2013-10-01

    Escape of prostate cancer (PCa) cells from ionizing radiation-induced (IR-induced) killing leads to disease progression and cancer relapse. The influence of sphingolipids, such as ceramide and its metabolite sphingosine 1-phosphate, on signal transduction pathways under cell stress is important to survival adaptation responses. In this study, we demonstrate that ceramide-deacylating enzyme acid ceramidase (AC) was preferentially upregulated in irradiated PCa cells. Radiation-induced AC gene transactivation by activator protein 1 (AP-1) binding on the proximal promoter was sensitive to inhibition of de novo ceramide biosynthesis, as demonstrated by promoter reporter and ChIP-qPCR analyses. Our data indicate that a protective feedback mechanism mitigates the apoptotic effect of IR-induced ceramide generation. We found that deregulation of c-Jun induced marked radiosensitization in vivo and in vitro, which was rescued by ectopic AC overexpression. AC overexpression in PCa clonogens that survived a fractionated 80-Gy IR course was associated with increased radioresistance and proliferation, suggesting a role for AC in radiotherapy failure and relapse. Immunohistochemical analysis of human PCa tissues revealed higher levels of AC after radiotherapy failure than those in therapy-naive PCa, prostatic intraepithelial neoplasia, or benign tissues. Addition of an AC inhibitor to an animal model of xenograft irradiation produced radiosensitization and prevention of relapse. These data indicate that AC is a potentially tractable target for adjuvant radiotherapy. PMID:24091326

  8. Recognition determinants of broadly neutralizing human antibodies against dengue viruses.

    PubMed

    Rouvinski, Alexander; Guardado-Calvo, Pablo; Barba-Spaeth, Giovanna; Duquerroy, Stéphane; Vaney, Marie-Christine; Kikuti, Carlos M; Navarro Sanchez, M Erika; Dejnirattisai, Wanwisa; Wongwiwat, Wiyada; Haouz, Ahmed; Girard-Blanc, Christine; Petres, Stéphane; Shepard, William E; Desprès, Philippe; Arenzana-Seisdedos, Fernando; Dussart, Philippe; Mongkolsapaya, Juthathip; Screaton, Gavin R; Rey, Félix A

    2015-04-01

    Dengue disease is caused by four different flavivirus serotypes, which infect 390 million people yearly with 25% symptomatic cases and for which no licensed vaccine is available. Recent phase III vaccine trials showed partial protection, and in particular no protection for dengue virus serotype 2 (refs 3, 4). Structural studies so far have characterized only epitopes recognized by serotype-specific human antibodies. We recently isolated human antibodies potently neutralizing all four dengue virus serotypes. Here we describe the X-ray structures of four of these broadly neutralizing antibodies in complex with the envelope glycoprotein E from dengue virus serotype 2, revealing that the recognition determinants are at a serotype-invariant site at the E-dimer interface, including the exposed main chain of the E fusion loop and the two conserved glycan chains. This 'E-dimer-dependent epitope' is also the binding site for the viral glycoprotein prM during virus maturation in the secretory pathway of the infected cell, explaining its conservation across serotypes and highlighting an Achilles' heel of the virus with respect to antibody neutralization. These findings will be instrumental for devising novel immunogens to protect simultaneously against all four serotypes of dengue virus. PMID:25581790

  9. Development of a poliovirus neutralization test with poliovirus pseudovirus for measurement of neutralizing antibody titer in human serum.

    PubMed

    Arita, Minetaro; Iwai, Masae; Wakita, Takaji; Shimizu, Hiroyuki

    2011-11-01

    In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying poliovirus (PV) from the stool samples from acute flaccid paralysis (AFP) cases. In recent years, reestablishment of PV circulation in countries where PV was previously eliminated has occurred because of decreased herd immunity, possibly due to poor vaccination coverage. To monitor the vulnerability of countries to PV circulation, surveillance of neutralizing-antibody titers against PV in susceptible populations is essential in the end game of the polio eradication program. In this study, we have developed a PV neutralization test with type 1, 2, and 3 PV pseudoviruses to determine the neutralizing-antibody titer against PV in human serum samples. With this test, the neutralizing-antibody titer against PV could be determined within 2 days by automated interpretation of luciferase signals without using infectious PV strains. We validated the pseudovirus PV neutralization test with 131 human serum samples collected from a wide range of age groups (ages 1 to >60 years) by comparison with a conventional neutralization test. We found good correlation in the neutralizing-antibody titers determined by these tests. These results suggest that a pseudovirus PV neutralization test would serve as a safe and simple procedure for the measurement of the neutralizing-antibody titer against PV. PMID:21880850

  10. Development of a Coxsackievirus A16 neutralization assay based on pseudoviruses for measurement of neutralizing antibody titer in human serum.

    PubMed

    Jin, Jun; Ma, Hongxia; Xu, Lin; An, Dong; Sun, Shiyang; Huang, Xueyong; Kong, Wei; Jiang, Chunlai

    2013-02-01

    Serum neutralizing antibody titers are indicative of protective immunity against Coxsackievirus A16 (CV-A16) and Enterovirus 71 (EV71), the two main etiological agents of hand, foot and mouth disease (HFMD), and provide the basis for evaluating vaccine efficacy. The current CV-A16 neutralization assay based on inhibition of cytopathic effects requires manual microscopic examination, which is time-consuming and labor-intensive. In this study, a high-throughput neutralization assay was developed by employing CV-A16 pseudoviruses expressing luciferase for detecting infectivity in rhabdomyosarcoma (RD) cells and measuring serum viral neutralizing antibodies. Without the need to use infectious CV-A16 strains, the neutralizing antibody titer against CV-A16 could be determined within 15h by measuring luciferase signals by this assay. The pseudovirus CV-A16 neutralization assay (pCNA) was validated by comparison with a conventional CV-A16 neutralization assay (cCNA) in testing 174 human serum samples collected from children (age <5 years). The neutralizing antibody titers determined by these two assays were well correlated (R(2)=0.7689). These results suggest that the pCNA can serve as a rapid and objective procedure for the measurement of neutralizing antibodies against CV-A16. PMID:23178532

  11. Acid Ceramidase (ASAH1) Is a Global Regulator of Steroidogenic Capacity and Adrenocortical Gene Expression

    PubMed Central

    Lucki, Natasha C.; Bandyopadhyay, Sibali; Wang, Elaine; Merrill, Alfred H.

    2012-01-01

    In H295R human adrenocortical cells, ACTH rapidly activates ceramide (Cer) and sphingosine (SPH) turnover with a concomitant increase in SPH-1-phosphate secretion. These bioactive lipids modulate adrenocortical steroidogenesis, primarily by acting as second messengers in the protein kinase A/cAMP-dependent pathway. Acid ceramidase (ASAH1) directly regulates the intracellular balance of Cer, SPH, and SPH-1-phosphate by catalyzing the hydrolysis of Cer into SPH. ACTH/cAMP signaling stimulates ASAH1 transcription and activity, supporting a role for this enzyme in glucocorticoid production. Here, the role of ASAH1 in regulating steroidogenic capacity was examined using a tetracycline-inducible ASAH1 short hairpin RNA H295R human adrenocortical stable cell line. We show that ASAH1 suppression increases the transcription of multiple steroidogenic genes, including Cytochrome P450 monooxygenase (CYP)17A1, CYP11B1/2, CYP21A2, steroidogenic acute regulatory protein, hormone-sensitive lipase, 18-kDa translocator protein, and the melanocortin-2 receptor. Induced gene expression positively correlated with enhanced histone H3 acetylation at target promoters. Repression of ASAH1 expression also induced the expression of members of the nuclear receptor nuclear receptor subfamily 4 (NR4A) family while concomitantly suppressing the expression of dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1. ASAH1 knockdown altered the expression of genes involved in sphingolipid metabolism and changed the cellular amounts of distinct sphingolipid species. Finally, ASAH1 silencing increased basal and cAMP-dependent cortisol and dehydroepiandrosterone secretion, establishing ASAH1 as a pivotal regulator of steroidogenic capacity in the human adrenal cortex. PMID:22261821

  12. Human cell lines used in a micro neutralization test for measuring influenza-neutralizing antibodies.

    PubMed

    Mittelholzer, C M; Brokstad, K A; Pauksens, K; Jonsson, R; Brytting, M; Linde, A

    2006-04-01

    An in situ neutralization test (NT) including ELISA for the measurement of influenza antigen was developed and evaluated. Two human cell lines, fibroblasts (HS27) cells and salivary gland epithelial duct (HSG) cells, were compared with Madin-Darby Canine Kidney (MDCK) cells. The viral production in the human cell lines was lower than that for MDCK cells, which influenced the results of the assay in the HSG and HS27 cells. However, when lowering the infectious dose, the NT using HS27 cells gave a sensitive and stable assay with low background in the ELISA. The NT titres were very low when using HSG cells compared to MDCK cells. The HS27 NT was used to analyze the humoral response after an influenza A infection in patients from a placebo-controlled zanamivir study. We found no differences in NT titres between patients treated with zanamivir or placebo. The MDCK and HS27 NT gave higher titres and more pronounced titre differences than the gold standard haemagglutinin inhibition (HAI) assay. Compared to the HAI assay, the sensitive NT using HS27 cells also revealed heterologous NT-titre rises after influenza infection in the patients. PMID:16623925

  13. Alkaline ceramidase 2 and its bioactive product sphingosine are novel regulators of the DNA damage response

    PubMed Central

    Xu, Ruijuan; Wang, Kai; Mileva, Izolda; Hannun, Yusuf A.; Obeid, Lina M.; Mao, Cungui

    2016-01-01

    Human cells respond to DNA damage by elevating sphingosine, a bioactive sphingolipid that induces programmed cell death (PCD) in response to various forms of stress, but its regulation and role in the DNA damage response remain obscure. Herein we demonstrate that DNA damage increases sphingosine levels in tumor cells by upregulating alkaline ceramidase 2 (ACER2) and that the upregulation of the ACER2/sphingosine pathway induces PCD in response to DNA damage by increasing the production of reactive oxygen species (ROS). Treatment with the DNA damaging agent doxorubicin increased both ACER2 expression and sphingosine levels in HCT116 cells in a dose-dependent manner. ACER2 overexpression increased sphingosine in HeLa cells whereas knocking down ACER2 inhibited the doxorubicin-induced increase in sphingosine in HCT116 cells, suggesting that DNA damage elevates sphingosine by upregulating ACER2. Knocking down ACER2 inhibited an increase in the apoptotic and necrotic cell population and the cleavage of poly ADP ribose polymerase (PARP) in HCT116 cells in response to doxorubicin as well as doxorubicin-induced release of lactate dehydrogenase (LDH) from these cells. Similar to treatment with doxorubicin, ACER2 overexpression induced an increase in the apoptotic and necrotic cell population and PARP cleavage in HeLa cells and LDH release from cells, suggesting that ACER2 upregulation mediates PCD in response to DNA damage through sphingosine. Mechanistic studies demonstrated that the upregulation of the ACER2/sphingosine pathway induces PCD by increasing ROS levels. Taken together, these results suggest that the ACER2/sphingosine pathway mediates PCD in response to DNA damage through ROS production. PMID:26943039

  14. Both Neutralizing and Non-Neutralizing Human H7N9 Influenza Vaccine-Induced Monoclonal Antibodies Confer Protection.

    PubMed

    Henry Dunand, Carole J; Leon, Paul E; Huang, Min; Choi, Angela; Chromikova, Veronika; Ho, Irvin Y; Tan, Gene S; Cruz, John; Hirsh, Ariana; Zheng, Nai-Ying; Mullarkey, Caitlin E; Ennis, Francis A; Terajima, Masanori; Treanor, John J; Topham, David J; Subbarao, Kanta; Palese, Peter; Krammer, Florian; Wilson, Patrick C

    2016-06-01

    Pathogenic H7N9 avian influenza viruses continue to represent a public health concern, and several candidate vaccines are currently being developed. It is vital to assess if protective antibodies are induced following vaccination and to characterize the diversity of epitopes targeted. Here we characterized the binding and functional properties of twelve H7-reactive human antibodies induced by a candidate A/Anhui/1/2013 (H7N9) vaccine. Both neutralizing and non-neutralizing antibodies protected mice in vivo during passive transfer challenge experiments. Mapping the H7 hemagglutinin antigenic sites by generating escape mutant variants against the neutralizing antibodies identified unique epitopes on the head and stalk domains. Further, the broadly cross-reactive non-neutralizing antibodies generated in this study were protective through Fc-mediated effector cell recruitment. These findings reveal important properties of vaccine-induced antibodies and provide a better understanding of the human monoclonal antibody response to influenza in the context of vaccines. PMID:27281570

  15. Alkaline Ceramidase 3 Deficiency Results in Purkinje Cell Degeneration and Cerebellar Ataxia Due to Dyshomeostasis of Sphingolipids in the Brain

    PubMed Central

    Preston, Chet; Wang, Louis; Yi, Jae Kyo; Lin, Chih-Li; Sun, Wei; Spyropoulos, Demetri D.; Rhee, Soyoung; Li, Mingsong; Zhou, Jie; Ge, Shaoyu; Zhang, Guofeng; Snider, Ashley J.; Hannun, Yusuf A.; Obeid, Lina M.; Mao, Cungui

    2015-01-01

    Dyshomeostasis of both ceramides and sphingosine-1-phosphate (S1P) in the brain has been implicated in aging-associated neurodegenerative disorders in humans. However, mechanisms that maintain the homeostasis of these bioactive sphingolipids in the brain remain unclear. Mouse alkaline ceramidase 3 (Acer3), which preferentially catalyzes the hydrolysis of C18:1-ceramide, a major unsaturated long-chain ceramide species in the brain, is upregulated with age in the mouse brain. Acer3 knockout causes an age-dependent accumulation of various ceramides and C18:1-monohexosylceramide and abolishes the age-related increase in the levels of sphingosine and S1P in the brain; thereby resulting in Purkinje cell degeneration in the cerebellum and deficits in motor coordination and balance. Our results indicate that Acer3 plays critically protective roles in controlling the homeostasis of various sphingolipids, including ceramides, sphingosine, S1P, and certain complex sphingolipids in the brain and protects Purkinje cells from premature degeneration. PMID:26474409

  16. Alkaline Ceramidase 3 Deficiency Results in Purkinje Cell Degeneration and Cerebellar Ataxia Due to Dyshomeostasis of Sphingolipids in the Brain.

    PubMed

    Wang, Kai; Xu, Ruijuan; Schrandt, Jennifer; Shah, Prithvi; Gong, Yong Z; Preston, Chet; Wang, Louis; Yi, Jae Kyo; Lin, Chih-Li; Sun, Wei; Spyropoulos, Demetri D; Rhee, Soyoung; Li, Mingsong; Zhou, Jie; Ge, Shaoyu; Zhang, Guofeng; Snider, Ashley J; Hannun, Yusuf A; Obeid, Lina M; Mao, Cungui

    2015-10-01

    Dyshomeostasis of both ceramides and sphingosine-1-phosphate (S1P) in the brain has been implicated in aging-associated neurodegenerative disorders in humans. However, mechanisms that maintain the homeostasis of these bioactive sphingolipids in the brain remain unclear. Mouse alkaline ceramidase 3 (Acer3), which preferentially catalyzes the hydrolysis of C18:1-ceramide, a major unsaturated long-chain ceramide species in the brain, is upregulated with age in the mouse brain. Acer3 knockout causes an age-dependent accumulation of various ceramides and C18:1-monohexosylceramide and abolishes the age-related increase in the levels of sphingosine and S1P in the brain; thereby resulting in Purkinje cell degeneration in the cerebellum and deficits in motor coordination and balance. Our results indicate that Acer3 plays critically protective roles in controlling the homeostasis of various sphingolipids, including ceramides, sphingosine, S1P, and certain complex sphingolipids in the brain and protects Purkinje cells from premature degeneration. PMID:26474409

  17. Safety, pharmacokinetics and neutralization of the broadly neutralizing HIV-1 human monoclonal antibody VRC01 in healthy adults.

    PubMed

    Ledgerwood, J E; Coates, E E; Yamshchikov, G; Saunders, J G; Holman, L; Enama, M E; DeZure, A; Lynch, R M; Gordon, I; Plummer, S; Hendel, C S; Pegu, A; Conan-Cibotti, M; Sitar, S; Bailer, R T; Narpala, S; McDermott, A; Louder, M; O'Dell, S; Mohan, S; Pandey, J P; Schwartz, R M; Hu, Z; Koup, R A; Capparelli, E; Mascola, J R; Graham, B S

    2015-12-01

    VRC-HIVMAB060-00-AB (VRC01) is a broadly neutralizing HIV-1 monoclonal antibody (mAb) isolated from the B cells of an HIV-infected patient. It is directed against the HIV-1 CD4 binding site and is capable of potently neutralizing the majority of diverse HIV-1 strains. This Phase I dose-escalation study in healthy adults was conducted at the National Institutes of Health (NIH) Clinical Center (Bethesda, MD, USA). Primary objectives were the safety, tolerability and pharmacokinetics (PK) of VRC01 intravenous (i.v.) infusion at 5, 20 or 40 mg/kg, given either once (20 mg/kg) or twice 28 days apart (all doses), and of subcutaneous (s.c.) delivery at 5 mg/kg compared to s.c. placebo given twice, 28 days apart. Cumulatively, 28 subjects received 43 VRC01 and nine received placebo administrations. There were no serious adverse events or dose-limiting toxicities. Mean 28-day serum trough concentrations after the first infusion were 35 and 57 μg/ml for groups infused with 20 mg/kg (n = 8) and 40 mg/kg (n = 5) doses, respectively. Mean 28-day trough concentrations after the second infusion were 56 and 89 μg/ml for the same two doses. Over the 5-40 mg/kg i.v. dose range (n = 18), the clearance was 0.016 l/h and terminal half-life was 15 days. After infusion VRC01 retained expected neutralizing activity in serum, and anti-VRC01 antibody responses were not detected. The human monoclonal antibody (mAb) VRC01 was well tolerated when delivered i.v. or s.c. The mAb demonstrated expected half-life and pharmacokinetics for a human immunoglobulin G. The safety and PK results support and inform VRC01 dosing schedules for planning HIV-1 prevention efficacy studies. PMID:26332605

  18. Serum Neutralization Assay Can Efficiently Replace Plaque Reduction Neutralization Test for Detection and Quantitation of West Nile Virus Antibodies in Human and Animal Serum Samples

    PubMed Central

    Di Gennaro, Annapia; Casaccia, Claudia; Conte, Annamaria; Monaco, Federica; Savini, Giovanni

    2014-01-01

    A serum neutralization assay (SN) was compared with the official plaque reduction neutralization test for the quantitation of West Nile virus antibodies. A total of 1,348 samples from equid sera and 38 from human sera were tested by these two methods. Statistically significant differences were not observed, thus supporting the use of SN for routine purposes. PMID:25100824

  19. Alkaline ceramidase 1 is essential for mammalian skin homeostasis and regulating whole-body energy expenditure.

    PubMed

    Liakath-Ali, Kifayathullah; Vancollie, Valerie E; Lelliott, Christopher J; Speak, Anneliese O; Lafont, David; Protheroe, Hayley J; Ingvorsen, Camilla; Galli, Antonella; Green, Angela; Gleeson, Diane; Ryder, Ed; Glover, Leanne; Vizcay-Barrena, Gema; Karp, Natasha A; Arends, Mark J; Brenn, Thomas; Spiegel, Sarah; Adams, David J; Watt, Fiona M; van der Weyden, Louise

    2016-07-01

    The epidermis is the outermost layer of skin that acts as a barrier to protect the body from the external environment and to control water and heat loss. This barrier function is established through the multistage differentiation of keratinocytes and the presence of bioactive sphingolipids such as ceramides, the levels of which are tightly regulated by a balance of ceramide synthase and ceramidase activities. Here we reveal the essential role of alkaline ceramidase 1 (Acer1) in the skin. Acer1-deficient (Acer1(-/-) ) mice showed elevated levels of ceramide in the skin, aberrant hair shaft cuticle formation and cyclic alopecia. We demonstrate that Acer1 is specifically expressed in differentiated interfollicular epidermis, infundibulum and sebaceous glands and consequently Acer1(-/-) mice have significant alterations in infundibulum and sebaceous gland architecture. Acer1(-/-) skin also shows perturbed hair follicle stem cell compartments. These alterations result in Acer1(-/-) mice showing increased transepidermal water loss and a hypermetabolism phenotype with associated reduction of fat content with age. We conclude that Acer1 is indispensable for mammalian skin homeostasis and whole-body energy homeostasis. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. PMID:27126290

  20. A High-Affinity Native Human Antibody Neutralizes Human Cytomegalovirus Infection of Diverse Cell Types

    PubMed Central

    Liu, Keyi; Park, Minha; DeChene, Neal; Stephenson, Robert; Tenorio, Edgar; Ellsworth, Stote L.; Tabata, Takako; Petitt, Matthew; Tsuge, Mitsuru; Fang-Hoover, June; Adler, Stuart P.; Cui, Xiaohong; McVoy, Michael A.; Pereira, Lenore

    2014-01-01

    Human cytomegalovirus (HCMV) is the most common infection causing poor outcomes among transplant recipients. Maternal infection and transplacental transmission are major causes of permanent birth defects. Although no active vaccines to prevent HCMV infection have been approved, passive immunization with HCMV-specific immunoglobulin has shown promise in the treatment of both transplant and congenital indications. Antibodies targeting the viral glycoprotein B (gB) surface protein are known to neutralize HCMV infectivity, with high-affinity binding being a desirable trait, both to compete with low-affinity antibodies that promote the transmission of virus across the placenta and to displace nonneutralizing antibodies binding nearby epitopes. Using a miniaturized screening technology to characterize secreted IgG from single human B lymphocytes, 30 antibodies directed against gB were previously cloned. The most potent clone, TRL345, is described here. Its measured affinity was 1 pM for the highly conserved site I of the AD-2 epitope of gB. Strain-independent neutralization was confirmed for 15 primary HCMV clinical isolates. TRL345 prevented HCMV infection of placental fibroblasts, smooth muscle cells, endothelial cells, and epithelial cells, and it inhibited postinfection HCMV spread in epithelial cells. The potential utility for preventing congenital transmission is supported by the blockage of HCMV infection of placental cell types central to virus transmission to the fetus, including differentiating cytotrophoblasts, trophoblast progenitor cells, and placental fibroblasts. Further, TRL345 was effective at controlling an ex vivo infection of human placental anchoring villi. TRL345 has been utilized on a commercial scale and is a candidate for clinical evaluation. PMID:25534746

  1. Generation and Characterization of Neutralizing Human Monoclonal Antibodies against Human Immunodeficiency Virus Type 1 Tat Antigen

    PubMed Central

    Moreau, Emmanuel; Hoebeke, Johan; Zagury, Daniel; Muller, Sylviane; Desgranges, Claude

    2004-01-01

    The human immunodeficiency virus Tat regulatory protein is essential for virus replication and pathogenesis. From human peripheral blood mononuclear cells of three Tat toxoid-immunized volunteers, we isolated five Tat-specific human monoclonal antibodies (HMAbs): two full-length immunoglobulin G (IgG) antibodies and three single-chain fragment-variable (scFv) antibodies. The two IgGs were mapped to distinct epitopes within the basic region of Tat, and the three scFvs were mapped to the N-terminal domain of Tat. The three scFvs were highly reactive with recombinant Tat in Western blotting or immunoprecipitation, but results were in contrast to those for the two IgGs, which are sensitive to a particular folding of the protein. In transactivation assays, scFvs were able to inhibit both active recombinant Tat and native Tat secreted by a transfected CEM cell line while IgGs neutralized only native Tat. These HMAbs were able to reduce viral p24 production in human immunodeficiency virus type 1 strain IIIB chronically infected cell lines in a dose-dependent manner. PMID:15016898

  2. Structural comparison of four different antibodies interacting with human papillomavirus 16 and mechanisms of neutralization

    SciTech Connect

    Guan, Jian; Bywaters, Stephanie M.; Brendle, Sarah A.; Lee, Hyunwook; Ashley, Robert E.; Makhov, Alexander M.; Conway, James F.; Christensen, Neil D.; Hafenstein, Susan

    2015-09-15

    Cryo-electron microscopy (cryo-EM) was used to solve the structures of human papillomavirus type 16 (HPV16) complexed with fragments of antibody (Fab) from three different neutralizing monoclonals (mAbs): H16.1A, H16.14J, and H263.A2. The structure-function analysis revealed predominantly monovalent binding of each Fab with capsid interactions that involved multiple loops from symmetry related copies of the major capsid protein. The residues identified in each Fab-virus interface map to a conformational groove on the surface of the capsomer. In addition to the known involvement of the FG and HI loops, the DE loop was also found to constitute the core of each epitope. Surprisingly, the epitope mapping also identified minor contributions by EF and BC loops. Complementary immunological assays included mAb and Fab neutralization. The specific binding characteristics of mAbs correlated with different neutralizing behaviors in pre- and post-attachment neutralization assays. - Highlights: • We present HPV16-Fab complexes from neutralizing mAbs: H16.1A, H16.14J, and H263.A2. • The structure-function analysis revealed predominantly monovalent binding of each mAb. • Capsid–Fab interactions involved multiple loops from symmetry related L1 proteins. • Besides the known FG and HI loops, epitope mapping also identified DE, EF, and BC loops. • Neutralizing assays complement the structures to show multiple neutralization mechanisms.

  3. Pseudotypes of human T-cell leukemia virus types 1 and 2: neutralization by patients' sera.

    PubMed Central

    Clapham, P; Nagy, K; Weiss, R A

    1984-01-01

    Pseudotypes of vesicular stomatitis virus (VSV) bearing envelope antigens of human T-cell leukemia virus (HTLV) types 1 and 2 were prepared by propagating VSV in cells lines productively infected with HTLV. Plaque assays of VSV (HTLV) pseudotypes were employed to determine the presence of (i) HTLV receptors on cells and (ii) neutralizing antibodies in the serum of patients with adult T-cell leukemia-lymphoma (ATLL). Cell surface receptors for HTLV-1 and HTLV-2 were found on nonlymphoid cells of human and mammalian origin. Neutralizing antibodies specific to VSV(HTLV-1) were found in sera of ATLL patients in titers varying from 1:50 to 1:30,000 and did not correlate closely with antibody titers for internal viral antigens. Sera from ATLL patients in the United Kingdom (Caribbean immigrants), United States, and Japan completely neutralized VSV (HTLV-1), indicating that the HTLV isolates from these distinct geographic regions represent a single envelope serotype. Neutralization of VSV (HTLV-1) was more specific and more sensitive than assays of syncytium inhibition. No cross-neutralization was observed between bovine leukosis virus and HTLV, and only limited cross-reaction was found for envelope antigens of HTLV-1 and HTLV-2. These studies show that VSV (HTLV) pseudotypes can be readily used to screen for neutralizing antibodies in patients' sera and to distinguish HTLV envelope serotypes. PMID:6326149

  4. A Novel Humanized Antibody Neutralizes H5N1 Influenza Virus via Two Different Mechanisms

    PubMed Central

    Tan, Yunrui; Ng, Qingyong; Jia, Qiang

    2015-01-01

    ABSTRACT Highly pathogenic avian influenza virus subtype H5N1 continues to be a severe threat to public health, as well as the poultry industry, because of its high lethality and antigenic drift rate. Neutralizing monoclonal antibodies (MAbs) can serve as a useful tool for preventing, treating, and detecting H5N1. In the present study, humanized H5 antibody 8A8 was developed from a murine H5 MAb. Both the humanized and mouse MAbs presented positive activity in hemagglutination inhibition (HI), virus neutralization, and immunofluorescence assays against a wide range of H5N1 strains. Interestingly, both human and murine 8A8 antibodies were able to detect H5 in Western blot assays under reducing conditions. Further, by sequencing of escape mutants, the conformational epitope of 8A8 was found to be located within the receptor binding domain (RBD) of H5. The linear epitope of 8A8 was identified by Western blotting of overlapping fragments and substitution mutant forms of HA1. Reverse genetic H5N1 strains with individual mutations in either the conformational or the linear epitope were generated and characterized in a series of assays, including HI, postattachment, and cell-cell fusion inhibition assays. The results indicate that for 8A8, virus neutralization mediated by RBD blocking relies on the conformational epitope while binding to the linear epitope contributes to the neutralization by inhibiting membrane fusion. Taken together, the results of this study show that a novel humanized H5 MAb binds to two types of epitopes on HA, leading to virus neutralization via two mechanisms. IMPORTANCE Recurrence of the highly pathogenic avian influenza virus subtype H5N1 in humans and poultry continues to be a serious public health concern. Preventive and therapeutic measures against influenza A viruses have received much interest in the context of global efforts to combat the current and future pandemics. Passive immune therapy is considered to be the most effective and

  5. Characterization of novel neutralizing monoclonal antibodies specific to human neurturin.

    PubMed

    Hongo, J A; Tsai, S P; Moffat, B; Schroeder, K A; Jung, C; Chuntharapai, A; Lampe, P A; Johnson, E M; de Sauvage, F J; Armanini, M; Phillips, H; Devaux, B

    2000-08-01

    Neurturin (NTN) a structural and functional relative of glial cell line-derived neurotrophic factor, was originally identified based on its ability to support the survival of sympathetic neurons in culture. Similar to glial cell line-derived neurotrophic factor (GDNF), Neurturin has been shown to bind to a high affinity glycosylphosphatidylinositol (GPI)-linked receptor (GFRalpha2) and induce phosphorylation of the tyrosine kinase receptor Ret, resulting in the activation of the mitogen activated protein kinase (MAPK) signalling pathway. A panel of six novel murine monoclonal antibodies (MAbs) specific to human Neurturin has been developed and characterized. Four of the MAbs tested inhibit, to varying degrees, binding of NTN to the GPI-linked GFRalpha2 receptor. Three MAbs cross-react with the murine homolog. These antibodies have been shown to be useful reagents for Western blotting, immunohistochemistry, and also for the development of a sensitive, quantitative enzyme-linked immunosorbent assay (ELISA) for human NTN. Novel, specific MAbs with varying epitope specificities and blocking activity will be valuable tools for both the in vitro and in vivo characterization of NTN and its relationship to the GFRalpha2 and Ret receptors. PMID:11001403

  6. Benzoxazolone Carboxamides as Potent Acid Ceramidase Inhibitors: Synthesis and Structure-Activity Relationship (SAR) Studies.

    PubMed

    Bach, Anders; Pizzirani, Daniela; Realini, Natalia; Vozella, Valentina; Russo, Debora; Penna, Ilaria; Melzig, Laurin; Scarpelli, Rita; Piomelli, Daniele

    2015-12-10

    Ceramides are lipid-derived intracellular messengers involved in the control of senescence, inflammation, and apoptosis. The cysteine amidase, acid ceramidase (AC), hydrolyzes these substances into sphingosine and fatty acid and, by doing so, regulates their signaling activity. AC inhibitors may be useful in the treatment of pathological conditions, such as cancer, in which ceramide levels are abnormally reduced. Here, we present a systematic SAR investigation of the benzoxazolone carboxamides, a recently described class of AC inhibitors that display high potency and systemic activity in mice. We examined a diverse series of substitutions on both benzoxazolone ring and carboxamide side chain. Several modifications enhanced potency and stability, and one key compound with a balanced activity-stability profile (14) was found to inhibit AC activity in mouse lungs and cerebral cortex after systemic administration. The results expand our arsenal of AC inhibitors, thereby facilitating the use of these compounds as pharmacological tools and their potential development as drug leads. PMID:26560855

  7. Acid ceramidase upregulation in prostate cancer: role in tumor development and implications for therapy.

    PubMed

    Liu, Xiang; Cheng, Joseph C; Turner, Lorianne S; Elojeimy, Saeed; Beckham, Thomas H; Bielawska, Alicja; Keane, Thomas E; Hannun, Yusuf A; Norris, James S

    2009-12-01

    Bioactive sphingolipids, such as ceramide, sphingosine and sphingosine-1-phosphate are known bio-effector molecules which play important roles in various aspects of cancer biology including cell proliferation, growth arrest, apoptosis, metastasis, senescence and inflammation. Therefore, enzymes involved in ceramide metabolism are gaining recognition as being critical regulators of cancer cell growth and/or survival. We previously observed that the ceramide metabolizing enzyme, acid ceramidase (AC) is upregulated in tumor tissues. Studies have now concluded that this creates a dysfunctional ceramide pathway, which is responsible for tumor progression and resistance to chemotherapy and radiation. This suggests that development of small-molecule drugs that inhibit AC enzyme activity is a promising approach for improving standard cancer therapy and patient's clinical outcomes. PMID:19874262

  8. Cross-neutralizing human anti-poliovirus antibodies bind the recognition site for cellular receptor

    PubMed Central

    Chen, Zhaochun; Fischer, Elizabeth R.; Kouiavskaia, Diana; Hansen, Bryan T.; Ludtke, Steven J.; Bidzhieva, Bella; Makiya, Michelle; Agulto, Liane; Purcell, Robert H.; Chumakov, Konstantin

    2013-01-01

    Most structural information about poliovirus interaction with neutralizing antibodies was obtained in the 1980s in studies of mouse monoclonal antibodies. Recently we have isolated a number of human/chimpanzee anti-poliovirus antibodies and demonstrated that one of them, MAb A12, could neutralize polioviruses of both serotypes 1 and 2. This communication presents data on isolation of an additional cross-neutralizing antibody (F12) and identification of a previously unknown epitope on the surface of poliovirus virions. Epitope mapping was performed by sequencing of antibody-resistant mutants and by cryo-EM of complexes of virions with Fab fragments. The results have demonstrated that both cross-neutralizing antibodies bind the site located at the bottom of the canyon surrounding the fivefold axis of symmetry that was previously shown to interact with cellular poliovirus receptor CD155. However, the same antibody binds to serotypes 1 and 2 through different specific interactions. It was also shown to interact with type 3 poliovirus, albeit with about 10-fold lower affinity, insufficient for effective neutralization. Antibody interaction with the binding site of the cellular receptor may explain its broad reactivity and suggest that further screening or antibody engineering could lead to a universal antibody capable of neutralizing all three serotypes of poliovirus. PMID:24277851

  9. Neutral versus Emotional Human Stimuli Processing in Children with Pervasive Developmental Disorders not Otherwise Specified

    ERIC Educational Resources Information Center

    Vannetzel, Leonard; Chaby, Laurence; Cautru, Fabienne; Cohen, David; Plaza, Monique

    2011-01-01

    Pervasive developmental disorder not otherwise specified (PDD-NOS) represents up to two-thirds of autism spectrum disorders; however, it is usually described in terms of the symptoms not shared by autism. The study explores processing of neutral and emotional human stimuli (by auditory, visual and multimodal channels) in children with PDD-NOS (n =…

  10. Development of human neutralizing monoclonal antibodies for prevention and therapy of MERS-CoV infections

    PubMed Central

    Ying, Tianlei; Li, Haoyang; Lu, Lu; Dimitrov, Dimiter S; Jiang, Shibo

    2014-01-01

    The recent Middle East respiratory syndrome coronavirus (MERS-CoV) outbreak poses a serious threat to public health. Here, we summarize recent advances in identifying human neutralizing monoclonal antibodies (mAbs) against MERS-CoV, describe their mechanisms of action, and analyze their potential for treatment of MERS-CoV infections. PMID:25456101

  11. Affinity Maturation to Improve Human Monoclonal Antibody Neutralization Potency and Breadth against Hepatitis C Virus*

    PubMed Central

    Wang, Yong; Keck, Zhen-yong; Saha, Anasuya; Xia, Jinming; Conrad, Fraser; Lou, Jianlong; Eckart, Michael; Marks, James D.; Foung, Steven K. H.

    2011-01-01

    A potent neutralizing antibody to a conserved hepatitis C virus (HCV) epitope might overcome its extreme variability, allowing immunotherapy. The human monoclonal antibody HC-1 recognizes a conformational epitope on the HCV E2 glycoprotein. Previous studies showed that HC-1 neutralizes most HCV genotypes but has modest potency. To improve neutralization, we affinity-matured HC-1 by constructing a library of yeast-displayed HC-1 single chain Fv (scFv) mutants, using for selection an E2 antigen from one of the poorly neutralized HCVpp. We developed an approach by parallel mutagenesis of the heavy chain variable (VH) and κ-chain variable (Vk) genes separately, then combining the optimized VH and Vk mutants. This resulted in the generation of HC-1-related scFv variants exhibiting improved affinities. The best scFv variant had a 92-fold improved affinity. After conversion to IgG1, some of the antibodies exhibited a 30-fold improvement in neutralization activity. Both surface plasmon resonance and solution kinetic exclusion analysis showed that the increase in affinity was largely due to a lowering of the dissociation rate constant, Koff. Neutralization against a panel of HCV pseudoparticles and infectious 2a HCV virus improved with the affinity-matured IgG1 antibodies. Interestingly, some of these antibodies neutralized a viral isolate that was not neutralized by wild-type HC-1. Moreover, propagating 2a HCVcc under the selective pressure of WT HC-1 or affinity-matured HC-1 antibodies yielded no viral escape mutants and, with the affinity-matured IgG1, needed 100-fold less antibody to achieve complete virus elimination. Taken together, these findings suggest that affinity-matured HC-1 antibodies are excellent candidates for therapeutic development. PMID:22002064

  12. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library.

    PubMed

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-01-01

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective. PMID:27626445

  13. Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits

    PubMed Central

    Robinson, James E.; Hastie, Kathryn M.; Cross, Robert W.; Yenni, Rachael E.; Elliott, Deborah H.; Rouelle, Julie A.; Kannadka, Chandrika B.; Smira, Ashley A.; Garry, Courtney E.; Bradley, Benjamin T.; Yu, Haini; Shaffer, Jeffrey G.; Boisen, Matt L.; Hartnett, Jessica N.; Zandonatti, Michelle A.; Rowland, Megan M.; Heinrich, Megan L.; Martínez-Sobrido, Luis; Cheng, Benson; de la Torre, Juan C.; Andersen, Kristian G.; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Gbakie, Michael; Kanneh, Lansana; Koroma, Veronica J.; Fonnie, Richard; Jalloh, Simbirie C.; Kargbo, Brima; Vandi, Mohamed A.; Gbetuwa, Momoh; Ikponmwosa, Odia; Asogun, Danny A.; Okokhere, Peter O.; Follarin, Onikepe A.; Schieffelin, John S.; Pitts, Kelly R.; Geisbert, Joan B.; Kulakoski, Peter C.; Wilson, Russell B.; Happi, Christian T.; Sabeti, Pardis C.; Gevao, Sahr M.; Khan, S. Humarr; Grant, Donald S.; Geisbert, Thomas W.; Saphire, Erica Ollmann; Branco, Luis M.; Garry, Robert F.

    2016-01-01

    Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design. PMID:27161536

  14. Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits.

    PubMed

    Robinson, James E; Hastie, Kathryn M; Cross, Robert W; Yenni, Rachael E; Elliott, Deborah H; Rouelle, Julie A; Kannadka, Chandrika B; Smira, Ashley A; Garry, Courtney E; Bradley, Benjamin T; Yu, Haini; Shaffer, Jeffrey G; Boisen, Matt L; Hartnett, Jessica N; Zandonatti, Michelle A; Rowland, Megan M; Heinrich, Megan L; Martínez-Sobrido, Luis; Cheng, Benson; de la Torre, Juan C; Andersen, Kristian G; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Gbakie, Michael; Kanneh, Lansana; Koroma, Veronica J; Fonnie, Richard; Jalloh, Simbirie C; Kargbo, Brima; Vandi, Mohamed A; Gbetuwa, Momoh; Ikponmwosa, Odia; Asogun, Danny A; Okokhere, Peter O; Follarin, Onikepe A; Schieffelin, John S; Pitts, Kelly R; Geisbert, Joan B; Kulakoski, Peter C; Wilson, Russell B; Happi, Christian T; Sabeti, Pardis C; Gevao, Sahr M; Khan, S Humarr; Grant, Donald S; Geisbert, Thomas W; Saphire, Erica Ollmann; Branco, Luis M; Garry, Robert F

    2016-01-01

    Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design. PMID:27161536

  15. A Trimeric, V2-Deleted HIV-1 Envelope Glycoprotein Vaccine Elicits Potent Neutralizing Antibodies but Limited Breadth of Neutralization in Human Volunteers

    PubMed Central

    Lally, Michelle A.; Elizaga, Marnie; Montefiori, David; Tomaras, Georgia D.; McElrath, M. Juliana; Hural, John; De Rosa, Stephen C.; Sato, Alicia; Huang, Yunda; Frey, Sharon E.; Sato, Paul; Donnelly, John; Barnett, Susan; Corey, Lawrence J.

    2011-01-01

    Background. A key missing element in the development of a successful human immunodeficiency virus (HIV) vaccine is an immunogen that can generate broadly cross-neutralizing antibodies against primary isolates of the virus. Methods. This phase 1 clinical trial employed a DNA prime and subunit envelope protein boost in an attempt to generate cellular and humoral immune responses that might be desirable in a protective HIV vaccine. Priming was performed via intramuscular injection with gag and env DNA adsorbed to polylactide coglycolide microspheres, followed by boosting with a recombinant trimeric envelope (Env) glycoprotein delivered in MF59 adjuvant. Results. The DNA prime and protein boost were generally safe and well-tolerated. Env-specific CD4+ cellular responses were generated that were predominantly detected after Env protein boosting. Neutralizing antibody responses against the homologous SF162 viral isolate were remarkably strong and were present in the majority of vaccine recipients, including a strong response against CD4-induced epitopes on gp120. Despite the promising potency of this vaccine approach, neutralization breadth against heterologous tier 2 strains of HIV-1 was minimal. Conclusions. Potent neutralization against neutralization-sensitive strains of HIV is achievable in humans through a DNA prime, recombinant oligomeric Env protein boost regimen. Eliciting substantial breadth of neutralization remains an elusive goal.  Clinical Trials Registration. NCT00073216. PMID:21451004

  16. Expression of Ceramide Synthase 6 Transcriptionally Activates Acid Ceramidase in a c-Jun N-terminal Kinase (JNK)-dependent Manner.

    PubMed

    Tirodkar, Tejas S; Lu, Ping; Bai, Aiping; Scheffel, Matthew J; Gencer, Salih; Garrett-Mayer, Elizabeth; Bielawska, Alicja; Ogretmen, Besim; Voelkel-Johnson, Christina

    2015-05-22

    A family of six ceramide synthases with distinct but overlapping substrate specificities is responsible for generation of ceramides with acyl chains ranging from ∼14-26 carbons. Ceramide synthase 6 (CerS6) preferentially generates C14- and C16-ceramides, and we have previously shown that down-regulation of this enzyme decreases apoptotic susceptibility. In this study, we further evaluated how increased CerS6 expression impacts sphingolipid composition and metabolism. Overexpression of CerS6 in HT29 colon cancer cells resulted in increased apoptotic susceptibility and preferential generation of C16-ceramide, which occurred at the expense of very long chain, saturated ceramides. These changes were also reflected in sphingomyelin composition. HT-CerS6 cells had increased intracellular levels of sphingosine, which is generated by ceramidases upon hydrolysis of ceramide. qRT-PCR analysis revealed that only expression of acid ceramidase (ASAH1) was increased. The increase in acid ceramidase was confirmed by expression and activity analyses. Pharmacological inhibition of JNK (SP600125) or curcumin reduced transcriptional up-regulation of acid ceramidase. Using an acid ceramidase promoter driven luciferase reporter plasmid, we demonstrated that CerS1 has no effect on transcriptional activation of acid ceramidase and that CerS2 slightly but significantly decreased the luciferase signal. Similar to CerS6, overexpression of CerS3-5 resulted in an ∼2-fold increase in luciferase reporter gene activity. Exogenous ceramide failed to induce reporter activity, while a CerS inhibitor and a catalytically inactive mutant of CerS6 failed to reduce it. Taken together, these results suggest that increased expression of CerS6 can mediate transcriptional activation of acid ceramidase in a JNK-dependent manner that is independent of CerS6 activity. PMID:25839235

  17. Isolation and characterization of novel human monoclonal antibodies possessing neutralizing ability against rabies virus.

    PubMed

    Matsumoto, Takashi; Yamada, Kentaro; Noguchi, Kazuko; Nakajima, Kantou; Takada, Kenzo; Khawplod, Pakamatz; Nishizono, Akira

    2010-11-01

    Rabies is a fatal viral encephalitis which is transmitted by exposure to the bite of rabid animals. Human and equine rabies immunoglobulins are indispensable pharmacological agents for severe bite exposure, as is vaccine. However, several disadvantages, including limited supply, adverse reactions, and high cost, hamper their wide application in developing countries. In the present study, two novel huMabs which neutralize rabies virus were established from vaccinated hyperimmune volunteers using the Epstein-Barr virus transformation method. One MAb (No. 254), which was subclass IgG3, effectively neutralized fixed rabies viruses of CVS, ERA, HEP-Flury, and Nishigahara strains and recognized a well-conserved epitope located in antigenic site II of the rabies virus glycoprotein. No. 254 possessed 68 ng/ml of FRNT₅₀ activity against CVS, 3.7 × 10⁻⁷ M of the Kd value, and the enhancing effect of complement-dependent virolysis. In addition, No. 254 showed effective neutralization potency in vivo in the mouse challenge test. The other MAb, 4D4, was subclass IgM and showed neutralizing activity against CVS and Nishigahara strains. 4D4 recognized a novel antigenic site which is associated with the neurovirulence of rabies, a glycoprotein located between antigenic site I and VI. Both human MAbs against rabies are expected to be utilized as a tool for future post-exposure prophylaxis. PMID:21044141

  18. Recognition of influenza H3N2 variant virus by human neutralizing antibodies

    PubMed Central

    Bangaru, Sandhya; Nieusma, Travis; Kose, Nurgun; Thornburg, Natalie J.; Finn, Jessica A.; Kaplan, Bryan S.; King, Hannah G.; Singh, Vidisha; Lampley, Rebecca M.; Sapparapu, Gopal; Cisneros, Alberto; Edwards, Kathryn M.; Slaughter, James C.; Edupuganti, Srilatha; Lai, Lilin; Richt, Juergen A.; Webby, Richard J.; Ward, Andrew B.; Crowe, James E.

    2016-01-01

    Since 2011, over 300 human cases of infection, especially in exposed children, with the influenza A H3N2 variant (H3N2v) virus that circulates in swine in the US have been reported. The structural and genetic basis for the lack of protection against H3N2v induced by vaccines containing seasonal H3N2 antigens is poorly understood. We isolated 17 human monoclonal antibodies (mAbs) that neutralized H3N2v virus from subjects experimentally immunized with an H3N2v candidate vaccine. Six mAbs exhibited very potent neutralizing activity (IC50 < 200 ng/ml) against the H3N2v virus but not against current human H3N2 circulating strains. Fine epitope mapping and structural characterization of antigen-antibody complexes revealed that H3N2v specificity was attributable to amino acid polymorphisms in the 150-loop and the 190-helix antigenic sites on the hemagglutinin protein. H3N2v-specific antibodies also neutralized human H3N2 influenza strains naturally circulating between 1995 and 2005. These results reveal a high level of antigenic relatedness between the swine H3N2v virus and previously circulating human strains, consistent with the fact that early human H3 seasonal strains entered the porcine population in the 1990s and reentered the human population, where they had not been circulating, as H3N2v about a decade later. The data also explain the increased susceptibility to H3N2v viruses in young children, who lack prior exposure to human seasonal strains from the 1990s. PMID:27482543

  19. Identification and Characterization of a New Cross-Reactive Human Immunodeficiency Virus Type 1-Neutralizing Human Monoclonal Antibody

    PubMed Central

    Zhang, Mei-Yun; Xiao, Xiaodong; Sidorov, Igor A.; Choudhry, Vidita; Cham, Fatim; Zhang, Peng Fei; Bouma, Peter; Zwick, Michael; Choudhary, Anil; Montefiori, David C.; Broder, Christopher C.; Burton, Dennis R.; Quinnan, Gerald V.; Dimitrov, Dimiter S.

    2004-01-01

    The identification and characterization of new human monoclonal antibodies (hMAbs) able to neutralize primary human immunodeficiency virus type 1 (HIV-1) isolates from different subtypes may help in our understanding of the mechanisms of virus entry and neutralization and in the development of entry inhibitors and vaccines. For enhanced selection of broadly cross-reactive antibodies, soluble HIV-1 envelope glycoproteins (Envs proteins) from two isolates complexed with two-domain soluble CD4 (sCD4) were alternated during panning of a phage-displayed human antibody library; these two Env proteins (89.6 and IIIB gp140s), and one additional Env (JR-FL gp120) alone and complexed with sCD4 were used for screening. An antibody with relatively long HCDR3 (17 residues), designated m14, was identified that bound to all antigens and neutralized heterologous HIV-1 isolates in multiple assay formats. Fab m14 potently neutralized selected well-characterized subtype B isolates, including JRCSF, 89.6, IIIB, and Yu2. Immunoglobulin G1 (IgG1) m14 was more potent than Fab m14 and neutralized 7 of 10 other clade B isolates; notably, although the potency was on average significantly lower than that of IgG1 b12, IgG1 m14 neutralized two of the isolates with significantly lower 50% inhibitory concentrations than did IgG1 b12. IgG1 m14 neutralized four of four selected clade C isolates with potency higher than that of IgG1 b12. It also neutralized 7 of 17 clade C isolates from southern Africa that were difficult to neutralize with other hMAbs and sCD4. IgG1 m14 neutralized four of seven primary HIV-1 isolates from other clades (A, D, E, and F) much more efficiently than did IgG1 b12; for the other three isolates, IgG b12 was much more potent. Fab m14 bound with high (nanomolar range) affinity to gp120 and gp140 from various isolates; its binding was reduced by soluble CD4 and antibodies recognizing the CD4 binding site (CD4bs) on gp120, and its footprint as defined by alanine

  20. Development of Human-Like scFv-Fc Neutralizing Botulinum Neurotoxin E

    PubMed Central

    Miethe, Sebastian; Rasetti-Escargueil, Christine; Avril, Arnaud; Liu, Yvonne; Chahboun, Siham; Korkeala, Hannu; Mazuet, Christelle; Popoff, Michel-Robert; Pelat, Thibaut; Thullier, Philippe; Sesardic, Dorothea; Hust, Michael

    2015-01-01

    Background Botulinum neurotoxins (BoNTs) are considered to be the most toxic substances known on earth and are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food-poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agent by the Centers of Disease Control and Prevention (CDC) and are listed among the six agents with the highest risk to be used as bioweapons. Neutralizing antibodies are required for the development of effective anti-botulism therapies to deal with the potential risk of exposure. Results In this study, a macaque (Macaca fascicularis) was immunized with recombinant light chain of BoNT/E3 and an immune phage display library was constructed. After a multi-step panning, several antibody fragments (scFv, single chain fragment variable) with nanomolar affinities were isolated, that inhibited the endopeptidase activity of pure BoNT/E3 in vitro by targeting its light chain. Furthermore, three scFv were confirmed to neutralize BoNT/E3 induced paralysis in an ex vivo mouse phrenic nerve-hemidiaphragm assay. The most effective neutralization (20LD50/mL, BoNT/E3) was observed with scFv ELC18, with a minimum neutralizing concentration at 0.3 nM. Furthermore, ELC18 was highly effective in vivo when administered as an scFv-Fc construct. Complete protection of 1LD50 BoNT/E3 was observed with 1.6 ng/dose in the mouse flaccid paralysis assay. Conclusion These scFv-Fcs antibodies are the first recombinant antibodies neutralizing BoNT/E by targeting its light chain. The human-like nature of the isolated antibodies is predicting a good tolerance for further clinical development. PMID:26440796

  1. Development and characterization of human monoclonal antibodies that neutralize multiple TGFβ isoforms

    PubMed Central

    Bedinger, Daniel; Lao, Llewelyn; Khan, Shireen; Lee, Steve; Takeuchi, Toshihiko; Mirza, Amer M.

    2016-01-01

    ABSTRACT Transforming growth factor (TGF)β levels are elevated in, and drive the progression of, numerous disease states such as advanced metastatic cancer and systemic and ocular fibrosis. There are 3 main isoforms, TGFβ1, 2, and 3. As multiple TGFβ isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGFβ isoforms. Fully human antibody phage display libraries were used to discover a number of antibodies that bind and neutralize various combinations of TGFβ1, 2 or 3. The primary panning did not yield any uniformly potent pan-isoform neutralizing antibodies; therefore, an antibody that displayed potent TGFβ 1, 2 inhibition, but more modest affinity versus TGFβ3, was affinity matured by shuffling with a light chain sub-library and further screening. This process yielded a high affinity pan-isoform neutralizing clone. Antibodies were analyzed and compared by binding affinity, as well as receptor and epitope competition by surface plasmon resonance methods. The antibodies were also shown to neutralize TGFβ effects in vitro in 3 assays: 1) interleukin (IL)-4 induced HT-2 cell proliferation; 2) TGFβ-mediated IL-11 release by A549 cells; and 3) decreasing SMAD2 phosphorylation in Detroit 562 cells. The antibodies’ potency in these in vitro assays correlated well with their isoform-specific affinities. Furthermore, the ability of the affinity-matured clone to decrease tumor burden in a Detroit 562 xenograft study was superior to that of the parent clone. This affinity-matured antibody acts as a very potent inhibitor of all 3 main isoforms of TGFβ and may have utility for therapeutic intervention in human disease. PMID:26563652

  2. Structural comparison of four different antibodies interacting with human papillomavirus 16 and mechanisms of neutralization.

    PubMed

    Guan, Jian; Bywaters, Stephanie M; Brendle, Sarah A; Lee, Hyunwook; Ashley, Robert E; Makhov, Alexander M; Conway, James F; Christensen, Neil D; Hafenstein, Susan

    2015-09-01

    Cryo-electron microscopy (cryo-EM) was used to solve the structures of human papillomavirus type 16 (HPV16) complexed with fragments of antibody (Fab) from three different neutralizing monoclonals (mAbs): H16.1A, H16.14J, and H263.A2. The structure-function analysis revealed predominantly monovalent binding of each Fab with capsid interactions that involved multiple loops from symmetry related copies of the major capsid protein. The residues identified in each Fab-virus interface map to a conformational groove on the surface of the capsomer. In addition to the known involvement of the FG and HI loops, the DE loop was also found to constitute the core of each epitope. Surprisingly, the epitope mapping also identified minor contributions by EF and BC loops. Complementary immunological assays included mAb and Fab neutralization. The specific binding characteristics of mAbs correlated with different neutralizing behaviors in pre- and post-attachment neutralization assays. PMID:25996608

  3. Characterization of Two Human Monoclonal Antibodies Neutralizing Influenza A H7N9 Viruses

    PubMed Central

    Wang, Jianmin; Chen, Zhe; Bao, Linlin; Zhang, Weijia; Xue, Ying; Pang, XingHuo; Zhang, Xi

    2015-01-01

    H7N9 was a cause of significant global health concern due to its severe infection and approximately 35% mortality in humans. By screening a Fab antibody phage library derived from patients who recovered from H7N9 infections, we characterized two human monoclonal antibodies (HuMAbs), HNIgGD5 and HNIgGH8. The epitope of these two antibodies was dependent on two residues in the receptor binding site at positions V186 and L226 of the hemagglutinin glycoprotein. Both antibodies possessed high neutralizing activity. PMID:26063436

  4. Identification and Application of Neutralizing Epitopes of Human Adenovirus Type 55 Hexon Protein

    PubMed Central

    Tian, Xingui; Ma, Qiang; Jiang, Zaixue; Huang, Junfeng; Liu, Qian; Lu, Xiaomei; Luo, Qingming; Zhou, Rong

    2015-01-01

    Human adenovirus type 55 (HAdV55) is a newly identified re-emergent acute respiratory disease (ARD) pathogen with a proposed recombination of hexon gene between HAdV11 and HAdV14 strains. The identification of the neutralizing epitopes is important for the surveillance and vaccine development against HAdV55 infection. In this study, four type-specific epitope peptides of HAdV55 hexon protein, A55R1 (residues 138 to 152), A55R2 (residues 179 to 187), A55R4 (residues 247 to 259) and A55R7 (residues 429 to 443), were predicted by multiple sequence alignment and homology modeling methods, and then confirmed with synthetic peptides by enzyme-linked immunosorbent assay (ELISA) and neutralization tests (NT). Finally, the A55R2 was incorporated into human adenoviruses 3 (HAdV3) and a chimeric adenovirus rAd3A55R2 was successfully obtained. The chimeric rAd3A55R2 could induce neutralizing antibodies against both HAdV3 and HAdV55. This current study will contribute to the development of novel adenovirus vaccine candidate and adenovirus structural analysis. PMID:26516903

  5. Preexisting human antibodies neutralize recently emerged H7N9 influenza strains

    PubMed Central

    Henry Dunand, Carole J.; Leon, Paul E.; Kaur, Kaval; Tan, Gene S.; Zheng, Nai-Ying; Andrews, Sarah; Huang, Min; Qu, Xinyan; Huang, Yunping; Salgado-Ferrer, Marlene; Ho, Irvin Y.; Taylor, William; Hai, Rong; Wrammert, Jens; Ahmed, Rafi; García-Sastre, Adolfo; Palese, Peter; Krammer, Florian; Wilson, Patrick C.

    2015-01-01

    The emergence and seasonal persistence of pathogenic H7N9 influenza viruses in China have raised concerns about the pandemic potential of this strain, which, if realized, would have a substantial effect on global health and economies. H7N9 viruses are able to bind to human sialic acid receptors and are also able to develop resistance to neuraminidase inhibitors without a loss in fitness. It is not clear whether prior exposure to circulating human influenza viruses or influenza vaccination confers immunity to H7N9 strains. Here, we demonstrate that 3 of 83 H3 HA-reactive monoclonal antibodies generated by individuals that had previously undergone influenza A virus vaccination were able to neutralize H7N9 viruses and protect mice against homologous challenge. The H7N9-neutralizing antibodies bound to the HA stalk domain but exhibited a difference in their breadth of reactivity to different H7 influenza subtypes. Mapping viral escape mutations suggested that these antibodies bind at least two different epitopes on the stalk region. Together, these results indicate that these broadly neutralizing antibodies may contribute to the development of therapies against H7N9 strains and may also be effective against pathogenic H7 strains that emerge in the future. PMID:25689254

  6. Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin

    SciTech Connect

    Whittle, James R.R.; Zhang, Ruijun; Khurana, Surender; King, Lisa R.; Manischewitz, Jody; Golding, Hana; Dormitzer, Philip R.; Haynes, Barton F.; Walter, Emmanuel B.; Moody, M. Anthony; Kepler, Thomas B.; Liao, Hua-Xin; Harrison, Stephen C.

    2011-09-20

    Seasonal antigenic drift of circulating influenza virus leads to a requirement for frequent changes in vaccine composition, because exposure or vaccination elicits human antibodies with limited cross-neutralization of drifted strains. We describe a human monoclonal antibody, CH65, obtained by isolating rearranged heavy- and light-chain genes from sorted single plasma cells, coming from a subject immunized with the 2007 trivalent influenza vaccine. The crystal structure of a complex of the hemagglutinin (HA) from H1N1 strain A/Solomon Islands/3/2006 with the Fab of CH65 shows that the tip of the CH65 heavy-chain complementarity determining region 3 (CDR3) inserts into the receptor binding pocket on HA1, mimicking in many respects the interaction of the physiological receptor, sialic acid. CH65 neutralizes infectivity of 30 out of 36 H1N1 strains tested. The resistant strains have a single-residue insertion near the rim of the sialic-acid pocket. We conclude that broad neutralization of influenza virus can be achieved by antibodies with contacts that mimic those of the receptor.

  7. Structure of HCMV glycoprotein B in the postfusion conformation bound to a neutralizing human antibody

    PubMed Central

    Chandramouli, Sumana; Ciferri, Claudio; Nikitin, Pavel A.; Caló, Stefano; Gerrein, Rachel; Balabanis, Kara; Monroe, James; Hebner, Christy; Lilja, Anders E.; Settembre, Ethan C.; Carfi, Andrea

    2015-01-01

    Human cytomegalovirus (HCMV) poses a significant threat to immunocompromised individuals and neonates infected in utero. Glycoprotein B (gB), the herpesvirus fusion protein, is a target for neutralizing antibodies and a vaccine candidate due to its indispensable role in infection. Here we show the crystal structure of the HCMV gB ectodomain bound to the Fab fragment of 1G2, a neutralizing human monoclonal antibody isolated from a seropositive subject. The gB/1G2 interaction is dominated by aromatic residues in the 1G2 heavy chain CDR3 protruding into a hydrophobic cleft in the gB antigenic domain 5 (AD-5). Structural analysis and comparison with HSV gB suggest the location of additional neutralizing antibody binding sites on HCMV gB. Finally, immunoprecipitation experiments reveal that 1G2 can bind to HCMV virion gB suggesting that its epitope is exposed and accessible on the virus surface. Our data will support the development of vaccines and therapeutic antibodies against HCMV infection. PMID:26365435

  8. Acid ceramidase upregulation in prostate cancer cells confers resistance to radiation: AC inhibition, a potential radiosensitizer.

    PubMed

    Mahdy, Ayman E M; Cheng, Joseph C; Li, Jun; Elojeimy, Saeed; Meacham, William D; Turner, Lorianne S; Bai, Aiping; Gault, Christopher R; McPherson, Alex S; Garcia, Nicole; Beckham, Thomas H; Saad, Antonio; Bielawska, Alicja; Bielawski, Jacek; Hannun, Yusuf A; Keane, Thomas E; Taha, Mohhammed I; Hammouda, Hisham M; Norris, James S; Liu, Xiang

    2009-03-01

    Radiation resistance in a subset of prostate tumors remains a challenge to prostate cancer radiotherapy. The current study on the effects of radiation on prostate cancer cells reveals that radiation programs an unpredicted resistance mechanism by upregulating acid ceramidase (AC). Irradiated cells demonstrated limited changes of ceramide levels while elevating levels of sphingosine and sphingosine-1-phosphate. By genetically downregulating AC with small interfering RNA (siRNA), we observed radiosensitization of cells using clonogenic and cytotoxicity assays. Conversely, AC overexpression further decreased sensitivity to radiation. We also observed that radiation-induced AC upregulation was sufficient to create cross-resistance to chemotherapy as demonstrated by decreased sensitivity to Taxol and C(6) ceramide compared to controls. Lower levels of caspase 3/7 activity were detected in cells pretreated with radiation, also indicating increased resistance. Finally, utilization of the small molecule AC inhibitor, LCL385, sensitized PPC-1 cells to radiation and significantly decreased tumor xenograft growth. These data suggest a new mechanism of cancer cell resistance to radiation, through upregulation of AC that is, in part, mediated by application of the therapy itself. An improved understanding of radiotherapy and the application of combination therapy achieved in this study offer new opportunities for the modulation of radiation effects in the treatment of cancer. PMID:19107118

  9. Acid ceramidase as a therapeutic target in metastatic prostate cancer[S

    PubMed Central

    Camacho, Luz; Meca-Cortés, Óscar; Abad, José Luis; García, Simón; Rubio, Nuria; Díaz, Alba; Celià-Terrassa, Toni; Cingolani, Francesca; Bermudo, Raquel; Fernández, Pedro L.; Blanco, Jerónimo; Delgado, Antonio; Casas, Josefina; Fabriàs, Gemma; Thomson, Timothy M.

    2013-01-01

    Acid ceramidase (AC) catalyzes the hydrolysis of ceramide into sphingosine, in turn a substrate of sphingosine kinases that catalyze its conversion into the mitogenic sphingosine-1-phosphate. AC is expressed at high levels in several tumor types and has been proposed as a cancer therapeutic target. Using a model derived from PC-3 prostate cancer cells, the highly tumorigenic, metastatic, and chemoresistant clone PC-3/Mc expressed higher levels of the AC ASAH1 than the nonmetastatic clone PC-3/S. Stable knockdown of ASAH1 in PC-3/Mc cells caused an accumulation of ceramides, inhibition of clonogenic potential, increased requirement for growth factors, and inhibition of tumorigenesis and lung metastases. We developed de novo ASAH1 inhibitors, which also caused a dose-dependent accumulation of ceramides in PC-3/Mc cells and inhibited their growth and clonogenicity. Finally, immunohistochemical analysis of primary prostate cancer samples showed that higher levels of ASAH1 were associated with more advanced stages of this neoplasia. These observations confirm ASAH1 as a therapeutic target in advanced and chemoresistant forms of prostate cancer and suggest that our new potent and specific AC inhibitors could act by counteracting critical growth properties of these highly aggressive tumor cells. PMID:23423838

  10. Prefusion F–specific antibodies determine the magnitude of RSV neutralizing activity in human sera

    PubMed Central

    Ngwuta, Joan O.; Chen, Man; Modjarrad, Kayvon; Joyce, M. Gordon; Kanekiyo, Masaru; Kumar, Azad; Yassine, Hadi M.; Moin, Syed M.; Killikelly, April M.; Chuang, Gwo-Yu; Druz, Aliaksandr; Georgiev, Ivelin S.; Rundlet, Emily J.; Sastry, Mallika; Stewart-Jones, Guillaume B. E.; Yang, Yongping; Zhang, Baoshan; Nason, Martha C.; Capella, Cristina; Peeples, Mark E.; Ledgerwood, Julie E.; McLellan, Jason S.; Kwong, Peter D.; Graham, Barney S.

    2015-01-01

    Respiratory syncytial virus (RSV) is estimated to claim more lives among infants <1 year old than any other single pathogen, except malaria, and poses a substantial global health burden. Viral entry is mediated by a type I fusion glycoprotein (F) that transitions from a metastable prefusion (pre-F) to a stable postfusion (post-F) trimer. A highly neutralization-sensitive epitope, antigenic site Ø, is found only on pre-F. We determined what fraction of neutralizing (NT) activity in human sera is dependent on antibodies specific for antigenic site Ø or other antigenic sites on F in healthy subjects from ages 7 to 93 years. Adsorption of individual sera with stabilized pre-F protein removed >90% of NT activity and depleted binding antibodies to both F conformations. In contrast, adsorption with post-F removed ~30% of NT activity, and binding antibodies to pre-F were retained. These findings were consistent across all age groups. Protein competition neutralization assays with pre-F mutants in which sites Ø or II were altered to knock out binding of antibodies to the corresponding sites showed that these sites accounted for ~35 and <10% of NT activity, respectively. Binding competition assays with monoclonal antibodies (mAbs) indicated that the amount of site Ø–specific antibodies correlated with NT activity, whereas the magnitude of binding competed by site II mAbs did not correlate with neutralization. Our results indicate that RSV NT activity in human sera is primarily derived from pre-F–specific antibodies, and therefore, inducing or boosting NT activity by vaccination will be facilitated by using pre-F antigens that preserve site Ø. PMID:26468324

  11. Mechanistic Study of Broadly Neutralizing Human Monoclonal Antibodies against Dengue Virus That Target the Fusion Loop

    PubMed Central

    Costin, Joshua M.; Zaitseva, Elena; Kahle, Kristen M.; Nicholson, Cindo O.; Rowe, Dawne K.; Graham, Amanda S.; Bazzone, Lindsey E.; Hogancamp, Greg; Figueroa Sierra, Marielys; Fong, Rachel H.; Yang, Sung-Tae; Lin, Li; Robinson, James E.; Doranz, Benjamin J.; Chernomordik, Leonid V.; Michael, Scott F.; Schieffelin, John S.

    2013-01-01

    There are no available vaccines for dengue, the most important mosquito-transmitted viral disease. Mechanistic studies with anti-dengue virus (DENV) human monoclonal antibodies (hMAbs) provide a rational approach to identify and characterize neutralizing epitopes on DENV structural proteins that can serve to inform vaccine strategies. Here, we report a class of hMAbs that is likely to be an important determinant in the human humoral response to DENV infection. In this study, we identified and characterized three broadly neutralizing anti-DENV hMAbs: 4.8A, D11C, and 1.6D. These antibodies were isolated from three different convalescent patients with distinct histories of DENV infection yet demonstrated remarkable similarities. All three hMAbs recognized the E glycoprotein with high affinity, neutralized all four serotypes of DENV, and mediated antibody-dependent enhancement of infection in Fc receptor-bearing cells at subneutralizing concentrations. The neutralization activities of these hMAbs correlated with a strong inhibition of virus-liposome and intracellular fusion, not virus-cell binding. We mapped epitopes of these antibodies to the highly conserved fusion loop region of E domain II. Mutations at fusion loop residues W101, L107, and/or G109 significantly reduced the binding of the hMAbs to E protein. The results show that hMAbs directed against the highly conserved E protein fusion loop block viral entry downstream of virus-cell binding by inhibiting E protein-mediated fusion. Characterization of hMAbs targeting this region may provide new insights into DENV vaccine and therapeutic strategies. PMID:23077306

  12. Neutralizing Human Fab Fragments against Measles Virus Recovered by Phage Display

    PubMed Central

    de Carvalho Nicacio, Cristina; Williamson, R. Anthony; Parren, Paul W. H. I.; Lundkvist, Åke; Burton, Dennis R.; Björling, Ewa

    2002-01-01

    Five human recombinant Fab fragments (Fabs) specific for measles virus (MV) proteins were isolated from three antibody phage display libraries generated from RNAs derived from bone marrow or splenic lymphocytes from three MV-immune individuals. All Fabs reacted in an enzyme-linked immunosorbent assay with MV antigens. In radioimmunoprecipitation assays two of the Fabs, MV12 and MT14, precipitated an ⊘80-kDa protein band corresponding to the hemagglutinin (H) protein from MV-infected Vero cell cultures, while two other Fabs, MT64 and GL29, precipitated an ⊘60-kDa protein corresponding the nucleocapsid (N) protein. In competition studies with MV fusion, H- and N protein-specific monoclonal antibodies (MAbs), the H-specific Fabs predominantly blocked the binding of H-specific MAbs, while the N-specific Fabs blocked MAbs to N. In addition, N-specific Fabs bound to denatured MV N protein in Western blotting. The specificity of the fifth Fab, MV4, could not be determined. By plaque reduction assays, three of the five Fabs, MV4, MV12, and MT14, exhibited neutralizing activity (80% cutoff) against MV (LEC-KI strain) at concentrations ranging between ≈2 and 7 μg ml−1. Neutralization capacity against MV strains Edmonston and Schwarz was also detected, albeit at somewhat higher Fab concentrations. In conclusion, three neutralizing Fabs were isolated, two of them reactive against the H glycoprotein of MV and another reactive against an undefined epitope. This is the first study in which MV-neutralizing human recombinant Fab antibodies have been isolated from phage display libraries. PMID:11739690

  13. Relationship of the quaternary structure of human secretory IgA to neutralization of influenza virus

    PubMed Central

    Suzuki, Tadaki; Kawaguchi, Akira; Ainai, Akira; Tamura, Shin-ichi; Ito, Ryo; Multihartina, Pretty; Setiawaty, Vivi; Pangesti, Krisna Nur Andriana; Odagiri, Takato; Tashiro, Masato; Hasegawa, Hideki

    2015-01-01

    Secretory IgA (S-IgA) antibodies, the major contributors to humoral mucosal immunity to influenza virus infection, are polymeric Igs present in many external secretions. In the present study, the quaternary structures of human S-IgA induced in nasal mucosa after administration of intranasal inactivated influenza vaccines were characterized in relation to neutralization potency against influenza A viruses. Human nasal IgA antibodies have been shown to contain at least five quaternary structures. Direct and real-time visualization of S-IgA using high-speed atomic force microscopy (AFM) demonstrated that trimeric and tetrameric S-IgA had six and eight antigen-binding sites, respectively, and that these structures exhibited large-scale asynchronous conformational changes while capturing influenza HA antigens in solution. Furthermore, trimeric, tetrameric, and larger polymeric structures, which are minor fractions in human nasal IgA, displayed increased neutralizing potency against influenza A viruses compared with dimeric S-IgA, suggesting that the larger polymeric than dimeric forms of S-IgA play some important roles in protection against influenza A virus infection in the human upper respiratory tract. PMID:26056267

  14. Isolation of a human-like antibody fragment (scFv) that neutralizes ricin biological activity

    PubMed Central

    Pelat, Thibaut; Hust, Michael; Hale, Martha; Lefranc, Marie-Paule; Dübel, Stefan; Thullier, Philippe

    2009-01-01

    Background Ricin is a lethal toxin that inhibits protein synthesis. It is easily extracted from a ubiquitously grown plant, Ricinus communis, and thus readily available for use as a bioweapon (BW). Anti-ricin antibodies provide the only known therapeutic against ricin intoxication. Results In this study, after immunizing a non-human primate (Macaca fascicularis) with the ricin chain A (RTA), a phage-displayed immune library was built (2 × 108 clones), that included the λ light chain fragment. The library was screened against ricin, and specific binders were sequenced and further analyzed. The best clone, 43RCA, was isolated using a new, stringent neutralization test. 43RCA had a high, picomolar affinity (41 pM) and neutralized ricin efficiently (IC50 = 23 ± 3 ng/ml, corresponding to a [scFv]/[ricin] molar ratio of 4). The neutralization capacity of 43RCA compared favourably with that of polyclonal anti-deglycosylated A chain (anti-dgRCA) IgGs, obtained from hyperimmune mouse serum, which were more efficient than any monoclonal at our disposal. The 43RCA sequence is very similar to that for human IgG germline genes, with 162 of 180 identical amino acids for the VH and VL (90% sequence identity). Conclusion Results of the characterization studies, and the high degree of identity with human germline genes, altogether make this anti-ricin scFv, or an IgG derived from it, a likely candidate for use in humans to minimize effects caused by ricin intoxication. PMID:19563687

  15. Epitope Mapping of Neutralizing Monoclonal Antibodies to Human Interferon-γ Using Human-Bovine Interferon-γ Chimeras

    PubMed Central

    Zuber, Bartek; Rudström, Karin; Ehrnfelt, Cecilia

    2016-01-01

    Our aim was to identify conformational epitopes, recognized by monoclonal antibodies (mAbs) made against human (h) interferon (IFN)-γ. Based on the mAbs' (n = 12) ability to simultaneously bind hIFN-γ in ELISA, 2 epitope clusters with 5 mAbs in each were defined; 2 mAbs recognized unique epitopes. Utilizing the mAbs' lack of reactivity with bovine (b) IFN-γ, epitopes were identified using 7 h/bIFN-γ chimeras where the helical regions (A-F) or the C terminus were substituted with bIFN-γ residues. Chimeras had a N-terminal peptide tag enabling the analysis of mAb recognition of chimeras in ELISA. The 2 mAb clusters mapped to region A and E, respectively; the epitopes of several mAbs also involved additional regions. MAbs in cluster A neutralized, to various degrees, IFN-γ-mediated activation of human cells, in line with the involvement of region A in the IFN-γ receptor interaction. MAbs mapping to region E displayed a stronger neutralizing capacity although this region has not been directly implicated in the receptor interaction. The results corroborate earlier studies and provide a detailed picture of the link between the epitope specificity and neutralizing capacity of mAbs. They further demonstrate the general use of peptide-tagged chimeric proteins as a powerful and straightforward method for efficient mapping of conformational epitopes. PMID:27336613

  16. Neutralizing human monoclonal antibodies to conformational epitopes of human T-cell lymphotropic virus type 1 and 2 gp46.

    PubMed Central

    Hadlock, K G; Rowe, J; Perkins, S; Bradshaw, P; Song, G Y; Cheng, C; Yang, J; Gascon, R; Halmos, J; Rehman, S M; McGrath, M S; Foung, S K

    1997-01-01

    Ten human monoclonal antibodies derived from peripheral B cells of a patient with human T-cell lymphotropic virus (HTLV)-associated myelopathy are described. One monoclonal antibody recognized a linear epitope within the carboxy-terminal 43 amino acids of HTLV gp21, and two monoclonal antibodies recognized linear epitopes within HTLV type 1 (HTLV-1) gp46. The remaining seven monoclonal antibodies recognized denaturation-sensitive epitopes within HTLV-1 gp46 that were expressed on the surfaces of infected cells. Two of these antibodies also bound to viable HTLV-2 infected cells and immunoprecipitated HTLV-2 gp46. Virus neutralization was determined by syncytium inhibition assays. Eight monoclonal antibodies, including all seven that recognized denaturation-sensitive epitopes within HTLV-1 gp46, possessed significant virus neutralization activity. By competitive inhibition analysis it was determined that these antibodies recognized at least four distinct conformational epitopes within HTLV-1 gp46. These findings indicate the importance of conformational epitopes within HTLV-1 gp46 in mediating a neutralizing antibody response to HTLV infection. PMID:9223472

  17. Epitope Mapping of Neutralizing Monoclonal Antibodies to Human Interferon-γ Using Human-Bovine Interferon-γ Chimeras.

    PubMed

    Zuber, Bartek; Rudström, Karin; Ehrnfelt, Cecilia; Ahlborg, Niklas

    2016-09-01

    Our aim was to identify conformational epitopes, recognized by monoclonal antibodies (mAbs) made against human (h) interferon (IFN)-γ. Based on the mAbs' (n = 12) ability to simultaneously bind hIFN-γ in ELISA, 2 epitope clusters with 5 mAbs in each were defined; 2 mAbs recognized unique epitopes. Utilizing the mAbs' lack of reactivity with bovine (b) IFN-γ, epitopes were identified using 7 h/bIFN-γ chimeras where the helical regions (A-F) or the C terminus were substituted with bIFN-γ residues. Chimeras had a N-terminal peptide tag enabling the analysis of mAb recognition of chimeras in ELISA. The 2 mAb clusters mapped to region A and E, respectively; the epitopes of several mAbs also involved additional regions. MAbs in cluster A neutralized, to various degrees, IFN-γ-mediated activation of human cells, in line with the involvement of region A in the IFN-γ receptor interaction. MAbs mapping to region E displayed a stronger neutralizing capacity although this region has not been directly implicated in the receptor interaction. The results corroborate earlier studies and provide a detailed picture of the link between the epitope specificity and neutralizing capacity of mAbs. They further demonstrate the general use of peptide-tagged chimeric proteins as a powerful and straightforward method for efficient mapping of conformational epitopes. PMID:27336613

  18. Patterns of proteoglycan degradation by a neutral protease from human growth-plate epiphyseal cartilage

    SciTech Connect

    Ehrlich, M.G.; Armstrong, A.L.; Neuman, R.G.; Davis, M.W.; Mankin, H.J.

    1982-12-01

    The hypothesis is widely held that proteolytic degradation of proteoglycans in the lower hypertrophic zone of the growth plate may be involved in the initiation of mineralization in the zone of provisional calcification. However, a neutral protease that is responsible for the degradation of proteoglycans in the growth plate has not been identified, isolated, and characterized. In the work reported here, neutral protease activity in the growth plate is demonstrated for the first time, and some of the properties of the enzyme are described. Proteoglycans subunits were prepared from bovine nasal cartilage and calf costal cartilage by equilibrium density-gradient centrifugation under dissociative conditions. The proteoglycan subunits were labeled with /sup 14/C-formaldehyde. Homogenates from human growth plates were examined for neutral protease activity using the proteoglycan subunits as substrates. Following incubation of the proteoglycan subunits with growth-plate homogenates at pH 5.3 and at pH 7.5 in the presence and absence of ten-millimolar magnesium chloride and calcium chloride, the digestion products were examined by gel chromatography on Sepharose-2B and 6B columns. Column eluants containing proteoglycan-subunit degradation products were monitored for uronic acid, hexose, and radio-activity. Maximum extensive degradation of proteoglycan subunits occurred at pH 7.5 in the presence of ten-millimolar magnesium chloride and calcium chloride.

  19. Neutralizing antibodies are unable to inhibit direct viral cell-to-cell spread of human cytomegalovirus.

    PubMed

    Jacob, Christian L; Lamorte, Louie; Sepulveda, Eliud; Lorenz, Ivo C; Gauthier, Annick; Franti, Michael

    2013-09-01

    Infection with human cytomegalovirus (CMV) during pregnancy is the most common cause of congenital disorders, and can lead to severe life-long disabilities with associated high cost of care. Since there is no vaccine or effective treatment, current efforts are focused on identifying potent neutralizing antibodies. A panel of CMV monoclonal antibodies identified from patent applications, was synthesized and expressed in order to reproduce data from the literature showing that anti-glycoprotein B antibodies neutralized virus entry into all cell types and that anti-pentameric complex antibodies are highly potent in preventing virus entry into epithelial cells. It had not been established whether antibodies could prevent subsequent rounds of infection that are mediated primarily by direct cell-to-cell transmission. A thorough validation of a plaque reduction assay to monitor cell-to-cell spread led to the conclusion that neutralizing antibodies do not significantly inhibit plaque formation or reduce plaque size when they are added post-infection. PMID:23849792

  20. Human Antibody Neutralizes Severe Fever with Thrombocytopenia Syndrome Virus, an Emerging Hemorrhagic Fever Virus

    PubMed Central

    Guo, Xiling; Zhang, Li; Zhang, Wenshuai; Chi, Ying; Zeng, Xiaoyan; Li, Xian; Qi, Xian; Jin, Qiu; Zhang, Xiao; Huang, Mingming; Wang, Hua; Chen, Yin; Bao, Changjun; Hu, Jianli; Liang, Shuyi; Bao, Lin; Wu, Tao

    2013-01-01

    Severe fever with thrombocytopenia syndrome virus (SFTSV), a newly discovered member of the Bunyaviridae family, is the causative agent of an emerging hemorrhagic fever, SFTS, in China. Currently, there are no vaccines or effective therapies against SFTS. In this study, a combinatorial human antibody library was constructed from the peripheral lymphocytes of 5 patients who had recovered from SFTS. The library was screened against purified virions for the production of single-chain variable-region fragments (ScFv). Of the 6 positive clones, one clone (monoclonal antibody [MAb] 4-5) showed neutralizing activity against SFTSV infection in Vero cells. MAb 4-5 was found to effectively neutralize all of the clinical isolates of SFTSV tested, which were isolated from patients in China from 2010 to 2012. MAb 4-5 was found to bind a linear epitope in the ectodomain of glycoprotein Gn. Its neutralizing activity is attributed to blockage of the interactions between the Gn protein and the cellular receptor, indicating that inhibition of virus-cell attachment is its main mechanism. These data suggest that MAb 4-5 can be used as a promising candidate molecule for immunotherapy against SFTSV infection. PMID:23863504

  1. Broad neutralizing human monoclonal antibodies against influenza virus from vaccinated healthy donors

    SciTech Connect

    Kubota-Koketsu, Ritsuko; Mizuta, Hiroyuki; Oshita, Masatoshi; Ideno, Shoji; Yunoki, Mikihiro; Kuhara, Motoki; Yamamoto, Naomasa; Okuno, Yoshinobu; Ikuta, Kazuyoshi

    2009-09-11

    Human monoclonal antibodies (HuMAbs) prepared from patients with viral infections could provide information on human epitopes important for the development of vaccines as well as potential therapeutic applications. Through the fusion of peripheral blood mononuclear cells from a total of five influenza-vaccinated volunteers, with newly developed murine-human chimera fusion partner cells, named SPYMEG, we obtained 10 hybridoma clones stably producing anti-influenza virus antibodies: one for influenza A H1N1, four for influenza A H3N2 and five for influenza B. Surprisingly, most of the HuMAbs showed broad reactivity within subtype and four (two for H3N2 and two for B) showed broad neutralizing ability. Importantly, epitope mapping revealed that the two broad neutralizing antibodies to H3N2 derived from different donors recognized the same epitope located underneath the receptor-binding site of the hemagglutinin globular region that is highly conserved among H3N2 strains.

  2. Invariant natural killer T-cell neutralization is a possible novel therapy for human eosinophilic esophagitis

    PubMed Central

    Rayapudi, Madhavi; Rajavelu, Priya; Zhu, Xiang; Kaul, Ajay; Niranjan, Rituraj; Dynda, Scott; Mishra, Akanksha; Mattner, Jochen; Zaidi, Asifa; Dutt, Parmesh; Mishra, Anil

    2014-01-01

    Eosinophilic esophagitis (EoE) is a recently recognized inflammatory disorder that needs a potential therapeutic strategy. We earlier showed that iNKT cell-deficient mice are protected from allergen-induced EoE. Therefore, we now tested the hypothesis that iNKT cells are induced in the human EoE and is a novel possible target for the treatment of human EoE. Accordingly, we examine number of iNKT cells and eosinophils and expression of iNKT-associated cell surface receptors and chemokines by performing immunofluorescence, qPCR and ELISA in the esophageal biopsies and blood samples of normal subjects (comparison control) and EoE patients. Herein, we show that iNKT cell number, their receptor subcomponents Vα24 and Vβ11 expression, and associated chemokine CXCL16 levels (or expression) are induced significantly in EoE patients compared with normal individuals. In addition, we show that CXCL16 levels (or expression) correlate with the mRNA levels of Vα24 receptor but not well with esophageal eosinophilia in human EoE. Of note, we show that in vivo activation of iNKT cells is sufficient to induce EoE in mice. Furthermore, we show that anti-mCD1d- and anti-hVα24Jα18-neutralizing antibody treatment protects allergen-induced experimental EoE. Taken together, we have shown first time that iNKT cells have a critical pathogenic role in human and experimental EoE. iNKT cell neutralization by humanized anti-CD1d and anti-Vα24Jα18 antibodies might be a novel and potential therapy for human EoE. PMID:25505954

  3. Development of Germline-Humanized Antibodies Neutralizing Botulinum Neurotoxin A and B.

    PubMed

    Miethe, Sebastian; Mazuet, Christelle; Liu, Yvonne; Tierney, Robert; Rasetti-Escargueil, Christine; Avril, Arnaud; Frenzel, André; Thullier, Philippe; Pelat, Thibaut; Urbain, Remi; Fontayne, Alexandre; Sesardic, Dorothea; Hust, Michael; Popoff, Michel Robert

    2016-01-01

    Botulinum neurotoxins (BoNTs) are counted among the most toxic substances known and are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. To date, 7 serologically distinct serotypes of BoNT (serotype A-G) are known. Due to the high toxicity of BoNTs the Centers for Disease Control and Prevention (CDC) have classified BoNTs as category A agent, including the six biological agents with the highest potential risk of use as bioweapons. Well tolerated antibodies neutralizing BoNTs are required to deal with the potential risk. In a previous work, we described the development of scFv and scFv-Fc (Yumab) from macaque origin (Macaca fascicularis) neutralizing BoNT/A and B by targeting the heavy and light chain of each serotype. In the present study, we humanized the macaque antibodies SEM120-IIIC1 (anti-BoNT/A light chain), A1HC38 (anti-BoNT/A heavy chain), BLC3 (anti-BoNT/B light chain) and B2-7 (anti-BoNT/B heavy chain) by germline-humanization to obtain a better potential immunotolerance in humans. We increased the Germinality Index (GI) of SEM120-IIIC1 to 94.5%, for A1HC38, to 95% for BLC3 and to 94.4% for B2-7. Furthermore, the neutralization efficacies of the germline-humanized antibodies were analyzed in lethal and non-lethal in vivo mouse assays as full IgG. The germline-humanized IgGs hu8SEM120-IIIC1, hu8A1HC38, hu8BLC3 and hu8B2-7 were protective in vivo, when anti-heavy and anti-light chain antibodies were combined. The synergistic effect and high humanness of the selected IgGs makes them promising lead candidates for further clinical development. PMID:27560688

  4. Development of Germline-Humanized Antibodies Neutralizing Botulinum Neurotoxin A and B

    PubMed Central

    Liu, Yvonne; Tierney, Robert; Rasetti-Escargueil, Christine; Avril, Arnaud; Frenzel, André; Thullier, Philippe; Pelat, Thibaut; Urbain, Remi; Fontayne, Alexandre; Sesardic, Dorothea; Hust, Michael; Popoff, Michel Robert

    2016-01-01

    Botulinum neurotoxins (BoNTs) are counted among the most toxic substances known and are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. To date, 7 serologically distinct serotypes of BoNT (serotype A-G) are known. Due to the high toxicity of BoNTs the Centers for Disease Control and Prevention (CDC) have classified BoNTs as category A agent, including the six biological agents with the highest potential risk of use as bioweapons. Well tolerated antibodies neutralizing BoNTs are required to deal with the potential risk. In a previous work, we described the development of scFv and scFv-Fc (Yumab) from macaque origin (Macaca fascicularis) neutralizing BoNT/A and B by targeting the heavy and light chain of each serotype. In the present study, we humanized the macaque antibodies SEM120-IIIC1 (anti-BoNT/A light chain), A1HC38 (anti-BoNT/A heavy chain), BLC3 (anti-BoNT/B light chain) and B2-7 (anti-BoNT/B heavy chain) by germline-humanization to obtain a better potential immunotolerance in humans. We increased the Germinality Index (GI) of SEM120-IIIC1 to 94.5%, for A1HC38, to 95% for BLC3 and to 94.4% for B2-7. Furthermore, the neutralization efficacies of the germline-humanized antibodies were analyzed in lethal and non-lethal in vivo mouse assays as full IgG. The germline-humanized IgGs hu8SEM120-IIIC1, hu8A1HC38, hu8BLC3 and hu8B2-7 were protective in vivo, when anti-heavy and anti-light chain antibodies were combined. The synergistic effect and high humanness of the selected IgGs makes them promising lead candidates for further clinical development. PMID:27560688

  5. Neutralization of Japanese Encephalitis Virus by heme-induced broadly reactive human monoclonal antibody

    PubMed Central

    Gupta, Nimesh; de Wispelaere, Mélissanne; Lecerf, Maxime; Kalia, Manjula; Scheel, Tobias; Vrati, Sudhanshu; Berek, Claudia; Kaveri, Srinivas V.; Desprès, Philippe; Lacroix-Desmazes, Sébastien; Dimitrov, Jordan D.

    2015-01-01

    Geographical expansion and re-emerging new genotypes of the Japanese encephalitis virus (JEV) require the development of novel therapeutic approaches. Here, we studied a non-conventional approach for antibody therapy and show that, upon exposure to heme, a fraction of natural human immunoglobulins acquires high-affinity reactivity with the antigenic domain-III of JEV E glycoprotein. These JEV-reactive antibodies exhibited neutralizing activity against recently dominant JEV genotypes. This study opens new therapeutic options for Japanese encephalitis. PMID:26542535

  6. Immunologic analysis of anaphylaxis to protamine component in neutral protamine Hagedorn human insulin.

    PubMed

    Dykewicz, M S; Kim, H W; Orfan, N; Yoo, T J; Lieberman, P

    1994-01-01

    We report the clinical and immunologic analysis of two patients with diabetes who had anaphylaxis to neutral protamine Hagedorn (NPH) human insulin in the absence of allergy to regular insulin. A 36-year-old woman without a recent history of local insulin reactions or interruption of insulin therapy experienced anaphylaxis within 15 minutes of her usual morning dose of subcutaneously administered NPH human insulin. A 62-year-old man with a history of generalized reactions to NPH human insulin and of anaphylaxis to intravenously administered protamine had generalized urticaria after injection of NPH human insulin. Both patients subsequently tolerated Lente human insulin. Skin test results in both patients were negative to regular and Lente insulin preparations but positive to NPH insulin and to protamine at concentrations tested. In vitro assays demonstrated that both patients had markedly elevated serum levels of IgE and IgG to protamine, but not to regular human insulin, and that their IgE antibodies to protamine recognized protamine antigenic determinants in NPH human insulin. We conclude that the anaphylactic reactions to NPH insulin in our patients were mediated by IgE to protamine, which should be a pathogenetic consideration in the evaluation of immediate-type reactions to protamine-containing insulins. PMID:8308177

  7. A Human Monoclonal Antibody with Neutralizing Activity against Highly Divergent Influenza Subtypes

    PubMed Central

    Solforosi, Laura; Moreno, Guisella J.; Gubareva, Larisa V.; Mishin, Vasiliy; Di Pietro, Andrea; Vicenzi, Elisa; Siccardi, Antonio G.; Clementi, Massimo; Burioni, Roberto

    2011-01-01

    The interest in broad-range anti-influenza A monoclonal antibodies (mAbs) has recently been strengthened by the identification of anti-hemagglutinin (HA) mAbs endowed with heterosubtypic neutralizing activity to be used in the design of “universal” prophylactic or therapeutic tools. However, the majority of the single mAbs described to date do not bind and neutralize viral isolates belonging to highly divergent subtypes clustering into the two different HA-based influenza phylogenetic groups: the group 1 including, among others, subtypes H1, H2, H5 and H9 and the group 2 including, among others, H3 subtype. Here, we describe a human mAb, named PN-SIA28, capable of binding and neutralizing all tested isolates belonging to phylogenetic group 1, including H1N1, H2N2, H5N1 and H9N2 subtypes and several isolates belonging to group 2, including H3N2 isolates from the first period of the 1968 pandemic. Therefore, PN-SIA28 is capable of neutralizing isolates belonging to subtypes responsible of all the reported pandemics, as well as other subtypes with pandemic potential. The region recognized by PN-SIA28 has been identified on the stem region of HA and includes residues highly conserved among the different influenza subtypes. A deep characterization of PN-SIA28 features may represent a useful help in the improvement of available anti-influenza therapeutic strategies and can provide new tools for the development of universal vaccinal strategies. PMID:22162996

  8. Neutralization breadth and potency of serum derived from recently human immunodeficiency virus type 1-infected Thai individuals.

    PubMed

    Chaitaveep, Nithinart; Utachee, Piraporn; Chuenchitra, Thippawan; Karasavvan, Nicos; Takeda, Naokazu; Kameoka, Masanori

    2016-05-01

    Neutralizing antibody responses play important roles in controlling several viral infections including human immunodeficiency virus type 1 (HIV-1). Potent and broad neutralizing antibody responses have been reported in some HIV-1-infected individuals; therefore, elucidating the mechanisms underlying neutralizing antibody responses will provide important information for the development of anti-HIV-1 vaccines. We herein performed a comparative study on the neutralization breadth and potency of serum samples collected from Thai individuals recently and chronically infected with HIV-1. Neutralization tests using a series of envelope glycoproteins (Env)-recombinant viruses revealed that although several serum samples derived from recently infected individuals did not show any HIV-1-specific neutralizing activity, the remaining serum samples exhibited neutralizing activity not only for recombinant viruses with CRF01_AE Env, but also for viruses with subtypes B and C Env. Furthermore, some serum samples derived from recently infected individuals showed the neutralization potency. Our results may provide a deeper insight into the characteristics of neutralizing antibody responses that develop during the course of HIV-1 infection among individuals in Thailand. PMID:26774333

  9. Germline V-genes sculpt the binding site of a family of antibodies neutralizing human cytomegalovirus

    SciTech Connect

    Thomson, Christy A.; Bryson, Steve; McLean, Gary R.; Creagh, A. Louise; Pai, Emil F.; Schrader, John W.

    2008-10-17

    Immunoglobulin genes are generated somatically through specialized mechanisms resulting in a vast repertoire of antigen-binding sites. Despite the stochastic nature of these processes, the V-genes that encode most of the antigen-combining site are under positive evolutionary selection, raising the possibility that V-genes have been selected to encode key structural features of binding sites of protective antibodies against certain pathogens. Human, neutralizing antibodies to human cytomegalovirus that bind the AD-2S1 epitope on its gB envelope protein repeatedly use a pair of well-conserved, germline V-genes IGHV3-30 and IGKV3-11. Here, we present crystallographic, kinetic and thermodynamic analyses of the binding site of such an antibody and that of its primary immunoglobulin ancestor. These show that these germline V-genes encode key side chain contacts with the viral antigen and thereby dictate key structural features of the hypermutated, high-affinity neutralizing antibody. V-genes may thus encode an innate, protective immunological memory that targets vulnerable, invariant sites on multiple pathogens.

  10. Prediction of optimal peptide mixtures to induce broadly neutralizing antibodies to human immunodeficiency virus type 1.

    PubMed Central

    Holley, L H; Goudsmit, J; Karplus, M

    1991-01-01

    Sequences of the principal neutralizing determinant (PND) of the external envelope protein, gp120, from 245 isolates of human immunodeficiency virus type 1 are analyzed. The minimal set of peptides that would elicit antibodies to neutralize a majority of U.S. and European isolates of human immunodeficiency virus type 1 is determined with the assumption that peptides of a given length including the central Gly-Pro-Gly triad are required. In spite of the hypervariability of the PND, 90% of these 245 sequences include peptides from a set of 7 pentapeptides, 13 hexapeptides, or 17 heptapeptides. Tests of these peptide sets on 78 additional PND sequences show that 95% are covered by the 7 pentapeptides, 94% by the 13 hexapeptides, and 86% by the 17 heptapeptides. To anticipate variants not yet observed, single amino acid mutation frequencies from the 245 isolates are used to calculate an expanded set of the 10,000 most probable PND sequences. These sequences cover 86% of the total distribution expected for the central portion of the PND. Peptide lists derived from this expanded set when tested on the 78 additional sequences show that 7 pentapeptides cover 95%, 13 hexapeptides cover 94%, and 17 heptapeptides cover 94%. These results suggest that peptide cocktails of limited size with the potential to cover a large fraction of PND sequence variation may be feasible vaccine candidates. PMID:1862103

  11. Structural Insights into the Neutralization Properties of the Fully Human, Anti-interferon Monoclonal Antibody Sifalimumab.

    PubMed

    Oganesyan, Vaheh; Peng, Li; Woods, Robert M; Wu, Herren; Dall'Acqua, William F

    2015-06-12

    We report the three-dimensional structure of human interferon α-2A (IFN-α2A) bound to the Fab fragment of a therapeutic monoclonal antibody (sifalimumab; IgG1/κ). The structure of the corresponding complex was solved at a resolution of 3.0 Å using molecular replacement and constitutes the first reported structure of a human type I IFN bound to a therapeutic antibody. This study revealed the major contribution made by the first complementarity-determining region in each of sifalimumab light and heavy chains. These data also provided the molecular basis for sifalimumab mechanism of action. We propose that its interferon-neutralizing properties are the result of direct competition for IFN-α2A binding to the IFN receptor subunit 1 (IFNAR1) and do not involve inhibiting IFN-α2A binding to the IFN receptor subunit 2 (IFNAR2). PMID:25925951

  12. Comprehensive Cross-Clade Neutralization Analysis of a Panel of Anti-Human Immunodeficiency Virus Type 1 Monoclonal Antibodies

    PubMed Central

    Binley, James M.; Wrin, Terri; Korber, Bette; Zwick, Michael B.; Wang, Meng; Chappey, Colombe; Stiegler, Gabriela; Kunert, Renate; Zolla-Pazner, Susan; Katinger, Hermann; Petropoulos, Christos J.; Burton, Dennis R.

    2004-01-01

    Broadly neutralizing monoclonal antibodies (MAbs) are potentially important tools in human immunodeficiency virus type 1 (HIV-1) vaccine design. A few rare MAbs have been intensively studied, but we still have a limited appreciation of their neutralization breadth. Using a pseudovirus assay, we evaluated MAbs from clade B-infected donors and a clade B HIV+ plasma against 93 viruses from diverse backgrounds. Anti-gp120 MAbs exhibited greater activity against clade B than non-B viruses, whereas anti-gp41 MAbs exhibited broad interclade activity. Unexpectedly, MAb 4E10 (directed against the C terminus of the gp41 ectodomain) neutralized all 90 viruses with moderate potency. MAb 2F5 (directed against an epitope adjacent to that of 4E10) neutralized 67% of isolates, but none from clade C. Anti-gp120 MAb b12 (directed against an epitope overlapping the CD4 binding site) neutralized 50% of viruses, including some from almost every clade. 2G12 (directed against a high-mannose epitope on gp120) neutralized 41% of the viruses, but none from clades C or E. MAbs to the gp120 V3 loop, including 447-52D, neutralized a subset of clade B viruses (up to 45%) but infrequently neutralized other clades (≤7%). MAbs b6 (directed against the CD4 binding site) and X5 (directed against a CD4-induced epitope of gp120) neutralized only sensitive primary clade B viruses. The HIV+ plasma neutralized 70% of the viruses, including some from all major clades. Further analysis revealed five neutralizing immunotypes that were somewhat associated with clades. As well as the significance for vaccine design, our data have implications for passive-immunization studies in countries where clade C viruses are common, given that only MAbs b12 and 4E10 were effective against viruses from this clade. PMID:15542675

  13. Development of human-like scFv-Fc antibodies neutralizing Botulinum toxin serotype B

    PubMed Central

    Rasetti-Escargueil, Christine; Avril, Arnaud; Chahboun, Siham; Tierney, Rob; Bak, Nicola; Miethe, Sebastian; Mazuet, Christelle; Popoff, Michel R; Thullier, Philippe; Hust, Michael; Pelat, Thibaut; Sesardic, Dorothea

    2015-01-01

    Botulinum neurotoxins (BoNTs) are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agents by the Centers for Disease Control and Prevention. To date, 7 subtypes of BoNT/B were identified showing that subtypes B1 (16 strains) and B2 (32 strains) constitute the vast majority of BoNT/B strains. Neutralizing antibodies are required for the development of anti-botulism drugs to deal with the potential risk. In this study, macaques (Macaca fascicularis) were immunized with recombinant light chain (LC) or heavy chain (HC) of BoNT/B2, followed by the construction of 2 hyper-immune phage display libraries. The best single-chain variable fragments (scFvs) isolated from each library were selected according to their affinities and cross reactivity with BoNT/B1 toxin subtype. These scFvs against LC and HC were further analyzed by assessing the inhibition of in vitro endopeptidase activity of BoNT/B1 and B2 and neutralization of BoNT/B1 and B2 toxin-induced paralysis in the mouse ex vivo phrenic nerve assay. The antibodies B2–7 (against HC) and BLC3 (against LC) were produced as scFv-Fc, and, when tested individually, neutralized BoNT/B1 and BoNT/B2 in a mouse ex vivo phrenic nerve assay. Whereas only scFv-Fc BLC3 alone protected mice against BoNT/B2-induced paralysis in vivo, when B2–7 and BLC3 were combined they exhibited potent synergistic protection. The present study provided an opportunity to assess the extent of antibody-mediated neutralization of BoNT/B1 and BoNT/B2 subtypes in ex vivo and in vitro assays, and to confirm the benefit of the synergistic effect of antibodies targeting the 2 distinct functional domains of the toxin in vivo. Notably, the framework regions of the most promising antibodies (B2–7 and BLC3) are close to the human germline sequences

  14. Development of human-like scFv-Fc antibodies neutralizing Botulinum toxin serotype B.

    PubMed

    Rasetti-Escargueil, Christine; Avril, Arnaud; Chahboun, Siham; Tierney, Rob; Bak, Nicola; Miethe, Sebastian; Mazuet, Christelle; Popoff, Michel R; Thullier, Philippe; Hust, Michael; Pelat, Thibaut; Sesardic, Dorothea

    2015-01-01

    Botulinum neurotoxins (BoNTs) are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agents by the Centers for Disease Control and Prevention. To date, 7 subtypes of BoNT/B were identified showing that subtypes B1 (16 strains) and B2 (32 strains) constitute the vast majority of BoNT/B strains. Neutralizing antibodies are required for the development of anti-botulism drugs to deal with the potential risk. In this study, macaques (Macaca fascicularis) were immunized with recombinant light chain (LC) or heavy chain (HC) of BoNT/B2, followed by the construction of 2 hyper-immune phage display libraries. The best single-chain variable fragments (scFvs) isolated from each library were selected according to their affinities and cross reactivity with BoNT/B1 toxin subtype. These scFvs against LC and HC were further analyzed by assessing the inhibition of in vitro endopeptidase activity of BoNT/B1 and B2 and neutralization of BoNT/B1 and B2 toxin-induced paralysis in the mouse ex vivo phrenic nerve assay. The antibodies B2-7 (against HC) and BLC3 (against LC) were produced as scFv-Fc, and, when tested individually, neutralized BoNT/B1 and BoNT/B2 in a mouse ex vivo phrenic nerve assay. Whereas only scFv-Fc BLC3 alone protected mice against BoNT/B2-induced paralysis in vivo, when B2-7 and BLC3 were combined they exhibited potent synergistic protection. The present study provided an opportunity to assess the extent of antibody-mediated neutralization of BoNT/B1 and BoNT/B2 subtypes in ex vivo and in vitro assays, and to confirm the benefit of the synergistic effect of antibodies targeting the 2 distinct functional domains of the toxin in vivo. Notably, the framework regions of the most promising antibodies (B2-7 and BLC3) are close to the human germline sequences, which

  15. Targeting (cellular) lysosomal acid ceramidase by B13: Design, synthesis and evaluation of novel DMG-B13 ester prodrugs

    PubMed Central

    Bai, Aiping; Szulc, Zdzislaw, M.; Bielawski, Jacek; Pierce, Jason S.; Rembisa, Barbara; Terzieva, Silva; Mao, Cungui; Xu, Ruijuan; Wu, Bill; Clarke, Christopher J.; Newcomb, Benjamin; Liu, Xiang; Norris, James; Hannun, Yusuf A.; Bielawska, Alicja

    2015-01-01

    Acid ceramidase (ACDase) is being recognized as a therapeutic target for cancer. B13 represents a moderate inhibitor of ACDase. The present study concentrates on the lysosomal targeting of B13 via its N, N-dimethylglycine (DMG) esters (DMG-B13 prodrugs). Novel analogs, the isomeric mono-DMG-B13, LCL522 (3-O-DMG-B13•HCl) and LCL596 (1-O-DMG-B13•HCl) and di-DMG-B13, LCL521 (1,3-O, O-DMG-B13•2HCl) conjugates, were designed and synthesized through N, N-dimethyl glycine (DMG) esterification of the hydroxyl groups of B13. In MCF7 cells, DMG-B13 prodrugs were efficiently metabolized to B13. The early inhibitory effect of DMG-B13 prodrugs on cellular ceramidases was ACDase specific by their lysosomal targeting. The corresponding dramatic decrease of cellular Sph (80-97% Control/1h) by DMG-B13 prodrugs was mainly from the inhibition of the lysosomal ACDase. PMID:25456083

  16. Cleavage of fibrinogen by the human neutrophil neutral peptide-generating protease.

    PubMed Central

    Wintroub, B U; Coblyn, J S; Kaempfer, C E; Austen, K F

    1980-01-01

    The human neutrophil peptide-generating protease, which generates a low molecular weight vasoactive peptide from a plasma protein substrate, is directly fibrinolytic and cleaves human fibrinogen in a manner distinct from plasmin. Fibrinogen was reduced from 340,000 Mr to derivatives of 270,000-325,000 Mr during interaction with the protease at enzyme-to-substrate ratios of 0.3 or 1.0 microgram/1.0 mg. The 310,000-325,000 Mr cleavage fragments exhibited prolonged thrombin-induced clotting activity but were able to be coagulated, whereas the 270,000-290,000 Mr fragments were not able to be coagulated. Anticoagulants were not generated at either enzyme dose. As analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis in 4-30% gradient gels and 10% gels stained for protein and carbohydrate, the diminution to 310,000-325,000 Mr and the prolongation of thrombin-induced clotting time resulted from cleavage of the fibrinogen A alpha chain. The further decrease in size to 270,000-290,000 Mr was associated with B beta-chain and gamma-chain cleavage and an inability to form gamma-gamma dimers. The neutral peptide-generating protease, a distinct human neutrophil neutral protease with fibrinolytic and fibrinogenolytic activities comparable to those of plasmin on a weight basis, cleaves fibrinogen in a manner that is distinct from the action of plasmin, leukocyte elastase, and leukocyte granule extracts. It may be that the concerted action of this neutrophil protease to generate a vasoactive peptide and to digest fibrinogen and fibrin facilitates neutrophil movement through vascular and extravascular sites. Images PMID:7001479

  17. Reconstituted high-density lipoprotein neutralizes gram-negative bacterial lipopolysaccharides in human whole blood.

    PubMed Central

    Parker, T S; Levine, D M; Chang, J C; Laxer, J; Coffin, C C; Rubin, A L

    1995-01-01

    We have tested hypotheses relating lipoprotein structure to function as measured by the relative ability to neutralize endotoxin by comparing natural human lipoproteins, a chemically defined form of reconstituted high-density lipoprotein (R-HDL), and a lipid emulsion (Intralipid). The human whole-blood system was used as an in vitro model of lipopolysaccharide (LPS) binding protein and CD14-dependent activation of cytokine production. When lipoproteins were compared on the basis of protein content, R-HDL was most effective in reducing tumor necrosis factor alpha (TNF-alpha) production followed in order by very low density lipoprotein, low-density lipoprotein, Intralipid, and natural HDL. However, when these particles were compared by protein, phospholipid, cholesterol, or triglyceride content by stepwise linear regression analysis, only phospholipid was correlated to effectiveness (r2 = 0.873; P < 0.0001). Anti-CD14 monoclonal antibodies MY4 and 3C10 inhibited LPS binding protein and CD14-dependent activation of TNF-alpha production by LPS at LPS concentrations up to approximately 1.0 ng/ml. R-HDL (2 mg of protein per ml) blocked TNF-alpha production by LPS from both smooth- and rough-type gram-negative bacteria at concentrations up to 100 ng of LPS per ml but had little effect on heat-killed gram-positive Staphylococcus aureus and no effect on other LPS-independent stimuli tested. These results support our hypothesis that LPS is neutralized by binding to phospholipid on the surface of R-HDL and demonstrate that R-HDL is a potent inhibitor of the induction of TNF-alpha by LPS from both rough- and smooth-form gram-negative bacteria in whole human blood. PMID:7528733

  18. Neutralizing human anti crotoxin scFv isolated from a nonimmunized phage library.

    PubMed

    Cardoso, D F; Nato, F; England, P; Ferreira, M L; Vaughan, T J; Mota, I; Mazie, J C; Choumet, V; Lafaye, P

    2000-04-01

    Combinatorial phage display technology offers a new possibility for making human antibodies which could be used in immune therapy. We explored the use of this technology to make human scFvs specific for crotoxin, the main toxic component of the venom of the South-American rattlesnake Crotalus durissus terrificus. Crotoxin, a phospholipase A2 neurotoxin constituted by the association of two subunits, exerts its lethal action by blocking neuromuscular transmission. This is the first report of human anticrotoxin scFvs (scFv 1, scFv 6 and scFv 8) isolated from a naive library of more than 1010 scFv clones with in vivo neutralizing activity. Nevertheless, differences are observed at the level of biological and immunological effects. Only scFv 8 is able to reduce the myotoxicity induced by crotoxin and scFv 1 is capable of altering the in vitro enzymatic activity of this toxin. All three scFvs recognize a region of one subunit located at the junction with the other one. Moreover these scFvs share strong amino acid homologies at the level of either the heavy or the light chain. Taken together, our results suggest that the use of human anticrotoxin scFvs may lead to a new and less aggressive passive immune therapy against poisoning by the venom of Crotalus durissus terrificus. PMID:10736105

  19. A novel human anti-interleukin-1β neutralizing monoclonal antibody showing in vivo efficacy

    PubMed Central

    Goh, Angeline XH; Bertin-Maghit, Sebastien; Ping Yeo, Siok; Ho, Adrian; Derks, Heidi; Mortellaro, Alessandra; Wang, Cheng-I

    2014-01-01

    The pro-inflammatory cytokine interleukin (IL)-1β is a clinical target in many conditions involving dysregulation of the immune system; therapeutics that block IL-1β have been approved to treat diseases such as rheumatoid arthritis (RA), neonatal onset multisystem inflammatory diseases, cryopyrin-associated periodic syndromes, active systemic juvenile idiopathic arthritis. Here, we report the generation and engineering of a new fully human antibody that binds tightly to IL-1β with a neutralization potency more than 10 times higher than that of the marketed antibody canakinumab. After affinity maturation, the derived antibody shows a >30-fold increased affinity to human IL-1β compared with its parent antibody. This anti-human IL-1β IgG also cross-reacts with mouse and monkey IL-1β, hence facilitating preclinical development. In a number of mouse models, this antibody efficiently reduced or abolished signs of disease associated with IL-1β pathology. Due to its high affinity for the cytokine and its potency both in vitro and in vivo, we propose that this novel fully human anti-IL-1β monoclonal antibody is a promising therapeutic candidate and a potential alternative to the current therapeutic arsenal. PMID:24671001

  20. Increased BLSS closure using mineralized human waste in plant cultivation on a neutral substrate

    NASA Astrophysics Data System (ADS)

    Ushakova, S.; Tikhomirov, A.; Shikhov, V.; Kudenko, Yu.; Anischenko, O.; Gros, J.-B.; Lasseur, Ch.

    2009-10-01

    The purpose of this work was to study the full-scale potential use of human mineralized waste (feces and urine) as a source of mineral elements for plant cultivation in a biological life support system (BLSS). Plants that are potential candidates for a photosynthesizing link were grown on a neutral solution containing human mineralized waste. Spring wheat Triticum aestivum L., peas Pisum sativum L. Ambrosia cultivar and leaf lettuce Lactuca sativa L., Vitaminny variety, were used. The plants were grown hydroponically on expanded clay aggregates in a vegetation chamber in constant environmental conditions. During plant growth, a determined amount of human mineralized waste was added daily to the nutrient solution. The nutrient solution remained unchanged throughout the vegetation period. Estimated plant requirements for macro-elements were based on a total biological productivity of 0.04 kg day -1 m -2. As the plant requirements for potassium exceeded the potassium content of human waste, a water extract of wheat straw containing the required amount of potassium was added to the nutrient solution. The Knop's solution was used in the control experiments. The experimental and control plants showed no significant differences in state or productivity of their photosynthetic apparatus. A small decrease in total productivity of the experimental plants was observed, which might result in some reduction of О 2 production in a BLSS.

  1. Segmenting the human genome based on states of neutral genetic divergence.

    PubMed

    Kuruppumullage Don, Prabhani; Ananda, Guruprasad; Chiaromonte, Francesca; Makova, Kateryna D

    2013-09-01

    Many studies have demonstrated that divergence levels generated by different mutation types vary and covary across the human genome. To improve our still-incomplete understanding of the mechanistic basis of this phenomenon, we analyze several mutation types simultaneously, anchoring their variation to specific regions of the genome. Using hidden Markov models on insertion, deletion, nucleotide substitution, and microsatellite divergence estimates inferred from human-orangutan alignments of neutrally evolving genomic sequences, we segment the human genome into regions corresponding to different divergence states--each uniquely characterized by specific combinations of divergence levels. We then parsed the mutagenic contributions of various biochemical processes associating divergence states with a broad range of genomic landscape features. We find that high divergence states inhabit guanine- and cytosine (GC)-rich, highly recombining subtelomeric regions; low divergence states cover inner parts of autosomes; chromosome X forms its own state with lowest divergence; and a state of elevated microsatellite mutability is interspersed across the genome. These general trends are mirrored in human diversity data from the 1000 Genomes Project, and departures from them highlight the evolutionary history of primate chromosomes. We also find that genes and noncoding functional marks [annotations from the Encyclopedia of DNA Elements (ENCODE)] are concentrated in high divergence states. Our results provide a powerful tool for biomedical data analysis: segmentations can be used to screen personal genome variants--including those associated with cancer and other diseases--and to improve computational predictions of noncoding functional elements. PMID:23959903

  2. Insight into Neutral and Disease-Associated Human Genetic Variants through Interpretable Predictors

    PubMed Central

    van den Berg, Bastiaan A.; Reinders, Marcel J. T.; de Ridder, Dick; de Beer, Tjaart A. P.

    2015-01-01

    A variety of methods that predict human nonsynonymous single nucleotide polymorphisms (SNPs) to be neutral or disease-associated have been developed over the last decade. These methods are used for pinpointing disease-associated variants in the many variants obtained with next-generation sequencing technologies. The high performances of current sequence-based predictors indicate that sequence data contains valuable information about a variant being neutral or disease-associated. However, most predictors do not readily disclose this information, and so it remains unclear what sequence properties are most important. Here, we show how we can obtain insight into sequence characteristics of variants and their surroundings by interpreting predictors. We used an extensive range of features derived from the variant itself, its surrounding sequence, sequence conservation, and sequence annotation, and employed linear support vector machine classifiers to enable extracting feature importance from trained predictors. Our approach is useful for providing additional information about what features are most important for the predictions made. Furthermore, for large sets of known variants, it can provide insight into the mechanisms responsible for variants being disease-associated. PMID:25826299

  3. Cross-Reactive and Potent Neutralizing Antibody Responses in Human Survivors of Natural Ebolavirus Infection.

    PubMed

    Flyak, Andrew I; Shen, Xiaoli; Murin, Charles D; Turner, Hannah L; David, Joshua A; Fusco, Marnie L; Lampley, Rebecca; Kose, Nurgun; Ilinykh, Philipp A; Kuzmina, Natalia; Branchizio, Andre; King, Hannah; Brown, Leland; Bryan, Christopher; Davidson, Edgar; Doranz, Benjamin J; Slaughter, James C; Sapparapu, Gopal; Klages, Curtis; Ksiazek, Thomas G; Saphire, Erica Ollmann; Ward, Andrew B; Bukreyev, Alexander; Crowe, James E

    2016-01-28

    Recent studies have suggested that antibody-mediated protection against the Ebolaviruses may be achievable, but little is known about whether or not antibodies can confer cross-reactive protection against viruses belonging to diverse Ebolavirus species, such as Ebola virus (EBOV), Sudan virus (SUDV), and Bundibugyo virus (BDBV). We isolated a large panel of human monoclonal antibodies (mAbs) against BDBV glycoprotein (GP) using peripheral blood B cells from survivors of the 2007 BDBV outbreak in Uganda. We determined that a large proportion of mAbs with potent neutralizing activity against BDBV bind to the glycan cap and recognize diverse epitopes within this major antigenic site. We identified several glycan cap-specific mAbs that neutralized multiple ebolaviruses, including SUDV, and a cross-reactive mAb that completely protected guinea pigs from the lethal challenge with heterologous EBOV. Our results provide a roadmap to develop a single antibody-based treatment effective against multiple Ebolavirus infections. PMID:26806128

  4. Dense nanoparticles exhibit enhanced vascular wall targeting over neutrally buoyant nanoparticles in human blood flow.

    PubMed

    Thompson, Alex J; Eniola-Adefeso, Omolola

    2015-07-01

    For vascular-targeting carrier (VTC) systems to be effective, carriers must be able to localize and adhere to the vascular wall at the target site. Research suggests that neutrally buoyant nanoparticles are limited by their inability to localize to the endothelium, making them sub-optimal as carriers. This study examines whether particle density can be exploited to improve the targeting (localization and adhesion) efficiency of nanospheres to the vasculature. Silica spheres with 500 nm diameter, which have a density roughly twice that of blood, exhibit improved adhesion to inflamed endothelium in an in vitro model of human vasculature compared to neutrally buoyant polystyrene spheres of the same size. Silica spheres also display better near-wall localization in the presence of red blood cells than they do in pure buffer, likely resulting in the observed improvement in adhesion. Titania spheres (4 times more dense than blood) adhere at levels higher than polystyrene, but only in conditions when gravity or centrifugal force acts in the direction of adhesion. In light of the wide array of materials proposed for use as carrier systems for drug delivery and diagnostics, particle density may be a useful tool for improving the targeting of diseased tissues. PMID:25870170

  5. Insight into neutral and disease-associated human genetic variants through interpretable predictors.

    PubMed

    van den Berg, Bastiaan A; Reinders, Marcel J T; de Ridder, Dick; de Beer, Tjaart A P

    2015-01-01

    A variety of methods that predict human nonsynonymous single nucleotide polymorphisms (SNPs) to be neutral or disease-associated have been developed over the last decade. These methods are used for pinpointing disease-associated variants in the many variants obtained with next-generation sequencing technologies. The high performances of current sequence-based predictors indicate that sequence data contains valuable information about a variant being neutral or disease-associated. However, most predictors do not readily disclose this information, and so it remains unclear what sequence properties are most important. Here, we show how we can obtain insight into sequence characteristics of variants and their surroundings by interpreting predictors. We used an extensive range of features derived from the variant itself, its surrounding sequence, sequence conservation, and sequence annotation, and employed linear support vector machine classifiers to enable extracting feature importance from trained predictors. Our approach is useful for providing additional information about what features are most important for the predictions made. Furthermore, for large sets of known variants, it can provide insight into the mechanisms responsible for variants being disease-associated. PMID:25826299

  6. Neutralization of Botulinum Neurotoxin by a Human Monoclonal Antibody Specific for the Catalytic Light Chain

    PubMed Central

    Adekar, Sharad P.; Takahashi, Tsuyoshi; Jones, R. Mark; Al-Saleem, Fetweh H.; Ancharski, Denise M.; Root, Michael J.; Kapadnis, B. P.; Simpson, Lance L.; Dessain, Scott K.

    2008-01-01

    Background Botulinum neurotoxins (BoNT) are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC), a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored. Methods and Findings We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A). The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro. Conclusions An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components of an antibody antidote for BoNT exposure. PMID:18714390

  7. Development of neutralizing monoclonal antibodies for oncogenic human papillomavirus types 31, 33, 45, 52, and 58.

    PubMed

    Brown, Martha J; Seitz, Hanna; Towne, Victoria; Müller, Martin; Finnefrock, Adam C

    2014-04-01

    Human papillomavirus (HPV) is the etiological agent for all cervical cancers, a significant number of other anogenital cancers, and a growing number of head and neck cancers. Two licensed vaccines offer protection against the most prevalent oncogenic types, 16 and 18, responsible for approximately 70% of cervical cancer cases worldwide and one of these also offers protection against types 6 and 11, responsible for 90% of genital warts. The vaccines are comprised of recombinantly expressed major capsid proteins that self-assemble into virus-like particles (VLPs) and prevent infection by eliciting neutralizing antibodies. Adding the other frequently identified oncogenic types 31, 33, 45, 52, and 58 to a vaccine would increase the coverage against HPV-induced cancers to approximately 90%. We describe the generation and characterization of panels of monoclonal antibodies to these five additional oncogenic HPV types, and the selection of antibody pairs that were high affinity and type specific and recognized conformation-dependent neutralizing epitopes. Such characteristics make these antibodies useful tools for monitoring the production and potency of a prototype vaccine as well as monitoring vaccine-induced immune responses in the clinic. PMID:24574536

  8. Global Geometric Morphometric Analyses of the Human Pelvis Reveal Substantial Neutral Population History Effects, Even across Sexes

    PubMed Central

    Betti, Lia; von Cramon-Taubadel, Noreen; Manica, Andrea; Lycett, Stephen J.

    2013-01-01

    Recent applications of population genetic models to human craniodental traits have revealed a strong neutral component to patterns of global variation. However, little work has been undertaken to determine whether neutral processes might also be influencing the postcranium, perhaps due to substantial evidence for selection and plastic environmental responses in these regions. Recent work has provided evidence for neutral effects in the pelvis, but has been limited in regard to shape data (small numbers of linear measurements) and restricted only to males. Here, we use geometric morphometric methods to examine population variation in the human os coxae (pelvic bone) in both males and females. Neutrality is examined via apportionment of variance patterns and fit to an Out-of-Africa serial founder effect model, which is known to structure neutral genetic patterns. Moreover, we compare males and females directly, and the true versus false pelvis, in order to examine potential obstetrical effects. Our results indicate evidence for substantial neutral population history effects on pelvic shape variation. They also reveal evidence for the effect of obstetrical constraints, but these affect males and females to equivalent extents. Our results do not deny an important role for selection in regard to specific aspects of human pelvic variation, especially in terms of features associated with body size and proportions. However, our analyses demonstrate that at a global level, the shape of the os coxae reveals substantial evidence for neutral variation. Our analyses thus indicate that population variation in the human pelvis might be used to address important questions concerning population history, just as the human cranium has done. PMID:23409086

  9. Selection and characterization of a human neutralizing antibody to human fibroblast growth factor-2

    SciTech Connect

    Tao, Jun; Xiang, Jun-Jian; Li, Dan; Deng, Ning; Wang, Hong; Gong, Yi-Ping

    2010-04-09

    Compelling evidences suggest that fibroblast growth factor-2 (FGF-2) plays important roles in tumor growth, angiogenesis and metastasis. Molecules blocking the FGF-2 signaling have been proposed as anticancer agents. Through screening of a human scFv phage display library, we have isolated several human single-chain Fv fragments (scFvs) that bind to human FGF-2. After expression and purification in bacteria, one scFv, named 1A2, binds to FGF-2 with a high affinity and specificity, and completes with FGF-2 binding to its receptor. This 1A2 scFv was then cloned into the pIgG1 vector and expressed in 293T cells. The purified hIgG1-1A2 antibody showed a high binding affinity of 8 x 10{sup -9} M to rhFGF-2. In a set of vitro assays, it inhibited various biological activities of FGF-2 such as the proliferation, migration and tube formation of human umbilical vein endothelial cells. More importantly, hIgG1-1A2 antibody also efficiently blocked the growth while inducing apoptosis of glioma cells. For the first time, we generated a human anti-FGF-2 antibody with proven in vitro anti-tumor activity. It may therefore present a new therapeutic candidate for the treatment of cancers that are dependent on FGF-2 signaling for growth and survival.

  10. Increase of a BLSS closure using mineralized human waste in plant cultivation on a neutral substrate

    NASA Astrophysics Data System (ADS)

    Gros, Jean-Bernard; Ushakova, Sofya; Tikhomirov, Alexander A.; Kudenko, Yurii; Lasseur, Christophe; Shikhov, V.; Anischenko, O.

    The purpose of this work was to study the full-scale potential use of human mineralized waste (feces and urine) as a source of mineral elements for plants cultivation in a Biological Life Support System. The plants which are potential candidates for a photosynthesizing link were grown on a neutral solution containing human mineralized waste. Spring wheat Triticum aestivum L., peas Pisum sativum L. Ambrosia cultivar and leaf lettuce Lactuca sativa L., Vitamin variety, were taken as the investigation objects. The plants were grown by hydroponics method on expanded clay aggregates in a vegetation chamber in constant environmental conditions. During the plants growth a definite amount of human mineralized waste was added daily in the nutrient solution. The nutrient solution was not changed during the entire vegetation period. Estimation of the plant needs in macro elements was based on a total biological productivity equal to 0.04 kg.day--1 .m-2 . As the plant requirements in potassium exceeded the potassium content in human waste, water extract of wheat straw containing the required potassium amount was added to the nutrient solution. Knop's solution was used in the control experiments. The experiment and control plants did not show significant differences in their photosynthetic apparatus state and productivity. A small decrease in total productivity of the experimental plants was observed which can result in some reduction of ˆ2 production in a BLSS. Most I probably it is due to the reduced nitrogen use. Therefore in a real BLSS after the mineralization of human feces and urine, it will be efficient to implement a more complete oxidation of nitrogencontaining compounds system, including nitrification. In this case the plants, prospective representatives of the BLSS photosynthesizing unit, could be cultivated on the solutions mainly based on human mineralized waste.

  11. Structural and molecular basis for Ebola virus neutralization by protective human antibodies.

    PubMed

    Misasi, John; Gilman, Morgan S A; Kanekiyo, Masaru; Gui, Miao; Cagigi, Alberto; Mulangu, Sabue; Corti, Davide; Ledgerwood, Julie E; Lanzavecchia, Antonio; Cunningham, James; Muyembe-Tamfun, Jean Jacques; Baxa, Ulrich; Graham, Barney S; Xiang, Ye; Sullivan, Nancy J; McLellan, Jason S

    2016-03-18

    Ebola virus causes hemorrhagic fever with a high case fatality rate for which there is no approved therapy. Two human monoclonal antibodies, mAb100 and mAb114, in combination, protect nonhuman primates against all signs of Ebola virus disease, including viremia. Here, we demonstrate that mAb100 recognizes the base of the Ebola virus glycoprotein (GP) trimer, occludes access to the cathepsin-cleavage loop, and prevents the proteolytic cleavage of GP that is required for virus entry. We show that mAb114 interacts with the glycan cap and inner chalice of GP, remains associated after proteolytic removal of the glycan cap, and inhibits binding of cleaved GP to its receptor. These results define the basis of neutralization for two protective antibodies and may facilitate development of therapies and vaccines. PMID:26917592

  12. De Novo Sequencing and Resurrection of a Human Astrovirus-Neutralizing Antibody

    PubMed Central

    2016-01-01

    Monoclonal antibody (mAb) therapeutics targeting cancer, autoimmune diseases, inflammatory diseases, and infectious diseases are growing exponentially. Although numerous panels of mAbs targeting infectious disease agents have been developed, their progression into clinically useful mAbs is often hindered by the lack of sequence information and/or loss of hybridoma cells that produce them. Here we combine the power of crystallography and mass spectrometry to determine the amino acid sequence and glycosylation modification of the Fab fragment of a potent human astrovirus-neutralizing mAb. We used this information to engineer a recombinant antibody single-chain variable fragment that has the same specificity as the parent monoclonal antibody to bind to the astrovirus capsid protein. This antibody can now potentially be developed as a therapeutic and diagnostic agent. PMID:27213181

  13. Departure from neutrality at the mitochondrial NADH dehydrogenase subunit 2 gene in humans, but not in chimpanzees.

    PubMed

    Wise, C A; Sraml, M; Easteal, S

    1998-01-01

    To test whether patterns of mitochondrial DNA (mtDNA) variation are consistent with a neutral model of molecular evolution, nucleotide sequences were determined for the 1041 bp of the NADH dehydrogenase subunit 2 (ND2) gene in 20 geographically diverse humans and 20 common chimpanzees. Contingency tests of neutrality were performed using four mutational categories for the ND2 molecule: synonymous and nonsynonymous mutations in the transmembrane regions, and synonymous and nonsynonymous mutations in the surface regions. The following three topological mutational categories were also used: intraspecific tips, intraspecific interiors, and interspecific fixed differences. The analyses reveal a significantly greater number of nonsynonymous polymorphisms within human transmembrane regions than expected based on interspecific comparisons, and they are inconsistent with a neutral equilibrium model. This pattern of excess nonsynonymous polymorphism is not seen within chimpanzees. Statistical tests of neutrality, such as TAJIMA's D test, and the D and F tests proposed by FU and LI, indicate an excess of low frequency polymorphisms in the human data, but not in the chimpanzee data. This is consistent with recent directional selection, a population bottleneck or background selection of slightly deleterious mutations in human mtDNA samples. The analyses further support the idea that mitochondrial genome evolution is governed by selective forces that have the potential to affect its use as a "neutral" marker in evolutionary and population genetic studies. PMID:9475751

  14. Human monoclonal antibodies targeting the haemagglutinin glycoprotein can neutralize H7N9 influenza virus.

    PubMed

    Chen, Zhe; Wang, Jianmin; Bao, Linlin; Guo, Li; Zhang, Weijia; Xue, Ying; Zhou, Hongli; Xiao, Yan; Wang, Jianwei; Wu, Fan; Deng, Ying; Qin, Chuan; Jin, Qi

    2015-01-01

    The recently identified avian-originated influenza H7N9 virus causes severe pulmonary disease and may lead to death in humans. Currently, treatment options for the prevention and control of fatal H7N9 infections in humans remain limited. Here we characterize two human monoclonal antibodies (HuMAbs), HNIgGA6 and HNIgGB5, by screening a Fab antibody phage library derived from patients who recovered from H7N9 infection. Both antibodies exhibit high neutralizing activity against H7N9 virus in cells. Two amino acids in the receptor-binding site, 186V and 226L, are crucial for the binding of these two HuMAbs to viral haemagglutinin antigens. Prophylaxis with HNIgGA6 and HNIgGB5 confers significant immunity against H7N9 virus in a mouse model and significantly reduces the pulmonary virus titre. When administered post infection, therapeutic doses of the HuMAbs also provide robust protection against lethality. These antibodies might represent a potential alternative or adjunct to H7N9 pandemic interventions. PMID:25819694

  15. 3BNC117 a Broadly Neutralizing Antibody Suppresses Viremia in HIV-1-Infected Humans

    PubMed Central

    Caskey, Marina; Klein, Florian; Lorenzi, Julio C. C.; Seaman, Michael S.; West, Anthony P.; Buckley, Noreen; Kremer, Gisela; Nogueira, Lilian; Braunschweig, Malte; Scheid, Johannes F.; Horwitz, Joshua A.; Shimeliovich, Irina; Ben Avraham-Shulman, Sivan; Witmer-Pack, Maggi; Platten, Martin; Lehmann, Clara; Burke, Leah A.; Hawthorne, Thomas; Gorelick, Robert J.; Walker, Bruce D.; Keler, Tibor; Gulick, Roy M.; Fätkenheuer, Gerd; Schlesinger, Sarah J.; Nussenzweig, Michel C.

    2016-01-01

    HIV-1 immunotherapy with a combination of first generation monoclonal antibodies was largely ineffective in pre-clinical and clinical settings and was therefore abandoned1–3. However, recently developed single cell based antibody cloning methods have uncovered a new generation of far more potent broadly neutralizing antibodies (bNAbs) to HIV-14,5. These antibodies can prevent infection and suppress viremia in humanized mice (hu-mice) and nonhuman primates, but their potential for human HIV-1 immunotherapy has not been evaluated6–10. Here we report the results of a first-in-man dose escalation phase 1 clinical trial of 3BNC117, a potent human CD4 binding site antibody11, in uninfected and HIV-1-infected individuals. 3BNC117 infusion was well tolerated and demonstrated favorable pharmacokinetics. A single 30 mg/kg infusion of 3BNC117 reduced the viral load (VL) in HIV-1-infected individuals by 0.8 – 2.5 log10 and viremia remained significantly reduced for 28 days. Emergence of resistant viral strains was variable, with some individuals remaining sensitive to 3BNC117 for a period of 28 days. We conclude that as a single agent 3BNC117 is safe and effective in reducing HIV-1 viremia, and that immunotherapy should be explored as a new modality for HIV-1 prevention, therapy, and cure. PMID:25855300

  16. Efficacy of broadly neutralizing monoclonal antibody PG16 in HIV-infected humanized mice.

    PubMed

    Stoddart, Cheryl A; Galkina, Sofiya A; Joshi, Pheroze; Kosikova, Galina; Long, Brian R; Maidji, Ekaterina; Moreno, Mary E; Rivera, Jose M; Sanford, Ukina R; Sloan, Barbara; Cieplak, Witold; Wrin, Terri; Chan-Hui, Po-Ying

    2014-08-01

    Highly potent broadly neutralizing human monoclonal antibodies hold promise for HIV prophylaxis and treatment. We used the SCID-hu Thy/Liv and BLT humanized mouse models to study the efficacy of these antibodies, primarily PG16, against HIV-1 clades A, B, and C. PG16 targets a conserved epitope in the V1/V2 region of gp120 common to 70-80% of HIV-1 isolates from multiple clades and has extremely potent in vitro activity against HIVJR-CSF. PG16 was highly efficacious in SCID-hu mice as a single intraperitoneal administration the day before inoculation of R5-tropic HIV directly into their Thy/Liv implants and demonstrated even greater efficacy if PG16 administration was continued after Thy/Liv implant HIV inoculation. However, PG16 as monotherapy had no activity in humanized mice with established R5-tropic HIV infection. These results provide evidence of tissue penetration of the antibodies, which could aid in their ability to prevent infection if virus crosses the mucosal barrier. PMID:24971704

  17. Efficacy of Broadly Neutralizing Monoclonal Antibody PG16 in HIV-infected Humanized Mice

    PubMed Central

    Stoddart, Cheryl A.; Galkina, Sofiya A.; Joshi, Pheroze; Kosikova, Galina; Long, Brian R.; Maidji, Ekaterina; Moreno, Mary E.; Rivera, Jose M.; Sanford, Ukina R.; Sloan, Barbara; Cieplak, Witold; Wrin, Terri; Chan-Hui, Po-Ying

    2014-01-01

    Highly potent broadly neutralizing human monoclonal antibodies hold promise for HIV prophylaxis and treatment. We used the SCID-hu Thy/Liv and BLT humanized mouse models to study the efficacy of these antibodies, primarily PG16, against HIV-1 clade A, B, and C. PG16 targets a conserved epitope in the V1/V2 region of gp120 common to 70–80% of HIV-1 isolates from multiple clades and has extremely potent in vitro activity against HIVJR-CSF. PG16 was highly efficacious in SCID-hu mice as a single intraperitoneal administration the day before inoculation of R5-tropic HIV-1 directly into their Thy/Liv implants and demonstrated even greater efficacy if PG16 administration was continued after Thy/Liv implant HIV-1 infection. However, PG16 as monotherapy had no activity in humanized mice with established R5-tropic HIV-1 infection. These results provide evidence of tissue penetration of the antibodies, which could aid in their ability to prevent infection if virus crosses the mucosal barrier. PMID:24971704

  18. Mutations in the neutral sphingomyelinase gene SMPD3 implicate the ceramide pathway in human leukemias.

    PubMed

    Kim, Woo Jae; Okimoto, Ross A; Purton, Louise E; Goodwin, Meagan; Haserlat, Sara M; Dayyani, Farshid; Sweetser, David A; McClatchey, Andrea I; Bernard, Olivier A; Look, A Thomas; Bell, Daphne W; Scadden, David T; Haber, Daniel A

    2008-05-01

    Ceramide is a lipid second messenger derived from the hydrolysis of sphingomyelin by sphingomyelinases (SMases) and implicated in diverse cellular responses, including growth arrest, differentiation, and apoptosis. Defects in the neutral SMase (nSMase) gene Smpd3, the primary regulator of ceramide biosynthesis, are responsible for developmental defects of bone; regulation of ceramide levels have been implicated in macrophage differentiation, but this pathway has not been directly implicated in human cancer. In a genomic screen for gene copy losses contributing to tumorigenesis in a mouse osteosarcoma model, we identified a somatic homozygous deletion specifically targeting Smpd3. Reconstitution of SMPD3 expression in mouse tumor cells lacking the endogenous gene enhanced tumor necrosis factor (TNF)-induced reduction of cell viability. Nucleotide sequencing of the highly conserved SMPD3 gene in a large panel of human cancers revealed mutations in 5 (5%) of 92 acute myeloid leukemias (AMLs) and 8 (6%) of 131 acute lymphoid leukemias (ALLs), but not in other tumor types. In a subset of these mutations, functional analysis indicated defects in protein stability and localization. Taken together, these observations suggest that disruption of the ceramide pathway may contribute to a subset of human leukemias. PMID:18299447

  19. Mutations in the neutral sphingomyelinase gene SMPD3 implicate the ceramide pathway in human leukemias

    PubMed Central

    Kim, Woo Jae; Okimoto, Ross A.; Purton, Louise E.; Goodwin, Meagan; Haserlat, Sara M.; Dayyani, Farshid; Sweetser, David A.; McClatchey, Andrea I.; Bernard, Olivier A.; Look, A. Thomas; Bell, Daphne W.; Scadden, David T.

    2008-01-01

    Ceramide is a lipid second messenger derived from the hydrolysis of sphingomyelin by sphingomyelinases (SMases) and implicated in diverse cellular responses, including growth arrest, differentiation, and apoptosis. Defects in the neutral SMase (nSMase) gene Smpd3, the primary regulator of ceramide biosynthesis, are responsible for developmental defects of bone; regulation of ceramide levels have been implicated in macrophage differentiation, but this pathway has not been directly implicated in human cancer. In a genomic screen for gene copy losses contributing to tumorigenesis in a mouse osteosarcoma model, we identified a somatic homozygous deletion specifically targeting Smpd3. Reconstitution of SMPD3 expression in mouse tumor cells lacking the endogenous gene enhanced tumor necrosis factor (TNF)–induced reduction of cell viability. Nucleotide sequencing of the highly conserved SMPD3 gene in a large panel of human cancers revealed mutations in 5 (5%) of 92 acute myeloid leukemias (AMLs) and 8 (6%) of 131 acute lymphoid leukemias (ALLs), but not in other tumor types. In a subset of these mutations, functional analysis indicated defects in protein stability and localization. Taken together, these observations suggest that disruption of the ceramide pathway may contribute to a subset of human leukemias. PMID:18299447

  20. Stochastic homeostasis in human airway epithelium is achieved by neutral competition of basal cell progenitors

    PubMed Central

    Teixeira, Vitor H; Nadarajan, Parthiban; Graham, Trevor A; Pipinikas, Christodoulos P; Brown, James M; Falzon, Mary; Nye, Emma; Poulsom, Richard; Lawrence, David; Wright, Nicholas A; McDonald, Stuart; Giangreco, Adam; Simons, Benjamin D; Janes, Sam M

    2013-01-01

    Lineage tracing approaches have provided new insights into the cellular mechanisms that support tissue homeostasis in mice. However, the relevance of these discoveries to human epithelial homeostasis and its alterations in disease is unknown. By developing a novel quantitative approach for the analysis of somatic mitochondrial mutations that are accumulated over time, we demonstrate that the human upper airway epithelium is maintained by an equipotent basal progenitor cell population, in which the chance loss of cells due to lineage commitment is perfectly compensated by the duplication of neighbours, leading to “neutral drift” of the clone population. Further, we show that this process is accelerated in the airways of smokers, leading to intensified clonal consolidation and providing a background for tumorigenesis. This study provides a benchmark to show how somatic mutations provide quantitative information on homeostatic growth in human tissues, and a platform to explore factors leading to dysregulation and disease. DOI: http://dx.doi.org/10.7554/eLife.00966.001 PMID:24151545

  1. Autologous antibody response against the principal neutralizing domain of human immunodeficiency virus type 1 isolated from infected humans.

    PubMed Central

    Holmbäck, K; Kusk, P; Hulgaard, E F; Bugge, T H; Scheibel, E; Lindhardt, B O

    1993-01-01

    High titers of neutralizing antibodies in human immunodeficiency virus type 1 (HIV-1) infection are directed primarily against the third hypervariable domain (V3) of the virion envelope glycoprotein gp120. This region has been designated the principal neutralizing domain of HIV-1. Because the frequency and significance of autologous V3 antibodies in natural infection are not fully clarified, we have cloned, sequenced, and expressed the V3 domain from virus of HIV-1-infected patients to test the autologous and heterologous V3 antibody response. The resulting recombinant Escherichia coli V3 fusion proteins reacted strongly with both autologous and heterologous patient antibodies in Western blots. Thirty-one different V3 fragments were cloned from 24 hemophiliac patients with different immunological and clinical statuses. Antibody reactivity against the autologous V3 fusion proteins was detected in all serum samples except one; moreover, all serum samples contained antibody reactivity against a vast majority of heterologous fusion proteins despite significant amino acid variability in V3. The results suggest that V3 antibodies are highly prevalent; further, we find no association between the stage of the HIV-1 infection and the presence of V3 antibodies. Images PMID:8437232

  2. G glycoprotein amino acid residues required for human monoclonal antibody RAB1 neutralization are conserved in rabies virus street isolates.

    PubMed

    Wang, Yang; Rowley, Kirk J; Booth, Brian J; Sloan, Susan E; Ambrosino, Donna M; Babcock, Gregory J

    2011-08-01

    Replacement of polyclonal anti-rabies immunoglobulin (RIG) used in rabies post-exposure prophylaxis (PEP) with a monoclonal antibody will eliminate cost and availability constraints that currently exist using RIG in the developing world. The human monoclonal antibody RAB1 has been shown to neutralize all rabies street isolates tested; however for the laboratory-adapted fixed strain, CVS-11, mutation in the G glycoprotein of amino acid 336 from asparagine (N) to aspartic acid (D) resulted in resistance to neutralization. Interestingly, this same mutation in the G glycoprotein of a second laboratory-adapted fixed strain (ERA) did not confer resistance to RAB1 neutralization. Using cell surface staining and lentivirus pseudotyped with rabies virus G glycoprotein (RABVpp), we identified an amino acid alteration in CVS-11 (K346), not present in ERA (R346), which was required in combination with D336 to confer resistance to RAB1. A complete analysis of G glycoprotein sequences from GenBank demonstrated that no identified rabies isolates contain the necessary combination of G glycoprotein mutations for resistance to RAB1 neutralization, consistent with the broad neutralization of RAB1 observed in direct viral neutralization experiments with street isolates. All combinations of amino acids 336 and 346 reported in the sequence database were engineered into the ERA G glycoprotein and RAB1 was able to neutralize RABVpp bearing ERA G glycoprotein containing all known combinations at these critical residues. These data demonstrate that RAB1 has the capacity to neutralize all identified rabies isolates and a minimum of two distinct mutations in the G glycoprotein are required for abrogation of RAB1 neutralization. PMID:21693135

  3. Neutralization of Human Serum β-Lysin by Sodium Polyanetholsulfonate and Sodium Amylosulfate

    PubMed Central

    Traub, Walter H.; Ima, Paula I. Fukushi

    1979-01-01

    Normal fresh and heat-inactivated (56°C, 30 min) human sera (80 vol%, i.e., 80% [vol/vol] of a 2-ml assay volume) killed Bacillus subtilis ATCC 6633 cell inocula of 1.5 × 104 colony-forming units per ml within 1 to 2 h after exposure. The B. subtilis assay strain proved slightly and reversibly susceptible to 5 μg of egg white lysozyme per ml. Seitz filtration of fresh human serum completely removed β-lysin activity; significant amounts of serum lysozyme were removed as well, as determined with the bioassay strain Micrococcus lysodeikticus ATCC 4698. However, bactericidal activity of human serum via classical or alternative complement pathway activation remained intact. Addition of 0.01 M dithiothreitol to fresh human serum abolished β-lysin activity, but not that of serum lysozyme. Chelation of fresh and heat-inactivated human serum with 0.01 M MgCl2 + 0.01 M ethylene glycol tetraacetic acid, but not with 0.01 M ethylenediaminetetraacetic acid, markedly retarded β-lysin activity; however, lysozyme activity remained unaffected. Chelation of serum with 0.01 M MgCl2 + 0.01 M ethylene glycol tetraacetic acid + 0.01 M CaCl2 completely abrogated β-lysin activity, but not that of lysozyme. Absorption of human serum with 10 mg of bentonite per ml (10 min, 37°C) completely removed β-lysin and lysozyme activity, but failed to affect serum bactericidal activity against Escherichia coli control strain C. Reconstitution of 50 vol% of bentonite-absorbed serum with 40 vol% of heat-inactivated human serum restored both β-lysin and lysozyme activity. Addition of either 63 to 500 μg of sodium polyanetholsulfonate per ml or 63 to 500 μg of sodium amylosulfate per ml to 80 vol% of fresh human serum completely neutralized β-lysin activity for the entire observation period of 22 h. PMID:227918

  4. Generation and characterization of ixekizumab, a humanized monoclonal antibody that neutralizes interleukin-17A

    PubMed Central

    Liu, Ling; Lu, Jirong; Allan, Barrett W; Tang, Ying; Tetreault, Jonathan; Chow, Chi-kin; Barmettler, Barbra; Nelson, James; Bina, Holly; Huang, Lihua; Wroblewski, Victor J; Kikly, Kristine

    2016-01-01

    Interleukin (IL)-17A exists as a homodimer (A/A) or as a heterodimer (A/F) with IL-17F. IL-17A is expressed by a subset of T-cells, called Th17 cells, at inflammatory sites. Most cell types can respond to the local production of IL-17A because of the near ubiquitous expression of IL-17A receptors, IL-17RA and IL-17RC. IL-17A stimulates the release of cytokines and chemokines designed to recruit and activate both neutrophils and memory T-cells to the site of injury or inflammation and maintain a proinflammatory state. IL-17A-producing pathogenic T-cells contribute to the pathogenesis of autoimmune diseases, including psoriasis, psoriatic arthritis, rheumatoid arthritis, and ankylosing spondylitis. This study describes the generation and characterization of ixekizumab, a humanized IgG4 variant IL-17A-neutralizing antibody. Ixekizumab binds human and cynomolgus monkey IL-17A with high affinity and binds rabbit IL-17A weakly but does not bind to rodent IL-17A or other IL-17 family members. Ixekizumab effectively inhibits the interaction between IL-17A and its receptor in binding assays and potently blocks IL-17A-induced GRO or KC secretion in cell-based assays. In an in vivo mouse pharmcodynamic model, ixekizumab blocks human IL-17A-induced mouse KC secretion. These data provide a comprehensive preclinical characterization of ixekizumab, for which the efficacy and safety have been demonstrated in human clinical trials in psoriasis and psoriatic arthritis. PMID:27143947

  5. Quantification of neutral human milk oligosaccharides by graphitic carbon HPLC with tandem mass spectrometry

    PubMed Central

    Bao, Yuanwu; Chen, Ceng; Newburg, David S.

    2012-01-01

    Defining the biologic roles of human milk oligosaccharides (HMOS) requires an efficient, simple, reliable, and robust analytical method for simultaneous quantification of oligosaccharide profiles from multiple samples. The HMOS fraction of milk is a complex mixture of polar, highly branched, isomeric structures that contain no intrinsic facile chromophore, making their resolution and quantification challenging. A liquid chromatography-mass spectrometry (LC-MS) method was devised to resolve and quantify 11 major neutral oligosaccharides of human milk simultaneously. Crude HMOS fractions are reduced, resolved by porous graphitic carbon HPLC with a water/acetonitrile gradient, detected by mass spectrometric specific ion monitoring, and quantified. The HPLC separates isomers of identical molecular weights allowing 11 peaks to be fully resolved and quantified by monitoring mass to charge (m/z) ratios of the deprotonated negative ions. The standard curves for each of the 11 oligosaccharides is linear from 0.078 or 0.156 to 20 μg/mL (R2 > 0.998). Precision (CV) ranges from 1% to 9%. Accuracy is from 86% to 104%. This analytical technique provides sensitive, precise, accurate quantification for each of the 11 milk oligosaccharides and allows measurement of differences in milk oligosaccharide patterns between individuals and at different stages of lactation. PMID:23068043

  6. Are we ignoring neutral and negative human-animal relationships in zoos?

    PubMed

    Hosey, Geoff; Melfi, Vicky

    2015-01-01

    Human-animal interactions (HAI), which may lead to human-animal relationships (HAR), may be positive, neutral, or negative in nature. Zoo studies show that visitors may be stressful, may have no effect, or may be enriching. There is also evidence that good HARs set up between animals and their keepers can have positive effects on animal welfare. However, we need to know more about negative HARs, and as a first step we attempt to do this here by considering cases where animals attack people in the zoo. Due to the sensitivity and rarity of these events data appear sparse and unsystematically collected. Here, information available in the public domain about the circumstances of these attacks has been collated to test hypotheses about negative HAIs derived from a model of zoo HARs. The limited data presented here broadly support the zoo HAR model, and suggest that attacks usually happen in unusual circumstances, where there may be a failure by the animal to recognise the HAR, or where the relationship, if there is one, does not hold; and give some support to the prediction that exposure to many keepers may impair the development of a positive HAR. This study may provide useful information for the zoo community to proactively collect systematic standardised records, which will enable a fuller understanding of zoo HARs, upon which appropriate measures might be adopted to build better zoo HARs, which are likely to positively impact zoo animal welfare, and reduce these rare incidences further. PMID:25328013

  7. Broadly Neutralizing Antibodies and Viral inducers decrease rebound from HIV-1 latent reservoirs in humanized mice

    PubMed Central

    Halper-Stromberg, Ariel; Lu, Ching-Lan; Klein, Florian; Horwitz, Joshua A.; Bournazos, Stylianos; Nogueira, Lillian; Eisenreich, Thomas R.; Liu, Cassie; Gazumyan, Anna; Schaefer, Uwe; Furze, Rebecca C.; Seaman, Michael S.; Prinjha, Rab; Tarakhovsky, Alexander; Ravetch, Jeffrey V.; Nussenzweig, Michel C.

    2014-01-01

    Summary Latent reservoirs of HIV-1 infected cells are refractory to antiretroviral therapies (ART) and remain the major barrier to curing HIV-1. Because latently infected cells are long-lived, immunologically invisible, and may undergo homeostatic proliferation, a “shock and kill” approach has been proposed to eradicate this reservoir by combining ART with inducers of viral transcription. However, all attempts to alter the HIV-1 reservoir in vivo have failed to date. Using humanized mice, we show that broadly neutralizing antibodies (bNAbs) can interfere with establishment of a silent reservoir by Fc-FcR mediated mechanisms. In established infection, bNAbs or bNAbs plus single inducers are ineffective in preventing viral rebound. However, bNAbs plus a combination of inducers that act by independent mechanisms synergize to decrease the reservoir as measured by viral rebound. Thus, combinations of inducers and bNAbs constitute a therapeutic strategy that impacts the establishment and maintenance of the HIV-1 reservoir in humanized mice. PMID:25131989

  8. Development of human neutralizing antibody to ADAMTS4 (aggrecanase-1) and ADAMTS5 (aggrecanase-2).

    PubMed

    Shiraishi, Aya; Mochizuki, Satsuki; Miyakoshi, Akira; Kojoh, Kanehisa; Okada, Yasunori

    2016-01-01

    ADAMTS4 (aggrecanase-1) and ADAMTS5 (aggrecanase-2), members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family, are considered to play a key role in aggrecan degradation of articular cartilage in human osteoarthritis. Here, we developed a neutralizing antibody to these aggrecanases by screening human combinatorial antibody library. Among the five candidate antibodies, one antibody was immunoreactive with both ADAMTS4 and ADAMTS5, showing no or negligible cross-reactivity with 10 different related metalloproteinases of the ADAMTS, ADAM (a disintegrin and metalloproteinase) and MMP (matrix metalloproteinase) gene families. This antibody almost completely and partially inhibited aggrecanase activity of ADAMTS4 and ADAMTS5, respectively. It also suppressed the aggrecanase activity derived from interleukin-1-stimulated osteoarthritic chondrocytes. These data demonstrate that the antibody is specific to ADAMTS4 and ADAMTS5 and inhibits their aggrecanase activity at molecular and cellular levels, and suggest that this antibody may be useful for treatment of pathological conditions such as osteoarthritis. PMID:26612259

  9. Early short-term treatment with neutralizing human monoclonal antibodies halts SHIV infection in infant macaques.

    PubMed

    Hessell, Ann J; Jaworski, J Pablo; Epson, Erin; Matsuda, Kenta; Pandey, Shilpi; Kahl, Christoph; Reed, Jason; Sutton, William F; Hammond, Katherine B; Cheever, Tracy A; Barnette, Philip T; Legasse, Alfred W; Planer, Shannon; Stanton, Jeffrey J; Pegu, Amarendra; Chen, Xuejun; Wang, Keyun; Siess, Don; Burke, David; Park, Byung S; Axthelm, Michael K; Lewis, Anne; Hirsch, Vanessa M; Graham, Barney S; Mascola, John R; Sacha, Jonah B; Haigwood, Nancy L

    2016-04-01

    Prevention of mother-to-child transmission (MTCT) of HIV remains a major objective where antenatal care is not readily accessible. We tested HIV-1-specific human neutralizing monoclonal antibodies (NmAbs) as a post-exposure therapy in an infant macaque model for intrapartum MTCT. One-month-old rhesus macaques were inoculated orally with the simian-human immunodeficiency virus SHIVSF162P3. On days 1, 4, 7 and 10 after virus exposure, we injected animals subcutaneously with NmAbs and quantified systemic distribution of NmAbs in multiple tissues within 24 h after antibody administration. Replicating virus was found in multiple tissues by day 1 in animals that were not treated. All NmAb-treated macaques were free of virus in blood and tissues at 6 months after exposure. We detected no anti-SHIV T cell responses in blood or tissues at necropsy, and no virus emerged after CD8(+) T cell depletion. These results suggest that early passive immunotherapy can eliminate early viral foci and thereby prevent the establishment of viral reservoirs. PMID:26998834

  10. Immune escape by human immunodeficiency virus type 1 from neutralizing antibodies: evidence for multiple pathways.

    PubMed Central

    Watkins, B A; Reitz, M S; Wilson, C A; Aldrich, K; Davis, A E; Robert-Guroff, M

    1993-01-01

    Sera from many HIV-1-infected individuals contain broadly reactive, specific neutralizing antibodies. Despite their broad reactivity, variant viruses, resistant to neutralization, can be selected in vitro in the presence of such antisera. We have previously shown that neutralization resistance of an escape mutant with an amino acid substitution in the transmembrane protein (A582T) occurs because of alteration of a conformational epitope that is recognized by neutralizing antibodies directed against the CD4 binding site. In this report we demonstrate that immune escape via a single-amino-acid substitution (A281V) within a conserved region of the envelope glycoprotein gp120 confers neutralization resistance against a broadly reactive neutralizing antiserum from a seropositive individual. We show this alteration affects V3 and additional regions unrelated to V3 or the CD4 binding site. Together with previous studies on escape mutants selected in vitro, our findings suggest that immune-selective pressure can arise by multiple pathways. PMID:7693973

  11. A broadly neutralizing human monoclonal antibody is effective against H7N9

    PubMed Central

    Tharakaraman, Kannan; Subramanian, Vidya; Viswanathan, Karthik; Sloan, Susan; Yen, Hui-Ling; Barnard, Dale L.; Leung, Y. H. Connie; Szretter, Kristy J.; Koch, Tyree J.; Delaney, James C.; Babcock, Gregory J.; Wogan, Gerald N.; Sasisekharan, Ram; Shriver, Zachary

    2015-01-01

    Emerging strains of influenza represent a significant public health threat with potential pandemic consequences. Of particular concern are the recently emerged H7N9 strains which cause pneumonia with acute respiratory distress syndrome. Estimates are that nearly 80% of hospitalized patients with H7N9 have received intensive care unit support. VIS410, a human antibody, targets a unique conserved epitope on influenza A. We evaluated the efficacy of VIS410 for neutralization of group 2 influenza strains, including H3N2 and H7N9 strains in vitro and in vivo. VIS410, administered at 50 mg/kg, protected DBA mice infected with A/Anhui/2013 (H7N9), resulting in significant survival benefit upon single-dose (−24 h) or double-dose (−12 h, +48 h) administration (P < 0.001). A single dose of VIS410 at 50 mg/kg (−12 h) combined with oseltamivir at 50 mg/kg (−12 h, twice daily for 7 d) in C57BL/6 mice infected with A/Shanghai 2/2013 (H7N9) resulted in significant decreased lung viral load (P = 0.002) and decreased lung cytokine responses for nine of the 11 cytokines measured. Based on these results, we find that VIS410 may be effective either as monotherapy or combined with antivirals in treating H7N9 disease, as well as disease from other influenza strains. PMID:26283346

  12. Reduction Impairs the Antibacterial Activity but Benefits the LPS Neutralization Ability of Human Enteric Defensin 5.

    PubMed

    Wang, Cheng; Shen, Mingqiang; Zhang, Naixin; Wang, Song; Xu, Yang; Chen, Shilei; Chen, Fang; Yang, Ke; He, Ting; Wang, Aiping; Su, Yongping; Cheng, Tianmin; Zhao, Jinghong; Wang, Junping

    2016-01-01

    Oxidized human defensin 5 (HD5OX), a Paneth cell-secreted antibacterial peptide with three characteristic disulfide bonds, protects the host from invasion by morbigenous microbes in the small intestine. HD5OX can be reduced by thioredoxin (Trx) in vitro, while the biochemical properties of the reduced linear peptide, HD5RED, remain unclear. Here, we first confirm that HD5RED does exist in vivo. Furthermore, we reveal that the recruitment of HD5RED to the outer membrane of Gram-negative bacteria and to the anionic lipid A is lower than that of HD5OX, and HD5RED is less efficient in penetrating bacterial outer and inner membranes and inducing membrane depolarization, which confers an attenuated antibacterial activity to HD5RED. However, due to its higher structural flexibility, the binding of HD5RED to bacterial lipopolysaccharide (LPS) is markedly stronger than that of HD5OX. Consequently, HD5RED is more effective in suppressing the production of the pro-inflammatory cytokine TNF-α in LPS-stimulated macrophages by blocking the interaction between LPS and LPS-binding protein, thus suggesting that HD5RED might act as a scavenger to neutralize LPS in the gut. This study provides insights into the antibacterial and immunoregulatory effects of HD5RED and expands the known repertoire of the enteric defensins. PMID:26960718

  13. Reduction Impairs the Antibacterial Activity but Benefits the LPS Neutralization Ability of Human Enteric Defensin 5

    PubMed Central

    Wang, Cheng; Shen, Mingqiang; Zhang, Naixin; Wang, Song; Xu, Yang; Chen, Shilei; Chen, Fang; Yang, Ke; He, Ting; Wang, Aiping; Su, Yongping; Cheng, Tianmin; Zhao, Jinghong; Wang, Junping

    2016-01-01

    Oxidized human defensin 5 (HD5OX), a Paneth cell-secreted antibacterial peptide with three characteristic disulfide bonds, protects the host from invasion by morbigenous microbes in the small intestine. HD5OX can be reduced by thioredoxin (Trx) in vitro, while the biochemical properties of the reduced linear peptide, HD5RED, remain unclear. Here, we first confirm that HD5RED does exist in vivo. Furthermore, we reveal that the recruitment of HD5RED to the outer membrane of Gram-negative bacteria and to the anionic lipid A is lower than that of HD5OX, and HD5RED is less efficient in penetrating bacterial outer and inner membranes and inducing membrane depolarization, which confers an attenuated antibacterial activity to HD5RED. However, due to its higher structural flexibility, the binding of HD5RED to bacterial lipopolysaccharide (LPS) is markedly stronger than that of HD5OX. Consequently, HD5RED is more effective in suppressing the production of the pro-inflammatory cytokine TNF-α in LPS-stimulated macrophages by blocking the interaction between LPS and LPS-binding protein, thus suggesting that HD5RED might act as a scavenger to neutralize LPS in the gut. This study provides insights into the antibacterial and immunoregulatory effects of HD5RED and expands the known repertoire of the enteric defensins. PMID:26960718

  14. Comparison of Neutral Proteases and Collagenase Class I as Essential Enzymes for Human Islet Isolation

    PubMed Central

    Brandhorst, Heide; Kurfürst, Manfred; Johnson, Paul R.; Korsgren, Olle; Brandhorst, Daniel

    2016-01-01

    Background Efficient islet isolation requires synergistic interaction between collagenase class I (CI) and class II (CII). The CI degradation alters the ratio between CI and CII and is responsible for batch-to-batch variations. This study compares the role of neutral protease (NP) plus clostripain (CP) with CI as essential enzymes for human islet isolation. Methods Human islets were isolated using 4 different enzyme mixtures composed of CII plus either intact (CI-115) or degraded CI (CI-100). Blends were administered either with or without NP/CP. Purified islets were cultured for 3 to 4 days before islet quality assessment. Results Whereas using intact CI-115 without NP/CP did not significantly reduce islet yield (3429 ± 631 vs 3087 ± 970 islet equivalent/g, nonsignificant), administration of degraded CI-100 without NP/CP decreased islet yield from 3501 ± 580 to 1312 ± 244 islet equivalent/g (P < 0.01), doubled the amount of undigested tissue from 11.8 ± 1.6 to 24.4 ± 1.2% (P < 0.01) and triplicated the percentage of trapped islets from 7.7 ± 2.8 to 22.5 ± 3.6% (P < 0.05). Islet yield did not vary between supplemented CI-115 and CI-100, but was increased using CI-115 when NP/CP was omitted (P < 0.05). A trend toward higher viability and increased secretory insulin response was noted in both CI-100 and CI-115 when NP/CP was not added. Conclusions This study suggests that NP/CP can compensate reduced CI activity. Future attempts to optimize enzyme blends should consider the possibility to increase the proportion of collagenase CI to reduce the need for potentially harmful NPs. PMID:27500241

  15. Mass spectrometric detection of multiple extended series of neutral highly fucosylated N-acetyllactosamine oligosaccharides in human milk

    NASA Astrophysics Data System (ADS)

    Pfenninger, Anja; Chan, Shiu-Yung; Karas, Michael; Finke, Berndt; Stahl, Bernd; Costello, Catherine E.

    2008-12-01

    Complex mixtures of high-molecular weight fractions of pooled neutral human milk oligosaccharides (obtained via gel permeation chromatography) have been investigated. The subfractions were each permethylated and analyzed by high-resolution mass spectrometry, using matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry, in order to investigate their oligosaccharide compositions. The obtained spectra reveal that human milk contains more complex neutral oligosaccharides than have been described previously; the data show that these oligosaccharides can be highly fucosylated, and that their poly-N-acetyllactosamine cores are substituted with up to 10 fucose residues on an oligosaccharide that has 7-N-acetyllactosamine units. This is the first report of the existence in human milk of this large range of highly fucosylated oligosaccharides which possess novel, potentially immunologically active structures.

  16. Global Survey of Variation in a Human Olfactory Receptor Gene Reveals Signatures of Non-Neutral Evolution.

    PubMed

    Hoover, Kara C; Gokcumen, Omer; Qureshy, Zoya; Bruguera, Elise; Savangsuksa, Aulaphan; Cobb, Matthew; Matsunami, Hiroaki

    2015-09-01

    Allelic variation at 4 loci in the human olfactory receptor gene OR7D4 is associated with perceptual variation in the sex steroid-derived odorants, androstenone, and androstadienone. Androstadienone has been linked with chemosensory identification whereas androstenone makes pork from uncastrated pigs distasteful ("boar taint"). In a sample of 2224 individuals from 43 populations, we identified 45 OR7D4 single nucleotide polymorphisms. Coalescent modeling of frequency-site-spectrum-based statistics identified significant deviation from neutrality in human OR7D4; individual populations with statistically significant deviations from neutrality include Gujarati, Beijing Han, Great Britain, Iberia, and Puerto Rico. Analysis of molecular variation values indicated statistically significant population differentiation driven mainly by the 4 alleles associated with androstenone perception variation; however, fixation values were low suggesting that genetic structure may not have played a strong role in creating these group divisions. We also studied OR7D4 in the genomes of extinct members of the human lineage: Altai Neandertal and Denisovan. No variants were identified in Altai but 2 were in Denisova, one of which is shared by modern humans and one of which is novel. A functional test of modern human and a synthesized mutant Denisova OR7D4 indicated no statistically significant difference in responses to androstenone between the 2 species. Our results suggest non-neutral evolution for an olfactory receptor gene. PMID:26072518

  17. Continuous viral escape and selection by autologous neutralizing antibodies in drug-naive human immunodeficiency virus controllers.

    PubMed

    Mahalanabis, Madhumita; Jayaraman, Pushpa; Miura, Toshiyuki; Pereyra, Florencia; Chester, E Michael; Richardson, Barbra; Walker, Bruce; Haigwood, Nancy L

    2009-01-01

    We assessed differences in the character and specificity of autologous neutralizing antibodies (ANAbs) against individual viral variants of the quasispecies in a cohort of drug-naïve subjects with long-term controlled human immunodeficiency virus type 1 (HIV-1) infection and moderate levels of broad heterologous neutralizing antibodies (HNAb). Functional plasma virus showed continuous env evolution despite a short time frame and low levels of viral replication. Neutralization-sensitive variants dominated in subjects with intermittent viral blips, while neutralization-resistant variants predominated in elite controllers. By sequence analysis of this panel of autologous variants with various sensitivities to neutralization, we identified more than 30 residues in envelope proteins (Env) associated with resistance or sensitivity to ANAbs. The appearance of new sensitive variants is consistent with a model of continuous selection and turnover. Strong ANAb responses directed against autologous Env variants are present in long-term chronically infected individuals, suggesting a role for these responses in contributing to the durable control of HIV replication. PMID:18987151

  18. Continuous Viral Escape and Selection by Autologous Neutralizing Antibodies in Drug-Naïve Human Immunodeficiency Virus Controllers▿

    PubMed Central

    Mahalanabis, Madhumita; Jayaraman, Pushpa; Miura, Toshiyuki; Pereyra, Florencia; Chester, E. Michael; Richardson, Barbra; Walker, Bruce; Haigwood, Nancy L.

    2009-01-01

    We assessed differences in the character and specificity of autologous neutralizing antibodies (ANAbs) against individual viral variants of the quasispecies in a cohort of drug-naïve subjects with long-term controlled human immunodeficiency virus type 1 (HIV-1) infection and moderate levels of broad heterologous neutralizing antibodies (HNAb). Functional plasma virus showed continuous env evolution despite a short time frame and low levels of viral replication. Neutralization-sensitive variants dominated in subjects with intermittent viral blips, while neutralization-resistant variants predominated in elite controllers. By sequence analysis of this panel of autologous variants with various sensitivities to neutralization, we identified more than 30 residues in envelope proteins (Env) associated with resistance or sensitivity to ANAbs. The appearance of new sensitive variants is consistent with a model of continuous selection and turnover. Strong ANAb responses directed against autologous Env variants are present in long-term chronically infected individuals, suggesting a role for these responses in contributing to the durable control of HIV replication. PMID:18987151

  19. Virus-neutralizing antibody response of mice to consecutive infection with human and avian influenza A viruses.

    PubMed

    Janulíková, J; Stropkovská, A; Bobišová, Z; Košík, I; Mucha, V; Kostolanský, F; Varečková, E

    2015-06-01

    In this work we simulated in a mouse model a naturally occurring situation of humans, who overcame an infection with epidemic strains of influenza A, and were subsequently exposed to avian influenza A viruses (IAV). The antibody response to avian IAV in mice previously infected with human IAV was analyzed. We used two avian IAV (A/Duck/Czechoslovakia/1956 (H4N6) and the attenuated virus rA/Viet Nam/1203-2004 (H5N1)) as well as two human IAV isolates (virus A/Mississippi/1/1985 (H3N2) of medium virulence and A/Puerto Rico/8/1934 (H1N1) of high virulence). Two repeated doses of IAV of H4 or of H5 virus elicited virus-specific neutralizing antibodies in mice. Exposure of animals previously infected with human IAV (of H3 or H1 subtype) to IAV of H4 subtype led to the production of antibodies neutralizing H4 virus in a level comparable with the level of antibodies against the human IAV used for primary infection. In contrast, no measurable levels of virus-neutralizing (VN) antibodies specific to H5 virus were detected in mice infected with H5 virus following a previous infection with human IAV. In both cases the secondary infection with avian IAV led to a significant increase of the titer of VN antibodies specific to the corresponding human virus used for primary infection. Moreover, cross-reactive HA2-specific antibodies were also induced by sequential infection. By virtue of these results we suggest that the differences in the ability of avian IAV to induce specific antibodies inhibiting virus replication after previous infection of mice with human viruses can have an impact on the interspecies transmission and spread of avian IAV in the human population. PMID:26104333

  20. The site of an immune-selected point mutation in the transmembrane protein of human immunodeficiency virus type 1 does not constitute the neutralization epitope.

    PubMed Central

    Wilson, C; Reitz, M S; Aldrich, K; Klasse, P J; Blomberg, J; Gallo, R C; Robert-Guroff, M

    1990-01-01

    We previously reported the in vitro generation of a neutralization-resistant variant of the molecularly cloned isolate of human immunodeficiency virus type 1 (HIV-1), HXB2D. The molecular basis for the resistance was shown to be a point mutation in the env gene, causing the substitution of threonine for alanine at position 582 of gp41. Here, we show the variant to be resistant to syncytium inhibition as well as to neutralization by the immune-selecting serum. Moreover, 30% of HIV-positive human sera able to neutralize the parental virus have significantly decreased ability to neutralize the variant. As the A-to-T substitution thus has general relevance to the interaction of HIV-1 with the host immune system, we investigated further the biologic and immunologic bases for the altered properties. Synthetic peptides corresponding to the 582 region failed to compete in infectivity, neutralization, or syncytium inhibition assays and did not elicit neutralizing antibodies. Furthermore, human antibodies, affinity purified on synthetic peptide resins, bound to gp41 and peptides from the 582 region but did not possess neutralizing antibody activity. Some viral constructs in which the AVERY sequence in the 582 region was altered by site-directed mutagenesis were not infectious, indicating that the primary structure in this region is crucial for viral infectivity. Constructs predicted to possess a local secondary structure similar to that of the variant nevertheless behaved like the parental virus and remained neutralization sensitive. These results suggest that the requirements for neutralization resistance in this region are very precise. Our results with synthetic peptides show that the 582 region does not by itself constitute a neutralization epitope. Moreover, the degree of flexibility in amino acid substitution which allows maintenance of neutralization sensitivity suggests that position 582 does not form part of a noncontiguous neutralization epitope. The basis for

  1. Human Antibody Titers to Epstein-Barr Virus (EBV) gp350 Correlate with Neutralization of Infectivity Better than Antibody Titers to EBV gp42 Using a Rapid Flow Cytometry-Based EBV Neutralization Assay

    PubMed Central

    Sashihara, Junji; Burbelo, Peter D.; Savoldo, Barbara; Pierson, Theodore C.; Cohen, Jeffrey I.

    2009-01-01

    Measurement of neutralizing antibodies to Epstein-Barr virus (EBV) is important for evaluation of candidate vaccines. The current neutralization assay is based on antibody inhibition of EBV transformation of B cells and requires 6 weeks to perform. We developed a rapid, quantitative flow cytometry assay and show that neutralizing antibody titers measured by the new assay strongly correlate with antibody titers in the standard transformation-based assay. Antibodies to EBV gp350 and gp42 have been shown to block infection of B cells by EBV. Using new assays to quantify antibodies to these glycoproteins, we show for the first time that that human plasma contains high titers of antibody to gp42; these titers correlate with neutralization of EBV infectivity or transformation. Furthermore, we show that antibody titers to EBV gp350 correlate more strongly with neutralization than antibody titers to gp42. These assays should be useful in accessing antibody responses to candidate EBV vaccines. PMID:19584018

  2. Enveloped Virus-Like Particle Expression of Human Cytomegalovirus Glycoprotein B Antigen Induces Antibodies with Potent and Broad Neutralizing Activity

    PubMed Central

    Kirchmeier, Marc; Fluckiger, Anne-Catherine; Soare, Catalina; Bozic, Jasminka; Ontsouka, Barthelemy; Ahmed, Tanvir; Diress, Abebaw; Pereira, Lenore; Schödel, Florian; Plotkin, Stanley; Dalba, Charlotte; Klatzmann, David

    2014-01-01

    A prophylactic vaccine to prevent the congenital transmission of human cytomegalovirus (HCMV) in newborns and to reduce life-threatening disease in immunosuppressed recipients of HCMV-infected solid organ transplants is highly desirable. Neutralizing antibodies against HCMV confer significant protection against infection, and glycoprotein B (gB) is a major target of such neutralizing antibodies. However, one shortcoming of past HCMV vaccines may have been their failure to induce high-titer persistent neutralizing antibody responses that prevent the infection of epithelial cells. We used enveloped virus-like particles (eVLPs), in which particles were produced in cells after the expression of murine leukemia virus (MLV) viral matrix protein Gag, to express either full-length CMV gB (gB eVLPs) or the full extracellular domain of CMV gB fused with the transmembrane and cytoplasmic domains from vesicular stomatitis virus (VSV)-G protein (gB-G eVLPs). gB-G-expressing eVLPs induced potent neutralizing antibodies in mice with a much greater propensity toward epithelial cell-neutralizing activity than that induced with soluble recombinant gB protein. An analysis of gB antibody binding titers and T-helper cell responses demonstrated that high neutralizing antibody titers were not simply due to enhanced immunogenicity of the gB-G eVLPs. The cells transiently transfected with gB-G but not gB plasmid formed syncytia, consistent with a prefusion gB conformation like those of infected cells and viral particles. Two of the five gB-G eVLP-induced monoclonal antibodies we examined in detail had neutralizing activities, one of which possessed particularly potent epithelial cell-neutralizing activity. These data differentiate gB-G eVLPs from gB antigens used in the past and support their use in a CMV vaccine candidate with improved neutralizing activity against epithelial cell infection. PMID:24334684

  3. Enveloped virus-like particle expression of human cytomegalovirus glycoprotein B antigen induces antibodies with potent and broad neutralizing activity.

    PubMed

    Kirchmeier, Marc; Fluckiger, Anne-Catherine; Soare, Catalina; Bozic, Jasminka; Ontsouka, Barthelemy; Ahmed, Tanvir; Diress, Abebaw; Pereira, Lenore; Schödel, Florian; Plotkin, Stanley; Dalba, Charlotte; Klatzmann, David; Anderson, David E

    2014-02-01

    A prophylactic vaccine to prevent the congenital transmission of human cytomegalovirus (HCMV) in newborns and to reduce life-threatening disease in immunosuppressed recipients of HCMV-infected solid organ transplants is highly desirable. Neutralizing antibodies against HCMV confer significant protection against infection, and glycoprotein B (gB) is a major target of such neutralizing antibodies. However, one shortcoming of past HCMV vaccines may have been their failure to induce high-titer persistent neutralizing antibody responses that prevent the infection of epithelial cells. We used enveloped virus-like particles (eVLPs), in which particles were produced in cells after the expression of murine leukemia virus (MLV) viral matrix protein Gag, to express either full-length CMV gB (gB eVLPs) or the full extracellular domain of CMV gB fused with the transmembrane and cytoplasmic domains from vesicular stomatitis virus (VSV)-G protein (gB-G eVLPs). gB-G-expressing eVLPs induced potent neutralizing antibodies in mice with a much greater propensity toward epithelial cell-neutralizing activity than that induced with soluble recombinant gB protein. An analysis of gB antibody binding titers and T-helper cell responses demonstrated that high neutralizing antibody titers were not simply due to enhanced immunogenicity of the gB-G eVLPs. The cells transiently transfected with gB-G but not gB plasmid formed syncytia, consistent with a prefusion gB conformation like those of infected cells and viral particles. Two of the five gB-G eVLP-induced monoclonal antibodies we examined in detail had neutralizing activities, one of which possessed particularly potent epithelial cell-neutralizing activity. These data differentiate gB-G eVLPs from gB antigens used in the past and support their use in a CMV vaccine candidate with improved neutralizing activity against epithelial cell infection. PMID:24334684

  4. Fatty acids and glucose increase neutral endopeptidase activity in human microvascular endothelial cells.

    PubMed

    Muangman, Pornprom; Spenny, Michelle L; Tamura, Richard N; Gibran, Nicole S

    2003-06-01

    Neutral endopeptidase (NEP), a membrane-bound metallopeptidase enzyme that degrades neuropeptides, bradykinin, atrial natriuretic factor, enkephalins, and endothelin may regulate response to injury. We have previously demonstrated increased NEP localization and enzyme activity in diabetic wounds and skin compared with normal controls. We hypothesized that hyperlipidemia and hyperglycemia associated with type 2 diabetes mellitus may induce excessive NEP activity and thereby diminish normal response to injury. Human microvascular endothelial cells were treated with five different fatty acids (40 microM) with varying degrees of saturation, including oleic acid, linoleic acid, palmitic acid, stearic acid, and linolenic acid and/or glucose (40 mM) for 48 h. The effect of the antioxidative agents vitamin E and C on NEP enzyme activation was determined by treating the cultured cells with alpha-tocopherol succinate and/or L-ascorbic acid. Cell membrane preparations were assayed for NEP activity by incubation with glutaryl-Ala-Ala-Phe-4-methoxy-beta naphthylamide to generate a fluorescent degradation product methoxy 2 naphthylamine. High glucose or fatty acid concentration upregulated NEP activity. The highest NEP activity was observed with combined elevated glucose, linoleic acid, and oleic acid (P < 0.05). Antioxidant vitamin E and C treatment significantly reduced NEP enzyme activity after fatty acid exposure (P < 0.05). Thus, hyperglycemia and hyperlipidemia associated with type 2 diabetes mellitus may increase endothelial cell NEP activity and thereby decrease early pro-inflammatory responses. The modulator effect of vitamin E and C on NEP membrane enzyme activity after exposure to fatty acid stimulation suggests that lipid oxidation may activate NEP. PMID:12785004

  5. Pseudovirion Particles Bearing Native HIV Envelope Trimers Facilitate a Novel Method for Generating Human Neutralizing Monoclonal Antibodies against HIV

    PubMed Central

    Hicar, Mark D.; Chen, Xuemin; Briney, Bryan; Hammonds, Jason; Wang, Jaang-Jiun; Kalams, Spyros; Spearman, Paul W.; Crowe, James E.

    2010-01-01

    Monomeric HIV envelope vaccines fail to elicit broadly neutralizing antibodies or to protect against infection. Neutralizing antibodies against HIV bind to native, functionally active Env trimers on the virion surface. Gag-Env pseudovirions recapitulate the native trimer, and could serve as an effective epitope presentation platform for study of the neutralizing antibody response in HIV-infected individuals. To address if pseudovirions can recapitulate native HIV virion epitope structures, we carefully characterized these particles, concentrating on the antigenic structure of the coreceptor binding site. By blue native gel shift assays, Gag-Env pseudovirions were shown to contain native trimers that were competent for binding to neutralizing monoclonal antibodies. In ELISA, pseudovirions exhibited increased binding of known CD4-induced antibodies following addition of CD4. Using flow cytometric analysis, fluorescently labeled pseudovirions specifically identified a subset of antigen-specific B cells in HIV-infected subjects. Interestingly, the sequence of one of these novel human antibodies, identified during cloning of single HIV-specific B cells and designated 2C6, exhibited homology to mAb 47e, a known anti-CD4-induced coreceptor binding site antibody. The secreted monoclonal antibody 2C6 did not bind monomeric gp120, but specifically bound envelope on pseudovirions. A recombinant form of the antibody 2C6 acted as a CD4-induced epitope-specific antibody in neutralization assays, yet did not bind monomeric gp120. These findings imply specificity against a quaternary epitope presented on the pseudovirion envelope spike. These data demonstrate that Gag-Env pseudovirions recapitulate CD4 and coreceptor binding pocket antigenic structures and can facilitate identification of B cell clones that secrete neutralizing antibodies. PMID:20531016

  6. Identification of human neutralizing antibodies against MERS-CoV and their role in virus adaptive evolution

    PubMed Central

    Tang, Xian-Chun; Agnihothram, Sudhakar S.; Jiao, Yongjun; Stanhope, Jeremy; Graham, Rachel L.; Peterson, Eric C.; Avnir, Yuval; Tallarico, Aimee St. Clair; Sheehan, Jared; Zhu, Quan; Baric, Ralph S.; Marasco, Wayne A.

    2014-01-01

    The newly emerging Middle East Respiratory Syndrome coronavirus (MERS-CoV) causes a Severe Acute Respiratory Syndrome-like disease with ∼43% mortality. Given the recent detection of virus in dromedary camels, zoonotic transfer of MERS-CoV to humans is suspected. In addition, little is known about the role of human neutralizing Ab (nAb) pressure as a driving force in MERS-CoV adaptive evolution. Here, we used a well-characterized nonimmune human Ab-phage library and a panning strategy with proteoliposomes and cells to identify seven human nAbs against the receptor-binding domain (RBD) of the MERS-CoV Spike protein. These nAbs bind to three different epitopes in the RBD and human dipeptidyl peptidase 4 (hDPP4) interface with subnanomolar/nanomolar binding affinities and block the binding of MERS-CoV Spike protein with its hDPP4 receptor. Escape mutant assays identified five amino acid residues that are critical for neutralization escape. Despite the close proximity of the three epitopes on the RBD interface, escape from one epitope did not have a major impact on neutralization with Abs directed to a different epitope. Importantly, the majority of escape mutations had negative impacts on hDPP4 receptor binding and viral fitness. To our knowledge, these results provide the first report on human nAbs against MERS-CoV that may contribute to MERS-CoV clearance and evolution. Moreover, in the absence of a licensed vaccine or antiviral for MERS, this panel of nAbs offers the possibility of developing human mAb-based immunotherapy, especially for health-care workers. PMID:24778221

  7. Novel Rabies Virus-Neutralizing Epitope Recognized by Human Monoclonal Antibody: Fine Mapping and Escape Mutant Analysis†

    PubMed Central

    Marissen, Wilfred E.; Kramer, R. Arjen; Rice, Amy; Weldon, William C.; Niezgoda, Michael; Faber, Milosz; Slootstra, Jerry W.; Meloen, Rob H.; Clijsters-van der Horst, Marieke; Visser, Therese J.; Jongeneelen, Mandy; Thijsse, Sandra; Throsby, Mark; de Kruif, John; Rupprecht, Charles E.; Dietzschold, Bernhard; Goudsmit, Jaap; Bakker, Alexander B. H.

    2005-01-01

    Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We produced two previously described potent rabies virus-neutralizing human MAbs, CR57 and CRJB, in human PER.C6 cells. The two MAbs competed for binding to rabies virus glycoprotein. Using CR57 and a set of 15-mer overlapping peptides covering the glycoprotein ectodomain, a neutralization domain was identified between amino acids (aa) 218 and 240. The minimal binding region was identified as KLCGVL (aa 226 to 231), with key residues K-CGV- identified by alanine replacement scanning. The critical binding region of this novel nonconformational rabies virus epitope is highly conserved within rabies viruses of genotype 1. Subsequently, we generated six rabies virus variants escaping neutralization by CR57 and six variants escaping CRJB. The CR57 escape mutants were only partially covered by CRJB, and all CRJB-resistant variants completely escaped neutralization by CR57. Without exception, the CR57-resistant variants showed a mutation at key residues within the defined minimal binding region, while the CRJB escape viruses showed a single mutation distant from the CR57 epitope (N182D) combined with mutations in the CR57 epitope. The competition between CR57 and CRJB, the in vitro escape profile, and the apparent overlap between the recognized epitopes argues against including both CR57 and CRJB in a MAb cocktail aimed at replacing classical immunoglobulin preparations. PMID:15795253

  8. [The coagulation characteristics of human oxyhemoglobin in the presence of a mercury (II) ion in a neutral phosphate buffer].

    PubMed

    Bogdanova, L D; Myshkin, A E

    1990-01-01

    The kinetics of human oxyhemoglobin coagulation in neutral phosphate buffer in the presence of mercury acetate at 20 degrees has been studied using turbidimetric methods. The addition of small amounts of concentrated Hg2+ solution leads to rapid local protein coagulation with subsequent dissolution of the formed coagulate. Coagulation can be inhibited by addition of Tris that binds to mercury ions. The pattern of oxyhemoglobin coagulation is determined by molar Hg2+/protein ration rather than by total Hg2+ concentration. PMID:2362035

  9. Antibodies that neutralize human beta interferon biologic activity recognize a linear epitope: analysis by synthetic peptide mapping.

    PubMed Central

    Redlich, P N; Hoeprich, P D; Colby, C B; Grossberg, S E

    1991-01-01

    The location of biologically relevant epitopes on recombinant human beta interferon in which Ser-17 replaces Cys-17 (rh[Ser17]IFN-beta) was evaluated by testing the immunoreactivity of antibodies against 159 sequential, overlapping octamer peptides. Three monoclonal antibodies (mAbs) that neutralize rh[Ser17]IFN-beta biologic activity, designated A1, A5, and A7, bound to peptides spanning only residues 39-48, whereas nonneutralizing mAb bound less specifically at multiple sites near the amino terminus. The immunoreactivity of peptides spanning residues 40-47 that contained a series of single amino acid substitutions suggested that residues 41-43 (Pro-Glu-Glu) and 46 (Gln) are important for the binding of neutralizing mAbs. The reactivity of mAbs to larger synthetic peptides containing rh[Ser17]IFN-beta sequences from residue 32 through residue 56 was evaluated. All mAbs except A7 reacted with synthetic peptides representing rh[Ser17]IFN-beta residues 32-47, 40-56, and 32-56, but only mAbs A1 and A5 bound to the core peptide composed of residues 40-47. Peptide 32-56 effectively blocked the binding of mAbs A1 and A5 to rh[Ser17]IFN-beta and markedly inhibited their neutralizing activity. Biologic activity of the peptides was undetectable. Rabbit antisera raised against peptides 32-47 and 40-56 recognized rh[Ser17]IFN-beta but did not neutralize its antiviral activity. Thus, structure-function analysis by peptide mapping has permitted the identification of a linear epitope recognized by neutralizing antibody on a biologically active cytokine. We conclude that the region spanning residues 32-56 is of major importance in the expression of the biologic activity of human IFN-beta. Images PMID:1708891

  10. Human immunodeficiency virus type 1 neutralization epitope with conserved architecture elicits early type-specific antibodies in experimentally infected chimpanzees.

    PubMed Central

    Goudsmit, J; Debouck, C; Meloen, R H; Smit, L; Bakker, M; Asher, D M; Wolff, A V; Gibbs, C J; Gajdusek, D C

    1988-01-01

    Chimpanzees are susceptible to infection by divergent strains of human immunodeficiency virus type 1 (HIV-1), none of which cause clinical or immunological abnormalities. Chimpanzees were inoculated with one of four strains of HIV-1: human T-lymphotropic virus (HTLV) type IIIB, lymphadenopathy virus (LAV) type 1, HTLV type IIIRF, or an isolate from the brain of a patient with acquired immunodeficiency syndrome. Within 6 months after inoculation with the closely related strains HTLV-IIIB or LAV-1, six chimpanzees developed serum antibodies to the C-terminal half (amino acids 288-467) of the HTLV-IIIB external envelope glycoprotein gp120. Sera from five of those chimpanzees had HTLV-IIIB cell-fusion-inhibiting antibody titers greater than or equal to 20 at that time, indicating that they neutralized the infecting strain of HIV-1 in vitro. No antibodies to the carboxyl terminus of HTLV-IIIB gp120 were observed in sera of chimpanzees inoculated with HTLV-IIIRF or with the brain-tissue strain, and those sera did not neutralize HTLV-IIIB. A rabbit immunized with the C-terminal portion of gp120 acquired neutralizing antibodies that bound to four domains of the HTLV-IIIB external envelope as analyzed by reactivity to 536 overlapping nonapeptides of gp120. One of these domains in the variable region V3, with the amino acid sequence IRIQRGPGRAFVTIG (amino acids 307-321), bound to all chimpanzee sera that neutralized HTLV-IIIB but not to the serum of the HTLV-IIIRF-inoculated chimpanzee that did not neutralize HTLV-IIIB. The HTLV-IIIRF sequence at the same location, ITKGPGRVIYA, was recognized by the serum of the HTLV-IIIRF-inoculated chimpanzee but not by any sera of the HTLV-IIIB-inoculated or LAV-1-inoculated chimpanzees. The HTLV-IIIB residues RIQR and AFV and the HTLV-IIIRF residues lysine and VIYA, flanking a highly conserved beta-turn (GPGR), appear to be critical for antibody binding and subsequent type-specific virus neutralization. This neutralization epitope

  11. Rapid Generation of Human-Like Neutralizing Monoclonal Antibodies in Urgent Preparedness for Influenza Pandemics and Virulent Infectious Diseases

    PubMed Central

    Meng, Weixu; Pan, Weiqi; Zhang, Anna J. X.; Li, Zhengfeng; Wei, Guowei; Feng, Liqiang; Dong, Zhenyuan; Li, Chufang; Hu, Xiangjing; Sun, Caijun; Luo, Qinfang; Yuen, Kwok-Yung; Zhong, Nanshan; Chen, Ling

    2013-01-01

    Background The outbreaks of emerging infectious diseases caused by pathogens such as SARS coronavirus, H5N1, H1N1, and recently H7N9 influenza viruses, have been associated with significant mortality and morbidity in humans. Neutralizing antibodies from individuals who have recovered from an infection confer therapeutic protection to others infected with the same pathogen. However, survivors may not always be available for providing plasma or for the cloning of monoclonal antibodies (mAbs). Methodology/Principal Findings The genome and the immunoglobulin genes in rhesus macaques and humans are highly homologous; therefore, we investigated whether neutralizing mAbs that are highly homologous to those of humans (human-like) could be generated. Using the H5N1 influenza virus as a model, we first immunized rhesus macaques with recombinant adenoviruses carrying a synthetic gene encoding hemagglutinin (HA). Following screening an antibody phage display library derived from the B cells of immunized monkeys, we cloned selected macaque immunoglobulin heavy chain and light chain variable regions into the human IgG constant region, which generated human-macaque chimeric mAbs exhibiting over 97% homology to human antibodies. Selected mAbs demonstrated potent neutralizing activities against three clades (0, 1, 2) of the H5N1 influenza viruses. The in vivo protection experiments demonstrated that the mAbs effectively protected the mice even when administered up to 3 days after infection with H5N1 influenza virus. In particular, mAb 4E6 demonstrated sub-picomolar binding affinity to HA and superior in vivo protection efficacy without the loss of body weight and obvious lung damage. The analysis of the 4E6 escape mutants demonstrated that the 4E6 antibody bound to a conserved epitope region containing two amino acids on the globular head of HA. Conclusions/Significance Our study demonstrated the generation of neutralizing mAbs for potential application in humans in urgent

  12. RELATIVE CONCENTRATIONS OF SERUM NEUTRALIZING ANTIBODY TO VP3 AND VP7 PROTEINS IN ADULTS INFECTED WITH A HUMAN ROTAVIRUS (JOURNAL VERSION)

    EPA Science Inventory

    Two outer capsid rotavirus proteins, VP3 and VP7, have been found to elicit neutralizing antibody production, but the immunogenicity of these proteins during human rotavirus infection has not been determined. The relative amounts of serum neutralizing antibody against the VP3 and...

  13. Development of a high-throughput β-Gal-based neutralization assay for quantitation of herpes simplex virus-neutralizing antibodies in human samples.

    PubMed

    Baccari, Amy; Cooney, Michael; Blevins, Tamara P; Morrison, Lynda A; Larson, Shane; Skoberne, Mojca; Belshe, Robert B; Flechtner, Jessica B; Long, Deborah

    2016-07-19

    Measurement of neutralizing antibodies against herpes simplex virus (HSV) is important for evaluation of candidate vaccines. The established plaque-reduction neutralization assay is time consuming, labor intensive, and difficult to validate and transfer. Here, we describe the characterization of a HSV-neutralization assay based on the expression of a reporter gene, β-galactosidase (β-Gal). Using previously constructed HSV-β-Gal recombinant viruses, HSV-2/Gal and HSV-1/tk12, we developed a colorimetric β-Gal-based neutralization assay that is sensitive and highly reproducible, and performed in less than 48h. HSV-1 and HSV-2 neutralizing titers measured by the β-Gal-based neutralization assay were equivalent to those obtained by a plaque reduction neutralization assay. Intra- and inter-assay precision studies demonstrated that the β-Gal-based assay was repeatable and yielded low and acceptable variation. In addition, comparison of HSV-2 neutralizing antibody (NAb) titers measured in two independent laboratories by two unique β-Gal-based assays showed a highly significant correlation (r=0.9499, p<0.0001) between the two assays. The new assay will serve as an important tool both for preclinical and clinical trials of new HSV vaccines. PMID:27265458

  14. Structural basis for the neutralization of MERS-CoV by a human monoclonal antibody MERS-27

    PubMed Central

    Yu, Xiaojuan; Zhang, Senyan; Jiang, Liwei; Cui, Ye; Li, Dongxia; Wang, Dongli; Wang, Nianshuang; Fu, Lili; Shi, Xuanlin; Li, Ziqiang; Zhang, Linqi; Wang, Xinquan

    2015-01-01

    The recently reported Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory illness in humans with an approximately 30% mortality rate. The envelope spike glycoprotein on the surface of MERS-CoV mediates receptor binding, membrane fusion, and viral entry. We previously reported two human monoclonal antibodies that target the receptor binding domain (RBD) of the spike and exhibit strong neutralization activity against live and pesudotyped MERS-CoV infection. Here we determined the crystal structure of MERS-CoV RBD bound to the Fab fragment of MERS-27 antibody at 3.20 Å resolution. The MERS-27 epitope in the RBD overlaps with the binding site of the MERS-CoV receptor DPP4. Further biochemical, viral entry, and neutralization analyses identified two critical residues in the RBD for both MERS-27 recognition and DPP4 binding. One of the residues, Trp535, was found to function as an anchor residue at the binding interface with MERS-27. Upon receptor binding, Trp535 interacts with the N-linked carbohydrate moiety of DPP4. Thus, MERS-27 inhibits MERS-CoV infection by directly blocking both protein-protein and protein-carbohydrate interactions between MERS-CoV RBD and DPP4. These results shed light on the molecular basis of MERS-27 neutralization and will assist in the optimization of MERS-27 as a tool to combat MERS-CoV infection. PMID:26281793

  15. Mining the human autoantibody repertoire: Isolation of potent IL17A-neutralizing monoclonal antibodies from a patient with thymoma

    PubMed Central

    Beerli, Roger R; Bauer, Monika; Fritzer, Andrea; Rosen, Lindsey B; Buser, Regula B; Hanner, Markus; Maudrich, Melanie; Nebenfuehr, Mario; Toepfer, Jorge Alejandro Sepulveda; Mangold, Susanne; Bauer, Anton; Holland, Steven M; Browne, Sarah K; Meinke, Andreas

    2014-01-01

    Anti-cytokine autoantibodies have been widely reported to be present in human plasma, both in healthy subjects and in patients with underlying autoimmune conditions, such as autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) or thymic epithelial neoplasms. While often asymptomatic, they can cause or facilitate a wide range of diseases including opportunistic infections. The potential therapeutic value of specific neutralizing anti-cytokine autoantibodies has not been thoroughly investigated. Here we used mammalian cell display to isolate IL17A-specific antibodies from a thymoma patient with proven high-titer autoantibodies against the same. We identified 3 distinct clonotypes that efficiently neutralized IL17A in a cell-based in vitro assay. Their potencies were comparable to those of known neutralizing antibodies, including 2, AIN457 (secukinumab) and ixekizumab that are currently in clinical development for the treatment of various inflammatory disorders. These data clearly demonstrate that the human autoantibody repertoire can be mined for antibodies with high therapeutic potential for clinical development. PMID:25484038

  16. Structure-Function Analysis of the Epitope for 4E10, a Broadly Neutralizing Human Immunodeficiency Virus Type 1 Antibody†

    PubMed Central

    Brunel, Florence M.; Zwick, Michael B.; Cardoso, Rosa M. F.; Nelson, Josh D.; Wilson, Ian A.; Burton, Dennis R.; Dawson, Philip E.

    2006-01-01

    The human immunodeficiency virus type 1 (HIV-1) neutralizing antibody 4E10 binds to a linear, highly conserved epitope within the membrane-proximal external region of the HIV-1 envelope glycoprotein gp41. We have delineated the peptide epitope of the broadly neutralizing 4E10 antibody to gp41 residues 671 to 683, using peptides with different lengths encompassing the previously suggested core epitope (NWFDIT). Peptide binding to the 4E10 antibody was assessed by competition enzyme-linked immunosorbent assay, and the Kd values of selected peptides were determined using surface plasmon resonance. An Ala scan of the epitope indicated that several residues, W672, F673, and T676, are essential (>1,000-fold decrease in binding upon replacement with alanine) for 4E10 recognition. In addition, five other residues, N671, D674, I675, W680, and L679, make significant contributions to 4E10 binding. In general, the Ala scan results agree well with the recently reported crystal structure of 4E10 in complex with a 13-mer peptide and with our circular dichroism analyses. Neutralization competition assays confirmed that the peptide NWFDITNWLWYIKKKK-NH2 could effectively inhibit 4E10 neutralization. Finally, to limit the conformational flexibility of the peptides, helix-promoting 2-aminoisobutyric acid residues and helix-inducing tethers were incorporated. Several peptides have significantly improved affinity (>1,000-fold) over the starting peptide and, when used as immunogens, may be more likely to elicit 4E10-like neutralizing antibodies. Hence, this study represents the first stage toward iterative development of a vaccine based on the 4E10 epitope. PMID:16439525

  17. Epitopes for Broad and Potent Neutralizing Antibody Responses during Chronic Infection with Human Immunodeficiency Virus Type 1

    PubMed Central

    Nandi, Avishek; Lavine, Christine L.; Wang, Pengcheng; Lipchina, Inna; Goepfert, Paul A.; Shaw, George M.; Tomaras, Georgia D.; Montefiori, David C.; Haynes, Barton F.; Easterbrook, Philippa; Robinson, James E.; Sodroski, Joseph G.; Yang, Xinzhen

    2009-01-01

    Neutralizing antibody (nAb) response is sporadic and has limited potency and breadth during infection with human immunodeficiency virus type 1 (HIV-1). In rare cases, broad and potent nAbs are actually induced in vivo. Identifying specific epitopes targeted by such broad and potent nAb response is valuable in guiding the design of a prophylactic vaccine aimed to induce nAb. In this study, we have defined neutralizing epitope usage in 7 out of 17 subjects with broad and potent nAbs by using targeted mutagenesis in known neutralizing epitopes of HIV-1 glycoproteins and by using in vitro depletion of serum neutralizing activity by various recombinant HIV-1 glycoproteins. Consistent with recent reports, the CD4 binding site (CD4BS) is targeted by nAbs in vivo (4 of the 7 subjects with defined neutralizing epitopes). The new finding from this study is that epitopes in the gp120 outer domain are also targeted by nAbs in vivo (5 of the 7 subjects). The outer domain epitopes include glycan-dependent epitopes (2 subjects), conserved non-linear epitope in the V3 region (2 subjects), and a CD4BS epitope composed mainly of the elements in the outer domain (1 subject). Importantly, we found indication for epitope poly-specificity, a dual usage of the V3 and CD4BS epitopes, in only one subject. This study provides a more complete profile of epitope usage for broad and potent nAb responses during HIV-1 infection. PMID:19922969

  18. Interactions between Lipids and Human Anti-HIV Antibody 4E10 Can Be Reduced without Ablating Neutralizing Activity▿

    PubMed Central

    Xu, Hengyu; Song, Likai; Kim, Mikyung; Holmes, Margaret A.; Kraft, Zane; Sellhorn, George; Reinherz, Ellis L.; Stamatatos, Leonidas; Strong, Roland K.

    2010-01-01

    Human 4E10 is one of the broadest-specificity, HIV-1-neutralizing monoclonal antibodies known, recognizing a membrane-proximal linear epitope on gp41. The lipid cross-reactivity of 4E10 has been alternately suggested either to contribute to the apparent rarity of 4E10-like antibody responses in HIV infections, through elimination by B-cell tolerance mechanisms to self-antigens, or to contribute to neutralization potency by virus-specific membrane binding outside of the membrane-proximal external region (MPER). To investigate how 4E10 interacts with membrane and protein components, and whether such interactions contribute to neutralization mechanisms, we introduced two mutations into 4E10 Fv constructs, Trp to Ala at position 100 in the heavy chain [W(H100)A] and Gly to Glu at position 50 in the light chain [G(L50)E], selected to disrupt potential lipid interactions via different mechanisms. Wild-type and mutant Fvs all bound with the same affinity to peptides and monomeric and trimeric gp140s, but the affinities for gp140s were uniformly 10-fold weaker than to peptides. 4E10 Fv binding responses to liposomes in the presence or absence of MPER peptides were weak in absolute terms, consistent with prior observations, and both mutations attenuated interactions even further, as predicted. The W(H100)A mutation reduced neutralization efficiency against four HIV-1 isolates, but the G(L50)E mutation increased potency across the same panel. Electron paramagnetic resonance experiments showed that the W(H100)A mutation, but not the G(L50)E mutation, reduced the ability of 4E10 to extract MPER peptides from membranes. These results show that 4E10 nonspecific membrane binding is separable from neutralization, which is achieved through specific peptide/lipid orientation changes. PMID:19906921

  19. Interaction of acid ceramidase inhibitor LCL521 with tumor response to photodynamic therapy and photodynamic therapy-generated vaccine.

    PubMed

    Korbelik, Mladen; Banáth, Judit; Zhang, Wei; Saw, Kyi Min; Szulc, Zdzislaw M; Bielawska, Alicja; Separovic, Duska

    2016-09-15

    Acid ceramidase has been identified as a promising target for cancer therapy. One of its most effective inhibitors, LCL521, was examined as adjuvant to photodynamic therapy (PDT) using mouse squamous cell carcinoma SCCVII model of head and neck cancer. Lethal effects of PDT, assessed by colony forming ability of in vitro treated SCCVII cells, were greatly enhanced when combined with 10 µM LCL521 treatment particularly when preceding PDT. When PDT-treated SCCVII cells are used to vaccinate SCCVII tumor-bearing mice (PDT vaccine protocol), adjuvant LCL521 treatment (75 mg/kg) resulted in a marked retardation of tumor growth. This effect can be attributed to the capacity of LCL521 to effectively restrict the activity of two main immunoregulatory cell populations (Tregs and myeloid-derived suppressor cells, MDSCs) that are known to hinder the efficacy of PDT vaccines. The therapeutic benefit with adjuvant LCL521 was also achieved with SCCVII tumors treated with standard PDT when using immunocompetent mice but not with immunodeficient hosts. The interaction of LCL521 with PDT-based antitumor mechanisms is dominated by immune system contribution that includes overriding the effects of immunoregulatory cells, but could also include a tacit contribution from boosting direct tumor cell kill. PMID:27136745

  20. Canine distemper virus neutralization activity is low in human serum and it is sensitive to an amino acid substitution in the hemagglutinin protein

    SciTech Connect

    Zhang, Xinsheng; Wallace, Olivia L.; Domi, Arban; Wright, Kevin J.; Driscoll, Jonathan; Anzala, Omu; Sanders, Eduard J.; Kamali, Anatoli; Allen, Susan; Fast, Pat; Gilmour, Jill; Price, Matt A.; Parks, Christopher L.

    2015-08-15

    Serum was analyzed from 146 healthy adult volunteers in eastern Africa to evaluate measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb) prevalence and potency. MV plaque reduction neutralization test (PRNT) results indicated that all sera were positive for MV nAbs. Furthermore, the 50% neutralizing dose (ND50) for the majority of sera corresponded to antibody titers induced by MV vaccination. CDV nAbs titers were low and generally were detected in sera with high MV nAb titers. A mutant CDV was generated that was less sensitive to neutralization by human serum. The mutant virus genome had 10 nucleotide substitutions, which coded for single amino acid substitutions in the fusion (F) and hemagglutinin (H) glycoproteins and two substitutions in the large polymerase (L) protein. The H substitution occurred in a conserved region involved in receptor interactions among morbilliviruses, implying that this region is a target for cross-reactive neutralizing antibodies. - Highlights: • Screened 146 serum samples for measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb). • MV nAb is prevalent in the sera. • CDV neutralizing activity is generally low or absent and when detected it is present in sera with high MV nAb titers. • A neutralization-resistant CDV mutant was isolated using human serum selection. • A mutation was identified in the receptor-binding region of CDV hemagglutinin protein that confers the neutralization resistance.

  1. Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody

    PubMed Central

    Ku, Zhiqiang; Zuo, Teng; Kong, Liangliang; Zhang, Chao; Shi, Jinping; Liu, Qingwei; Chen, Tan; Zhang, Yingyi; Jiang, Wen; Zhang, Linqi; Huang, Zhong; Cong, Yao

    2016-01-01

    Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection. PMID:26938634

  2. Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody.

    PubMed

    Ye, Xiaohua; Fan, Chen; Ku, Zhiqiang; Zuo, Teng; Kong, Liangliang; Zhang, Chao; Shi, Jinping; Liu, Qingwei; Chen, Tan; Zhang, Yingyi; Jiang, Wen; Zhang, Linqi; Huang, Zhong; Cong, Yao

    2016-03-01

    Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection. PMID:26938634

  3. Human domain antibodies to conserved sterically restricted regions on gp120 as exceptionally potent cross-reactive HIV-1 neutralizers

    PubMed Central

    Chen, Weizao; Zhu, Zhongyu; Feng, Yang; Dimitrov, Dimiter S.

    2008-01-01

    The antibody access to some conserved structures on the HIV-1 envelope glycoprotein (Env) is sterically restricted. We have hypothesized that the smallest independently folded antibody fragments (domains) could exhibit exceptionally potent and broadly cross-reactive neutralizing activity by targeting hidden conserved epitopes that are not accessible by larger antibodies. To test this hypothesis, we constructed a large (size 2.5 × 1010), highly diversified library of human antibody variable domains (domain antibodies) and used it for selection of binders to conserved Env structures by panning sequentially against Envs from different isolates. The highest affinity binder, m36, neutralized all tested HIV-1 isolates from clades A– D with an activity on average higher than that of C34, a peptide similar to the fusion inhibitor T20, which is in clinical use, and that of m9, which exhibits a neutralizing activity superior to known potent cross-reactive antibodies. Large-size fusion proteins of m36 exhibited diminished neutralizing activity but preincubation of virions with soluble CD4 restored it, suggesting that m36 epitope is sterically restricted and induced by CD4 (CD4i). M36 bound to gp120-CD4 complexes better than to gp120 alone and competed with CD4i antibodies. M36 is the only reported representative of a promising class of potent, broadly cross-reactive HIV-1 inhibitors based on human domain antibodies. It has potential for prevention and therapy and as an agent for exploration of the closely guarded conserved Env structures with implications for design of small molecule inhibitors and elucidation of mechanisms of virus entry and evasion of immune responses. PMID:18957538

  4. Crystal structure of the Hendra virus attachment G glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody.

    PubMed

    Xu, Kai; Rockx, Barry; Xie, Yihu; DeBuysscher, Blair L; Fusco, Deborah L; Zhu, Zhongyu; Chan, Yee-Peng; Xu, Yan; Luu, Truong; Cer, Regina Z; Feldmann, Heinz; Mokashi, Vishwesh; Dimitrov, Dimiter S; Bishop-Lilly, Kimberly A; Broder, Christopher C; Nikolov, Dimitar B

    2013-01-01

    The henipaviruses, represented by Hendra (HeV) and Nipah (NiV) viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb) have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4) was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines. PMID:24130486

  5. Quantitative analysis of serum neutralization of human immunodeficiency virus type 1 from subtypes A, B, C, D, E, F, and I: lack of direct correlation between neutralization serotypes and genetic subtypes and evidence for prevalent serum-dependent infectivity enhancement.

    PubMed Central

    Kostrikis, L G; Cao, Y; Ngai, H; Moore, J P; Ho, D D

    1996-01-01

    Human immunodeficiency virus type 1 (HIV-1) M group strains have been assigned to date to nine distinct genetic subtypes, designated A through I, according to phylogenetic analyses of nucleotide sequences of their env or gag genes. Whether there is any relationship between phylogenetic subtypes and the neutralization serotypes is not clear, yet defining the nature of any such relationship by mathematical means would be of major importance for the development of globally effective HIV-1 vaccines. We have therefore developed a quantitative method to analyze serum neutralization of HIV-1 isolates and to identify HIV-1 neutralization serotypes. This method involves calculations of the neutralization index, N(i), a newly defined parameter derived from plots generated from in vitro neutralization assays, calculations of pairwise serum-virus vector distances, and cluster analyses. We have applied this approach to analyze three independent neutralization matrices involving primary HIV-1 strains and sera from genetic subtypes A, B, C, D, E, F, and I. Detailed serum and HIV-1 isolate cluster analyses have shown that in general, the identified neutralization serotypes do not directly correlate with HIV-1 genetic subtypes. These results suggest that neutralization serotypes do not during natural HIV-1 infection are not governed by antibodies directed against simple epitopes within gp120 monomers. A significant proportion (28%) of 1,213 combinations of sera and HIV-1 isolates caused serum-dependent infectivity enhancement [negative N(i) values] rather than neutralization. We also noted that negative N(i) values tended to correlate better with certain HIV-1 isolates rather than with HIV-1-positive sera. Syncytium-inducing variants of HIV-1 were slightly more likely than non-syncytium-inducing variants to undergo serum-dependent infectivity enhancement, although the latter variants could clearly be susceptible to enhancement. PMID:8523557

  6. Protection of Humanized Mice From Repeated Intravaginal HIV Challenge by Passive Immunization: A Model for Studying the Efficacy of Neutralizing Antibodies In Vivo.

    PubMed

    Deruaz, Maud; Moldt, Brian; Le, Khoa M; Power, Karen A; Vrbanac, Vladimir D; Tanno, Serah; Ghebremichael, Musie S; Allen, Todd M; Tager, Andrew M; Burton, Dennis R; Luster, Andrew D

    2016-08-15

    Humanized mice reconstituted with a human immune system can be mucosally infected with human immunodeficiency virus (HIV), opening up the possibility of studying HIV transmission in a small-animal model. Here we report that passive immunization with the broadly neutralizing antibody b12 protected humanized mice against repetitive intravaginal infection in a dose-dependent manner. In addition, treatment with the antibody PGT126, which is more potent in vitro, was more efficacious in vivo and provided sterilizing protection. Our results demonstrate that humanized mice can be used as a small-animal model to study the efficacy and mechanism of broadly neutralizing antibody protection against HIV acquisition. PMID:27357340

  7. Type-specific neutralization of the human immunodeficiency virus with antibodies to env-encoded synthetic peptides.

    PubMed Central

    Palker, T J; Clark, M E; Langlois, A J; Matthews, T J; Weinhold, K J; Randall, R R; Bolognesi, D P; Haynes, B F

    1988-01-01

    A synthetic peptide (SP-10-IIIB) with an amino acid sequence [Cys-Thr-Arg-Pro-Asn-Asn-Asn-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-Gly-Pro -Pro-Gly-(Tyr); amino acids 303-321] from the human immunodeficiency virus (HIV) isolate human T-cell lymphotropic virus type III (HTLV-III) HTLV-IIIB envelope glycoprotein gp120 was coupled to tetanus toxoid and used to raise goat antibodies to HIV gp120. Goat anti-SP-10-IIIB serum bound to the surface of HTLV-IIIB-infected CEM T cells but not to the surface of HTLV-IIIRF-infected or uninfected CEM T cells. Anti-SP-10-IIIB antibodies also selectively bound to gp120 from lysates of HTLV-IIIB cells in immunoblot assays. Twenty-one percent of sera (28 of 175) from patients seropositive for HIV contained antibodies that reacted with SP-10-IIIB in RIA. Human anti-SP-10-IIIB antibodies affinity purified from acquired immunodeficiency syndrome (AIDS) patient serum bound to HTLV-IIIB-infected cells and immunoprecipitated gp120. Goat antibodies to SP-10-IIIB neutralized HTLV-IIIB (80% neutralization titer of 1/600), inhibited HTLV-IIIB-induced syncytium formation, but did not neutralize HIV isolates HTLV-IIIRF or HTLV-IIIMN or inhibit syncytium formation with these isolates. Also, goat antiserum to an homologous synthetic peptide [SP-10-IIIRF(A), (Cys)-Arg-Lys-Ser-Ile-Thr-Lys-Gly-Pro-Gly-Arg-Val-Ile-Tyr] from gp120 of HIV isolate HTLV-IIIRF inhibited syncytium formation by HTLV-IIIRF, but did not inhibit syncytium formation by HTLV-IIIB or by HTLV-IIIMN. Thus, the amino acid sequences of SP-10-IIIB and SP-10-IIIRF(A) define homologous regions of gp120 that are important in type-specific virus neutralization. The identification of these type-specific neutralizing epitopes should facilitate the design of a polyvalent, synthetic vaccine for AIDS. Images PMID:2450351

  8. Isolation of HIV-1-Neutralizing Mucosal Monoclonal Antibodies from Human Colostrum

    PubMed Central

    Friedman, James; Alam, S. Munir; Shen, Xiaoying; Xia, Shi-Mao; Stewart, Shelley; Anasti, Kara; Pollara, Justin; Fouda, Genevieve G.; Yang, Guang; Kelsoe, Garnett; Ferrari, Guido; Tomaras, Georgia D.; Haynes, Barton F.; Liao, Hua-Xin

    2012-01-01

    Background Generation of potent anti-HIV antibody responses in mucosal compartments is a potential requirement of a transmission-blocking HIV vaccine. HIV-specific, functional antibody responses are present in breast milk, and these mucosal antibody responses may play a role in protection of the majority of HIV-exposed, breastfeeding infants. Therefore, characterization of HIV-specific antibodies produced by B cells in milk could guide the development of vaccines that elicit protective mucosal antibody responses. Methods We isolated B cells from colostrum of an HIV-infected lactating woman with a detectable neutralization response in milk and recombinantly produced and characterized the resulting HIV-1 Envelope (Env)-specific monoclonal antibodies (mAbs). Results The identified HIV-1 Env-specific colostrum mAbs, CH07 and CH08, represent two of the first mucosally-derived anti-HIV antibodies yet to be reported. Colostrum mAb CH07 is a highly-autoreactive, weakly-neutralizing gp140-specific mAb that binds to linear epitopes in the gp120 C5 region and gp41 fusion domain. In contrast, colostrum mAb CH08 is a nonpolyreactive CD4-inducible (CD4i) gp120-specific mAb with moderate breadth of neutralization. Conclusions These novel HIV-neutralizing mAbs isolated from a mucosal compartment provide insight into the ability of mucosal B cell populations to produce functional anti-HIV antibodies that may contribute to protection against virus acquisition at mucosal surfaces. PMID:22624058

  9. Neutral sphingomyelinase 2 modulates cytotoxic effects of protopanaxadiol on different human cancer cells

    PubMed Central

    2013-01-01

    Background Some of ginsenosides, root extracts from Panax ginseng, exert cytotoxicity against cancer cells through disruption of membrane subdomains called lipid rafts. Protopanaxadiol (PPD) exhibits the highest cytotoxic effect among 8 ginsenosides which we evaluated for anti-cancer activity. We investigated if PPD disrupts lipid rafts in its cytotoxic effects and also the possible mechanisms. Methods Eight ginsenosides were evaluated using different cancer cells and cell viability assays. The potent ginsenoside, PPD was investigated for its roles in lipid raft disruption and downstream pathways to apoptosis of cancer cells. Anti-cancer effects of PPD was also investigated in vivo using mouse xenograft model. Results PPD consistently exerts its potent cytotoxicity in 2 cell survival assays using 5 different cancer cell lines. PPD disrupts lipid rafts in different ways from methyl-β-cyclodextrin (MβCD) depleting cholesterol out of the subdomains, since lipid raft proteins were differentially modulated by the saponin. During disruption of lipid rafts, PPD activated neutral sphingomyelinase 2 (nSMase 2) hydrolyzing membrane sphingomyelins into pro-apoptotic intracellular ceramides. Furthermore, PPD demonstrated its anti-cancer activities against K562 tumor cells in mouse xenograft model, confirming its potential as an adjunct or chemotherapeutic agent by itself in vivo. Conclusions This study demonstrates that neutral sphingomyelinase 2 is responsible for the cytotoxicity of PPD through production of apoptotic ceramides from membrane sphingomyelins. Thus neutral sphingomyelinase 2 and its relevant mechanisms may potentially be employed in cancer chemotherapies. PMID:23889969

  10. Protection of Macaques against Pathogenic Simian/Human Immunodeficiency Virus 89.6PD by Passive Transfer of Neutralizing Antibodies

    PubMed Central

    Mascola, John R.; Lewis, Mark G.; Stiegler, Gabriela; Harris, Dawn; VanCott, Thomas C.; Hayes, Deborah; Louder, Mark K.; Brown, Charles R.; Sapan, Christine V.; Frankel, Sarah S.; Lu, Yichen; Robb, Merlin L.; Katinger, Hermann; Birx, Deborah L.

    1999-01-01

    The role of antibody in protection against human immunodeficiency virus (HIV-1) has been difficult to study in animal models because most primary HIV-1 strains do not infect nonhuman primates. Using a chimeric simian/human immunodeficiency virus (SHIV) based on the envelope of a primary isolate (HIV-89.6), we performed passive-transfer experiments in rhesus macaques to study the role of anti-envelope antibodies in protection. Based on prior in vitro data showing neutralization synergy by antibody combinations, we evaluated HIV immune globulin (HIVIG), and human monoclonal antibodies (MAbs) 2F5 and 2G12 given alone, compared with the double combination 2F5/2G12 and the triple combination HIVIG/2F5/2G12. Antibodies were administered 24 h prior to intravenous challenge with the pathogenic SHIV-89.6PD. Six control monkeys displayed high plasma viremia, rapid CD4+-cell decline, and clinical AIDS within 14 weeks. Of six animals given HIVIG/2F5/2G12, three were completely protected; the remaining three animals became SHIV infected but displayed reduced plasma viremia and near normal CD4+-cell counts. One of three monkeys given 2F5/2G12 exhibited only transient evidence of infection; the other two had marked reductions in viral load. All monkeys that received HIVIG, 2F5, or 2G12 alone became infected and developed high-level plasma viremia. However, compared to controls, monkeys that received HIVIG or MAb 2G12 displayed a less profound drop in CD4+ T cells and a more benign clinical course. These data indicate a general correlation between in vitro neutralization and protection and suggest that a vaccine that elicits neutralizing antibody should have a protective effect against HIV-1 infection or disease. PMID:10196297

  11. Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-gp120 interface

    SciTech Connect

    Huang, Jinghe; Kang, Byong H.; Pancera, Marie; Lee, Jeong Hyun; Tong, Tommy; Feng, Yu; Imamichi, Hiromi; Georgiev, Ivelin S.; Chuang, Gwo-Yu; Druz, Aliaksandr; Doria-Rose, Nicole A.; Laub, Leo; Sliepen, Kwinten; van Gils, Marit J.; de la Peña, Alba Torrents; Derking, Ronald; Klasse, Per-Johan; Migueles, Stephen A.; Bailer, Robert T.; Alam, Munir; Pugach, Pavel; Haynes, Barton F.; Wyatt, Richard T.; Sanders, Rogier W.; Binley, James M.; Ward, Andrew B.; Mascola, John R.; Kwong, Peter D.; Connors, Mark

    2015-10-15

    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) <50 μg ml-1. The median IC50 of neutralized viruses was 0.033 μg ml-1, among the most potent thus far described. 35O22 did not bind monomeric forms of Env tested, but did bind the trimeric BG505 SOSIP.664. Mutagenesis and a reconstruction by negative-stain electron microscopy of the Fab in complex with trimer revealed that it bound to a conserved epitope, which stretched across gp120 and gp41. The specificity of 35O22 represents a novel site of vulnerability on HIV Env, which serum analysis indicates to be commonly elicited by natural infection. Binding to this new site of vulnerability may thus be an important complement to current monoclonal-antibody-based approaches to immunotherapies, prophylaxis and vaccine design.

  12. Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-120 interface

    PubMed Central

    Huang, Jinghe; Kang, Byong H.; Pancera, Marie; Lee, Jeong Hyun; Tong, Tommy; Feng, Yu; Georgiev, Ivelin S.; Chuang, Gwo-Yu; Druz, Aliaksandr; Doria-Rose, Nicole A.; Laub, Leo; Sliepen, Kwinten; van Gils, Marit J.; de la Peña, Alba Torrents; Derking, Ronald; Klasse, Per-Johan; Migueles, Stephen A.; Bailer, Robert T.; Alam, Munir; Pugach, Pavel; Haynes, Barton F.; Wyatt, Richard T.; Sanders, Rogier W.; Binley, James M.; Ward, Andrew B.; Mascola, John R.; Kwong, Peter D.; Connors, Mark

    2014-01-01

    The isolation of human monoclonal antibodies (mAbs) is providing important insights regarding the specificities that underlie broad neutralization of HIV-1 (reviewed in1). Here we report a broad and extremely potent HIV-specific mAb, termed 35O22, which binds novel HIV-1 envelope glycoprotein (Env) epitope. 35O22 neutralized 62% of 181 pseudoviruses with an IC50<50 μg/ml. The median IC50 of neutralized viruses was 0.033 μg/ml, among the most potent thus far described. 35O22 did not bind monomeric forms of Env tested, but did bind the trimeric BG505 SOSIP.664. Mutagenesis and a reconstruction by negative-stain electron microscopy of the Fab in complex with trimer revealed it to bind a conserved epitope, which stretched across gp120 and gp41. The specificity of 35O22 represents a novel site of vulnerability on HIV Env, which serum analysis indicates to be commonly elicited by natural infection. Binding to this new site of vulnerability may thus be an important complement to current mAb-based approaches to immunotherapies, prophylaxis, and vaccine design. PMID:25186731

  13. Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-gp120 interface.

    PubMed

    Huang, Jinghe; Kang, Byong H; Pancera, Marie; Lee, Jeong Hyun; Tong, Tommy; Feng, Yu; Imamichi, Hiromi; Georgiev, Ivelin S; Chuang, Gwo-Yu; Druz, Aliaksandr; Doria-Rose, Nicole A; Laub, Leo; Sliepen, Kwinten; van Gils, Marit J; de la Peña, Alba Torrents; Derking, Ronald; Klasse, Per-Johan; Migueles, Stephen A; Bailer, Robert T; Alam, Munir; Pugach, Pavel; Haynes, Barton F; Wyatt, Richard T; Sanders, Rogier W; Binley, James M; Ward, Andrew B; Mascola, John R; Kwong, Peter D; Connors, Mark

    2014-11-01

    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) <50 μg ml(-1). The median IC50 of neutralized viruses was 0.033 μg ml(-1), among the most potent thus far described. 35O22 did not bind monomeric forms of Env tested, but did bind the trimeric BG505 SOSIP.664. Mutagenesis and a reconstruction by negative-stain electron microscopy of the Fab in complex with trimer revealed that it bound to a conserved epitope, which stretched across gp120 and gp41. The specificity of 35O22 represents a novel site of vulnerability on HIV Env, which serum analysis indicates to be commonly elicited by natural infection. Binding to this new site of vulnerability may thus be an important complement to current monoclonal-antibody-based approaches to immunotherapies, prophylaxis and vaccine design. PMID:25186731

  14. The Response of Human Thermal Sensation and Its Prediction to Temperature Step-Change (Cool-Neutral-Cool)

    PubMed Central

    Du, Xiuyuan; Li, Baizhan; Liu, Hong; Yang, Dong; Yu, Wei; Liao, Jianke; Huang, Zhichao; Xia, Kechao

    2014-01-01

    This paper reports on studies of the effect of temperature step-change (between a cool and a neutral environment) on human thermal sensation and skin temperature. Experiments with three temperature conditions were carried out in a climate chamber during the period in winter. Twelve subjects participated in the experiments simulating moving inside and outside of rooms or cabins with air conditioning. Skin temperatures and thermal sensation were recorded. Results showed overshoot and asymmetry of TSV due to the step-change. Skin temperature changed immediately when subjects entered a new environment. When moving into a neutral environment from cool, dynamic thermal sensation was in the thermal comfort zone and overshoot was not obvious. Air-conditioning in a transitional area should be considered to limit temperature difference to not more than 5°C to decrease the unacceptability of temperature step-change. The linear relationship between thermal sensation and skin temperature or gradient of skin temperature does not apply in a step-change environment. There is a significant linear correlation between TSV and Qloss in the transient environment. Heat loss from the human skin surface can be used to predict dynamic thermal sensation instead of the heat transfer of the whole human body. PMID:25136808

  15. Cryo-EM structures elucidate neutralizing mechanisms of anti-chikungunya human monoclonal antibodies with therapeutic activity

    PubMed Central

    Long, Feng; Fong, Rachel H.; Austin, Stephen K.; Chen, Zhenguo; Klose, Thomas; Fokine, Andrei; Liu, Yue; Porta, Jason; Sapparapu, Gopal; Akahata, Wataru; Doranz, Benjamin J.; Crowe, James E.; Diamond, Michael S.; Rossmann, Michael G.

    2015-01-01

    Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes severe acute and chronic disease in humans. Although highly inhibitory murine and human monoclonal antibodies (mAbs) have been generated, the structural basis of their neutralizing activity remains poorly characterized. Here, we determined the cryo-EM structures of chikungunya virus-like particles complexed with antibody fragments (Fab) of two highly protective human mAbs, 4J21 and 5M16, that block virus fusion with host membranes. Both mAbs bind primarily to sites within the A and B domains, as well as to the B domain’s β-ribbon connector of the viral glycoprotein E2. The footprints of these antibodies on the viral surface were consistent with results from loss-of-binding studies using an alanine scanning mutagenesis-based epitope mapping approach. The Fab fragments stabilized the position of the B domain relative to the virus, particularly for the complex with 5M16. This finding is consistent with a mechanism of neutralization in which anti-CHIKV mAbs that bridge the A and B domains impede movement of the B domain away from the underlying fusion loop on the E1 glycoprotein and therefore block the requisite pH-dependent fusion of viral and host membranes. PMID:26504196

  16. International Technology Transfer of a GCLP-Compliant HIV-1 Neutralizing Antibody Assay for Human Clinical Trials

    PubMed Central

    Todd, Christopher A.; Greene, Kelli M.; Montefiori, David C.; Sarzotti-Kelsoe, Marcella

    2012-01-01

    The Collaboration for AIDS Vaccine Discovery/Comprehensive Antibody – Vaccine Immune Monitoring Consortium (CAVD/CA-VIMC) assisted an international network of laboratories in transferring a validated assay used to judge HIV-1 vaccine immunogenicity in compliance with Good Clinical Laboratory Practice (GCLP) with the goal of adding quality to the conduct of endpoint assays for Human Immunodeficiency Virus I (HIV-1) vaccine human clinical trials. Eight Regional Laboratories in the international setting (Regional Laboratories), many located in regions where the HIV-1 epidemic is most prominent, were selected to implement the standardized, GCLP-compliant Neutralizing Antibody Assay for HIV-1 in TZM-bl Cells (TZM-bl NAb Assay). Each laboratory was required to undergo initial training and implementation of the immunologic assay on-site and then perform partial assay re-validation, competency testing, and undergo formal external audits for GCLP compliance. Furthermore, using a newly established external proficiency testing program for the TZM-bl NAb Assay has allowed the Regional Laboratories to assess the comparability of assay results at their site with the results of neutralizing antibody assays performed around the world. As a result, several of the CAVD/CA-VIMC Regional Laboratories are now in the process of conducting or planning to conduct the GCLP-compliant TZM-bl NAb Assay as an indicator of vaccine immunogenicity for ongoing human clinical trials. PMID:22303476

  17. Cryo-EM structures elucidate neutralizing mechanisms of anti-chikungunya human monoclonal antibodies with therapeutic activity.

    PubMed

    Long, Feng; Fong, Rachel H; Austin, Stephen K; Chen, Zhenguo; Klose, Thomas; Fokine, Andrei; Liu, Yue; Porta, Jason; Sapparapu, Gopal; Akahata, Wataru; Doranz, Benjamin J; Crowe, James E; Diamond, Michael S; Rossmann, Michael G

    2015-11-10

    Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes severe acute and chronic disease in humans. Although highly inhibitory murine and human monoclonal antibodies (mAbs) have been generated, the structural basis of their neutralizing activity remains poorly characterized. Here, we determined the cryo-EM structures of chikungunya virus-like particles complexed with antibody fragments (Fab) of two highly protective human mAbs, 4J21 and 5M16, that block virus fusion with host membranes. Both mAbs bind primarily to sites within the A and B domains, as well as to the B domain's β-ribbon connector of the viral glycoprotein E2. The footprints of these antibodies on the viral surface were consistent with results from loss-of-binding studies using an alanine scanning mutagenesis-based epitope mapping approach. The Fab fragments stabilized the position of the B domain relative to the virus, particularly for the complex with 5M16. This finding is consistent with a mechanism of neutralization in which anti-CHIKV mAbs that bridge the A and B domains impede movement of the B domain away from the underlying fusion loop on the E1 glycoprotein and therefore block the requisite pH-dependent fusion of viral and host membranes. PMID:26504196

  18. The response of human thermal sensation and its prediction to temperature step-change (cool-neutral-cool).

    PubMed

    Du, Xiuyuan; Li, Baizhan; Liu, Hong; Yang, Dong; Yu, Wei; Liao, Jianke; Huang, Zhichao; Xia, Kechao

    2014-01-01

    This paper reports on studies of the effect of temperature step-change (between a cool and a neutral environment) on human thermal sensation and skin temperature. Experiments with three temperature conditions were carried out in a climate chamber during the period in winter. Twelve subjects participated in the experiments simulating moving inside and outside of rooms or cabins with air conditioning. Skin temperatures and thermal sensation were recorded. Results showed overshoot and asymmetry of TSV due to the step-change. Skin temperature changed immediately when subjects entered a new environment. When moving into a neutral environment from cool, dynamic thermal sensation was in the thermal comfort zone and overshoot was not obvious. Air-conditioning in a transitional area should be considered to limit temperature difference to not more than 5°C to decrease the unacceptability of temperature step-change. The linear relationship between thermal sensation and skin temperature or gradient of skin temperature does not apply in a step-change environment. There is a significant linear correlation between TSV and Qloss in the transient environment. Heat loss from the human skin surface can be used to predict dynamic thermal sensation instead of the heat transfer of the whole human body. PMID:25136808

  19. Development of a Triple-Color Pseudovirion-Based Assay to Detect Neutralizing Antibodies against Human Papillomavirus

    PubMed Central

    Nie, Jianhui; Liu, Yangyang; Huang, Weijin; Wang, Youchun

    2016-01-01

    Pseudovirion-based neutralization assay is considered the gold standard method for evaluating the immune response to human papillomavirus (HPV) vaccines. In this study, we developed a multicolor neutralization assay to simultaneously detect the neutralizing antibodies against different HPV types. FluoroSpot was used to interpret the fluorescent protein expression instead of flow cytometry. The results of FluoroSpot and flow cytometry showed good consistency, with R2 > 0.98 for the log-transformed IC50 values. Regardless of the reporter color, the single-, dual-, and triple-color neutralization assays reported identical results for the same samples. In low-titer samples from naturally HPV-infected individuals, there was strong agreement between the single- and triple-color assays, with kappa scores of 0.92, 0.89, and 0.96 for HPV16, HPV18, and HPV58, respectively. Good reproducibility was observed for the triple-color assay, with coefficients of variation of 2.0%–41.5% within the assays and 8.3%–36.2% between the assays. Three triple-color systems, HPV16-18-58, HPV6-33-45, and HPV11-31-52, were developed that could evaluate the immunogenicity of a nonavalent vaccine in three rounds of the assay. With the advantages of an easy-to-use procedure and less sample consumption, the multiple-color assay is more suitable than classical assays for large sero-epidemiological studies and clinical trials and is more amenable to automation. PMID:27120611

  20. Development of a Triple-Color Pseudovirion-Based Assay to Detect Neutralizing Antibodies against Human Papillomavirus.

    PubMed

    Nie, Jianhui; Liu, Yangyang; Huang, Weijin; Wang, Youchun

    2016-04-01

    Pseudovirion-based neutralization assay is considered the gold standard method for evaluating the immune response to human papillomavirus (HPV) vaccines. In this study, we developed a multicolor neutralization assay to simultaneously detect the neutralizing antibodies against different HPV types. FluoroSpot was used to interpret the fluorescent protein expression instead of flow cytometry. The results of FluoroSpot and flow cytometry showed good consistency, with R² > 0.98 for the log-transformed IC50 values. Regardless of the reporter color, the single-, dual-, and triple-color neutralization assays reported identical results for the same samples. In low-titer samples from naturally HPV-infected individuals, there was strong agreement between the single- and triple-color assays, with kappa scores of 0.92, 0.89, and 0.96 for HPV16, HPV18, and HPV58, respectively. Good reproducibility was observed for the triple-color assay, with coefficients of variation of 2.0%-41.5% within the assays and 8.3%-36.2% between the assays. Three triple-color systems, HPV16-18-58, HPV6-33-45, and HPV11-31-52, were developed that could evaluate the immunogenicity of a nonavalent vaccine in three rounds of the assay. With the advantages of an easy-to-use procedure and less sample consumption, the multiple-color assay is more suitable than classical assays for large sero-epidemiological studies and clinical trials and is more amenable to automation. PMID:27120611

  1. Identification of amino acids in the V3 region of gp120 critical for virus neutralization by human HIV-1-specific antibodies.

    PubMed Central

    Broliden, P A; Mäkitalo, B; Akerblom, L; Rosen, J; Broliden, K; Utter, G; Jondal, M; Norrby, E; Wahren, B

    1991-01-01

    The importance of the dependence on single amino acids in the V3 region of HIV-1 gp120 was evaluated for virus neutralization and antibody-dependent cellular cytotoxicity (ADCC). Synthetic overlapping 15-mer peptides and a set of omission peptides covering amino acids 301-317 were used. Sera from 29 HIV-1-infected individuals at different stages of disease were tested for neutralization, ADCC and specific IgG reactivity. Six HIV-1 neutralizing monoclonal antibodies (mAb) acted as controls. All mAb reacted with a region (amino acids 304-318) of gp120, previously shown to induce neutralizing antibodies. The amino acids essential for reactivity were identified to be within the sequence GPGR (amino acids 312-315). The importance of this region for occurrence of neutralizing antibodies in infected humans was investigated using the same set of peptides. Out of 29 individuals, 21 were found to have neutralizing antibodies in titres between 100 and 1000. Among the neutralization-positive sera, 17/21 (81%) reacted with amino acids 304-318, compared with only one of eight sera (13%) negative in neutralization. When any of the four amino acids G, P, G or R were deleted, the seroreactivity decreased considerably. The conserved sequence GPGR was therefore considered to be the most important for neutralization in this region in human sera as well. Thus, the conserved sequence GPGR in the V3 region of gp120 is critical for virus neutralization by human HIV-1-specific antibodies. PMID:1916888

  2. Canine distemper virus neutralization activity is low in human serum and it is sensitive to an amino acid substitution in the hemagglutinin protein.

    PubMed

    Zhang, Xinsheng; Wallace, Olivia L; Domi, Arban; Wright, Kevin J; Driscoll, Jonathan; Anzala, Omu; Sanders, Eduard J; Kamali, Anatoli; Karita, Etienne; Allen, Susan; Fast, Pat; Gilmour, Jill; Price, Matt A; Parks, Christopher L

    2015-08-01

    Serum was analyzed from 146 healthy adult volunteers in eastern Africa to evaluate measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb) prevalence and potency. MV plaque reduction neutralization test (PRNT) results indicated that all sera were positive for MV nAbs. Furthermore, the 50% neutralizing dose (ND50) for the majority of sera corresponded to antibody titers induced by MV vaccination. CDV nAbs titers were low and generally were detected in sera with high MV nAb titers. A mutant CDV was generated that was less sensitive to neutralization by human serum. The mutant virus genome had 10 nucleotide substitutions, which coded for single amino acid substitutions in the fusion (F) and hemagglutinin (H) glycoproteins and two substitutions in the large polymerase (L) protein. The H substitution occurred in a conserved region involved in receptor interactions among morbilliviruses, implying that this region is a target for cross-reactive neutralizing antibodies. PMID:25880113

  3. Crystal structure of a human rhinovirus neutralizing antibody complexed with a peptide derived from viral capsid protein VP2.

    PubMed Central

    Tormo, J; Blaas, D; Parry, N R; Rowlands, D; Stuart, D; Fita, I

    1994-01-01

    The three-dimensional structure of the complex between the Fab fragment of an anti-human rhinovirus neutralizing antibody (8F5) and a cross-reactive synthetic peptide from the viral capsid protein VP2 has been determined at 2.5 A resolution by crystallographic methods. The refinement is presently at an R factor of 0.18 and the antigen-binding site and viral peptide are well defined. The peptide antigen adopts a compact fold by two tight turns and interacts through hydrogen bonds, some with ionic character, and van der Waals contacts with antibody residues from the six hypervariable loops as well as several framework amino acids. The conformation adopted by the peptide is closely related to the corresponding region of the viral protein VP2 on the surface of human rhinovirus 1A whose three-dimensional structure is known. Implications for the cross-reactivity between peptides and the viral capsid are discussed. The peptide-antibody interactions, together with the analysis of mutant viruses that escape neutralization by 8F5 suggest two different mechanisms for viral escape. The comparison between the complexed and uncomplexed antibody structures shows important conformational rearrangements, especially in the hypervariable loops of the heavy chain. Thus, it constitutes a clear example of the 'induced fit' molecular recognition mechanism. Images PMID:8194515

  4. Mucosal but Not Parenteral Immunization with Purified Human Papillomavirus Type 16 Virus-Like Particles Induces Neutralizing Titers of Antibodies throughout the Estrous Cycle of Mice

    PubMed Central

    Nardelli-Haefliger, Denise; Roden, Richard; Balmelli, Carole; Potts, Alexandra; Schiller, John; De Grandi, Pierre

    1999-01-01

    We have recently shown that nasal immunization of anesthetized mice with human papillomavirus type 16 (HPV16) virus-like particles (VLPs) is highly effective at inducing both neutralizing immunoglobulin A (IgA) and IgG in genital secretions, while parenteral immunization induced only neutralizing IgG. Our data also demonstrated that both isotypes are similarly neutralizing according to an in vitro pseudotyped neutralization assay. However, it is known that various amounts of IgA and IgG are produced in genital secretions along the estrous cycle. Therefore, we have investigated how this variation influences the amount of HPV16 neutralizing antibodies induced after immunization with VLPs. We have compared parenteral and nasal protocols of vaccination with daily samplings of genital secretions of mice. Enzyme-linked immunosorbent assay analysis showed that total IgA and IgG inversely varied along the estrous cycle, with the largest amounts of IgA in proestrus-estrus and the largest amount of IgG in diestrus. This resulted in HPV16 neutralizing titers of IgG only being achieved during diestrus upon parenteral immunization. In contrast, nasal vaccination induced neutralizing titers of IgA plus IgG throughout the estrous cycle, as confirmed by in vitro pseudotyped neutralization assays. Our data suggest that mucosal immunization might be more efficient than parenteral immunization at inducing continuous protection of the female genital tract. PMID:10516071

  5. Putative rhesus macaque germline predecessors of human broadly HIV-neutralizing antibodies: differences from the human counterparts and implications for HIV-1 vaccine development

    PubMed Central

    Yuan, Tingting; Li, Jingjing; Zhang, Yu; Wang, Yanping; Streaker, Emily; Dimitrov, Dimiter S.; Zhang, Mei-Yun

    2011-01-01

    Broadly neutralizing antibodies (bnAbs) are likely to be a key component of protective immunity conferred by an effective HIV-1 vaccine. We and others have reported that putative human germline predecessors of known human bnAbs lack measurable binding to HIV-1 envelope glycoproteins (Env), which could be a new challenge for eliciting human bnAbs. Rhesus macaques have been used as nonhuman primate models for testing vaccine candidates, but little is known about their germline Abs. Here we show the similarities and differences between putative rhesus macaque and human germline predecessors and possible intermediate antibodies of one of the best characterized bnAbs, b12. Similar to the human counterpart, a putative rhesus macaque b12 germline antibody lacks measurable binding to HIV-1 Envs, suggesting that initiation of somatic maturation of rhesus macaque germline b12 predecessor may also be a challenge. However, differences in sequence characteristics and binding properties between macaque and human b12 germline and intermediate antibodies suggest that the two germline predecessors may undergo different maturation pathways in rhesus macaques and in humans. These results indicate that immunogens that could initiate the immune responses and drive somatic mutations leading to elicitation of b12 or b12-like bnAbs in rhesus macaques and in humans are likely to be different. This has important implications for HIV-1 vaccine development. PMID:21807049

  6. Broadly neutralizing human monoclonal JC polyomavirus VP1–specific antibodies as candidate therapeutics for progressive multifocal leukoencephalopathy

    PubMed Central

    Jelcic, Ivan; Combaluzier, Benoit; Jelcic, Ilijas; Faigle, Wolfgang; Senn, Luzia; Reinhart, Brenda J.; Ströh, Luisa; Nitsch, Roger M.; Stehle, Thilo; Sospedra, Mireia; Grimm, Jan; Martin, Roland

    2016-01-01

    In immunocompromised individuals, JC polyomavirus (JCPyV) may mutate and gain access to the central nervous system resulting in progressive multifocal leukoencephalopathy (PML), an often fatal opportunistic infection for which no treatments are currently available. Despite recent progress, the contribution of JCPyV-specific humoral immunity to controlling asymptomatic infection throughout life and to eliminating JCPyV from the brain is poorly understood. We examined antibody responses against JCPyV major capsid protein VP1 (viral protein 1) variants in the serum and cerebrospinal fluid (CSF) of healthy donors (HDs), JCPyV-positive multiple sclerosis patients treated with the anti-VLA-4 monoclonal antibody natalizumab (NAT), and patients with NAT-associated PML. Before and during PML, CSF antibody responses against JCPyV VP1 variants show “recognition holes”; however, upon immune reconstitution, CSF antibody titers rise, then recognize PML-associated JCPyV VP1 variants, and may be involved in elimination of the virus. We therefore reasoned that the memory B cell repertoire of individuals who recovered from PML could be a source for the molecular cloning of broadly neutralizing antibodies for passive immunization. We generated a series of memory B cell-derived JCPyV VP1-specific human monoclonal antibodies from HDs and a patient with NAT-associated PML-immune reconstitution inflammatory syndrome (IRIS). These antibodies exhibited diverse binding affinity, cross-reactivity with the closely related BK polyomavirus, recognition of PML-causing VP1 variants, and JCPyV neutralization. Almost all antibodies with exquisite specificity for JCPyV, neutralizing activity, recognition of all tested JCPyV PML variants, and high affinity were derived from one patient who had recovered from PML. These antibodies are promising drug candidates for the development of a treatment of PML. PMID:26400911

  7. Use of monoclonal antibodies to identify four neutralization immunogens on a common cold picornavirus, human rhinovirus 14.

    PubMed Central

    Sherry, B; Mosser, A G; Colonno, R J; Rueckert, R R

    1986-01-01

    A collection of 35 mouse monoclonal antibodies, raised against human rhinovirus 14 (HRV-14), was used to isolate 62 neutralization-resistant mutants. When cross-tested against the antibodies in a neutralization assay, the mutants fell into four antigenic groups, here called neutralization immunogens: NIm-IA, -IB, -II, and -III. Sequencing the mutant RNA in segments corresponding to serotype-variable regions revealed that the amino acid substitutions segregated into clusters, which correlated exactly with the immunogenic groups (NIm-IA mutants at VP1 amino acid residue 91 or 95; NIm-II mutants at VP2 residue 158, 159, 161, or 162; NIm-III mutants at VP3 residue 72, 75, or 78; and NIm-IB mutants at two sites, either VP1 residue 83 or 85, or residue 138 or 139). Examination of the three-dimensional structure of the virus (M. G. Rossmann, E. Arnold, J. W. Erickson, E. A. Frankenberger, J. P. Griffith, H.-J. Hecht, J. E. Johnson, G. Kamer, M. Luo, A. G. Mosser, R. R. Rueckert, B. Sherry, and G. Vriend, Nature [London], 317:145-153, 1985) revealed that each of the substitution clusters formed a protrusion from the virus surface, and the side chains of the substituted amino acids pointed outward. Moreover, four of the amino acid substitutions, which initially appeared to be anomalous because they were encoded well outside the cluster groups, could be traced to surface positions immediately adjacent to the appropriate viral protrusions. We conclude that three of the four antigens, NIm-IB, -II, and -III, are discontinuous. Thus, the amino acid substitutions in all 62 mutants fell within the proposed immunogenic sites; there was no evidence for alteration of any antigenic site by a distal mutation. Images PMID:2416951

  8. Broadly neutralizing human monoclonal JC polyomavirus VP1-specific antibodies as candidate therapeutics for progressive multifocal leukoencephalopathy.

    PubMed

    Jelcic, Ivan; Combaluzier, Benoit; Jelcic, Ilijas; Faigle, Wolfgang; Senn, Luzia; Reinhart, Brenda J; Ströh, Luisa; Nitsch, Roger M; Stehle, Thilo; Sospedra, Mireia; Grimm, Jan; Martin, Roland

    2015-09-23

    In immunocompromised individuals, JC polyomavirus (JCPyV) may mutate and gain access to the central nervous system resulting in progressive multifocal leukoencephalopathy (PML), an often fatal opportunistic infection for which no treatments are currently available. Despite recent progress, the contribution of JCPyV-specific humoral immunity to controlling asymptomatic infection throughout life and to eliminating JCPyV from the brain is poorly understood. We examined antibody responses against JCPyV major capsid protein VP1 (viral protein 1) variants in the serum and cerebrospinal fluid (CSF) of healthy donors (HDs), JCPyV-positive multiple sclerosis patients treated with the anti-VLA-4 monoclonal antibody natalizumab (NAT), and patients with NAT-associated PML. Before and during PML, CSF antibody responses against JCPyV VP1 variants show "recognition holes"; however, upon immune reconstitution, CSF antibody titers rise, then recognize PML-associated JCPyV VP1 variants, and may be involved in elimination of the virus. We therefore reasoned that the memory B cell repertoire of individuals who recovered from PML could be a source for the molecular cloning of broadly neutralizing antibodies for passive immunization. We generated a series of memory B cell-derived JCPyV VP1-specific human monoclonal antibodies from HDs and a patient with NAT-associated PML-immune reconstitution inflammatory syndrome (IRIS). These antibodies exhibited diverse binding affinity, cross-reactivity with the closely related BK polyomavirus, recognition of PML-causing VP1 variants, and JCPyV neutralization. Almost all antibodies with exquisite specificity for JCPyV, neutralizing activity, recognition of all tested JCPyV PML variants, and high affinity were derived from one patient who had recovered from PML. These antibodies are promising drug candidates for the development of a treatment of PML. PMID:26400911

  9. A Neutralizing Human Monoclonal Antibody Protects against Lethal Disease in a New Ferret Model of Acute Nipah Virus Infection

    PubMed Central

    Bossart, Katharine N.; Zhu, Zhongyu; Middleton, Deborah; Klippel, Jessica; Crameri, Gary; Bingham, John; McEachern, Jennifer A.; Green, Diane; Hancock, Timothy J.; Chan, Yee-Peng; Hickey, Andrew C.; Dimitrov, Dimiter S.; Wang, Lin-Fa; Broder, Christopher C.

    2009-01-01

    Nipah virus is a broadly tropic and highly pathogenic zoonotic paramyxovirus in the genus Henipavirus whose natural reservoirs are several species of Pteropus fruit bats. Nipah virus has repeatedly caused outbreaks over the past decade associated with a severe and often fatal disease in humans and animals. Here, a new ferret model of Nipah virus pathogenesis is described where both respiratory and neurological disease are present in infected animals. Severe disease occurs with viral doses as low as 500 TCID50 within 6 to 10 days following infection. The underlying pathology seen in the ferret closely resembles that seen in Nipah virus infected humans, characterized as a widespread multisystemic vasculitis, with virus replicating in highly vascular tissues including lung, spleen and brain, with recoverable virus from a variety of tissues. Using this ferret model a cross-reactive neutralizing human monoclonal antibody, m102.4, targeting the henipavirus G glycoprotein was evaluated in vivo as a potential therapeutic agent. All ferrets that received m102.4 ten hours following a high dose oral-nasal Nipah virus challenge were protected from disease while all controls died. This study is the first successful post-exposure passive antibody therapy for Nipah virus using a human monoclonal antibody. PMID:19888339

  10. Vaccine-Derived Neutralizing Antibodies to the Human Cytomegalovirus gH/gL Pentamer Potently Block Primary Cytotrophoblast Infection

    PubMed Central

    Chiuppesi, Flavia; Wussow, Felix; Johnson, Erica; Bian, Chao; Zhuo, Meng; Rajakumar, Augustine; Barry, Peter A.; Britt, William J.; Chakraborty, Rana

    2015-01-01

    ABSTRACT Human cytomegalovirus (HCMV) elicits neutralizing antibodies (NAb) of various potencies and cell type specificities to prevent HCMV entry into fibroblasts (FB) and epithelial/endothelial cells (EpC/EnC). NAb targeting the major essential envelope glycoprotein complexes gB and gH/gL inhibit both FB and EpC/EnC entry. In contrast to FB infection, HCMV entry into EpC/EnC is additionally blocked by extremely potent NAb to conformational epitopes of the gH/gL/UL128/130/131A pentamer complex (PC). We recently developed a vaccine concept based on coexpression of all five PC subunits by a single modified vaccinia virus Ankara (MVA) vector, termed MVA-PC. Vaccination of mice and rhesus macaques with MVA-PC resulted in a high titer and sustained NAb that blocked EpC/EnC infection and lower-titer NAb that inhibited FB entry. However, antibody function responsible for the neutralizing activity induced by the MVA-PC vaccine is uncharacterized. Here, we demonstrate that MVA-PC elicits NAb with cell type-specific neutralization potency and antigen recognition pattern similar to human NAb targeting conformational and linear epitopes of the UL128/130/131A subunits or gH. In addition, we show that the vaccine-derived PC-specific NAb are significantly more potent than the anti-gH NAb to prevent HCMV spread in EpC and infection of human placental cytotrophoblasts, cell types thought to be of critical importance for HCMV transmission to the fetus. These findings further validate MVA-PC as a clinical vaccine candidate to elicit NAb that resembles those induced during HCMV infection and provide valuable insights into the potency of PC-specific NAb to interfere with HCMV cell-associated spread and infection of key placental cells. IMPORTANCE As a consequence of the leading role of human cytomegalovirus (HCMV) in causing permanent birth defects, developing a vaccine against HCMV has been assigned a major public health priority. We have recently introduced a vaccine strategy based

  11. Generation of a neutralization-resistant CCR5 tropic simian/human immunodeficiency virus (SHIV-MK38) molecular clone, a derivative of SHIV-89.6.

    PubMed

    Ishida, Yuki; Yoneda, Mai; Otsuki, Hiroyuki; Watanabe, Yuji; Kato, Fumihiro; Matsuura, Kanako; Kikukawa, Minako; Matsushita, Shuzo; Hishiki, Takayuki; Igarashi, Tatsuhiko; Miura, Tomoyuki

    2016-05-01

    Previously, we reported that a new genetically diverse CCR5 (R5) tropic simian/human immunodeficiency virus (SHIV-MK38) adapted to rhesus monkeys became more neutralization resistant to SHIV-infected plasma than did the parental SHIV-KS661 clone. Here, to clarify the significance of the neutralization-resistant phenotype of SHIV in a macaque model, we initially investigated the precise neutralization phenotype of the SHIVs, including SHIV-MK38 molecular clones, using SHIV-MK38-infected plasma, a pooled plasma of human immunodeficiency virus (HIV)-infected individuals, soluble CD4 and anti-HIV-1 neutralizing mAbs, the epitopes of which were known. The results show that SHIV-KS661 had tier 1 neutralization sensitivity, but monkey-adapted R5 tropic SHIV-MK38 acquired neutralization resistance similar to that of tier 2 or 3 as a clone virus. Sequence analysis of the env gene suggested that the neutralization-resistant phenotype of SHIV-MK38 was acquired by conformational changes in Env associated with the net charge and potential N-linked glycosylation sites. To examine the relationship between neutralization phenotype and stably persistent infection in monkeys, we performed in vivo rectal inoculation experiments using a SHIV-MK38 molecular clone. The results showed that one of three rhesus monkeys exhibited durable infection with a plasma viral load of 105 copies ml- 1 despite the high antibody responses that occurred in the host. Whilst further improvements are required in the development of a challenge virus, it will be useful to generate a neutralization-resistant R5 tropic molecular clone of the SHIV-89.6 lineage commonly used for vaccine development - a result that can be used to explore the foundation of AIDS pathogenesis. PMID:26850058

  12. HCMV Infection of Human Trophoblast Progenitor Cells of the Placenta Is Neutralized by a Human Monoclonal Antibody to Glycoprotein B and Not by Antibodies to the Pentamer Complex

    PubMed Central

    Zydek, Martin; Petitt, Matthew; Fang-Hoover, June; Adler, Barbara; Kauvar, Lawrence M.; Pereira, Lenore; Tabata, Takako

    2014-01-01

    Human cytomegalovirus (HCMV) is the major viral cause of congenital infection and birth defects. Primary maternal infection often results in virus transmission, and symptomatic babies can have permanent neurological deficiencies and deafness. Congenital infection can also lead to intrauterine growth restriction, a defect in placental transport. HCMV replicates in primary cytotrophoblasts (CTBs), the specialized cells of the placenta, and inhibits differentiation/invasion. Human trophoblast progenitor cells (TBPCs) give rise to the mature cell types of the chorionic villi, CTBs and multi-nucleated syncytiotrophoblasts (STBs). Here we report that TBPCs are fully permissive for pathogenic and attenuated HCMV strains. Studies with a mutant virus lacking a functional pentamer complex (gH/gL/pUL128-131A) showed that virion entry into TBPCs is independent of the pentamer. In addition, infection is blocked by a potent human neutralizing monoclonal antibody (mAb), TRL345, reactive with glycoprotein B (gB), but not mAbs to the pentamer proteins pUL130/pUL131A. Functional studies revealed that neutralization of infection preserved the capacity of TBPCs to differentiate and assemble into trophospheres composed of CTBs and STBs in vitro. Our results indicate that mAbs to gB protect trophoblast progenitors of the placenta and could be included in antibody treatments developed to suppress congenital infection and prevent disease. PMID:24651029

  13. Modeling neutralization of Shiga 2 toxin by A-and B-subunit-specific human monoclonal antibodies.

    PubMed

    Skakauskas, Vladas; Katauskis, Pranas

    2016-06-01

    A mathematical model for Shiga 2 toxin neutralization by A-and B-subunit-specific human monoclonal antibodies initially delivered in the extracellular domain is presented, taking into account toxin and antibodies interaction in the extracellular domain, diffusion of toxin, antibodies, and their reaction products toward the cell, the receptor-mediated toxin and complex composed of toxin and antibody to A-subunit internalization from the extracellular into the intracellular medium and excretion of this complex back to the extracellular environment via recycling endosomal carriers. The retrograde transport of the intact toxin to the endoplasmic reticulum and its anterograde movement back to the vicinity of the plasma membrane with its subsequent exocytotic removal to the extracellular space via the secretory vesicle pathway is also taken into account. The model is composed of a set of coupled PDEs. A mathematical model based on a system of ODEs for Shiga 2 toxin neutralization by antibodies in the absence of cell is also studied. Both PDE and ODE systems are solved numerically. Numerical results are illustrated by figures and discussed. PMID:27155978

  14. Chlamydial Plasmid-Encoded Virulence Factor Pgp3 Neutralizes the Antichlamydial Activity of Human Cathelicidin LL-37

    PubMed Central

    Hou, Shuping; Dong, Xiaohua; Yang, Zhangsheng; Li, Zhongyu; Liu, Quanzhong

    2015-01-01

    Chlamydia trachomatis infection in the lower genital tract can ascend to and cause pathologies in the upper genital tract, potentially leading to severe complications, such as tubal infertility. However, chlamydial organisms depleted of plasmid or deficient in the plasmid-encoded Pgp3 are attenuated in ascending infection and no longer are able to induce the upper genital tract pathologies, indicating a significant role of Pgp3 in chlamydial pathogenesis. We now report that C. trachomatis Pgp3 can neutralize the antichlamydial activity of human cathelicidin LL-37, a host antimicrobial peptide secreted by both genital tract epithelial cells and infiltrating neutrophils. Pgp3 bound to and formed stable complexes with LL-37. We further showed that the middle region of Pgp3 (Pgp3m) was responsible for both the binding to and neutralization of LL-37, suggesting that Pgp3m can be targeted for attenuating chlamydial pathogenicity or developed for blocking LL-37-involved non-genital-tract pathologies, such as rosacea and psoriasis. Thus, the current study has provided significant information for both understanding the mechanisms of chlamydial pathogenesis and developing novel therapeutic agents. PMID:26416907

  15. Characterization of the cDNA of a broadly reactive neutralizing human anti-gp120 monoclonal antibody.

    PubMed Central

    Marasco, W A; Bagley, J; Zani, C; Posner, M; Cavacini, L; Haseltine, W A; Sodroski, J

    1992-01-01

    The F105 mAb, identified in an HIV-1-infected individual, binds to a discontinuous epitope on the HIV-1 gp120 envelope glycoprotein, blocks the binding of gp120 to the CD4 viral receptor, and neutralizes a broad range of HIV-1 isolates. This study reports the primary nucleotide and deduced amino acid sequences of the rearranged heavy and light chains of the mAb F105. This IgG1k mAb uses a VH gene member of the VH4 gene family (V71-4) and is productively rearranged with a D-D fusion product of the dlr4 and da4 germline DH genes and the JH5 gene. This rearranged heavy chain gene expresses the VH4-HV2a idiotope, which is seen in human monoclonal IgM cold agglutinins. The F105 Vk appears to be derived from the Humvk325 germline gene and is rearranged with a Jk2 gene. For both chains, the mutational pattern in the rearranged VH and VL genes is indicative of an antigen-driven process. These studies show that production of a broadly neutralizing anti-HIV-1 antibody that recognizes determinants within the CD4 recognition site of the envelope glycoprotein is achieved by rearrangement of the V71-4 and Humvk325 germline variable region genes along with selected individual point mutations in the rearranged genes. PMID:1401079

  16. Partitioning and levels of neutral organochlorine compounds in human serum, blood cells, and adipose and liver tissue.

    PubMed

    Mussalo-Rauhamaa, H

    1991-04-15

    Concentrations of neutral organochlorine compounds (OCs) in blood compartments and adipose tissue were determined in three groups of subjects. In 12 healthy volunteers a positive correlation was found between DDT residue levels in paired serum/adipose tissue samples when the concentrations were calculated on a fat-weight basis (r = 0.74, p less than 0.05); positive correlations were also found for PCB and HCB when the calculations were based on a wet-weight basis (r = 0.68, p less than 0.01; r = 0.69, p less than 0.01). For lindane the correlation coefficient for paired serum/adipose tissue samples was -0.94 (p less than 0.01). The association between adipose tissue and blood cells was weaker than that obtained for serum. These readily obtainable samples are adequate for estimating, or monitoring, the total burden of neutral organochlorines in adipose tissue, especially in cases of low chronic exposure, such as those found in epidemiological studies. In paired liver-adipose tissue samples from 23 autopsy cases, no correlation was found either on a wet- or fat-weight basis. In 131 adults resident in southern and eastern Finland the concentration medians for serum were 1.8, 2.0 and 0.3 ng g-1 wet wt for DDT compounds. PCB and HCB, respectively. This study indicates that monitoring of fat/serum ratios may provide, with tissue concentrations, more information about human exposure to OCs. PMID:1909054

  17. Molecular Signatures of Hemagglutinin Stem-Directed Heterosubtypic Human Neutralizing Antibodies against Influenza A Viruses

    PubMed Central

    Avnir, Yuval; Tallarico, Aimee S.; Zhu, Quan; Bennett, Andrew S.; Connelly, Gene; Sheehan, Jared; Sui, Jianhua; Fahmy, Amr; Huang, Chiung-yu; Cadwell, Greg; Bankston, Laurie A.; McGuire, Andrew T.; Stamatatos, Leonidas; Wagner, Gerhard; Liddington, Robert C.; Marasco, Wayne A.

    2014-01-01

    Recent studies have shown high usage of the IGHV1-69 germline immunoglobulin gene for influenza hemagglutinin stem-directed broadly-neutralizing antibodies (HV1-69-sBnAbs). Here we show that a major structural solution for these HV1-69-sBnAbs is achieved through a critical triad comprising two CDR-H2 loop anchor residues (a hydrophobic residue at position 53 (Ile or Met) and Phe54), and CDR-H3-Tyr at positions 98±1; together with distinctive V-segment CDR amino acid substitutions that occur in positions sparse in AID/polymerase-η recognition motifs. A semi-synthetic IGHV1-69 phage-display library screen designed to investigate AID/polη restrictions resulted in the isolation of HV1-69-sBnAbs that featured a distinctive Ile52Ser mutation in the CDR-H2 loop, a universal CDR-H3 Tyr at position 98 or 99, and required as little as two additional substitutions for heterosubtypic neutralizing activity. The functional importance of the Ile52Ser mutation was confirmed by mutagenesis and by BCR studies. Structural modeling suggests that substitution of a small amino acid at position 52 (or 52a) facilitates the insertion of CDR-H2 Phe54 and CDR-H3-Tyr into adjacent pockets on the stem. These results support the concept that activation and expansion of a defined subset of IGHV1-69-encoded B cells to produce potent HV1-69-sBnAbs does not necessarily require a heavily diversified V-segment acquired through recycling/reentry into the germinal center; rather, the incorporation of distinctive amino acid substitutions by Phase 2 long-patch error-prone repair of AID-induced mutations or by random non-AID SHM events may be sufficient. We propose that these routes of B cell maturation should be further investigated and exploited as a pathway for HV1-69-sBnAb elicitation by vaccination. PMID:24788925

  18. Complement inhibition prevents oncolytic vaccinia virus neutralization in immune humans and cynomolgus macaques.

    PubMed

    Evgin, Laura; Acuna, Sergio A; Tanese de Souza, Christiano; Marguerie, Monique; Lemay, Chantal G; Ilkow, Carolina S; Findlay, C Scott; Falls, Theresa; Parato, Kelley A; Hanwell, David; Goldstein, Alyssa; Lopez, Roberto; Lafrance, Sandra; Breitbach, Caroline J; Kirn, David; Atkins, Harold; Auer, Rebecca C; Thurman, Joshua M; Stahl, Gregory L; Lambris, John D; Bell, John C; McCart, J Andrea

    2015-06-01

    Oncolytic viruses (OVs) have shown promising clinical activity when administered by direct intratumoral injection. However, natural barriers in the blood, including antibodies and complement, are likely to limit the ability to repeatedly administer OVs by the intravenous route. We demonstrate here that for a prototype of the clinical vaccinia virus based product Pexa-Vec, the neutralizing activity of antibodies elicited by smallpox vaccination, as well as the anamnestic response in hyperimmune virus treated cancer patients, is strictly dependent on the activation of complement. In immunized rats, complement depletion stabilized vaccinia virus in the blood and led to improved delivery to tumors. Complement depletion also enhanced tumor infection when virus was directly injected into tumors in immunized animals. The feasibility and safety of using a complement inhibitor, CP40, in combination with vaccinia virus was tested in cynomolgus macaques. CP40 pretreatment elicited an average 10-fold increase in infectious titer in the blood early after the infusion and prolonged the time during which infectious virus was detectable in the blood of animals with preexisting immunity. Capitalizing on the complement dependence of antivaccinia antibody with adjunct complement inhibitors may increase the infectious dose of oncolytic vaccinia virus delivered to tumors in virus in immune hosts. PMID:25807289

  19. Human symbionts inject and neutralize antibacterial toxins to persist in the gut.

    PubMed

    Wexler, Aaron G; Bao, Yiqiao; Whitney, John C; Bobay, Louis-Marie; Xavier, Joao B; Schofield, Whitman B; Barry, Natasha A; Russell, Alistair B; Tran, Bao Q; Goo, Young Ah; Goodlett, David R; Ochman, Howard; Mougous, Joseph D; Goodman, Andrew L

    2016-03-29

    The human gut microbiome is a dynamic and densely populated microbial community that can provide important benefits to its host. Cooperation and competition for nutrients among its constituents only partially explain community composition and interpersonal variation. Notably, certain human-associated Bacteroidetes--one of two major phyla in the gut--also encode machinery for contact-dependent interbacterial antagonism, but its impact within gut microbial communities remains unknown. Here we report that prominent human gut symbionts persist in the gut through continuous attack on their immediate neighbors. Our analysis of just one of the hundreds of species in these communities reveals 12 candidate antibacterial effector loci that can exist in 32 combinations. Through the use of secretome studies, in vitro bacterial interaction assays and multiple mouse models, we uncover strain-specific effector/immunity repertoires that can predict interbacterial interactions in vitro and in vivo, and find that some of these strains avoid contact-dependent killing by accumulating immunity genes to effectors that they do not encode. Effector transmission rates in live animals can exceed 1 billion events per minute per gram of colonic contents, and multiphylum communities of human gut commensals can partially protect sensitive strains from these attacks. Together, these results suggest that gut microbes can determine their interactions through direct contact. An understanding of the strategies human gut symbionts have evolved to target other members of this community may provide new approaches for microbiome manipulation. PMID:26957597

  20. Human symbionts inject and neutralize antibacterial toxins to persist in the gut

    PubMed Central

    Wexler, Aaron G.; Bao, Yiqiao; Whitney, John C.; Bobay, Louis-Marie; Xavier, Joao B.; Schofield, Whitman B.; Barry, Natasha A.; Russell, Alistair B.; Tran, Bao Q.; Goo, Young Ah; Goodlett, David R.; Ochman, Howard; Mougous, Joseph D.; Goodman, Andrew L.

    2016-01-01

    The human gut microbiome is a dynamic and densely populated microbial community that can provide important benefits to its host. Cooperation and competition for nutrients among its constituents only partially explain community composition and interpersonal variation. Notably, certain human-associated Bacteroidetes—one of two major phyla in the gut—also encode machinery for contact-dependent interbacterial antagonism, but its impact within gut microbial communities remains unknown. Here we report that prominent human gut symbionts persist in the gut through continuous attack on their immediate neighbors. Our analysis of just one of the hundreds of species in these communities reveals 12 candidate antibacterial effector loci that can exist in 32 combinations. Through the use of secretome studies, in vitro bacterial interaction assays and multiple mouse models, we uncover strain-specific effector/immunity repertoires that can predict interbacterial interactions in vitro and in vivo, and find that some of these strains avoid contact-dependent killing by accumulating immunity genes to effectors that they do not encode. Effector transmission rates in live animals can exceed 1 billion events per minute per gram of colonic contents, and multiphylum communities of human gut commensals can partially protect sensitive strains from these attacks. Together, these results suggest that gut microbes can determine their interactions through direct contact. An understanding of the strategies human gut symbionts have evolved to target other members of this community may provide new approaches for microbiome manipulation. PMID:26957597

  1. Measurement of neutralizing serum antibodies of patients vaccinated with human papillomavirus L1 or L2-based immunogens using furin-cleaved HPV Pseudovirions.

    PubMed

    Wang, Joshua W; Jagu, Subhashini; Wang, Chenguang; Kitchener, Henry C; Daayana, Sai; Stern, Peter L; Pang, Susana; Day, Patricia M; Huh, Warner K; Roden, Richard B S

    2014-01-01

    Antibodies specific for neutralizing epitopes in either Human papillomavirus (HPV) capsid protein L1 or L2 can mediate protection from viral challenge and thus their accurate and sensitive measurement at high throughput is likely informative for monitoring response to prophylactic vaccination. Here we compare measurement of L1 and L2-specific neutralizing antibodies in human sera using the standard Pseudovirion-Based Neutralization Assay (L1-PBNA) with the newer Furin-Cleaved Pseudovirion-Based Neutralization Assay (FC-PBNA), a modification of the L1-PBNA intended to improve sensitivity towards L2-specific neutralizing antibodies without compromising assay of L1-specific responses. For detection of L1-specific neutralizing antibodies in human sera, the FC- PBNA and L1-PBNA assays showed similar sensitivity and a high level of correlation using WHO standard sera (n = 2), and sera from patients vaccinated with Gardasil (n = 30) or an experimental human papillomavirus type 16 (HPV16) L1 VLP vaccine (n = 70). The detection of L1-specific cross-neutralizing antibodies in these sera using pseudovirions of types phylogenetically-related to those targeted by the L1 virus-like particle (VLP) vaccines was also consistent between the two assays. However, for sera from patients (n = 17) vaccinated with an L2-based immunogen (TA-CIN), the FC-PBNA was more sensitive than the L1-PBNA in detecting L2-specific neutralizing antibodies. Further, the neutralizing antibody titers measured with the FC-PBNA correlated with those determined with the L2-PBNA, another modification of the L1-PBNA that spacio-temporally separates primary and secondary receptor engagement, as well as the protective titers measured using passive transfer studies in the murine genital-challenge model. In sum, the FC-PBNA provided sensitive measurement for both L1 VLP and L2-specific neutralizing antibody in human sera. Vaccination with TA-CIN elicits weak cross-protective antibody in a subset of

  2. Alkaline ceramidase 3 deficiency aggravates colitis and colitis-associated tumorigenesis in mice by hyperactivating the innate immune system

    PubMed Central

    Wang, K; Xu, R; Snider, A J; Schrandt, J; Li, Y; Bialkowska, A B; Li, M; Zhou, J; Hannun, Y A; Obeid, L M; Yang, V W; Mao, C

    2016-01-01

    Increasing studies suggest that ceramides differing in acyl chain length and/or degree of unsaturation have distinct roles in mediating biological responses. However, still much remains unclear about regulation and role of distinct ceramide species in the immune response. Here, we demonstrate that alkaline ceramidase 3 (Acer3) mediates the immune response by regulating the levels of C18:1-ceramide in cells of the innate immune system and that Acer3 deficiency aggravates colitis in a murine model by augmenting the expression of pro-inflammatory cytokines in myeloid and colonic epithelial cells (CECs). According to the NCBI Gene Expression Omnibus (GEO) database, ACER3 is downregulated in immune cells in response to lipopolysaccharides (LPS), a potent inducer of the innate immune response. Consistent with these data, we demonstrated that LPS downregulated both Acer3 mRNA levels and its enzymatic activity while elevating C18:1-ceramide, a substrate of Acer3, in murine immune cells or CECs. Knocking out Acer3 enhanced the elevation of C18:1-ceramide and the expression of pro-inflammatory cytokines in immune cells and CECs in response to LPS challenge. Similar to Acer3 knockout, treatment with C18:1-ceramide, but not C18:0-ceramide, potentiated LPS-induced expression of pro-inflammatory cytokines in immune cells. In the mouse model of dextran sulfate sodium-induced colitis, Acer3 deficiency augmented colitis-associated elevation of colonic C18:1-ceramide and pro-inflammatory cytokines. Acer3 deficiency aggravated diarrhea, rectal bleeding, weight loss and mortality. Pathological analyses revealed that Acer3 deficiency augmented colonic shortening, immune cell infiltration, colonic epithelial damage and systemic inflammation. Acer3 deficiency also aggravated colonic dysplasia in a mouse model of colitis-associated colorectal cancer. Taken together, these results suggest that Acer3 has an important anti-inflammatory role by suppressing cellular or tissue C18:1-ceramide, a

  3. Neutralizer optimization

    NASA Technical Reports Server (NTRS)

    Patterson, Michael J.; Mohajeri, Kayhan

    1991-01-01

    The preliminary results of a test program to optimize a neutralizer design for 30 cm xenon ion thrusters are discussed. The impact of neutralizer geometry, neutralizer axial location, and local magnetic fields on neutralizer performance is discussed. The effect of neutralizer performance on overall thruster performance is quantified, for thruster operation in the 0.5-3.2 kW power range. Additionally, these data are compared to data published for other north-south stationkeeping (NSSK) and primary propulsion xenon ion thruster neutralizers.

  4. Antigenic Characterization of the HCMV gH/gL/gO and Pentamer Cell Entry Complexes Reveals Binding Sites for Potently Neutralizing Human Antibodies

    PubMed Central

    Ciferri, Claudio; Chandramouli, Sumana; Leitner, Alexander; Donnarumma, Danilo; Cianfrocco, Michael A.; Gerrein, Rachel; Friedrich, Kristian; Aggarwal, Yukti; Palladino, Giuseppe; Aebersold, Ruedi; Norais, Nathalie; Settembre, Ethan C.; Carfi, Andrea

    2015-01-01

    Human Cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and in fetuses following congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are required for HCMV entry in fibroblasts and endothelial/epithelial cells, respectively, and are targeted by potently neutralizing antibodies in the infected host. Using purified soluble forms of gH/gL/gO and Pentamer as well as a panel of naturally elicited human monoclonal antibodies, we determined the location of key neutralizing epitopes on the gH/gL/gO and Pentamer surfaces. Mass Spectrometry (MS) coupled to Chemical Crosslinking or to Hydrogen Deuterium Exchange was used to define residues that are either in proximity or part of neutralizing epitopes on the glycoprotein complexes. We also determined the molecular architecture of the gH/gL/gO- and Pentamer-antibody complexes by Electron Microscopy (EM) and 3D reconstructions. The EM analysis revealed that the Pentamer specific neutralizing antibodies bind to two opposite surfaces of the complex, suggesting that they may neutralize infection by different mechanisms. Together, our data identify the location of neutralizing antibodies binding sites on the gH/gL/gO and Pentamer complexes and provide a framework for the development of antibodies and vaccines against HCMV. PMID:26485028

  5. Isolation and characterization of human neutralizing antibodies to rabies virus derived from a recombinant immune antibody library.

    PubMed

    Houimel, Mehdi; Dellagi, Koussay

    2009-11-01

    A human immune Fab library was constructed using RNAs from peripheral blood lymphocytes obtained from rabies virus hyperimmune volunteers on phagemid vector. The size of the constructed Fab library was 2 x 10(7) Escherichia coli transformants. After four rounds of panning on whole inactivated rabies virus (PV-11), phage clones displaying rabies virus-specific human Fab were selected. The specificity of soluble Fab antibody fragments, derived from positive phage clones was verified by ELISA. Among 20 specific Fab clones, the genetic sequence of 6 of them (FabRV01, FabRV02, FabRV03, FabRV04, FabRV05, and FabRV06) was analyzed. The variable heavy (VH) and variable light (VL) domains were found to share 90% and 93% homology with sequences encoded by the corresponding human germline genes, respectively. The soluble Fab fragments, expressed in Escherichia coli were purified by a single step Nickel-NTA affinity chromatography via a hexa-histidine tag and their binding specificities to rabies virus were confirmed. Three of the Fab antibodies, FabRV01, FabRV02 and FabRV03, showed binding characteristics to rabies virus glycoprotein antigenic site III with affinities in the K(D) range 7 x 10(-9) to 5 x 10(-8)M. The Fab fragments showed dose-dependent neutralization properties for the challenge virus standard (CVS-11). PMID:19559727

  6. Biochemical and pharmacological characterization of human c-Met neutralizing monoclonal antibody CE-355621

    PubMed Central

    Michaud, Neil R.; Jani, Jitesh P.; Hillerman, Stephen; Tsaparikos, Konstantinos E.; Barbacci-Tobin, Elsa G.; Knauth, Elisabeth; Putz Jr., Henry; Campbell, Mary; Karam, George A.; Chrunyk, Boris; Gebhard, David F.; Green, Larry L.; Xu, Jinghai J.; Dunn, Margaret C.; Coskran, Tim M.; Lapointe, Jean-Martin; Cohen, Bruce D.; Coleman, Kevin G.; Bedian, Vahe; Vincent, Patrick; Kajiji, Shama; Steyn, Stefan J.; Borzillo, Gary V.; Los, Gerrit

    2012-01-01

    The c-Met proto-oncogene is a multifunctional receptor tyrosine kinase that is stimulated by its ligand, hepatocyte growth factor (HGF), to induce cell growth, motility and morphogenesis. Dysregulation of c-Met function, through mutational activation or overexpression, has been observed in many types of cancer and is thought to contribute to tumor growth and metastasis by affecting mitogenesis, invasion, and angiogenesis. We identified human monoclonal antibodies that bind to the extracellular domain of c-Met and inhibit tumor growth by interfering with ligand-dependent c-Met activation. We identified antibodies representing four independent epitope classes that inhibited both ligand binding and ligand-dependent activation of c-Met in A549 cells. In cells, the antibodies antagonized c-Met function by blocking receptor activation and by subsequently inducing downregulation of the receptor, translating to phenotypic effects in soft agar growth and tubular morphogenesis assays. Further characterization of the antibodies in vivo revealed significant inhibition of c-Met activity (≥ 80% lasting for 72–96 h) in excised tumors corresponded to tumor growth inhibition in multiple xenograft tumor models. Several of the antibodies identified inhibited the growth of tumors engineered to overexpress human HGF and human c-Met (S114 NIH 3T3) when grown subcutaneously in athymic mice. Furthermore, lead candidate antibody CE-355621 inhibited the growth of U87MG human glioblastoma and GTL-16 gastric xenografts by up to 98%. The findings support published pre-clinical and clinical data indicating that targeting c-Met with human monoclonal antibodies is a promising therapeutic approach for the treatment of cancer. PMID:23007574

  7. The molecular mode of action and species specificity of canakinumab, a human monoclonal antibody neutralizing IL-1β

    PubMed Central

    Rondeau, Jean-Michel; Ramage, Paul; Zurini, Mauro; Gram, Hermann

    2015-01-01

    Interleukin-1β (IL-1β) plays a key role in autoinflammatory diseases, such as systemic juvenile idiopathic arthritis (sJIA) or cryopyrin-associated periodic syndrome (CAPS). Canakinumab, a human monoclonal anti-IL-1β antibody, was recently approved for human use under the brand name Ilaris®. Canakinumab does not cross-react with IL-1β from mouse, rat, rabbit, or macaques. The crystal structure of the canakinumab Fab bound to human IL-1β was determined in an attempt to rationalize the species specificity. The X-ray analysis reveals a complex surface epitope with an intricate network of well-ordered water molecules at the antibody-antigen interface. The canakinumab paratope is largely pre-organized, as demonstrated by the structure determination of the free Fab. Glu 64 of human IL-1β is a pivotal epitope residue explaining the exquisite species specificity of canakinumab. We identified marmoset as the only non-human primate species that carries Glu 64 in its IL-1β and demonstrates full cross-reactivity of canakinumab, thereby enabling toxicological studies in this species. As demonstrated by the X-ray structure of the complex with IL-1β, canakinumab binds IL-1β on the opposite side with respect to the IL-1RAcP binding site, and in an approximately orthogonal orientation with respect to IL-1RI. However, the antibody and IL-1RI binding sites slightly overlap and the VH region of canakinumab would sterically interfere with the D1 domain of IL-1RI, as shown by a structural overlay with the IL-1β:IL-1RI complex. Therefore, direct competition with IL-1RI for IL-1β binding is the molecular mechanism of neutralization by canakinumab, which is also confirmed by competition assays with recombinant IL-1RI and IL-1RII. PMID:26284424

  8. Cross-protection of newly emerging HPAI H5 viruses by neutralizing human monoclonal antibodies: A viable alternative to oseltamivir.

    PubMed

    Ren, Huanhuan; Wang, Guiqin; Wang, Shuangshuang; Chen, Honglin; Chen, Zhiwei; Hu, Hongxing; Cheng, Genhong; Zhou, Paul

    2016-01-01

    Newly emerging highly pathogenic avian influenza (HPAI) H5N2, H5N3, H5N5, H5N6, H5N8 and H5N9 viruses have been spreading in poultry and wild birds. The H5N6 viruses have also caused 10 human infections with 4 fatal cases in China. Here, we assessed the cross-neutralization and cross-protection of human and mouse monoclonal antibodies against 2 viruses: a HPAI H5N8 virus, A/chicken/Netherlands/14015526/2014 (NE14) and a HPAI H5N6 virus, A/Sichuan/26221/2014 (SC14). The former was isolated from an infected chicken in Netherlands in 2014 and the latter was isolated from an infected human patient in Sichuan, China. We show that antibodies FLA5.10, FLD21.140, 100F4 and 65C6, but not AVFluIgG01, AVFluIgG03, S139/1 and the VRC01 control, potently cross-neutralize the H5N8 NE14 and H5N6 SC14 viruses. Furthermore, we show that a single injection of >1 mg/kg of antibody 100F4 at 4 hours before, or 20 mg/kg antibody 100F4 at 72 hours after, a lethal dose of H5N8 NE14 enables mice to withstand the infection. Finally, we show that a single injection of 0.5 or 1 mg/kg antibody 100F4 prophylactically or 10 mg/kg 100F4 therapeutically outperforms a 5-day course of 10 mg/kg/day oseltamivir treatment against lethal H5N8 NE14 or H5N6 SC14 infection in mice. Our results suggest that further preclinical evaluation of human monoclonal antibodies against newly emerging H5 viruses is warranted. PMID:27167234

  9. Neutralizing Antibodies in Sera from Macaques Infected with Chimeric Simian-Human Immunodeficiency Virus Containing the Envelope Glycoproteins of either a Laboratory-Adapted Variant or a Primary Isolate of Human Immunodeficiency Virus Type 1

    PubMed Central

    Montefiori, David C.; Reimann, Keith A.; Wyand, Michael S.; Manson, Kelledy; Lewis, Mark G.; Collman, Ronald G.; Sodroski, Joseph G.; Bolognesi, Dani P.; Letvin, Norman L.

    1998-01-01

    The magnitude and breadth of neutralizing antibodies raised in response to infection with chimeric simian-human immunodeficiency virus (SHIV) in rhesus macaques were evaluated. Infection with either SHIV-HXB2, SHIV-89.6, or SHIV-89.6PD raised high-titer neutralizing antibodies to the homologous SHIV (SHIV-89.6P in the case of SHIV-89.6PD-infected animals) and significant titers of neutralizing antibodies to human immunodeficiency virus type 1 (HIV-1) strains MN and SF-2. With few exceptions, however, titers of neutralizing antibodies to heterologous SHIV were low or undetectable. The antibodies occasionally neutralized heterologous primary isolates of HIV-1; these antibodies required >40 weeks of infection to reach detectable levels. Notable was the potent neutralization of the HIV-1 89.6 primary isolate by serum samples from SHIV-89.6-infected macaques. These results demonstrate that SHIV-HXB2, SHIV-89.6, and SHIV-89.6P possess highly divergent, strain-specific neutralization epitopes. The results also provide insights into the requirements for raising neutralizing antibodies to primary isolates of HIV-1. PMID:9525675

  10. Sequential Antigen Panning for Selection of Broadly Cross-Reactive HIV-1-Neutralizing Human Monoclonal Antibodies

    PubMed Central

    Zhang, Mei-Yun; Dimitrov, Dimiter S.

    2012-01-01

    Summary Many phage display techniques drive selection toward the isolation of highly specific antibodies. However, the identification of monoclonal antibodies that are cross-reactive has implications for the development of diagnostics, therapeutics, and vaccines against pathogens or cancer cells that are able to rapidly generate variants and escape mutants. To identify human monoclonal antibodies with high activity against HIV and broad-spectrum activity, we developed a technique termed sequential antigen panning. This methodology could be used to isolated recombinant antibodies against any antigen that shares epitopes with other antigens. PMID:19554293

  11. Individual contributions of the human metapneumovirus F, G, and SH surface glycoproteins to the induction of neutralizing antibodies and protective immunity

    SciTech Connect

    Skiadopoulos, Mario H. . E-mail: mskiadopoulos@niaid.nih.gov; Biacchesi, Stephane; Buchholz, Ursula J.; Amaro-Carambot, Emerito; Surman, Sonja R.; Collins, Peter L.; Murphy, Brian R.

    2006-02-20

    We evaluated the individual contributions of the three surface glycoproteins of human metapneumovirus (HMPV), namely the fusion F, attachment G, and small hydrophobic SH proteins, to the induction of serum HMPV-binding antibodies, serum HMPV-neutralizing antibodies, and protective immunity. Using reverse genetics, each HMPV protein was expressed individually from an added gene in recombinant human parainfluenza virus type 1 (rHPIV1) and used to infect hamsters once or twice by the intranasal route. The F protein was highly immunogenic and protective, whereas G and SH were only weakly or negligibly immunogenic and protective, respectively. Thus, in contrast to other paramyxoviruses, the HMPV attachment G protein is not a major neutralization or protective antigen. Also, although the SH protein of HMPV is a virion protein that is much larger than its counterparts in previously studied paramyxoviruses, it does not appear to be a significant neutralization or protective antigen.

  12. Expression and regulation of enzymes in the ceramide metabolic pathway in human retinal pigment epithelial cells and their relevance to retinal degeneration

    PubMed Central

    Zhu, DanHong; Sreekumar, Parameswaran G.; Hinton, David R.; Kannan, Ram

    2009-01-01

    Ceramide and its metabolic derivatives are important modulators of cellular apoptosis and proliferation. Dysregulation or imbalance of their metabolic pathways may promote the development of retinal degeneration. The aim of this study was to identify the expression and regulation of key enzymes of the ceramide pathway in retinal pigment epithelial (RPE) cells. RT-PCR was used to screen the enzymes involved in ceramide metabolism that are expressed in RPE. Over-expression of neutral sphingomyelinase-2 (SMPD3) or sphingosine kinase 1 (Sphk1) in ARPE-19 cells was achieved by transient transfection of SMPD3 or Sphk1 cDNA subcloned into an expression vector. The number of apoptotic or proliferating cells was determined using TUNEL and BrdU assays respectively. Neutral sphingomyelinase-1, neutral sphingomyelinase-2, acidic ceramidase, ceramide kinase, SphK1 and Sphk2 were expressed in both ARPE-19 and early passage human fetal RPE (fRPE) cells, while alkaline ceramidase 2 was only expressed in fRPE cells. Over-expression of SMPD3 decreased RPE cell proliferation and increased cell apoptosis. The percentage of apoptotic cells increased proportionally with the amount of transfected SMPD3 DNA. Over-expression of SphK1 promoted cell proliferation and protected ARPE-19 cells from ceramide-induced apoptosis. The effect of C2 ceramide on induction of apoptosis was evaluated in polarized vs. non-polarized RPE cultures; polarization of RPE was associated with much reduced apoptosis in response to ceramide. In conclusion, RPE cells possess the synthetic machinery for the production of ceramide, sphingosine, ceramide-1-phosphate (C1P), and sphingosine-1-phosphate (S1P). Overexpression of SMPD3 may increase cellular ceramide levels, leading to enhanced cell death and arrested cell proliferation. The selective induction of apoptosis in non-polarized RPE cultures by C2 ceramide suggests that increased ceramide levels will preferentially affect non-polarized RPE, as are found in late

  13. Generation of scFv specific to human VEGFR-3 from the neutralizing mAb BDD073.

    PubMed

    Chen, Hao; Wang, Chaoqun; Gao, Yuan; Gao, Jianen; Zhou, Xiaping; Cai, Zhiming; Sun, Qihong

    2015-01-01

    In our previous study, we have produced a neutralizing mAb of vascular endothelial growth factor receptor 3 (VEGFR-3), specifically BDD073, which could inhibit angiogenesis in the CAM model. However, the clinical application of BDD073 is restricted due to its mouse origin, which might cause human anti-mouse antibody reactions. Herein, we generated functional recombinant single-chain variable fragments (scFv) from mAb BDD073 producing mouse hybridoma cells. The scFv gene containing variable regions of heavy and light chains of BDD073 was cloned into an expression vector with trx tag and expressed in Escherichia coli BL21 (DE3). The recombinant scFv was purified and refolded with Ni-NTA agarose metal affinity column. The bacterially expressed scFv showed moderate potency and specificity to the human VEGFR-3. It may serve as a potential candidate of anti-VEGFR3 treatment for biotechnological and therapeutic applications. PMID:25428897

  14. HIV-1 suppression and durable control by combining single broadly neutralizing antibodies and antiretroviral drugs in humanized mice

    PubMed Central

    Horwitz, Joshua A.; Halper-Stromberg, Ariel; Mouquet, Hugo; Gitlin, Alexander D.; Tretiakova, Anna; Eisenreich, Thomas R.; Malbec, Marine; Gravemann, Sophia; Billerbeck, Eva; Dorner, Marcus; Büning, Hildegard; Schwartz, Olivier; Knops, Elena; Kaiser, Rolf; Seaman, Michael S.; Wilson, James M.; Rice, Charles M.; Ploss, Alexander; Bjorkman, Pamela J.; Klein, Florian; Nussenzweig, Michel C.

    2013-01-01

    Effective control of HIV-1 infection in humans is achieved using combinations of antiretroviral therapy (ART) drugs. In humanized mice (hu-mice), control of viremia can be achieved using either ART or by immunotherapy using combinations of broadly neutralizing antibodies (bNAbs). Here we show that treatment of HIV-1–infected hu-mice with a combination of three highly potent bNAbs not only resulted in complete viremic control but also led to a reduction in cell-associated HIV-1 DNA. Moreover, lowering the initial viral load by coadministration of ART and immunotherapy enabled prolonged viremic control by a single bNAb after ART was withdrawn. Similarly, a single injection of adeno-associated virus directing expression of one bNAb produced durable viremic control after ART was terminated. We conclude that immunotherapy reduces plasma viral load and cell-associated HIV-1 DNA and that decreasing the initial viral load enables single bNAbs to control viremia in hu-mice. PMID:24043801

  15. Immunodominant SARS Coronavirus Epitopes in Humans Elicited both Enhancing and Neutralizing Effects on Infection in Non-human Primates.

    PubMed

    Wang, Qidi; Zhang, Lianfeng; Kuwahara, Kazuhiko; Li, Li; Liu, Zijie; Li, Taisheng; Zhu, Hua; Liu, Jiangning; Xu, Yanfeng; Xie, Jing; Morioka, Hiroshi; Sakaguchi, Nobuo; Qin, Chuan; Liu, Gang

    2016-05-13

    Severe acute respiratory syndrome (SARS) is caused by a coronavirus (SARS-CoV) and has the potential to threaten global public health and socioeconomic stability. Evidence of antibody-dependent enhancement (ADE) of SARS-CoV infection in vitro and in non-human primates clouds the prospects for a safe vaccine. Using antibodies from SARS patients, we identified and characterized SARS-CoV B-cell peptide epitopes with disparate functions. In rhesus macaques, the spike glycoprotein peptides S471-503, S604-625, and S1164-1191 elicited antibodies that efficiently prevented infection in non-human primates. In contrast, peptide S597-603 induced antibodies that enhanced infection both in vitro and in non-human primates by using an epitope sequence-dependent (ESD) mechanism. This peptide exhibited a high level of serological reactivity (64%), which resulted from the additive responses of two tandem epitopes (S597-603 and S604-625) and a long-term human B-cell memory response with antisera from convalescent SARS patients. Thus, peptide-based vaccines against SARS-CoV could be engineered to avoid ADE via elimination of the S597-603 epitope. We provide herein an alternative strategy to prepare a safe and effective vaccine for ADE of viral infection by identifying and eliminating epitope sequence-dependent enhancement of viral infection. PMID:27627203

  16. Fully humanized neutralizing antibodies to interleukin-8 (ABX-IL8) inhibit angiogenesis, tumor growth, and metastasis of human melanoma.

    PubMed

    Huang, Suyun; Mills, Lisa; Mian, Badar; Tellez, Carmen; McCarty, Marya; Yang, X-D; Gudas, Jean M; Bar-Eli, Menashe

    2002-07-01

    Interleukin-8 (IL-8) has recently been shown to contribute to human melanoma progression by functioning as a mitogenic and angiogenic factor. In the present study, we investigated whether targeting IL-8 by a fully human anti-IL-8 antibody (ABX-IL8) could be a potential therapeutic strategy to control angiogenesis, growth, and metastasis of melanoma. The human melanoma cells A375SM (high IL-8 producer) and TXM-13 (intermediate IL-8 producer) were injected subcutaneously into nude mice, which were then treated with ABX-IL8 (1 mg/3 times weekly, i.p., for 3 weeks). Tumor growth of both melanomas in ABX-IL8-treated mice was significantly inhibited when compared with control IgG-treated animals. ABX-IL8 treatment also suppressed experimental metastasis when the melanoma cells were injected intravenously. IL-8 blockade by ABX-IL8 significantly inhibited the promoter activity and the collagenase activity of matrix metalloproteinase-2 in human melanoma cells, resulting in decreased invasion through reconstituted basement membrane in vitro. In vivo, ABX-IL8 treatment resulted in decreased expression of matrix metalloproteinase-2, and decreased vascularization (angiogenesis) of tumors concomitant with increased apoptosis of tumor cells. Moreover, in an in vitro vessel formation assay, ABX-IL8 directly interfered with the tubule formation by human umbilical vein endothelial cells. Taken together, these results point to the potential utility of ABX-IL8 as a modality to treat melanoma and other solid tumors either alone or in combination with conventional chemotherapy or other anti-tumor agents. PMID:12107097

  17. A human monoclonal antibody neutralizes diverse HIV-1 isolates by binding a critical gp41 epitope

    PubMed Central

    Miller, Michael D.; Geleziunas, Romas; Bianchi, Elisabetta; Lennard, Simon; Hrin, Renee; Zhang, Hangchun; Lu, Meiqing; An, Zhiqiang; Ingallinella, Paolo; Finotto, Marco; Mattu, Marco; Finnefrock, Adam C.; Bramhill, David; Cook, James; Eckert, Debra M.; Hampton, Richard; Patel, Mayuri; Jarantow, Stephen; Joyce, Joseph; Ciliberto, Gennaro; Cortese, Riccardo; Lu, Ping; Strohl, William; Schleif, William; McElhaugh, Michael; Lane, Steven; Lloyd, Christopher; Lowe, David; Osbourn, Jane; Vaughan, Tristan; Emini, Emilio; Barbato, Gaetano; Kim, Peter S.; Hazuda, Daria J.; Shiver, John W.; Pessi, Antonello

    2005-01-01

    HIV-1 entry into cells is mediated by the envelope glycoprotein receptor-binding (gp120) and membrane fusion-promoting (gp41) subunits. The gp41 heptad repeat 1 (HR1) domain is the molecular target of the fusion-inhibitor drug enfuvirtide (T20). The HR1 sequence is highly conserved and therefore considered an attractive target for vaccine development, but it is unknown whether antibodies can access HR1. Herein, we use gp41-based peptides to select a human antibody, 5H/I1-BMV-D5 (D5), that binds to HR1 and inhibits the assembly of fusion intermediates in vitro. D5 inhibits the replication of diverse HIV-1 clinical isolates and therefore represents a previously unknown example of a crossneutralizing IgG selected by binding to designed antigens. NMR studies and functional analyses map the D5-binding site to a previously identified hydrophobic pocket situated in the HR1 groove. This hydrophobic pocket was proposed as a drug target and subsequently identified as a common binding site for peptide and peptidomimetic fusion inhibitors. The finding that the D5 fusion-inhibitory antibody shares the same binding site suggests that the hydrophobic pocket is a “hot spot” for fusion inhibition and an ideal target on which to focus a vaccine-elicited antibody response. Our data provide a structural framework for the design of new immunogens and therapeutic antibodies with crossneutralizing potential. PMID:16203977

  18. Mass Spectrometric Collisional Activation and Product Ion Mobility of Human Serum Neutral Lipid Extracts

    PubMed Central

    Hankin, Joseph A.; Barkley, Robert M.; Zemski-Berry, Karin; Deng, Yiming; Murphy, Robert C.

    2016-01-01

    A novel method for lipid analysis called CTS (collisional activation and traveling wave mass spectrometry) involving tandem mass spectrometry of all precursor ions with ion mobility determinations of all product ions was applied to a sample of human serum. The resulting four dimensional data set (precursor ion, product ion, ion mobility values, and intensity) was found to be useful for characterization of lipids as classes as well as identification of specific species. Utilization of ion mobility measurements of the product ions is a novel approach for lipid analysis. The trends and patterns of product mobility values when visually displayed yield information on lipid classes and specific species independent of mass determination. The collection of a comprehensive set of data that incorporates all precursor-product relationships combined with ion mobility measurements of all products enables data analysis where different molecular properties can be juxtaposed and analyzed to assist with class and species identification. Overall, CTS is powerful, specific, and comprehensive method for lipid analysis. PMID:27213895

  19. Identification of amino acid substitutions associated with neutralization phenotype in the human immunodeficiency virus type-1 subtype C gp120

    PubMed Central

    Kirchherr, Jennifer L; Hamilton, Jennifer; Lu, Xiaozhi; Gnanakaran, S; Muldoon, Mark; Daniels, Marcus; Kasongo, Webster; Chalwe, Victor; Mulenga, Chanda; Mwananyanda, Lawrence; Musonda, Rosemary M; Yuan, Xing; Montefiori, David C; Korber, Bette T; Haynes, Barton F; Gao, Feng

    2010-01-01

    Neutralizing antibodies (Nabs) are thought to play an important role in prevention and control of HIV-1 infection and should be targeted by an AIDS vaccine. It is critical to understand how HIV-1 induces Nabs by analyzing viral sequences in both tested viruses and sera. Neutralization susceptibility to antibodies in autologous and heterologous plasma was determined for multiple Envs (3–6) from each of 15 subtype C infected-individuals. Heterologous neutralization was divided into two distinct groups: plasma with strong, cross-reactive neutralization (N=9) and plasma with weak neutralization (N=6). Plasma with cross-reactive heterologous Nabs also more potently neutralized contemporaneous autologous viruses. Analysis of Env sequences in plasma from both groups revealed a three-amino acid substitution pattern in the V4 region that was associated with greater neutralization potency and breadth. Identification of such potential neutralization signatures may have important implications for the development of HIV-1 vaccines capable of inducing Nabs to subtype C HIV-1. PMID:21036380

  20. Structures of complexes formed by H5 influenza hemagglutinin with a potent broadly neutralizing human monoclonal antibody

    PubMed Central

    Xiong, Xiaoli; Corti, Davide; Liu, Junfeng; Pinna, Debora; Foglierini, Mathilde; Calder, Lesley J.; Martin, Stephen R.; Lin, Yi Pu; Walker, Philip A.; Collins, Patrick J.; Monne, Isabella; Suguitan, Amorsolo L.; Santos, Celia; Temperton, Nigel J.; Subbarao, Kanta; Lanzavecchia, Antonio; Gamblin, Steven J.; Skehel, John J.

    2015-01-01

    H5N1 avian influenza viruses remain a threat to public health mainly because they can cause severe infections in humans. These viruses are widespread in birds, and they vary in antigenicity forming three major clades and numerous antigenic variants. The most important features of the human monoclonal antibody FLD194 studied here are its broad specificity for all major clades of H5 influenza HAs, its high affinity, and its ability to block virus infection, in vitro and in vivo. As a consequence, this antibody may be suitable for anti-H5 therapy and as a component of stockpiles, together with other antiviral agents, for health authorities to use if an appropriate vaccine was not available. Our mutation and structural analyses indicate that the antibody recognizes a relatively conserved site near the membrane distal tip of HA, near to, but distinct from, the receptor-binding site. Our analyses also suggest that the mechanism of infectivity neutralization involves prevention of receptor recognition as a result of steric hindrance by the Fc part of the antibody. Structural analyses by EM indicate that three Fab fragments are bound to each HA trimer. The structure revealed by X-ray crystallography is of an HA monomer bound by one Fab. The monomer has some similarities to HA in the fusion pH conformation, and the monomer’s formation, which results from the presence of isopropanol in the crystallization solvent, contributes to considerations of the process of change in conformation required for membrane fusion. PMID:26170284

  1. Structure, Aggregation, and Activity of a Covalent Insulin Dimer Formed During Storage of Neutral Formulation of Human Insulin.

    PubMed

    Hjorth, Christian Fogt; Norrman, Mathias; Wahlund, Per-Olof; Benie, Andrew J; Petersen, Bent O; Jessen, Christian M; Pedersen, Thomas Å; Vestergaard, Kirsten; Steensgaard, Dorte B; Pedersen, Jan Skov; Naver, Helle; Hubálek, František; Poulsen, Christian; Otzen, Daniel

    2016-04-01

    A specific covalently linked dimeric species of insulin high molecular weight products (HMWPs), formed during prolonged incubation of a neutral pharmaceutical formulation of human insulin, were characterized in terms of tertiary structure, self-association, biological activity, and fibrillation properties. The dimer was formed by a covalent link between A21Asn and B29Lys. It was analyzed using static and dynamic light scattering and small-angle X-ray scattering to evaluate its self-association behavior. The tertiary structure was obtained using nuclear magnetic resonance and X-ray crystallography. The biological activity of HMWP was determined using 2 in vitro assays, and its influence on fibrillation was investigated using Thioflavin T assays. The dimer's tertiary structure was nearly identical to that of the noncovalent insulin dimer, and it was able to form hexamers in the presence of zinc. The dimer exhibited reduced propensity for self-association in the absence of zinc but significantly postponed the onset of fibrillation in insulin formulations. Consistent with its dimeric state, the tested species of HMWP showed little to no biological activity in the used assays. This study is the first detailed characterization of a specific type of human insulin HMWP formed during storage of a marketed pharmaceutical formulation. These results indicate that this specific type of HMWP is unlikely to antagonize the physical stability of the formulation, as HMWP retained a tertiary structure similar to the noncovalent dimer and participated in hexamer assembly in the presence of zinc. In addition, increasing amounts of HMWP reduce the rate of insulin fibrillation. PMID:26921119

  2. Cross-Reactive HIV-1 Neutralizing Monoclonal Antibodies Selected by Screening of an Immune Human Phage Library against an Envelope Glycoprotein (gp140) Isolated from a Patient (R2) with Broadly HIV-1 Neutralizing Antibodies

    PubMed Central

    Choudhry, Vidita; Zhang, Mei-Yun; Sidorov, Igor A.; Louise, John M.; Harris, Ilia; Dimitrov, Antony S.; Bouma, Peter; Cham, Fatim; Choudhary, Anil; Rybak, Susanna M.; Fouts, Timothy; Montefiori, David C.; Broder, Christopher C.; Quinnan, Gerald V.; Dimitrov, Dimiter S.

    2009-01-01

    Elicitation of broadly cross-reactive neutralizing antibodies (bcnAbs) in HIV infections is rare. To test the hypothesis that such antibodies could be elicited by HIV envelope glycoproteins (Envs) with unusual immunogenic properties and to identify novel bcnAbs, we used a soluble Env ectodomain (gp140) from a donor (R2) with high level of bcnAbs as an antigen for panning of an immune phage-displayed antibody library. The panning with the R2 Env resulted in significantly higher number of cross-reactive antibody clones than by using Envs from two other isolates (89.6 and IIIB). Two of the identified human monoclonal antibodies (hmAbs), m22 and m24, had sequences, neutralizing and binding activities similar or identical to those of the gp120-specific bcnAbs m18 and m14. The use of the R2 Env but not other Envs for panning resulted in the identification of a novel gp41-specific hmAb, m46. For several of the tested HIV-1 primary isolates its potency on molar basis was comparable to that of T20. It inhibited entry of primary isolates from different clades with an increased activity for cell lines with low CCR5 surface concentrations. The m46 neutralizing activity against a panel of clade C isolates was significantly higher in an assay based on peripheral blood mononuclear cells (4 out of 5 isolates were neutralized with an IC50 in the range from 1.5 to 25 μg/ml) than in an assay based on a cell line with relatively high concentration of cell-surface associated CCR5. In contrast to 2F5 and Z13, this antibody did not bind to denatured gp140 and gp41-derived peptides indicating a conformational nature of its epitope. It bound to a 5-helix bundle but not to N-heptad repeat coiled coils and a 6-helix bundle construct indicating contribution of both gp41 heptad repeats to its epitope and to a possible mechanism of neutralization. These results indicate that the R2 Env may contain unique exposed conserved epitopes that could contribute to its ability to elicit broadly cross

  3. Structural Analysis of Human and Macaque Monoclonal Antibodies 2909 and 2.5B: Implications for the Configuration of the Quaternary Neutralizing Epitope of HIV-1 gp120

    SciTech Connect

    B Spurrier; J Sampson; M Totrov; H Li; T ONeal; C Williams; J Robinson; M Gorny; S Zolla-Pazner; X Kong

    2011-12-31

    The quaternary neutralizing epitope (QNE) of HIV-1 gp120 is preferentially expressed on the trimeric envelope spikes of intact HIV virions, and QNE-specific monoclonal antibodies (mAbs) potently neutralize HIV-1. Here, we present the crystal structures of the Fabs of human mAb 2909 and macaque mAb 2.5B. Both mAbs have long beta hairpin CDR H3 regions >20 {angstrom} in length that are each situated at the center of their respective antigen-binding sites. Computational analysis showed that the paratopes include the whole CDR H3, while additional CDR residues form shallow binding pockets. Structural modeling suggests a way to understand the configuration of QNEs and the antigen-antibody interaction for QNE mAbs. Our data will be useful in designing immunogens that may elicit potent neutralizing QNE Abs.

  4. A Cell Line-Based Neutralization Assay for Primary Human Immunodeficiency Virus Type 1 Isolates That Use either the CCR5 or the CXCR4 Coreceptor

    PubMed Central

    Trkola, Alexandra; Matthews, Jamie; Gordon, Cynthia; Ketas, Tom; Moore, John P.

    1999-01-01

    We describe here a cell line-based assay for the evaluation of human immunodeficiency virus type 1 (HIV-1) neutralization. The assay is based on CEM.NKR cells, transfected to express the HIV-1 coreceptor CCR5 to supplement the endogenous expression of CD4 and the CXCR4 coreceptor. The resulting CEM.NKR-CCR5 cells efficiently replicate primary HIV-1 isolates of both R5 and X4 phenotypes. A comparison of the CEM.NKR-CCR5 cells with mitogen-activated peripheral blood mononuclear cells (PBMC) in neutralization assays with sera from HIV-1-infected individuals or specific anti-HIV-1 monoclonal antibodies shows that the sensitivity of HIV-1 neutralization is similar in the two cell types. The CEM.NKR-CCR5 cell assay, however, is more convenient to perform and eliminates the donor-to-donor variation in HIV-1 replication efficiency, which is one of the principal drawbacks of the PBMC-based neutralization assay. We suggest that this new assay is suitable for the general measurement of HIV-1 neutralization by antibodies. PMID:10516002

  5. Fully human broadly neutralizing monoclonal antibodies against influenza A viruses generated from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient

    SciTech Connect

    Hu, Weibin; Chen, Aizhong; Miao, Yi; Xia, Shengli; Ling, Zhiyang; Xu, Ke; Wang, Tongyan; Xu, Ying; Cui, Jun; Wu, Hongqiang; Hu, Guiyu; Tian, Lin; Wang, Lingling; Shu, Yuelong; Ma, Xiaowei; Xu, Bianli; Zhang, Jin; Lin, Xiaojun; Bian, Chao; Sun, Bing

    2013-01-20

    Whether the 2009 pandemic H1N1 influenza vaccine can induce heterosubtypic cross-protective anti-hemagglutinin (HA) neutralizing antibodies is an important issue. We obtained a panel of fully human monoclonal antibodies from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient. Most of the monoclonal antibodies targeted the HA protein but not the HA1 fragment. Among the analyzed antibodies, seven mAbs exhibited neutralizing activity against several influenza A viruses of different subtypes. The conserved linear epitope targeted by the neutralizing mAbs (FIEGGWTGMVDGWYGYHH) is part of the fusion peptide on HA2. Our work suggests that a heterosubtypic neutralizing antibody response primarily targeting the HA stem region exists in recipients of the 2009 pandemic H1N1 influenza vaccine. The HA stem region contains various conserved neutralizing epitopes with the fusion peptide as an important one. This work may aid in the design of a universal influenza A virus vaccine.

  6. Broadly Neutralizing Human Immunodeficiency Virus Type 1 Antibody Gene Transfer Protects Nonhuman Primates from Mucosal Simian-Human Immunodeficiency Virus Infection

    PubMed Central

    Saunders, Kevin O.; Wang, Lingshu; Joyce, M. Gordon; Yang, Zhi-Yong; Balazs, Alejandro B.; Cheng, Cheng; Ko, Sung-Youl; Kong, Wing-Pui; Rudicell, Rebecca S.; Georgiev, Ivelin S.; Duan, Lijie; Foulds, Kathryn E.; Donaldson, Mitzi; Xu, Ling; Schmidt, Stephen D.; Todd, John-Paul; Baltimore, David; Roederer, Mario; Haase, Ashley T.; Kwong, Peter D.; Rao, Srinivas S.

    2015-01-01

    ABSTRACT Broadly neutralizing antibodies (bnAbs) can prevent lentiviral infection in nonhuman primates and may slow the spread of human immunodeficiency virus type 1 (HIV-1). Although protection by passive transfer of human bnAbs has been demonstrated in monkeys, durable expression is essential for its broader use in humans. Gene-based expression of bnAbs provides a potential solution to this problem, although immune responses to the viral vector or to the antibody may limit its durability and efficacy. Here, we delivered an adeno-associated viral vector encoding a simianized form of a CD4bs bnAb, VRC07, and evaluated its immunogenicity and protective efficacy. The expressed antibody circulated in macaques for 16 weeks at levels up to 66 μg/ml, although immune suppression with cyclosporine (CsA) was needed to sustain expression. Gene-delivered simian VRC07 protected against simian-human immunodeficiency virus (SHIV) infection in monkeys 5.5 weeks after treatment. Gene transfer of an anti-HIV antibody can therefore protect against infection by viruses that cause AIDS in primates when the host immune responses are controlled. IMPORTANCE Sustained interventions that can prevent HIV-1 infection are needed to halt the spread of the HIV-1 pandemic. The protective capacity of anti-HIV antibody gene therapy has been established in mouse models of HIV-1 infection but has not been established for primates. We show here a proof-of-concept that gene transfer of anti-HIV antibody genes can protect against infection by viruses that cause AIDS in primates when host immune responses are controlled. PMID:26041300

  7. Improved method for the determination of the major neutral steroids and unconjugated bile acids in human faeces using capillary gas chromatography.

    PubMed

    Bailey, E; Brooks, A G; Purchase, R; Meakings, M; Davies, M; Walters, D G

    1987-10-01

    An improved method has been developed for the determination of the major neutral steroids (cholesterol and 5 beta-cholestan-3 beta-ol) and unconjugated bile acids (deoxycholic acid and lithocholic acid) in human faeces, using capillary gas chromatography with flame ionization detection. The freeze-dried faecal sample was subjected to a two-stage Soxhlet extraction followed by an aqueous alkali-organic solvent partition step to separate neutral steroids from bile acids. The neutral steroids were analysed as their trimethylsilyl ether derivatives on an OV-1 capillary column. The bile acids were further purified on a Sep-Pak C18 cartridge and then fractionated on a Sep-Pak SIL cartridge. Unconjugated bile acids were analysed as their methyl ester-trimethylsilyl ether derivatives also on an OV-1 capillary column. Quantitation of neutral steroids and unconjugated bile acids was achieved by reference to appropriate internal standards, added to the faecal extract immediately after the Soxhlet extraction stage. The method is being used in a study of the effect of diet on the metabolic activity of human gut flora. PMID:3429569

  8. Antibodies with specificity to native gp120 and neutralization activity against primary human immunodeficiency virus type 1 isolates elicited by immunization with oligomeric gp160.

    PubMed Central

    VanCott, T C; Mascola, J R; Kaminski, R W; Kalyanaraman, V; Hallberg, P L; Burnett, P R; Ulrich, J T; Rechtman, D J; Birx, D L

    1997-01-01

    Current human immunodeficiency virus type 1 (HIV-1) envelope vaccine candidates elicit high antibody binding titers with neutralizing activity against T-cell line-adapted but not primary HIV-1 isolates. Serum antibodies from these human vaccine recipients were also found to be preferentially directed to linear epitopes within gp120 that are poorly exposed on native gp120. Systemic immunization of rabbits with an affinity-purified oligomeric gp160 protein formulated with either Alhydrogel or monophosphoryl lipid A-containing adjuvants resulted in the induction of high-titered serum antibodies that preferentially bound epitopes exposed on native forms of gp120 and gp160, recognized a restricted number of linear epitopes, efficiently bound heterologous strains of monomeric gp120 and cell surface-expressed oligomeric gp120/gp41, and neutralized several strains of T-cell line-adapted HIV-1. Additionally, those immune sera with the highest oligomeric gp160 antibody binding titers had neutralizing activity against some primary HIV-1 isolates, using phytohemagglutinin-stimulated peripheral blood mononuclear cell targets. Induction of an antibody response preferentially reactive with natively folded gp120/gp160 was dependent on the tertiary structure of the HIV-1 envelope immunogen as well as its adjuvant formulation, route of administration, and number of immunizations administered. These studies demonstrate the capacity of a soluble HIV-1 envelope glycoprotein vaccine to elicit an antibody response capable of neutralizing primary HIV-1 isolates. PMID:9151820

  9. A human antibody recognizing a conserved epitope of H5 hemagglutinin broadly neutralizes highly pathogenic avian influenza H5N1 viruses.

    PubMed

    Hu, Hongxing; Voss, Jarrod; Zhang, Guoliang; Buchy, Philippi; Zuo, Teng; Wang, Lulan; Wang, Feng; Zhou, Fan; Wang, Guiqing; Tsai, Cheguo; Calder, Lesley; Gamblin, Steve J; Zhang, Linqi; Deubel, Vincent; Zhou, Boping; Skehel, John J; Zhou, Paul

    2012-03-01

    Influenza A virus infection is a persistent threat to public health worldwide due to its ability to evade immune surveillance through rapid genetic drift and shift. Current vaccines against influenza A virus provide immunity to viral isolates that are similar to vaccine strains. High-affinity neutralizing antibodies against conserved epitopes could provide immunity to diverse influenza virus strains and protection against future pandemic viruses. In this study, by using a highly sensitive H5N1 pseudotype-based neutralization assay to screen human monoclonal antibodies produced by memory B cells from an H5N1-infected individual and molecular cloning techniques, we developed three fully human monoclonal antibodies. Among them, antibody 65C6 exhibited potent neutralization activity against all H5 clades and subclades except for subclade 7.2 and prophylactic and therapeutic efficacy against highly pathogenic avian influenza H5N1 viruses in mice. Studies on hemagglutinin (HA)-antibody complexes by electron microscopy and epitope mapping indicate that antibody 65C6 binds to a conformational epitope comprising amino acid residues at positions 118, 121, 161, 164, and 167 (according to mature H5 numbering) on the tip of the membrane-distal globular domain of HA. Thus, we conclude that antibody 65C6 recognizes a neutralization epitope in the globular head of HA that is conserved among almost all divergent H5N1 influenza stains. PMID:22238297

  10. Increased infectivity in human cells and resistance to antibody-mediated neutralization by truncation of the SIV gp41 cytoplasmic tail.

    PubMed

    Kuwata, Takeo; Kaori, Takaki; Enomoto, Ikumi; Yoshimura, Kazuhisa; Matsushita, Shuzo

    2013-01-01

    The role of antibodies in protecting the host from human immunodeficiency virus type 1 (HIV-1) infection is of considerable interest, particularly because the RV144 trial results suggest that antibodies contribute to protection. Although infection of non-human primates with simian immunodeficiency virus (SIV) is commonly used as an animal model of HIV-1 infection, the viral epitopes that elicit potent and broad neutralizing antibodies to SIV have not been identified. We isolated a monoclonal antibody (MAb) B404 that potently and broadly neutralizes various SIV strains. B404 targets a conformational epitope comprising the V3 and V4 loops of Env that intensely exposed when Env binds CD4. B404-resistant variants were obtained by passaging viruses in the presence of increasing concentration of B404 in PM1/CCR5 cells. Genetic analysis revealed that the Q733stop mutation, which truncates the cytoplasmic tail of gp41, was the first major substitution in Env during passage. The maximal inhibition by B404 and other MAbs were significantly decreased against a recombinant virus with a gp41 truncation compared with the parental SIVmac316. This indicates that the gp41 truncation was associated with resistance to antibody-mediated neutralization. The infectivities of the recombinant virus with the gp41 truncation were 7,900-, 1,000-, and 140-fold higher than those of SIVmac316 in PM1, PM1/CCR5, and TZM-bl cells, respectively. Immunoblotting analysis revealed that the gp41 truncation enhanced the incorporation of Env into virions. The effect of the gp41 truncation on infectivity was not obvious in the HSC-F macaque cell line, although the resistance of viruses harboring the gp41 truncation to neutralization was maintained. These results suggest that viruses with a truncated gp41 cytoplasmic tail were selected by increased infectivity in human cells and by acquiring resistance to neutralizing antibody. PMID:23717307

  11. A human monoclonal antibody against HPV16 recognizes an immunodominant and neutralizing epitope partially overlapping with that of H16.V5

    PubMed Central

    Xia, Lin; Xian, Yangfei; Wang, Daning; Chen, Yuanzhi; Huang, Xiaofen; Bi, Xingjian; Yu, Hai; Fu, Zheng; Liu, Xinlin; Li, Shaowei; An, Zhiqiang; Luo, Wenxin; Zhao, Qinjian; Xia, Ningshao

    2016-01-01

    The presence of neutralizing epitopes in human papillomavirus (HPV) L1 virus-like particles (VLPs) is the structural basis of prophylactic vaccines. An anti-HPV16 neutralizing monoclonal antibody (N-mAb) 26D1 was isolated from a memory B cell of a human vaccinee. The pre-binding of heparan sulfate to VLPs inhibited the binding of both N-mAbs to the antigen, indicating that the epitopes are critical for viral cell attachment/entry. Hybrid VLP binding with surface loop swapping between types indicated the essential roles of the DE and FG loops for both 26D1 (DEa in particular) and H16.V5 binding. Specifically, Tyr135 and Val141 on the DEa loop were shown to be critical residues for 26D1 binding via site-directed mutagenesis. Partially overlap between the epitopes between 26D1 and H16.V5 was shown using pairwise epitope mapping, and their binding difference is demonstrated to be predominantly in DE loop region. In addition, 26D1 epitope is immunodominant epitope recognized by both antibodies elicited by the authentic virus from infected individuals and polyclonal antibodies from vaccinees. Overall, a partially overlapping but distinct neutralizing epitope from that of H16.V5 was identified using a human N-mAb, shedding lights to the antibody arrays as part of human immune response to vaccination and infection. PMID:26750243

  12. A human monoclonal antibody against HPV16 recognizes an immunodominant and neutralizing epitope partially overlapping with that of H16.V5.

    PubMed

    Xia, Lin; Xian, Yangfei; Wang, Daning; Chen, Yuanzhi; Huang, Xiaofen; Bi, Xingjian; Yu, Hai; Fu, Zheng; Liu, Xinlin; Li, Shaowei; An, Zhiqiang; Luo, Wenxin; Zhao, Qinjian; Xia, Ningshao

    2016-01-01

    The presence of neutralizing epitopes in human papillomavirus (HPV) L1 virus-like particles (VLPs) is the structural basis of prophylactic vaccines. An anti-HPV16 neutralizing monoclonal antibody (N-mAb) 26D1 was isolated from a memory B cell of a human vaccinee. The pre-binding of heparan sulfate to VLPs inhibited the binding of both N-mAbs to the antigen, indicating that the epitopes are critical for viral cell attachment/entry. Hybrid VLP binding with surface loop swapping between types indicated the essential roles of the DE and FG loops for both 26D1 (DEa in particular) and H16.V5 binding. Specifically, Tyr(135) and Val(141) on the DEa loop were shown to be critical residues for 26D1 binding via site-directed mutagenesis. Partially overlap between the epitopes between 26D1 and H16.V5 was shown using pairwise epitope mapping, and their binding difference is demonstrated to be predominantly in DE loop region. In addition, 26D1 epitope is immunodominant epitope recognized by both antibodies elicited by the authentic virus from infected individuals and polyclonal antibodies from vaccinees. Overall, a partially overlapping but distinct neutralizing epitope from that of H16.V5 was identified using a human N-mAb, shedding lights to the antibody arrays as part of human immune response to vaccination and infection. PMID:26750243

  13. Breadth of neutralization and synergy of clinically relevant human monoclonal antibodies against HCV genotypes 1a, 1b, 2a, 2b, 2c, and 3a

    PubMed Central

    Carlsen, Thomas H.R.; Pedersen, Jannie; Prentoe, Jannick C.; Giang, Erick; Keck, Zhen-Yong; Mikkelsen, Lotte S.; Law, Mansun; Foung, Steven K. H.; Bukh, Jens

    2015-01-01

    Human monoclonal antibodies (HMAbs) with neutralizing capabilities constitute potential immune-based treatments or prophylaxis against hepatitis C virus (HCV). However, lack of cell culture-derived HCV (HCVcc) harboring authentic envelope proteins (E1/E2) has hindered neutralization investigations across genotypes, subtypes, and isolates. We investigated the breadth of neutralization of 10 HMAbs with therapeutic potential against a panel of 16 JFH1-based HCVcc expressing patient-derived Core-NS2 from genotypes 1a (strains H77, TN, and DH6), 1b (J4, DH1, and DH5), 2a (J6, JFH1, and T9), 2b (J8, DH8, and DH10), 2c (S83), and 3a (S52, DBN, and DH11). Virus stocks used for in vitro neutralization analysis contained authentic E1/E2, with the exception of full-length JFH1 that acquired the N417S substitution in E2. The 50% inhibition concentration (IC50) for each HMAb against the HCVcc panel was determined by dose-response neutralization assays in Huh7.5 cells with antibody concentrations ranging from 0.0012 to 100 μg/ml. Interestingly, IC50-values against the different HCVcc’s exhibited large variations among the HMAbs, and only three HMAbs (HC-1AM, HC84.24, and AR4A) neutralized all 16 HCVcc recombinants. Furthermore, the IC50-values for a given HMAb varied greatly with the HCVcc strain, which supports the use of a diverse virus panel. In cooperation analyses, HMAbs HC84.24, AR3A, and, especially HC84.26, demonstrated synergistic effects towards the majority of the HCVcc’s when combined individually with AR4A. Conclusion: Through a neutralization analysis of 10 clinically relevant HMAbs against 16 JFH1-based Core-NS2 recombinants from genotypes 1a, 1b, 2a, 2b, 2c, and 3a, we identified at least 3 HMAbs with potent and broad neutralization potential. The neutralization synergism obtained when pooling the most potent HMAbs could have significant implications for developing novel strategies to treat and control HCV. PMID:25043937

  14. Analysis of Cross-Reactive Neutralizing Antibodies in Human HFMD Serum with an EV71 Pseudovirus-Based Assay

    PubMed Central

    Jin, Jun; Xu, Lin; Sun, Shiyang; Jiang, Liping; Li, Xiaojun; Shao, Jie; Ma, Hongxia; Huang, Xueyong; Guo, Shijie; Chen, Haiying; Cheng, Tong; Yang, Lisheng; Su, Weiheng; Kong, Wei; Liang, Zhenglun; Jiang, Chunlai

    2014-01-01

    Hand, foot and mouth disease, associated with enterovirus 71 (EV71) infections, has recently become an important public health issue throughout the world. Serum neutralizing antibodies are major indicators of EV71 infection and protective immunity. However, the potential for cross-reactivity of neutralizing antibodies for different EV71 genotypes and subgenotypes is unclear. Here we measured the cross-reactive neutralizing antibody titers against EV71 of different genotypes or subgenotypes in sera collected from EV71-infected children and vaccine-inoculated children in a phase III clinical trial (ClinicalTrials.gov Identifier: NCT01636245) using a new pseudovirus-based neutralization assay. Antibodies induced by EV71-C4a were cross-reactive for different EV71 genotypes, demonstrating that C4a is a good candidate strain for an EV71 vaccine. Our study also demonstrated that this new assay is practical for analyses of clinical samples from epidemiological and vaccine studies. PMID:24964084

  15. Isolation, composition and reactivity of the neutral glycoproteins from human meconiums with specificities of the ABO and Lewis systems.

    PubMed Central

    Côté, R H; Valet, J P

    1976-01-01

    Blood-group-specific A1, B, AB, H and Lea neutral glycoproteins were isolated from suitable pools of human normal meconiums by a preliminary fractionation with a cationic detergent at pH5 and 9 (borate), followed by ion-exchange and gel chromatography. The ABH materials have sedimentation coefficients of about 10S-11S, whereas the Lea preparation, not strictly homogeneous, shows a coefficient of 7S. From the detailed analytical data collected, the following relations are deduced between these various substances; they all possess a common peptide core; there are stable ratios of N-acetylglucosamine/N-acetylgalactosamine in the B, H and Lea materials and of N-acetylglucosamine/galactose in A, H and Lea materials, from which the numbers of A and B determinants are estimated. In the ABH substances, the ratio of glucosamine to the sum of threonine and serine is stable. Presumably because of genetic factors, the amount of fucose varies among the different glycoproteins, but it is always definitely lower than in the average cyst substances. Various serological tests and precipitin methods were used to measure the potency, purity and integrity of the preparations, including comparisons between A1 and A2 substances from this source. The Leb activity did not appear as high as it is in glycoproteins from adults and a possible interpretation would be the immature Lewis system as observed on erythrocytes; this could explain their very strong inhibiting power towards iso-agglutinins. This family of substances with various specificities has common features with that prepared from ovarian cysts, but differs clearly on some points. PMID:1259715

  16. Body mapping of cutaneous wetness perception across the human torso during thermo-neutral and warm environmental exposures.

    PubMed

    Filingeri, Davide; Fournet, Damien; Hodder, Simon; Havenith, George

    2014-10-15

    Sensing skin wetness is linked to inputs arising from cutaneous cold-sensitive afferents. As thermosensitivity to cold varies significantly across the torso, we investigated whether similar regional differences in wetness perception exist. We also investigated the regional differences in thermal pleasantness and whether these sensory patterns are influenced by ambient temperature. Sixteen males (20 ± 2 yr) underwent a quantitative sensory test under thermo-neutral [air temperature (Tair) = 22°C; relative humidity (RH) = 50%] and warm conditions (Tair = 33°C; RH = 50%). Twelve regions of the torso were stimulated with a dry thermal probe (25 cm(2)) with a temperature of 15°C below local skin temperature (Tsk). Variations in Tsk, thermal, wetness, and pleasantness sensations were recorded. As a result of the same cold-dry stimulus, the skin-cooling response varied significantly by location (P = 0.003). The lateral chest showed the greatest cooling (-5 ± 0.4°C), whereas the lower back showed the smallest (-1.9 ± 0.4°C). Thermal sensations varied significantly by location and independently from regional variations in skin cooling with colder sensations reported on the lateral abdomen and lower back. Similarly, the frequency of perceived skin wetness was significantly greater on the lateral and lower back as opposed to the medial chest. Overall wetness perception was slightly higher under warm conditions. Significantly more unpleasant sensations were recorded when the lateral abdomen and lateral and lower back were stimulated. We conclude that humans present regional differences in skin wetness perception across the torso, with a pattern similar to the regional differences in thermosensitivity to cold. These findings indicate the presence of a heterogeneous distribution of cold-sensitive thermo-afferent information. PMID:25103965

  17. Quantitative Profiling of Major Neutral Lipid Classes in Human Meibum by Direct Infusion Electrospray Ionization Mass Spectrometry

    PubMed Central

    Chen, Jianzhong; Green, Kari B.; Nichols, Kelly K.

    2013-01-01

    Purpose. The purpose of this investigation was to better understand lipid composition in human meibum. Methods. Intact lipids in meibum samples were detected by direct infusion electrospray ionization mass spectrometry (ESI-MS) analysis in positive detection mode using sodium iodide (NaI) as an additive. The peak intensities of all major types of lipid species, that is, wax esters (WEs), cholesteryl esters (CEs), and diesters (DEs) were corrected for peak overlapping and isotopic distribution; an additional ionization efficiency correction was performed for WEs and CEs, which was simplified by the observation that the corresponding ionization efficiency was primarily dependent on the specific lipid class and saturation degree of the lipids while independent of the carbon chain length. A set of WE and CE standards was spiked in meibum samples for ionization efficiency determination and absolute quantitation. Results. The absolute amount (μmol/mg) for each of 51 WEs and 31 CEs in meibum samples was determined. The summed masses for 51 WEs and 31 CEs accounted for 48 ± 4% and 40 ± 2%, respectively, of the total meibum lipids. The mass percentages of saturated and unsaturated species were determined to be 75 ± 2% and 25 ± 1% for CEs and 14 ± 1% and 86 ± 1% for WEs. The profiles for two types of DEs were also obtained, which include 42 α,ω Type II DEs, and 21 ω Type I-St DEs. Conclusions. Major neutral lipid classes in meibum samples were quantitatively profiled by ESI-MS analysis with NaI additive. PMID:23847307

  18. A novel antibody discovery platform identifies anti-influenza A broadly neutralizing antibodies from human memory B cells.

    PubMed

    Xiao, Xiaodong; Chen, Yan; Varkey, Reena; Kallewaard, Nicole; Koksal, Adem C; Zhu, Qing; Wu, Herren; Chowdhury, Partha S; Dall'Acqua, William F

    2016-07-01

    Monoclonal antibody isolation directly from circulating human B cells is a powerful tool to delineate humoral responses to pathological conditions and discover antibody therapeutics. We have developed a platform aimed at improving the efficiencies of B cell selection and V gene recovery. Here, memory B cells are activated and amplified using Epstein-Barr virus infection, co-cultured with CHO-muCD40L cells, and then assessed by functional screenings. An in vitro transcription and translation (IVTT) approach was used to analyze variable (V) genes recovered from each B cell sample and identify the relevant heavy/light chain pair(s). We achieved efficient amplification and activation of memory B cells, and eliminated the need to: 1) seed B cells at clonal level (≤1 cell/well) or perform limited dilution cloning; 2) immortalize B cells; or 3) assemble V genes into an IgG expression vector to confirm the relevant heavy/light chain pairing. Cross-reactive antibodies targeting a conserved epitope on influenza A hemagglutinin were successfully isolated from a healthy donor. In-depth analysis of the isolated antibodies suggested their potential uses as anti-influenza A antibody therapeutics and uncovered a distinct affinity maturation pathway. Importantly, our results showed that cognate heavy/light chain pairings contributed to both the expression level and binding abilities of our newly isolated VH1-69 family, influenza A neutralizing antibodies, contrasting with previous observations that light chains do not significantly contribute to the function of this group of antibodies. Our results further suggest the potential use of the IVTT as a powerful antibody developability assessment tool. PMID:27049174

  19. Multiple Interactions across the Surface of the gp120 Core Structure Determine the Global Neutralization Resistance Phenotype of Human Immunodeficiency Virus Type 1

    PubMed Central

    Bouma, Peter; Leavitt, Maria; Zhang, Peng Fei; Sidorov, Igor A.; Dimitrov, Dimiter S.; Quinnan, Gerald V.

    2003-01-01

    Resistance to neutralization is an important characteristic of primary isolates of human immunodeficiency virus type 1 (HIV-1) that relates to the potential for successful vaccination to prevent infection and use of immunotherapeutics for treatment of established infection. In order to further elucidate mechanisms responsible for neutralization resistance, we studied the molecular mechanisms that determine the resistance of the primary virus isolate of the strain HIV-1 MN to neutralization by soluble CD4 (sCD4). As is the case for the global neutralization resistance phenotype, sCD4 resistance depended upon sequences in the amino-terminal heptad repeat region of gp41 (HR1), as well as on multiple functional interactions within the envelope complex. The functional interactions that determined the resistance included interactions between the variable loop 1 and 2 (V1/V2) region and sequences in or near the CD4 binding site (CD4bs) and with the V3 loop. Additionally, the V3 loop region was found to interact functionally with sequences in the outer domain of gp120, distant from the CD4bs and coreceptor-binding site, as well as with a residue thought to be located centrally in the coreceptor-binding site. These and previous results provide the basis for a model by which functional signals that determine the neutralization resistance, high-infectivity phenotype depend upon interactions occurring across the surface of the gp120 core structure and involving variable loop structures and gp41. This model should be useful in efforts to define epitopes that may be important for primary virus neutralization. PMID:12829845

  20. Development of a cell-based qualitative assay for detection of neutralizing anti-human interleukin-1 receptor antagonist (hIL-1Ra) antibodies in rats.

    PubMed

    Gao, Jin; Li, Jingjing; Yang, Minmin; Wu, Mingyuan; Tu, Ping; Yu, Yan; Han, Wei

    2015-01-01

    To determine the incidence of the positive neutralizing anti-human interleukin receptor antagonist (anti-IL-1Ra), a novel assay based on the proliferation of human melanoma A375.S2 cells was developed and validated. In the presence of a growth-limiting concentration of IL-1β, A375.S2 cells were able to regain proliferation following the addition of IL-1Ra in a concentration-dependent manner. This dose-response effect enabled the validation of a standard curve for calculation of the concentration of IL-1Ra or, inversely, the concentration of neutralizing anti-IL-1Ra antibodies in cell culture medium or sera. The assay used CCK-8 as an indicator of proliferation. The dose-response relationship between rhIL-1Ra (dose range of 5-75 ng/ml rhIL-1Ra) and A375.S2 cell proliferation was sigmoidal and fitted a four-parameter logistic model. The percent coefficients of variation (%CVs) of quality control samples were 12.5 and 11.9% for intra-assay repeatability and 14.5 and 19.5% for inter-assay repeatability, while the total accuracy was in the range of 97.2-103.6%. For the neutralization assay, the optimal sample dilution factor was found to be 40-fold and the reasonable standard for positive and negative decision was calculated to be 59.4% neutralization rate. The %CVs of quality control samples were 12.7 and 24.0% for intra-assay repeatability and 11.6 and 30.0% for inter-assay repeatability. Analysis using the assay showed that rats could produce neutralizing anti-IL-1Ra antibodies after repeated intramuscular injection with rhIL-1Ra, and this response was not significantly dependent on the dose injected. PMID:26153108

  1. An Active Form of Sphingosine Kinase-1 Is Released in the Extracellular Medium as Component of Membrane Vesicles Shed by Two Human Tumor Cell Lines

    PubMed Central

    Rigogliuso, Salvatrice; Donati, Chiara; Cassarà, Donata; Taverna, Simona; Salamone, Monica; Bruni, Paola; Vittorelli, Maria Letizia

    2010-01-01

    Expression of sphingosine kinase-1 (SphK-1) correlates with a poor survival rate of tumor patients. This effect is probably due to the ability of SphK-1 to be released into the extracellular medium where it catalyzes the biosynthesis of sphingosine-1-phosphate (S1P), a signaling molecule endowed with profound proangiogenic effects. SphK-1 is a leaderless protein which is secreted by an unconventional mechanism. In this paper, we will show that in human hepatocarcinoma Sk-Hep1 cells, extracellular signaling is followed by targeting the enzyme to the cell surface and parallels targeting of FGF-2 to the budding vesicles. We will also show that SphK-1 is present in a catalitycally active form in vesicles shed by SK-Hep1 and human breast carcinoma 8701-BC cells. The enzyme substrate sphingosine is present in shed vesicles where it is produced by neutral ceramidase. Shed vesicles are therefore a site for S1P production in the extracellular medium and conceivably also within host cell following vesicle endocytosis. PMID:20508814

  2. Neutralization sensitivity of human immunodeficiency virus type 1 is determined in part by the cell in which the virus is propagated.

    PubMed Central

    Sawyer, L S; Wrin, M T; Crawford-Miksza, L; Potts, B; Wu, Y; Weber, P A; Alfonso, R D; Hanson, C V

    1994-01-01

    Neutralizing antibody responses to human immunodeficiency virus type 1 (HIV-1) vary widely and have not been reproducibly associated with prognosis or disease progression. We have found that both low-passage clinical isolates and laboratory-adapted strains of HIV-1 have different sensitivities to neutralization by the same antiserum, depending on the host cell in which the viral stock is prepared. One such isolate (VL069) grown in H9 cells was neutralized by 20 human sera at a geometric mean titer of 1:2,047; this same isolate prepared in peripheral blood mononuclear cell (PBMC) culture was neutralized at a mean titer of < 1:10 by the same sera. Adsorption and mixing experiments indicated that neither antibody to H9 cell components nor blocking by excess viral antigen was responsible for the differences observed. This host cell effect is rapidly reversible upon passage of the virus from PBMCs to H9 cells and back into PBMCs. In contrast, the neutralization characteristics remained remarkably stable over extended culture in PBMCs. Two laboratory strains and five clinical isolates were evaluated in expanded studies of this phenomenon. While the neutralization characteristics of most of the strains studied were affected by the host cell in which the strain was propagated, two of the strains (one clinical isolate and one laboratory strain) appeared antigenically unaffected by their cell of origin. Host cell effect was also evident in neutralization by monoclonal antibodies directed against the CD4-binding region and the V2, V3, and gp41 regions. Possible mechanisms for this host cell effect include (i) mutation during passaging; (ii) selection in different host cells of different subpopulations of the (uncloned) viral stock; and (iii) cell-specific posttranslational modifications. To explore these possibilities, the V3 through V5 region of gp120 was sequenced in preparations made by passing VL069 into H9 cells and into PBMCs; HIVMN grown in CEM-SS cells and in PBMCs

  3. Tumor necrosis factor alpha neutralization has no direct effect on parasite burden, but causes impaired IFN-γ production by spleen cells from human visceral leishmaniasis patients.

    PubMed

    Singh, Neetu; Kumar, Rajiv; Engwerda, Christian; Sacks, David; Nylen, Susanne; Sundar, Shyam

    2016-09-01

    The pro-inflammatory cytokine tumor necrosis factor (TNF)-α has an important role in control of experimental Leishmania donovani infection. Less is known about the role of TNF-α in human visceral leishmaniasis (VL). Evidence for a protective role is primarily based on case reports of VL development in individuals treated with TNF-α neutralizing antibody. In this study, we have evaluated how TNF-α neutralization affects parasite replication and cytokine production in ex vivo splenic aspirates (SA) from active VL patients. The effect of TNF-α neutralization on cell mediated antigen specific responses were also evaluated using whole blood cultures. Neutralization of TNF-α did not affect parasite numbers in SA cultures. Interferon (IFN)-γ levels were significantly reduced, but interleukin (IL)-10 levels were unchanged in these cultures. Leishmania antigen stimulated SA produced significant TNF-α which suggests that TNF-α is actively produced in VL spleen. Further it stimulates IFN-γ production, but no direct effect on parasite replication. PMID:27372917

  4. A safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus MERS-CoV

    PubMed Central

    2013-01-01

    Background Evidence points to the emergence of a novel human coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), which causes a severe acute respiratory syndrome (SARS)-like disease. In response, the development of effective vaccines and therapeutics remains a clinical priority. To accomplish this, it is necessary to evaluate neutralizing antibodies and screen for MERS-CoV entry inhibitors. Methods In this study, we produced a pseudovirus bearing the full-length spike (S) protein of MERS-CoV in the Env-defective, luciferase-expressing HIV-1 backbone. We then established a pseudovirus-based inhibition assay to detect neutralizing antibodies and anti-MERS-CoV entry inhibitors. Results Our results demonstrated that the generated MERS-CoV pseudovirus allows for single-cycle infection of a variety of cells expressing dipeptidyl peptidase-4 (DPP4), the confirmed receptor for MERS-CoV. Consistent with the results from a live MERS-CoV-based inhibition assay, the antisera of mice vaccinated with a recombinant protein containing receptor-binding domain (RBD, residues 377–662) of MERS-CoV S fused with Fc of human IgG exhibited neutralizing antibody response against infection of MERS-CoV pseudovirus. Furthermore, one small molecule HIV entry inhibitor targeting gp41 (ADS-J1) and the 3-hydroxyphthalic anhydride-modified human serum albumin (HP-HSA) could significantly inhibit MERS-CoV pseudovirus infection. Conclusion Taken together, the established MERS-CoV inhibition assay is a safe and convenient pseudovirus-based alternative to BSL-3 live-virus restrictions and can be used to rapidly screen MERS-CoV entry inhibitors, as well as evaluate vaccine-induced neutralizing antibodies against the highly pathogenic MERS-CoV. PMID:23978242

  5. Accumulation of co-localised unesterified cholesterol and neutral lipids within vacuolised elastin fibres in athero-prone areas of the human aorta.

    PubMed

    Bobryshev, Y V; Lord, R S

    1999-01-01

    To investigate whether there are alterations of elastin fibres in the arterial intima at the pre-atherosclerotic stage, grossly normal areas of human thoracic aorta were taken soon after death from 13 healthy trauma victims whose ages ranged from 16 to 40 years. Two areas were compared: atherosclerosis-prone (AP) areas localised to the dorsal aspect of the aorta along the rows of intercostal branch origins, and atherosclerosis-resistant (AR) areas from the ventral aorta. Electron microscopic analysis combined with cytochemical staining was applied. Unesterified cholesterol was identified using the filipin-staining technique while neutral lipids were visualised by the OTO-technique. Intimal features were studied by combining the filipin-staining and the OTO-technique. Electron microscopical examination showed that in both AR and AP areas, some elastin fibres in the intima were vacuolised. Unesterified cholesterol was found to be predominantly localised in the musculoelastic layer, in particular, inside the vacuolised elastin fibres. This localisation was seen in all 13 AP areas studied in contrast to the AR areas where it was observed in only four of 13 aortas studied (P < 0.0005, chi2-test). Accumulation of neutral lipids inside vacuolised elastin fibres was found in five out of 13 AP areas but was not observed in any of the AR areas (P=0.01, chi2). A combination of the filipin-staining and OTO-techniques showed that some deposits of neutral lipids and unesterified cholesterol within vacuolised elastin fibres were independently located from each other, but more frequently, neutral lipids were co-located with unesterified cholesterol. The present observations indicate a difference between AP and AR intimal areas which, in particular, relates to the structure of elastin fibres in the musculoelastic layer. The observations suggest that alterations of the extracellular matrix are involved in the trapping and retention of cholesterol and neutral lipids within the intima

  6. Induction of antibody responses that neutralize human T-cell leukemia virus type I infection in vitro and in vivo by peptide immunization.

    PubMed Central

    Tanaka, Y; Tanaka, R; Terada, E; Koyanagi, Y; Miyano-Kurosaki, N; Yamamoto, N; Baba, E; Nakamura, M; Shida, H

    1994-01-01

    In order to define neutralization regions on the envelope antigen of human T-cell leukemia virus type I (HTLV-I), we have generated a number of new anti-envelope gp46 monoclonal antibodies from rats and mice. Epitopes recognized by new monoclonal antibodies which could neutralize HTLV-I in syncytium and transformation inhibition assays were localized to sequences in gp46 from amino acids 186 to 193, 190 to 195, 191 to 195, 191 to 196, and 194 to 199. Ovalbumin-conjugated synthetic gp46 peptides containing these neutralization epitopes, pep190-199 (a synthetic gp46 peptide containing amino acids 190 to 199) and pep180-204, but not pep185-194 or pep194-203, could give rise to HTLV-I-neutralizing antibody responses in rabbits. These immune or nonimmune rabbits were then challenged with HTLV-I by intravenous inoculation with 5 x 10(7) live HTLV-I-producing ILT-8M2 cells. By a PCR assay, it was revealed that HTLV-I provirus was detected in peripheral blood lymphocytes from nonimmune and pep288-312-immunized rabbits, whereas the provirus was not detected in peripheral blood lymphocytes from pep190-199- and pep180-204-immunized rabbits over an extended period. These results suggest that the induction of anti-gp46 neutralizing antibody responses by immunization with synthetic peptides has the potential to protect animals against HTLV-I infection in vivo. Images PMID:8083972

  7. Heat-labile- and heat-stable-toxoid fusions (LTR₁₉₂G-STaP₁₃F) of human enterotoxigenic Escherichia coli elicit neutralizing antitoxin antibodies.

    PubMed

    Liu, Mei; Ruan, Xiaosai; Zhang, Chengxian; Lawson, Steve R; Knudsen, David E; Nataro, James P; Robertson, Donald C; Zhang, Weiping

    2011-10-01

    Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and animals. Adhesins and enterotoxins, including heat-labile (LT) and heat-stable (STa) toxins, are the key virulence factors. Antigenic adhesin and LT antigens have been used in developing vaccines against ETEC diarrhea. However, STa has not been included because of its poor immunogenicity and potent toxicity. Our recent study showed that porcine-type STa toxoids became immunogenic and elicited neutralizing anti-STa antibodies after being genetically fused to a full-length porcine-type LT toxoid, LT(R₁₉₂G) (W. Zhang et al., Infect. Immun. 78:316-325, 2010). In this study, we mutated human-type LT and STa genes, which are highly homologous to porcine-type toxin genes, for a full-length LT toxoid (LT(R₁₉₂)) and a full-length STa toxoid (STa(P₁₃F)) and genetically fused them to produce LT₁₉₂-STa₁₃ toxoid fusions. Mice immunized with LT₁₉₂-STa₁₃ fusion antigens developed anti-LT and anti-STa IgG (in serum and feces) and IgA antibodies (in feces). Moreover, secretory IgA antibodies from immunized mice were shown to neutralize STa and cholera toxins in T-84 cells. In addition, we fused the STa₁₃ toxoid at the N terminus and C terminus, between the A1 and A2 peptides, and between the A and B subunits of LT₁₉₂ to obtain different fusions in order to explore strategies for enhancing STa immunogenicity. This study demonstrated that human-type LT₁₉₂-STa₁₃ fusions induce neutralizing antitoxin antibodies and provided important information for developing toxoid vaccines against human ETEC diarrhea. PMID:21788385

  8. Resistance of a human serum-selected human immunodeficiency virus type 1 escape mutant to neutralization by CD4 binding site monoclonal antibodies is conferred by a single amino acid change in gp120.

    PubMed Central

    McKeating, J A; Bennett, J; Zolla-Pazner, S; Schutten, M; Ashelford, S; Brown, A L; Balfe, P

    1993-01-01

    We have selected an HXB2 variant which can replicate in the presence of a neutralizing human serum. Sequencing of the gp120 region of the env gene from the variant and parental viruses identified a single amino acid substitution in the third conserved region of gp120 at residue 375 (AGT-->AAT, Ser-->Asn; designated 375 S/N). The escape mutant was found to be resistant to neutralization by soluble CD4 (sCD4) and four monoclonal antibodies (MAbs), 39.13g, 1.5e, G13, and 448, binding to epitopes overlapping that of the CD4 binding site (CD4 b.s.). Introduction of the 375 S/N mutation into HXB2 by site-directed mutagenesis confirmed that this mutation is responsible for the neutralization-resistant phenotype. Both sCD4 and three of the CD4 b.s. MAbs (39.13g, 1.5e, and G13) demonstrated reduced binding to the native 375 S/N mutant gp120. The ability to select for an escape variant resistant to multiple independent CD4 b.s. MAbs by a human serum confirms the reports that antibodies to the discontinuous CD4 b.s. are a major component of the group-specific neutralizing activity in human sera. PMID:7688820

  9. Structural Studies of Chikungunya Virus-Like Particles Complexed with Human Antibodies: Neutralization and Cell-to-Cell Transmission

    PubMed Central

    Mangala Prasad, Vidya; Wang, Cheng-I; Akahata, Wataru; Ng, Lisa F. P.

    2015-01-01

    ABSTRACT Chikungunya virus is a positive-stranded RNA alphavirus. Structures of chikungunya virus-like particles in complex with strongly neutralizing antibody Fab fragments (8B10 and 5F10) were determined using cryo-electron microscopy and X-ray crystallography. By fitting the crystallographically determined structures of these Fab fragments into the cryo-electron density maps, we show that Fab fragments of antibody 8B10 extend radially from the viral surface and block receptor binding on the E2 glycoprotein. In contrast, Fab fragments of antibody 5F10 bind the tip of the E2 B domain and lie tangentially on the viral surface. Fab 5F10 fixes the B domain rigidly to the surface of the virus, blocking exposure of the fusion loop on glycoprotein E1 and therefore preventing the virus from becoming fusogenic. Although Fab 5F10 can neutralize the wild-type virus, it can also bind to a mutant virus without inhibiting fusion or attachment. Although the mutant virus is no longer able to propagate by extracellular budding, it can, however, enter the next cell by traveling through junctional complexes without being intercepted by a neutralizing antibody to the wild-type virus, thus clarifying how cell-to-cell transmission can occur. IMPORTANCE Alphaviral infections are transmitted mainly by mosquitoes. Chikungunya virus (CHIKV), which belongs to the Alphavirus genus, has a wide distribution in the Old World that has expanded in recent years into the Americas. There are currently no vaccines or drugs against alphaviral infections. Therefore, a better understanding of CHIKV and its associated neutralizing antibodies will aid in the development of effective treatments. PMID:26537684

  10. Structural Basis of Neutralization of the Major Toxic Component from the Scorpion Centruroides noxius Hoffmann by a Human-derived Single-chain Antibody Fragment

    SciTech Connect

    Canul-Tec, Juan Carlos; Riaño-Umbarila, Lidia; Rudiño-Piñera, Enrique; Becerril, Baltazar; Possani, Lourival D.; Torres-Larios, Alfredo

    2011-08-09

    It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 {angstrom} resolution. A 15-residue span of the toxin is recognized by the antibody through a cleft formed by residues from five of the complementarity-determining regions of the scFv. Analysis of the interface of the complex reveals three features. First, the epitope of toxin Cn2 overlaps with essential residues for the binding of {beta}-toxins to its Na+ channel receptor site. Second, the putative recognition of Css2 involves mainly residues that are present in both Cn2 and Css2 toxins. Finally, the effect on the increase of affinity of previously reported key residues during the maturation process of different scFvs can be inferred from the structure. Taken together, these results provide the structural basis that explain the mechanism of the 9004G neutralizing activity and give insight into the process of directed evolution that gave rise to this family of neutralizing scFvs.

  11. A Conformational Switch in Human Immunodeficiency Virus gp41 Revealed by the Structures of Overlapping Epitopes Recognized by Neutralizing Antibodies ▿

    PubMed Central

    Pejchal, Robert; Gach, Johannes S.; Brunel, Florence M.; Cardoso, Rosa M.; Stanfield, Robyn L.; Dawson, Philip E.; Burton, Dennis R.; Zwick, Michael B.; Wilson, Ian A.

    2009-01-01

    The membrane-proximal external region (MPER) of the human immunodeficiency virus (HIV) envelope glycoprotein (gp41) is critical for viral fusion and infectivity and is the target of three of the five known broadly neutralizing HIV type 1 (HIV-1) antibodies, 2F5, Z13, and 4E10. Here, we report the crystal structure of the Fab fragment of Z13e1, an affinity-enhanced variant of monoclonal antibody Z13, in complex with a 12-residue peptide corresponding to the core epitope (W670NWFDITN677) at 1.8-Å resolution. The bound peptide adopts an S-shaped conformation composed of two tandem, perpendicular helical turns. This conformation differs strikingly from the α-helical structure adopted by an overlapping MPER peptide bound to 4E10. Z13e1 binds to an elbow in the MPER at the membrane interface, making relatively few interactions with conserved aromatics (Trp672 and Phe673) that are critical for 4E10 recognition. The comparison of the Z13e1 and 4E10 epitope structures reveals a conformational switch such that neutralization can occur by the recognition of the different conformations and faces of the largely amphipathic MPER. The Z13e1 structure provides significant new insights into the dynamic nature of the MPER, which likely is critical for membrane fusion, and it has significant implications for mechanisms of HIV-1 neutralization by MPER antibodies and for the design of HIV-1 immunogens. PMID:19515770

  12. Immune Response to Recombinant Capsid Proteins of Adenovirus in Humans: Antifiber and Anti-Penton Base Antibodies Have a Synergistic Effect on Neutralizing Activity

    PubMed Central

    Gahéry-Ségard, Hanne; Farace, Françoise; Godfrin, Dominique; Gaston, Jesintha; Lengagne, Renée; Tursz, Thomas; Boulanger, Pierre; Guillet, Jean-Gérard

    1998-01-01

    Replication-deficient adenovirus used in humans for gene therapy induces a strong immune response to the vector, resulting in transient recombinant protein expression and the blocking of gene transfer upon a second administration. Therefore, in this study we examined in detail the capsid-specific humoral immune response in sera of patients with lung cancer who had been given one dose of a replication-defective adenovirus. We analyzed the immune response to the three major components of the viral capsid, hexon (Hx), penton base (Pb), and fiber (Fi). A longitudinal study of the humoral response assayed on adenovirus particle-coated enzyme-linked immunosorbent assay plates showed that patients had preexisting immunity to adenovirus prior to the administration of adenovirus–β-gal. The level of the response increased in three patients after adenovirus administration and remained at a maximum after three months. One patient had a strong immune response to adenovirus prior to treatment, and this response was unaffected by adenovirus administration. Sera collected from the patients were assayed for recognition of each individual viral capsid protein to determine more precisely the molecular basis of the humoral immune response. Clear differences existed in the humoral response to the three major components of the viral capsid in serum from humans. Sequential appearance of these antibodies was observed: anti-Fi antibodies appeared first, followed by anti-Pb antibodies and then by anti-Hx antibodies. Moreover, anti-Fi antibodies preferentially recognized the native trimeric form of Fi protein, suggesting that they recognized conformational epitopes. Our results showed that sera with no neutralizing activity contained only anti-Fi antibodies. In contrast, neutralizing activity was only obtained with sera containing anti-Fi and anti-Pb antibodies. More importantly, we showed that anti-native Fi and anti-Pb antibodies had a synergistic effect on neutralization. The

  13. Characterization of the promoter region of the gene for the rat neutral and basic amino acid transporter and chromosomal localization of the human gene.

    PubMed Central

    Yan, N; Mosckovitz, R; Gerber, L D; Mathew, S; Murty, V V; Tate, S S; Udenfriend, S

    1994-01-01

    The promoter region of the rat kidney neutral and basic amino acid transporter (NBAT) gene has been isolated and sequenced. The major transcription initiation site was mapped by primer extension. The entire promoter region and a set of 5' deletions within it were expressed at a high level in LLC-PK1 cells using the luciferase indicator gene. Positive and negative regulatory elements in the promoter region were observed. A human genomic clone of the transporter was also obtained and was used to localize the NBAT gene at the p21 region of chromosome 2. Images PMID:8052618

  14. A humanized monoclonal antibody neutralizes yellow fever virus strain 17D-204 in vitro but does not protect a mouse model from disease.

    PubMed

    Calvert, Amanda E; Dixon, Kandice L; Piper, Joseph; Bennett, Susan L; Thibodeaux, Brett A; Barrett, Alan D T; Roehrig, John T; Blair, Carol D

    2016-07-01

    The yellow fever virus (YFV) vaccine 17D-204 is considered safe and effective, yet rare severe adverse events (SAEs), some resulting in death, have been documented following vaccination. Individuals exhibiting post-vaccinal SAEs are ideal candidates for antiviral monoclonal antibody (MAb) therapy; the time until appearance of clinical signs post-exposure is usually short and patients are quickly hospitalized. We previously developed a murine-human chimeric monoclonal antibody (cMAb), 2C9-cIgG, reactive with both virulent YFV and 17D-204, and demonstrated its ability to prevent and treat YF disease in both AG129 mouse and hamster models of infection. To counteract possible selection of 17D-204 variants that escape neutralization by treatment with a single MAb (2C9-cIgG), we developed a second cMAb, 864-cIgG, for use in combination with 2C9-cIgG in post-vaccinal therapy. MAb 864-cIgG recognizes/neutralizes only YFV 17D-204 vaccine substrain and binds to domain III (DIII) of the viral envelope protein, which is different from the YFV type-specific binding site of 2C9-cIgG in DII. Although it neutralized 17D-204 in vitro, administration of 864-cIgG had no protective capacity in the interferon receptor-deficient AG129 mouse model of 17D-204 infection. The data presented here show that although DIII-specific 864-cIgG neutralizes virus infectivity in vitro, it does not have the ability to abrogate disease in vivo. Therefore, combination of 864-cIgG with 2C9-cIgG for treatment of YF vaccination SAEs does not appear to provide an improvement on 2C9-cIgG therapy alone. PMID:27126613

  15. Discrimination of the toxic potential of chemically differing topical glucocorticoids using a neutral red release assay with human keratinocytes and fibroblasts.

    PubMed

    Korting, H C; Hülsebus, E; Kerscher, M; Greber, R; Schäfer-Korting, M

    1995-07-01

    In inflammatory skin disease, hydrocortisone and prednisolone double esters are about equipotent to conventional medium potency topical glucocorticoids, such as betamethasone valerate. Local adverse effects, in particular skin atrophy, are a potential problem with topical glucocorticoids. Recently, cell cultures have shown promise as a means of assessing local tolerance. To investigate the toxic potential of hydrocortisone, hydrocortisone-17-butyrate, hydrocortisone aceponate, prednicarbate, triamcinolone acetonide, betamethasone valerate and desoximethasone, human keratinocytes and fibroblasts were exposed to these agents in vitro, using a modified neutral red release assay. In addition, the morphology of these cells was assessed by light microscopy. Although all the topical glucocorticoids tested proved toxic to both cell types, there were major differences between glucocorticoids in their effect on fibroblasts. Hydrocortisone and the non-halogenated double-ester-type glucocorticoids were less toxic than the conventional medium potency topical glucocorticoids tested (betamethasone valerate and desoximethasone). In particular, hydrocortisone aceponate was less toxic than betamethasone valerate (P < or = 0.05). In general, the effect of topical glucocorticoids on the cells, based on neutral red release, was more marked with keratinocytes than with fibroblasts. Although the ranking order with respect to the toxic potential was similar, a clear-cut difference was not observed between non-halogenated double-ester-type glucocorticoids and betamethasone valerate. Morphological changes due to glucocorticoid exposure followed the same pattern with both keratinocytes and fibroblasts. The neutral red release assay is able to discriminate between the cytotoxic effects of chemically differing topical glucocorticoids on human keratinocytes and fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7669640

  16. Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354

    SciTech Connect

    Kaufmann, Bärbel; Vogt, Matthew R.; Goudsmit, Jaap; Holdaway, Heather A.; Aksyuk, Anastasia A.; Chipman, Paul R.; Kuhn, Richard J.; Diamond, Michael S.; Rossmann, Michael G.

    2010-11-15

    Many flaviviruses are significant human pathogens, with the humoral immune response playing an essential role in restricting infection and disease. CR4354, a human monoclonal antibody isolated from a patient, neutralizes West Nile virus (WNV) infection at a postattachment stage in the viral life-cycle. Here, we determined the structure of WNV complexed with Fab fragments of CR4354 using cryoelectron microscopy. The outer glycoprotein shell of a mature WNV particle is formed by 30 rafts of three homodimers of the viral surface protein E. CR4354 binds to a discontinuous epitope formed by protein segments from two neighboring E molecules, but does not cause any detectable structural disturbance on the viral surface. The epitope occurs at two independent positions within an icosahedral asymmetric unit, resulting in 120 binding sites on the viral surface. The cross-linking of the six E monomers within one raft by four CR4354 Fab fragments suggests that the antibody neutralizes WNV by blocking the pH-induced rearrangement of the E protein required for virus fusion with the endosomal membrane.

  17. Structural Basis of Neutralization by a Human Anti-severe Acute Respiratory Syndrome Spike Protein Antibody,80R.

    SciTech Connect

    Hwang,W.; Lin, Y.; Santelli, E.; Sui, J.; Jaroszewski, L.; Stec, B.; Farzan, M.; Marasco, W.; Liddington, R.

    2006-01-01

    Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease that caused pandemic spread in 2003. The etiological agent of SARS is a novel coronavirus (SARS-CoV). The coronaviral surface spike protein S is a type I transmembrane glycoprotein that mediates initial host binding via the cell surface receptor angiotensin-converting enzyme 2 (ACE2), as well as the subsequent membrane fusion events required for cell entry. Here we report the crystal structure of the S1 receptor binding domain (RBD) in complex with a neutralizing antibody, 80R, at 2.3 {angstrom} resolution, as well as the structure of the uncomplexed S1 RBD at 2.2 {angstrom} resolution. We show that the 80R-binding epitope on the S1 RBD overlaps very closely with the ACE2-binding site, providing a rationale for the strong binding and broad neutralizing ability of the antibody. We provide a structural basis for the differential effects of certain mutations in the spike protein on 80R versus ACE2 binding, including escape mutants, which should facilitate the design of immunotherapeutics to treat a future SARS outbreak. We further show that the RBD of S1 forms dimers via an extensive interface that is disrupted in receptor- and antibody-bound crystal structures, and we propose a role for the dimer in virus stability and infectivity.

  18. Targeting ceramide metabolic pathway induces apoptosis in human breast cancer cell lines

    SciTech Connect

    Vethakanraj, Helen Shiphrah; Babu, Thabraz Ahmed; Sudarsanan, Ganesh Babu; Duraisamy, Prabhu Kumar; Ashok Kumar, Sekar

    2015-08-28

    The sphingolipid ceramide is a pro apoptotic molecule of ceramide metabolic pathway and is hydrolyzed to proliferative metabolite, sphingosine 1 phosphate by the action of acid ceramidase. Being upregulated in the tumors of breast, acid ceramidase acts as a potential target for breast cancer therapy. We aimed at targeting this enzyme with a small molecule acid ceramidase inhibitor, Ceranib 2 in human breast cancer cell lines MCF 7 and MDA MB 231. Ceranib 2 effectively inhibited the growth of both the cell lines in dose and time dependant manner. Morphological apoptotic hallmarks such as chromatin condensation, fragmented chromatin were observed in AO/EtBr staining. Moreover, ladder pattern of fragmented DNA observed in DNA gel electrophoresis proved the apoptotic activity of Ceranib 2 in breast cancer cell lines. The apoptotic events were associated with significant increase in the expression of pro-apoptotic genes (Bad, Bax and Bid) and down regulation of anti-apoptotic gene (Bcl 2). Interestingly, increase in sub G1 population of cell cycle phase analysis and elevated Annexin V positive cells after Ceranib 2 treatment substantiated its apoptotic activity in MCF 7 and MDA MB 231 cell lines. Thus, we report Ceranib 2 as a potent therapeutic agent against both ER{sup +} and ER{sup −} breast cancer cell lines. - Highlights: • Acid Ceramidase inhibitor, Ceranib 2 induced apoptosis in Breast cancer cell lines (MCF 7 and MDA MB 231 cell lines). • Apoptosis is mediated by DNA fragmentation and cell cycle arrest. • Ceranib 2 upregulated the expression of pro-apoptotic genes and down regulated anti-apoptotic gene expression. • More potent compared to the standard drug Tamoxifen.

  19. Immunization of Rabbits with Highly Purified, Soluble, Trimeric Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Induces a Vigorous B Cell Response and Broadly Cross-Reactive Neutralization

    PubMed Central

    Quinnan, Gerald V.; Onabajo, Olusegun; Zhang, Pengfei; Yan, Lianying; Mattapallil, Joseph J.; Zhang, Zhiqiang; Dong, Ming; Lu, Min; Montefiori, David; LaBranche, Celia; Broder, Christopher C.

    2014-01-01

    Previously we described induction of cross-reactive HIV-1 neutralizing antibody responses in rabbits using a soluble HIV-1 gp140 envelope glycoprotein (Env) in an adjuvant containing monophosphoryl lipid A (MPL) and QS21 (AS02A). Here, we compared different forms of the same HIV-1 strain R2 Env for antigenic and biophysical characteristics, and in rabbits characterized the extent of B cell induction for specific antibody expression and secretion and neutralizing responses. The forms of this Env that were produced in and purified from stably transformed 293T cells included a primarily dimeric gp140, a trimeric gp140 appended to a GCN4 trimerization domain (gp140-GCN4), gp140-GCN4 with a 15 amino acid flexible linker between the gp120 and gp41 ectodomain (gp140-GCN4-L), also trimeric, and a gp140 with the flexible linker purified from cell culture supernatants as either dimer (gp140-L(D)) or monomer (gp140-L(M)). Multimeric states of the Env proteins were assessed by native gel electrophoresis and analytical ultracentrifugation. The different forms of gp140 bound broadly cross-reactive neutralizing (BCN) human monoclonal antibodies (mAbs) similarly in ELISA and immunoprecipitation assays. All Envs bound CD4i mAbs in the presence and absence of sCD4, as reported for the R2 Env. Weak neutralization of some strains of HIV-1 was seen after two additional doses in AS02A. Rabbits that were given a seventh dose of gp140-GCN4-L developed BCN responses that were weak to moderate, similar to our previous report. The specificity of these responses did not appear similar to that of any of the known BCN human mAbs. Induction of spleen B cell and plasma cells producing immunoglobulins that bound trimeric gp140-GCN4-L was vigorous, based on ELISpot and flow cytometry analyses. The results demonstrate that highly purified gp140-GCN4-L trimer in adjuvant elicits BCN responses in rabbits accompanied by vigorous B cell induction. PMID:24846288

  20. Naturally Occurring Antibodies in Humans Can Neutralize a Variety of Influenza Virus Strains, Including H3, H1, H2, and H5 ▿ §

    PubMed Central

    Ohshima, Nobuko; Iba, Yoshitaka; Kubota-Koketsu, Ritsuko; Asano, Yoshizo; Okuno, Yoshinobu; Kurosawa, Yoshikazu

    2011-01-01

    Influenza A viruses are classified into 16 subtypes according to the serotypes of hemagglutinin (HA). It is generally thought that neutralizing antibodies (Abs) are not broadly cross-reactive among HA subtypes. We examined the repertoire of neutralizing Abs against influenza viruses in humans. B lymphocytes were collected from donors by apheresis, and Ab libraries were constructed by using phage-display technology. Anti-HA clones were isolated by screening with H3N2 viruses. Their binding activity was examined, and four kinds of Abs showing broad strain specificity were identified from one donor. Two of the Abs, F045-092 and F026-427, were extensively analyzed. They neutralized not only H3N2 but also H1N1, H2N2, and H5N1 viruses, although the activities were largely varied. Flow cytometry suggested that they have the ability to bind to HA and HA1 artificially expressed on the cell surface. They show hemagglutination inhibition activity and do not compete with C179, an Ab thought to bind to the stalk region. F045-092 competes with Abs that recognize sites A and B for binding to HA. Furthermore, the serine at residue 136 in site A could be a part of the epitope. Thus, it is likely that F045-092 and F026-427 bind to a conserved epitope in the head region formed by HA1. Interestingly, while the VH1-69 gene can encode MAbs against the HA stem that are group 1 specific, F045-092 and its relatives that recognize the head region also use VH1-69. The possible epitope recognized by these clones is discussed. PMID:21865387

  1. Human rotavirus strains bearing VP4 gene P[6] allele recovered from asymptomatic or symptomatic infections share similar, if not identical, VP4 neutralization specificities.

    PubMed

    Hoshino, Yasutaka; Jones, Ronald W; Ross, Jerri; Santos, Norma; Kapikian, Albert Z

    2003-11-10

    A rotavirus VP4 gene P[6] allele has been documented in a number of countries to be characteristically associated with an endemic predominantly asymptomatic infection in neonates in maternity hospital nurseries. The mechanisms underlying the endemicity and asymptomatic nature of such neonatal infections remain unknown. Rotavirus strains sharing this same P genotype, however, have more recently been recovered from an increasing number of symptomatic diarrheal episodes in infants and young children in various parts of the world. Previously, we have shown that an asymptomatic P[6] rotavirus neonatal infection is not associated with a unique VP7 (G) serotype but may occur in conjunction with various G types. Although amino acid sequence comparisons of the VP4 gene between selected "asymptomatic" and "symptomatic" P[6] rotavirus strains have been reported and yielded information concerning their VP4 genotypes, serotypic comparisons of the outer capsid spike protein VP4 of such viruses have not been studied systematically by two-way cross-neutralizations. We determined the VP4 neutralization specificities of four asymptomatic and four symptomatic P[6] strains: two each of asymptomatic and symptomatic strains by two-way tests, and two each of additional asymptomatic and symptomatic strains by one-way tests. Both asymptomatic and symptomatic P[6] strains were shown to bear similar, if not identical, VP4 neutralization specificities. Thus, P[6] rotavirus strains causing asymptomatic or symptomatic infections did not appear to belong to unique P (VP4) serotypes. In addition, a close VP4 serotypic relationship between human P[6] rotavirus strains and the porcine P[6] rotavirus Gottfried strain was confirmed. PMID:14599785

  2. Humanized-Single Domain Antibodies (VH/VHH) that Bound Specifically to Naja kaouthia Phospholipase A2 and Neutralized the Enzymatic Activity

    PubMed Central

    Chavanayarn, Charnwit; Thanongsaksrikul, Jeeraphong; Thueng-in, Kanyarat; Bangphoomi, Kunan; Sookrung, Nitat; Chaicumpa, Wanpen

    2012-01-01

    Naja kaouthia (monocled cobra) venom contains many isoforms of secreted phospholipase A2 (sPLA2). The PLA2 exerts several pharmacologic and toxic effects in the snake bitten subject, dependent or independent on the enzymatic activity. N. kaouthia venom appeared in two protein profiles, P3 and P5, after fractionating the venom by ion exchange column chromatography. In this study, phage clones displaying humanized-camel single domain antibodies (VH/VHH) that bound specifically to the P3 and P5 were selected from a humanized-camel VH/VHH phage display library. Two phagemid transfected E. coli clones (P3-1 and P3-3) produced humanized-VHH, while another clone (P3-7) produced humanized-VH. At the optimal venom:antibody ratio, the VH/VHH purified from the E. coli homogenates neutralized PLA2 enzyme activity comparable to the horse immune serum against the N. kaouthia holo-venom. Homology modeling and molecular docking revealed that the VH/VHH covered the areas around the PLA2 catalytic groove and inserted their Complementarity Determining Regions (CDRs) into the enzymatic cleft. It is envisaged that the VH/VHH would ameliorate/abrogate the principal toxicity of the venom PLA2 (membrane phospholipid catabolism leading to cellular and subcellular membrane damage which consequently causes hemolysis, hemorrhage, and dermo-/myo-necrosis), if they were used for passive immunotherapy of the cobra bitten victim. The speculation needs further investigations. PMID:22852068

  3. Passive Transfer of A Germline-like Neutralizing Human Monoclonal Antibody Protects Transgenic Mice Against Lethal Middle East Respiratory Syndrome Coronavirus Infection

    PubMed Central

    Agrawal, Anurodh Shankar; Ying, Tianlei; Tao, Xinrong; Garron, Tania; Algaissi, Abdullah; Wang, Yanping; Wang, Lili; Peng, Bi-Hung; Jiang, Shibo; Dimitrov, Dimiter S.; Tseng, Chien-Te K.

    2016-01-01

    Middle East Respiratory Syndrome coronavirus (MERS-CoV) has repeatedly caused outbreaks in the Arabian Peninsula. To date, no approved medical countermeasures (MCM) are available to combat MERS-CoV infections. Several neutralizing human monoclonal antibodies (mAbs), including m336, a germline-like human mAb, have been chosen as promising MCM for MERS-CoV. However, their clinical development has been hindered by the lack of a robust animal model that recapitulate the morbidity and mortality of human infections. We assessed the prophylactic and therapeutic efficacy of m336 by using well-characterized transgenic mice shown to be highly sensitive to MERS-CoV infection and disease. We found that mice treated with m336 prior to or post lethal MERS-CoV challenging were fully protected, compared to control mice which sufferered from profound weight loss and uniform death within days after infection. Taken together, these results support further development of m336 and other human monoclonal antibodies as potential therapeutics for MERS-CoV infection. PMID:27538452

  4. Structural analysis of underivatized neutral human milk oligosaccharides in the negative ion mode by nano-electrospray MS(n) (part 1: methodology).

    PubMed

    Pfenninger, Anja; Karas, Michael; Finke, Berndt; Stahl, Bernd

    2002-11-01

    Underivatized neutral oligosaccharides from human milk were analyzed by nano-electrospray ionization (ESI) using a quadrupole ion trap mass spectrometer (QIT-MS) in the negative-ion mode. Under these conditions neutral oligosaccharides are observed as deprotonated molecules [M-H]- with high intensity. CID-experiments of these species with the charge localized at the reducing end lead to C-type fragment ions forming a "new" reducing end. Fragmentations are accompanied by cross-ring cleavages that yield information about linkages of internal monosaccharides. Several isomeric compounds with distinct structural features, such as different glycosidic linkages, fucosylation and branching sites were investigated. The rules governing the fragmentation behavior of this class of oligosaccharides were elucidated and tested for a representative number of certain isomeric glycoforms using the MS/MS and MS(n) capabilities of the QIT. On the basis of the specific fragmentation behavior of deprotonated molecules, the position of fucoses and the linkage type (Gal beta-->3 GlcNAc or Gal beta1-->4 GlcNAc) could be determined and linear and branched could be differentiated. Rules could be established which can be applied in further investigations of these types of oligosaccharides even from heterogenous mixtures. PMID:12443024

  5. Human non-neutralizing HIV-1 envelope monoclonal antibodies limit the number of founder viruses during SHIV mucosal infection in rhesus macaques

    SciTech Connect

    Santra, Sampa; Tomaras, Georgia D.; Warrier, Ranjit; Nicely, Nathan I.; Liao, Hua -Xin; Pollara, Justin; Liu, Pinghuang; Alam, S. Munir; Zhang, Ruijun; Cocklin, Sarah L.; Shen, Xiaoying; Duffy, Ryan; Xia, Shi -Mao; Schutte, Robert J.; Pemble IV, Charles W.; Dennison, S. Moses; Li, Hui; Chao, Andrew; Vidnovic, Kora; Evans, Abbey; Klein, Katja; Kumar, Amit; Robinson, James; Landucci, Gary; Forthal, Donald N.; Montefiori, David C.; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Rerks-Ngarm, Supachai; Robb, Merlin L.; Michael, Nelson L.; Kim, Jerome H.; Soderberg, Kelly A.; Giorgi, Elena E.; Blair, Lily; Korber, Bette T.; Moog, Christiane; Shattock, Robin J.; Letvin, Norman L.; Schmitz, Joern E.; Moody, M. A.; Gao, Feng; Ferrari, Guido; Shaw, George M.; Haynes, Barton F.; Douek, Daniel C.

    2015-08-03

    HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4⁺ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant region of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses.

  6. Human Non-neutralizing HIV-1 Envelope Monoclonal Antibodies Limit the Number of Founder Viruses during SHIV Mucosal Infection in Rhesus Macaques

    PubMed Central

    Liao, Hua-Xin; Pollara, Justin; Liu, Pinghuang; Alam, S. Munir; Zhang, Ruijun; Cocklin, Sarah L.; Shen, Xiaoying; Duffy, Ryan; Xia, Shi-Mao; Schutte, Robert J.; Pemble IV, Charles W.; Dennison, S. Moses; Li, Hui; Chao, Andrew; Vidnovic, Kora; Evans, Abbey; Klein, Katja; Kumar, Amit; Robinson, James; Landucci, Gary; Forthal, Donald N.; Montefiori, David C.; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Rerks-Ngarm, Supachai; Robb, Merlin L.; Michael, Nelson L.; Kim, Jerome H.; Soderberg, Kelly A.; Giorgi, Elena E.; Blair, Lily; Korber, Bette T.; Moog, Christiane; Shattock, Robin J.; Schmitz, Joern E.; Moody, M. A.; Gao, Feng; Ferrari, Guido; Shaw, George M.; Haynes, Barton F.

    2015-01-01

    HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4+ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant region of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Thus, some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses. PMID:26237403

  7. Evolution of the human immunodeficiency virus type 2 envelope in the first years of infection is associated with the dynamics of the neutralizing antibody response

    SciTech Connect

    Rocha, Cheila; Calado, Rita; Borrego, Pedro; Marcelino, José Maria; Bártolo, Inês; Rosado, Lino; Cavaco-Silva, Patrícia; Gomes, Perpétua; Família, Carlos; Quintas, Alexandre; Skar, Helena; Leitner, Thomas; Barroso, Helena; Taveira, Nuno

    2013-10-24

    Background: therapy and the majority of HIV-2 infected individuals survive as elite controllers with normal CD4+ T cell counts and low or undetectable plasma viral load. Neutralizing antibodies (Nabs) are thought to play a central role in HIV-2 evolution and pathogenesis. However, the dynamic of the Nab response and resulting HIV-2 escape during acute infection and their impact in HIV-2 evolution and disease progression remain largely unknown. Our objective was to characterize the Nab response and the molecular and phenotypic evolution of HIV-2 in association with Nab escape in the first years of infection in two children infected at birth. As a result, CD4+ T cells decreased from about 50% to below 30% in both children in the first five years of infection and the infecting R5 viruses were replaced by X4 viruses within the same period. With antiretroviral therapy, viral load in child 1 decreased to undetectable levels and CD4+ T cells recovered to normal levels, which have been sustained at least until the age of 12. In contrast, viral load increased in child 2 and she progressed to AIDS and death at age 9. Beginning in the first year of life, child 1 raised high titers of antibodies that neutralized primary R5 isolates more effectively than X4 isolates, both autologous and heterologous. Child 2 raised a weak X4-specific Nab response that decreased sharply as disease progressed. Rate of evolution, nucleotide and amino acid diversity, and positive selection, were significantly higher in the envelope of child 1 compared to child 2. Rates of R5-to-X4 tropism switch, of V1 and V3 sequence diversification, and of convergence of V3 to a β-hairpin structure were related with rate of escape from the neutralizing antibodies. Finally, our data suggests that the molecular and phenotypic evolution of the human immunodeficiency virus type 2 envelope are related with the dynamics of the neutralizing antibody response providing further support

  8. Human non-neutralizing HIV-1 envelope monoclonal antibodies limit the number of founder viruses during SHIV mucosal infection in rhesus macaques

    DOE PAGESBeta

    Santra, Sampa; Tomaras, Georgia D.; Warrier, Ranjit; Nicely, Nathan I.; Liao, Hua -Xin; Pollara, Justin; Liu, Pinghuang; Alam, S. Munir; Zhang, Ruijun; Cocklin, Sarah L.; et al

    2015-08-03

    HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4⁺ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant regionmore » of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses.« less

  9. Evolution of the human immunodeficiency virus type 2 envelope in the first years of infection is associated with the dynamics of the neutralizing antibody response

    DOE PAGESBeta

    Rocha, Cheila; Calado, Rita; Borrego, Pedro; Marcelino, José Maria; Bártolo, Inês; Rosado, Lino; Cavaco-Silva, Patrícia; Gomes, Perpétua; Família, Carlos; Quintas, Alexandre; et al

    2013-10-24

    Background: therapy and the majority of HIV-2 infected individuals survive as elite controllers with normal CD4+ T cell counts and low or undetectable plasma viral load. Neutralizing antibodies (Nabs) are thought to play a central role in HIV-2 evolution and pathogenesis. However, the dynamic of the Nab response and resulting HIV-2 escape during acute infection and their impact in HIV-2 evolution and disease progression remain largely unknown. Our objective was to characterize the Nab response and the molecular and phenotypic evolution of HIV-2 in association with Nab escape in the first years of infection in two children infected at birth.more » As a result, CD4+ T cells decreased from about 50% to below 30% in both children in the first five years of infection and the infecting R5 viruses were replaced by X4 viruses within the same period. With antiretroviral therapy, viral load in child 1 decreased to undetectable levels and CD4+ T cells recovered to normal levels, which have been sustained at least until the age of 12. In contrast, viral load increased in child 2 and she progressed to AIDS and death at age 9. Beginning in the first year of life, child 1 raised high titers of antibodies that neutralized primary R5 isolates more effectively than X4 isolates, both autologous and heterologous. Child 2 raised a weak X4-specific Nab response that decreased sharply as disease progressed. Rate of evolution, nucleotide and amino acid diversity, and positive selection, were significantly higher in the envelope of child 1 compared to child 2. Rates of R5-to-X4 tropism switch, of V1 and V3 sequence diversification, and of convergence of V3 to a β-hairpin structure were related with rate of escape from the neutralizing antibodies. Finally, our data suggests that the molecular and phenotypic evolution of the human immunodeficiency virus type 2 envelope are related with the dynamics of the neutralizing antibody response providing further support for a model in which Nabs

  10. Application of laser postionization secondary neutral mass spectrometry/time-of-flight secondary ion mass spectrometry in nanotoxicology: visualization of nanosilver in human macrophages and cellular responses.

    PubMed

    Haase, Andrea; Arlinghaus, Heinrich F; Tentschert, Jutta; Jungnickel, Harald; Graf, Philipp; Mantion, Alexandre; Draude, Felix; Galla, Sebastian; Plendl, Johanna; Goetz, Mario E; Masic, Admir; Meier, Wolfgang; Thünemann, Andreas F; Taubert, Andreas; Luch, Andreas

    2011-04-26

    Silver nanoparticles (SNP) are the subject of worldwide commercialization because of their antimicrobial effects. Yet only little data on their mode of action exist. Further, only few techniques allow for visualization and quantification of unlabeled nanoparticles inside cells. To study SNP of different sizes and coatings within human macrophages, we introduce a novel laser postionization secondary neutral mass spectrometry (Laser-SNMS) approach and prove this method superior to the widely applied confocal Raman and transmission electron microscopy. With time-of-flight secondary ion mass spectrometry (TOF-SIMS) we further demonstrate characteristic fingerprints in the lipid pattern of the cellular membrane indicative of oxidative stress and membrane fluidity changes. Increases of protein carbonyl and heme oxygenase-1 levels in treated cells confirm the presence of oxidative stress biochemically. Intriguingly, affected phagocytosis reveals as highly sensitive end point of SNP-mediated adversity in macrophages. The cellular responses monitored are hierarchically linked, but follow individual kinetics and are partially reversible. PMID:21456612

  11. The chimeric antibody chLpMab-7 targeting human podoplanin suppresses pulmonary metastasis via ADCC and CDC rather than via its neutralizing activity

    PubMed Central

    Ogasawara, Satoshi; Fujii, Yuki; Oki, Hiroharu; Fukayama, Masashi; Nishioka, Yasuhiko; Kaneko, Mika K.

    2015-01-01

    Podoplanin (PDPN/Aggrus/T1α) binds to C-type lectin-like receptor-2 (CLEC-2) and induces platelet aggregation. PDPN is associated with malignant progression, tumor metastasis, and poor prognosis in several types of cancer. Although many anti-human PDPN (hPDPN) monoclonal antibodies (mAbs), such as D2-40 and NZ-1, have been established, these epitopes are limited to the platelet aggregation-stimulating (PLAG) domain (amino acids 29-54) of hPDPN. Recently, we developed a novel mouse anti-hPDPN mAb, LpMab-7, which is more sensitive than D2-40 and NZ-1, using the Cancer-specific mAb (CasMab) method. The epitope of LpMab-7 was shown to be entirely different from that of NZ-1, a neutralizing mAb against the PLAG domain according to an inhibition assay and lectin microarray analysis. In the present study, we produced a mouse-human chimeric anti-hPDPN mAb, chLpMab-7. ChLpMab-7 showed high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Furthermore, chLpMab-7 inhibited the growth of hPDPN-expressing tumors in vivo. Although chLpMab-7 recognizes a non-PLAG domain of hPDPN, it suppressed the hematogenous metastasis of hPDPN-expressing tumors. These results indicated that chLpMab-7 suppressed tumor development and hematogenous metastasis in a neutralization-independent manner. In conclusion, hPDPN shows promise as a target in the development of a novel antibody-based therapy. PMID:26416352

  12. Addition of a Single gp120 Glycan Confers Increased Binding to Dendritic Cell-Specific ICAM-3-Grabbing Nonintegrin and Neutralization Escape to Human Immunodeficiency Virus Type 1

    PubMed Central

    Lue, James; Hsu, Mayla; Yang, David; Marx, Preston; Chen, Zhiwei; Cheng-Mayer, Cecilia

    2002-01-01

    The potential role of dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) binding in human immunodeficiency virus transmission across the mucosal barrier was investigated by assessing the ability of simian-human immunodeficiency chimeric viruses (SHIVs) showing varying degrees of mucosal transmissibility to bind the DC-SIGN expressed on the surface of transfected cells. We found that gp120 of the highly transmissible, pathogenic CCR5-tropic SHIVSF162P3 bound human and rhesus DC-SIGN with an efficiency threefold or greater than that of gp120 of the nonpathogenic, poorly transmissible parental SHIVSF162, and this increase in binding to the DC-SIGN of the SHIVSF162P3 envelope gp120 translated into an enhancement of T-cell infection in trans. The presence of an additional glycan at the N-terminal base of the V2 loop of SHIVSF162P3 gp120 compared to that of the parental virus was shown to be responsible for the increase in binding to DC-SIGN. Interestingly, this glycan also conferred escape from autologous neutralization, raising the possibility that the modification occurred as a result of immune selection. Our data suggest that more-efficient binding of envelope gp120 to DC-SIGN could be relevant to the enhanced mucosal transmissibility of SHIVSF162P3 compared to that of parental SHIVSF162. PMID:12239306

  13. Humanized-single domain antibodies (VH/VHH) that bound specifically to Naja kaouthia phospholipase A2 and neutralized the enzymatic activity.

    PubMed

    Chavanayarn, Charnwit; Thanongsaksrikul, Jeeraphong; Thueng-In, Kanyarat; Bangphoomi, Kunan; Sookrung, Nitat; Chaicumpa, Wanpen

    2012-07-01

    Naja kaouthia (monocled cobra) venom contains many isoforms of secreted phospholipase A2 (sPLA(2)). The PLA(2) exerts several pharmacologic and toxic effects in the snake bitten subject, dependent or independent on the enzymatic activity. N. kaouthia venom appeared in two protein profiles, P3 and P5, after fractionating the venom by ion exchange column chromatography. In this study, phage clones displaying humanized-camel single domain antibodies (VH/V(H)H) that bound specifically to the P3 and P5 were selected from a humanized-camel VH/V(H)H phage display library. Two phagemid transfected E. coli clones (P3-1 and P3-3) produced humanized-V(H)H, while another clone (P3-7) produced humanized-VH. At the optimal venom:antibody ratio, the VH/V(H)H purified from the E. coli homogenates neutralized PLA(2) enzyme activity comparable to the horse immune serum against the N. kaouthia holo-venom. Homology modeling and molecular docking revealed that the VH/V(H)H covered the areas around the PLA(2) catalytic groove and inserted their Complementarity Determining Regions (CDRs) into the enzymatic cleft. It is envisaged that the VH/V(H)H would ameliorate/abrogate the principal toxicity of the venom PLA(2) (membrane phospholipid catabolism leading to cellular and subcellular membrane damage which consequently causes hemolysis, hemorrhage, and dermo-/myo-necrosis), if they were used for passive immunotherapy of the cobra bitten victim. The speculation needs further investigations. PMID:22852068

  14. Human neutral amino acid transporter ASCT1: Structure of the gene (SLC1A4) and localization to chromosome 2p13-p15

    SciTech Connect

    Hofmann, K.; Dueker, M.; Stoffel, W.

    1994-11-01

    Screening for cDNAs encoding proteins similar to the sodium-coupled glutamate transporter GLAST1 led to the isolation of a cDNA clone coding for a protein that turned out to be identical to the recently described neutral amino acid transporter ASCT1. The new member of the GLAST-related transporter family does not transport glutamate or aspartate but alanine, serine, cysteine, and threonine instead. The expressed sequence tag EST02446, a short cDNA sequence found in the course of a large-scale sequencing project of human brain-derived cDNA, showed significant similarity to the eukaryotic glutamate transporter GLAST1 and was therefore used as probe in the search for further glutamate transporter cDNAs. Fragments of the cDNA were used for the isolation and characterization of human ASCT1 genomic clones. The ORF of 1572 bp encoding 524 amino acid residues is distributed over 8 exons, which span at least 40 kb of human chromosomal DNA. The ASCT1 gene locus was assigned to chromosome 2p13-p15 by chromosomal in situ suppression (CISS) studies. The gene structure is not related to any other previously characterized transporter gene. In contrast to the genes of the sodium-coupled nonglutamate neurotransmitter transporters, it shows no obvious correspondence between intron/exon structure and transmembrane organization. The transcription start site in human liver tissue was determined by primer extension analysis to be located 291 bp upstream of the initiating ATG codon. The DNA region immediately upstream of the transcription start lacks any TATA or CAAT boxes but contains several bindings sites for the transcription factors Sp1 and Egr-1. The ASCT1 gene (SLC1A4) structure reported here will facilitate the characterization of the genes of the other members of the GLAST-related transporter family and might be useful in the elucidation of amino acid transport-related defects. 36 refs., 5 figs., 1 tab.

  15. Potent neutralization of VEGF biological activities with a fully human antibody Fab fragment directed against VEGF receptor 2

    SciTech Connect

    Miao, H.-Q. . E-mail: hua-quan.miao@imclone.com; Hu, Kun; Jimenez, Xenia; Navarro, Elizabeth; Zhang, Haifan; Lu Dan; Ludwig, Dale L.; Balderes, Paul; Zhu Zhenping . E-mail: zhenping.zhu@imclone.com

    2006-06-23

    Compelling evidence suggest that vascular endothelial growth factor (VEGF) and its receptors, especially receptor 2 (VEGFR2, or kinase insert domain-containing receptor, KDR), play a critical role in angiogenesis under both physiological and pathological conditions, including cancer and angiogenic retinopathies such as age-related macular degeneration (AMD). To this end, inhibition of angiogenesis with antagonists to either VEGF or KDR has yielded significant therapeutic efficacy both in preclinical studies in animal models and in clinical trials in patients with cancer and AMD. We previously reported the identification of a high affinity, fully human anti-KDR antibody fragment, 1121B Fab, through a highly stringent affinity maturation process with a Fab originally isolated from a naive human antibody phage display library. In this study, we demonstrate that 1121B Fab is able to strongly block KDR/VEGF interaction, resulting in potent inhibition of an array of biological activities of VEGF, including activation of the receptor and its signaling pathway, intracellular calcium mobilization, and migration and proliferation of endothelial cells. Taken together, our data lend strong support to the further development of 1121B Fab fragment as an anti-angiogenesis agent in both cancer and angiogenic retinopathies.

  16. A recombinant mimetics of the HIV-1 gp41 prehairpin fusion intermediate fused with human IgG Fc fragment elicits neutralizing antibody response in the vaccinated mice

    SciTech Connect

    Qi, Zhi; Pan, Chungen; Lu, Hong; Shui, Yuan; Li, Lin; Li, Xiaojuan; Xu, Xueqing; Liu, Shuwen; Jiang, Shibo

    2010-07-30

    Research highlights: {yields} One recombinant mimetics of gp41 prehairpin fusion intermediate (PFI) consisting of gp41 N46 sequence, foldon and IgG Fc, designated N46FdFc, was expressed. {yields} N46FdFc-induced antibodies in mice that neutralized HIV-1 infection, inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. {yields} These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines. -- Abstract: HIV-1 gp41 prehairpin fusion intermediate (PFI) composed of three N-terminal heptad repeats (NHR) plays a crucial role in viral fusion and entry and represents an attractive target for anti-HIV therapeutics (e.g., enfuvirtide) and vaccines. In present study, we constructed and expressed two recombinant gp41 PFI mimetics, designated N46Fd and N46FdFc. N46Fd consists of N46 (residues 536-581) in gp41 NHR and foldon (Fd), a trimerization motif. N46FdFc is composed of N46Fd fused with human IgG Fc fragment as an immunoenhancer. We immunized mice with N46 peptide, N46Fd and N46FdFc, respectively, and found that only N46FdFc elicited neutralizing antibody response in mice against infection by HIV-1 strains IIIB (clade B, X4), 92US657 (clade B, R5), and 94UG103 (clade A, X4R5). Anti-N46FdFc antibodies inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines.

  17. Human Immunodeficiency Virus Type 1 Subtype B Ancestral Envelope Protein Is Functional and Elicits Neutralizing Antibodies in Rabbits Similar to Those Elicited by a Circulating Subtype B Envelope

    PubMed Central

    Doria-Rose, N. A.; Learn, G. H.; Rodrigo, A. G.; Nickle, D. C.; Li, F.; Mahalanabis, M.; Hensel, M. T.; McLaughlin, S.; Edmonson, P. F.; Montefiori, D.; Barnett, S. W.; Haigwood, N. L.; Mullins, J. I.

    2005-01-01

    Human immunodeficiency virus type 1 (HIV-1) is a difficult target for vaccine development, in part because of its ever-expanding genetic diversity and attendant capacity to escape immunologic recognition. Vaccine efficacy might be improved by maximizing immunogen antigenic similarity to viruses likely to be encountered by vaccinees. To this end, we designed a prototype HIV-1 envelope vaccine using a deduced ancestral state for the env gene. The ancestral state reconstruction method was shown to be >95% accurate by computer simulation and 99.8% accurate when estimating the known inoculum used in an experimental infection study in rhesus macaques. Furthermore, the deduced ancestor gene differed from the set of sequences used to derive the ancestor by an average of 12.3%, while these latter sequences were an average of 17.3% different from each other. A full-length ancestral subtype B HIV-1 env gene was constructed and shown to produce a glycoprotein of 160 kDa that bound and fused with cells expressing the HIV-1 coreceptor CCR5. This Env was also functional in a virus pseudotype assay. When either gp160- or gp140-expressing plasmids and recombinant gp120 were used to immunize rabbits in a DNA prime-protein boost regimen, the artificial gene induced immunoglobulin G antibodies capable of weakly neutralizing heterologous primary HIV-1 strains. The results were similar for rabbits immunized in parallel with a natural isolate, HIV-1 SF162. Further design efforts to better present conserved neutralization determinants are warranted. PMID:16103173

  18. Dengue virus neutralization and antibody-dependent enhancement activities of human monoclonal antibodies derived from dengue patients at acute phase of secondary infection.

    PubMed

    Sasaki, Tadahiro; Setthapramote, Chayanee; Kurosu, Takeshi; Nishimura, Mitsuhiro; Asai, Azusa; Omokoko, Magot D; Pipattanaboon, Chonlatip; Pitaksajjakul, Pannamthip; Limkittikul, Kriengsak; Subchareon, Arunee; Chaichana, Panjaporn; Okabayashi, Tamaki; Hirai, Itaru; Leaungwutiwong, Pornsawan; Misaki, Ryo; Fujiyama, Kazuhito; Ono, Ken-Ichiro; Okuno, Yoshinobu; Ramasoota, Pongrama; Ikuta, Kazuyoshi

    2013-06-01

    Public health concern about dengue diseases, caused by mosquito-borne infections with four serotypes of dengue virus (DENV-1-DENV-4), is escalating in tropical and subtropical countries. Most of the severe dengue cases occur in patients experiencing a secondary infection with a serotype that is different from the first infection. This is believed to be due to antibody-dependent enhancement (ADE), by which one DENV serotype uses pre-existing anti-DENV antibodies elicited in the primary infection to facilitate entry of a different DENV serotype into the Fc receptor-positive macrophages. Recently, we prepared a number of hybridomas producing human monoclonal antibodies (HuMAbs) by using peripheral blood lymphocytes from Thai patients at acute phase of secondary infection with DENV-2. Here, we characterized 17 HuMAbs prepared from two patients with dengue fever (DF) and one patient with dengue hemorrhagic fever (DHF) that were selected as antibodies recognizing viral envelope protein and showing higher neutralization activity to all serotypes. In vivo evaluation using suckling mice revealed near perfect activity to prevent mouse lethality following intracerebral DENV-2 inoculation. In a THP-1 cell assay, these HuMAbs showed ADE activities against DENV-2 at similar levels between HuMAbs derived from DF and DHF patients. However, the F(ab')2 fragment of the HuMAb showed a similar virus neutralization activity as original, with no ADE activity. Thus, these HuMAbs could be one of the therapeutic candidates against DENV infection. PMID:23545366

  19. A rapid immunization strategy with a live-attenuated tetravalent dengue vaccine elicits protective neutralizing antibody responses in non-human primates.

    PubMed

    Ambuel, Yuping; Young, Ginger; Brewoo, Joseph N; Paykel, Joanna; Weisgrau, Kim L; Rakasz, Eva G; Haller, Aurelia A; Royals, Michael; Huang, Claire Y-H; Capuano, Saverio; Stinchcomb, Dan T; Partidos, Charalambos D; Osorio, Jorge E

    2014-01-01

    Dengue viruses (DENVs) cause approximately 390 million cases of DENV infections annually and over 3 billion people worldwide are at risk of infection. No dengue vaccine is currently available nor is there an antiviral therapy for DENV infections. We have developed a tetravalent live-attenuated DENV vaccine tetravalent dengue vaccine (TDV) that consists of a molecularly characterized attenuated DENV-2 strain (TDV-2) and three chimeric viruses containing the pre-membrane and envelope genes of DENV-1, -3, and -4 expressed in the context of the TDV-2 genome. To impact dengue vaccine delivery in endemic areas and immunize travelers, a simple and rapid immunization strategy (RIS) is preferred. We investigated RIS consisting of two full vaccine doses being administered subcutaneously or intradermally on the initial vaccination visit (day 0) at two different anatomical locations with a needle-free disposable syringe jet injection delivery devices (PharmaJet) in non-human primates. This vaccination strategy resulted in efficient priming and induction of neutralizing antibody responses to all four DENV serotypes comparable to those elicited by the traditional prime and boost (2 months later) vaccination schedule. In addition, the vaccine induced CD4(+) and CD8(+) T cells producing IFN-γ, IL-2, and TNF-α, and targeting the DENV-2 NS1, NS3, and NS5 proteins. Moreover, vaccine-specific T cells were cross-reactive with the non-structural NS3 and NS5 proteins of DENV-4. When animals were challenged with DENV-2 they were protected with no detectable viremia, and exhibited sterilizing immunity (no increase of neutralizing titers post-challenge). RIS could decrease vaccination visits and provide quick immune response to all four DENV serotypes. This strategy could increase vaccination compliance and would be especially advantageous for travelers into endemic areas. PMID:24926294

  20. A Non-VH1-69 Heterosubtypic Neutralizing Human Monoclonal Antibody Protects Mice against H1N1 and H5N1 Viruses

    PubMed Central

    Solforosi, Laura; Moreno, Guisella J.; Sun, Xiangjie; Tumpey, Terrence M.; Gubareva, Larisa V.; Mishin, Vasiliy; Clementi, Massimo; Burioni, Roberto

    2012-01-01

    Influenza viruses are among the most important human pathogens and are responsible for annual epidemics and sporadic, potentially devastating pandemics. The humoral immune response plays an important role in the defense against these viruses, providing protection mainly by producing antibodies directed against the hemagglutinin (HA) glycoprotein. However, their high genetic variability allows the virus to evade the host immune response and the potential protection offered by seasonal vaccines. The emergence of resistance to antiviral drugs in recent years further limits the options available for the control of influenza. The development of alternative strategies for influenza prophylaxis and therapy is therefore urgently needed. In this study, we describe a human monoclonal antibody (PN-SIA49) that recognizes a highly conserved epitope located on the stem region of the HA and able to neutralize a broad spectrum of influenza viruses belonging to different subtypes (H1, H2 and H5). Furthermore, we describe its protective activity in mice after lethal challenge with H1N1 and H5N1 viruses suggesting a potential application in the treatment of influenza virus infections. PMID:22496802

  1. A non-VH1-69 heterosubtypic neutralizing human monoclonal antibody protects mice against H1N1 and H5N1 viruses.

    PubMed

    De Marco, Donata; Clementi, Nicola; Mancini, Nicasio; Solforosi, Laura; Moreno, Guisella J; Sun, Xiangjie; Tumpey, Terrence M; Gubareva, Larisa V; Mishin, Vasiliy; Clementi, Massimo; Burioni, Roberto

    2012-01-01

    Influenza viruses are among the most important human pathogens and are responsible for annual epidemics and sporadic, potentially devastating pandemics. The humoral immune response plays an important role in the defense against these viruses, providing protection mainly by producing antibodies directed against the hemagglutinin (HA) glycoprotein. However, their high genetic variability allows the virus to evade the host immune response and the potential protection offered by seasonal vaccines. The emergence of resistance to antiviral drugs in recent years further limits the options available for the control of influenza. The development of alternative strategies for influenza prophylaxis and therapy is therefore urgently needed. In this study, we describe a human monoclonal antibody (PN-SIA49) that recognizes a highly conserved epitope located on the stem region of the HA and able to neutralize a broad spectrum of influenza viruses belonging to different subtypes (H1, H2 and H5). Furthermore, we describe its protective activity in mice after lethal challenge with H1N1 and H5N1 viruses suggesting a potential application in the treatment of influenza virus infections. PMID:22496802

  2. Global CNS gene delivery and evasion of anti-AAV-neutralizing antibodies by intrathecal AAV administration in non-human primates.

    PubMed

    Gray, S J; Nagabhushan Kalburgi, S; McCown, T J; Jude Samulski, R

    2013-04-01

    Injection of adeno-associated virus (AAV) into the cerebrospinal fluid (CSF) offers a means to achieve widespread transgene delivery to the central nervous system, where the doses can be readily translated from small to large animals. In contrast to studies with other serotypes (AAV2, AAV4 and AAV5) in rodents, we report that a naturally occurring capsid (AAV9) and rationally engineered capsid (AAV2.5) are able to achieve broad transduction throughout the brain and spinal cord parenchyma following a single injection into the CSF (via cisterna magna or lumbar cistern) in non-human primates (NHP). Using either vector at a dose of ∼2 × 10(12) vector genome (vg) per 3-6 kg animal, approximately 2% of the entire brain and spinal cord was transduced, covering all regions of the central nervous system (CNS). AAV9 in particular displayed efficient transduction of spinal cord motor neurons. The peripheral organ biodistribution was highly reduced compared with intravascular delivery, and the presence of circulating anti-AAV-neutralizing antibodies up to a 1:128 titer had no inhibitory effect on CNS gene transfer. Intra-CSF delivery effectively translates from rodents to NHPs, which provides encouragement for the use of this approach in humans to treat motor neuron and lysosomal storage diseases. PMID:23303281

  3. Mucosal delivery of the human immunodeficiency virus-1 Tat protein in mice elicits systemic neutralizing antibodies, cytotoxic T lymphocytes and mucosal IgA.

    PubMed

    Marinaro, Mariarosaria; Riccomi, Antonella; Rappuoli, Rino; Pizza, Mariagrazia; Fiorelli, Valeria; Tripiciano, Antonella; Cafaro, Aurelio; Ensoli, Barbara; De Magistris, Maria Teresa

    2003-09-01

    Human immunodeficiency virus (HIV)-1 Tat protein induces protection in non-human primates upon systemic vaccination. In view of the design of mucosal vaccines against HIV-1 we studied the immune response to native Tat (aa 1-86) in mice following intranasal delivery of the protein with two mucosal adjuvants, Escherichia coli heat-labile enterotoxin (LT) and LT-R72, a non-toxic mutant of LT. Immunization with Tat and the two adjuvants induced in BALB/c but not in C57BL/6 mice high and persistent levels of serum IgG and secretory IgA in vaginal and intestinal fluids. Mice sera neutralized Tat and recognized two epitopes mapping in the regions 1-20 and 46-60. Furthermore, their splenocytes proliferated and secreted IFN-gamma and IL-6 in response to Tat. Finally, CTLs were also elicited and they recognized an epitope localized within aa 11-40 of Tat. PMID:12922133

  4. CD10/neutral endopeptidase 24.11 in developing human fetal lung. Patterns of expression and modulation of peptide-mediated proliferation.

    PubMed Central

    Sunday, M E; Hua, J; Torday, J S; Reyes, B; Shipp, M A

    1992-01-01

    The cell membrane-associated enzyme CD10/neutral endopeptidase 24.11 (CD10/NEP) functions in multiple organ systems to downregulate responses to peptide hormones. Recently, CD10/NEP was found to hydrolyze bombesin-like peptides (BLP), which are mitogens for normal bronchial epithelial cells and small cell lung carcinomas. Growth of BLP-responsive small cell lung carcinomas was potentiated by CD10/NEP inhibition, implicating CD10/NEP in regulation of BLP-mediated tumor growth. BLP are also likely to participate in normal lung development because high BLP levels are found in fetal lung, and bombesin induces proliferation and maturation of human fetal lung in organ cultures and murine fetal lung in utero. To explore potential roles for CD10/NEP in regulating peptide-mediated human fetal lung development, we have characterized temporal and cellular patterns of CD10/NEP expression and effects of CD10/NEP inhibition in organ cultures. Peak CD10/NEP transcript levels are identified at 11-13 wk gestation by Northern blots and localized to epithelial cells and mesenchyme of developing airways by in situ hybridization. CD10/NEP immunostaining is most intense in undifferentiated airway epithelium. In human fetal lung organ cultures, inhibition of CD10/NEP with either phosphoramidon or SCH32615 increases thymidine incorporation by 166-182% (P < 0.025). The specific BLP receptor antagonist, [Leu13-psi(CH2NH)Leu14]bombesin abolishes these effects on fetal lung growth, suggesting that CD10/NEP modulates BLP-mediated proliferation. CD10/NEP expression in the growing front of airway epithelium and the effects of CD10/NEP inhibitors in lung explants implicate the enzyme in the regulation of peptide-mediated fetal lung growth. Images PMID:1469102

  5. Human monoclonal antibodies derived from a patient infected with 2009 pandemic influenza A virus broadly cross-neutralize group 1 influenza viruses

    SciTech Connect

    Pan, Yang; Sasaki, Tadahiro; Du, Anariwa; and others

    2014-07-18

    Highlights: • Influenza infection can elicit heterosubtypic antibodies to group 1 influenza virus. • Three human monoclonal antibodies were generated from an H1N1-infected patient. • The antibodies predominantly recognized α-helical stem of viral hemagglutinin (HA). • The antibodies inhibited HA structural activation during the fusion process. • The antibodies are potential candidates for future antibody therapy to influenza. - Abstract: Influenza viruses are a continuous threat to human public health because of their ability to evolve rapidly through genetic drift and reassortment. Three human monoclonal antibodies (HuMAbs) were generated in this study, 1H11, 2H5 and 5G2, and they cross-neutralize a diverse range of group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H5N1 and H9N2. The three HuMAbs were prepared by fusing peripheral blood lymphocytes from an H1N1pdm-infected patient with a newly developed fusion partner cell line, SPYMEG. All the HuMAbs had little hemagglutination inhibition activity but had strong membrane-fusion inhibition activity against influenza viruses. A protease digestion assay showed the HuMAbs targeted commonly a short α-helix region in the stalk of the hemagglutinin. Furthermore, Ile45Phe and Glu47Gly double substitutions in the α-helix region made the HA unrecognizable by the HuMAbs. These two amino acid residues are highly conserved in the HAs of H1N1, H5N1 and H9N2 viruses. The HuMAbs reported here may be potential candidates for the development of therapeutic antibodies against group 1 influenza viruses.

  6. Five birds, one stone: neutralization of α-hemolysin and 4 bi-component leukocidins of Staphylococcus aureus with a single human monoclonal antibody.

    PubMed

    Rouha, Harald; Badarau, Adriana; Visram, Zehra C; Battles, Michael B; Prinz, Bianka; Magyarics, Zoltán; Nagy, Gábor; Mirkina, Irina; Stulik, Lukas; Zerbs, Manuel; Jägerhofer, Michaela; Maierhofer, Barbara; Teubenbacher, Astrid; Dolezilkova, Ivana; Gross, Karin; Banerjee, Srijib; Zauner, Gerhild; Malafa, Stefan; Zmajkovic, Jakub; Maier, Sabine; Mabry, Robert; Krauland, Eric; Wittrup, K Dane; Gerngross, Tillman U; Nagy, Eszter

    2015-01-01

    Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component leukocidins that share only ∼26% overall amino acid sequence identity. The molecular basis of cross-reactivity is the recognition of a conformational epitope shared by α-hemolysin and F-components of gamma-hemolysin (HlgAB and HlgCB), LukED and LukSF (Panton-Valentine Leukocidin). The amino acids predicted to form the epitope are conserved and known to be important for cytotoxic activity. We found that a single cross-reactive antibody prevented lysis of human phagocytes, epithelial and red blood cells induced by α-hemolysin and leukocidins in vitro, and therefore had superior effectiveness compared to α-hemolysin specific antibodies to protect from the combined cytolytic effect of secreted S. aureus toxins. Such mAb afforded high levels of protection in murine models of pneumonia and sepsis. PMID:25523282

  7. Heterosubtypic Neutralizing Monoclonal Antibodies Cross-Protective against H5N1 and H1N1 Recovered from Human IgM+ Memory B Cells

    PubMed Central

    Throsby, Mark; van den Brink, Edward; Jongeneelen, Mandy; Poon, Leo L. M.; Alard, Philippe; Cornelissen, Lisette; Bakker, Arjen; Cox, Freek; van Deventer, Els; Guan, Yi; Cinatl, Jindrich; ter Meulen, Jan; Lasters, Ignace; Carsetti, Rita; Peiris, Malik; de Kruif, John; Goudsmit, Jaap

    2008-01-01

    Background The hemagglutinin (HA) glycoprotein is the principal target of protective humoral immune responses to influenza virus infections but such antibody responses only provide efficient protection against a narrow spectrum of HA antigenic variants within a given virus subtype. Avian influenza viruses such as H5N1 are currently panzootic and pose a pandemic threat. These viruses are antigenically diverse and protective strategies need to cross protect against diverse viral clades. Furthermore, there are 16 different HA subtypes and no certainty the next pandemic will be caused by an H5 subtype, thus it is important to develop prophylactic and therapeutic interventions that provide heterosubtypic protection. Methods and Findings Here we describe a panel of 13 monoclonal antibodies (mAbs) recovered from combinatorial display libraries that were constructed from human IgM+ memory B cells of recent (seasonal) influenza vaccinees. The mAbs have broad heterosubtypic neutralizing activity against antigenically diverse H1, H2, H5, H6, H8 and H9 influenza subtypes. Restriction to variable heavy chain gene IGHV1-69 in the high affinity mAb panel was associated with binding to a conserved hydrophobic pocket in the stem domain of HA. The most potent antibody (CR6261) was protective in mice when given before and after lethal H5N1 or H1N1 challenge. Conclusions The human monoclonal CR6261 described in this study could be developed for use as a broad spectrum agent for prophylaxis or treatment of human or avian influenza infections without prior strain characterization. Moreover, the CR6261 epitope could be applied in targeted vaccine strategies or in the design of novel antivirals. Finally our approach of screening the IgM+ memory repertoire could be applied to identify conserved and functionally relevant targets on other rapidly evolving pathogens. PMID:19079604

  8. Neutralization-Sensitive R5-Tropic Simian-Human Immunodeficiency Virus SHIV-2873Nip, Which Carries env Isolated from an Infant with a Recent HIV Clade C Infection▿

    PubMed Central

    Siddappa, Nagadenahalli B.; Song, Ruijiang; Kramer, Victor G.; Chenine, Agnès-Laurence; Velu, Vijayakumar; Ong, Helena; Rasmussen, Robert A.; Grisson, Ricky D.; Wood, Charles; Zhang, Hong; Kankasa, Chipeppo; Amara, Rama Rao; Else, James G.; Novembre, Francis J.; Montefiori, David C.; Ruprecht, Ruth M.

    2009-01-01

    Human immunodeficiency virus clade C (HIV-C) accounts for >56% of all HIV infections worldwide. To investigate vaccine safety and efficacy in nonhuman primates, a pathogenic, R5-tropic, neutralization-sensitive simian-human immunodeficiency virus (SHIV) carrying HIV-C env would be desirable. We have constructed SHIV-2873Ni, an R5-tropic SHIV carrying a primary pediatric HIV-C env gene isolated from a 2-month-old Zambian infant, who died within 1 year of birth. SHIV-2873Ni was constructed using SHIV-1157ipd3N4 (R. J. Song, A. L. Chenine, R. A. Rasmussen, C. R. Ruprecht, S. Mirshahidi, R. D. Grisson, W. Xu, J. B. Whitney, L. M. Goins, H. Ong, P. L. Li, E. Shai-Kobiler, T. Wang, C. M. McCann, H. Zhang, C. Wood, C. Kankasa, W. E. Secor, H. M. McClure, E. Strobert, J. G. Else, and R. M. Ruprecht. J. Virol. 80:8729-8738, 2006) as the backbone, since the latter contains additional NF-κB sites in the long terminal repeats to enhance viral replicative capacity. The parental virus, SHIV-2873Ni, was serially passaged through five rhesus monkeys (RMs); SHIV-2873Nip, the resulting passaged virus, was reisolated from the fourth recipient about 1 year postinoculation. SHIV-2873Nip was replication competent in RM peripheral blood mononuclear cells of all random donors tested and was exclusively R5 tropic, and its env gene clustered with HIV-C by phylogenetic analysis; its high sensitivity to neutralization led to classification as a tier 1 virus. Indian-origin RMs were inoculated by different mucosal routes, resulting in high peak viral RNA loads. Signs of virus-induced disease include depletion of gut CD4+ T lymphocytes, loss of memory T cells in blood, and thrombocytopenia that resulted in fatal cerebral hemorrhage. SHIV-2873Nip is a highly replication-competent, mucosally transmissible, pathogenic R5-tropic virus that will be useful to study viral pathogenesis and to assess the efficacy of immunogens targeting HIV-C Env. PMID:19019970

  9. Human papillomavirus type 16 virus-like particles expressed in attenuated Salmonella typhimurium elicit mucosal and systemic neutralizing antibodies in mice.

    PubMed Central

    Nardelli-Haefliger, D; Roden, R B; Benyacoub, J; Sahli, R; Kraehenbuhl, J P; Schiller, J T; Lachat, P; Potts, A; De Grandi, P

    1997-01-01

    Attenuated strains of Salmonella are attractive live vaccine candidates for eliciting mucosal as well as systemic immune responses. The ability to induce immune responses in the reproductive tract may be critical for the effectiveness of a prophylactic vaccine against genital human papillomaviruses (HPV), which are important etiologic agents in the development of cervical cancer. To examine the potential of a live Salmonella-based vaccine to prevent genital HPV infection, the L1 major capsid protein from HPV type 16 (HPV16) was constitutively expressed in the PhoPc strain of Salmonella typhimurium. As demonstrated by electron microscopy, the L1 protein expressed in these bacteria assembled into virus-like particles (VLPs) that resemble authentic papillomavirus virions. This is the first demonstration that papillomavirus VLPs can self-assemble in prokaryotes. BALB/c mice were immunized with the HPV16 L1 recombinant PhoPc strain by the oral and nasal routes. Despite a low stability of the L1-expressing plasmid in vivo, a double nasal immunization was effective in inducing L1-specific serum antibodies that recognized mainly native, but not disassembled, VLPs. These antibodies effectively neutralized HPV16 pseudotyped virions in an in vitro infectivity assay. Conformationally dependent anti-VLP immunoglobulin A (IgA) and IgG were also detected in oral and vaginal secretions, indicating that potentially protective antibody responses were elicited at mucosal sites. Recombinant attenuated Salmonella expressing HPV capsids may represent a promising vaccine candidate against genital HPV infection. PMID:9234794

  10. Mass spectrometric analysis of neutral and anionic N-glycans from a Dictyostelium discoideum model for human congenital disorder of glycosylation CDG IL.

    PubMed

    Hykollari, Alba; Balog, Crina I A; Rendić, Dubravko; Braulke, Thomas; Wilson, Iain B H; Paschinger, Katharina

    2013-03-01

    The HL241 mutant strain of the cellular slime mold Dictyostelium discoideum is a potential model for human congenital disorder of glycosylation type IL (ALG9-CDG) and has been previously predicted to possess a lower degree of modification of its N-glycans with anionic moieties than the parental wild-type. In this study, we first showed that this strain has a premature stop codon in its alg9 mannosyltransferase gene compatible with the occurrence of truncated N-glycans. These were subject to an optimized analytical workflow, considering that the mass spectrometry of acidic glycans often presents challenges due to neutral loss and suppression effects. Therefore, the protein-bound N-glycans were first fractionated, after serial enzymatic release, by solid phase extraction. Then primarily single glycan species were isolated by mixed hydrophilic-interaction/anion-exchange or reversed-phase HPLC and analyzed using chemical and enzymatic treatments and MS/MS. We show that protein-linked N-glycans of the mutant are of reduced size as compared to those of wild-type AX3, but still contain core α1,3-fucose, intersecting N-acetylglucosamine, bisecting N-acetylglucosamine, methylphosphate, phosphate, and sulfate residues. We observe that a single N-glycan can carry up to four of these six possible modifications. Due to the improved analytical procedures, we reveal fuller details regarding the N-glycomic potential of this fascinating model organism. PMID:23320427

  11. Structural Characterization of Humanized Nanobodies with Neutralizing Activity against the Bordetella pertussis CyaA-Hemolysin: Implications for a Potential Epitope of Toxin-Protective Antigen

    PubMed Central

    Malik, Aijaz Ahmad; Imtong, Chompounoot; Sookrung, Nitat; Katzenmeier, Gerd; Chaicumpa, Wanpen; Angsuthanasombat, Chanan

    2016-01-01

    Previously, the 126-kDa CyaA-hemolysin (CyaA-Hly) fragment cloned from Bordetella pertussis—the causative agent of whooping cough—and functionally expressed in Escherichia coli was revealed as a key determinant for CyaA-mediated hemolysis against target erythrocytes. Here, phagemid-transfected E. coli clones producing nanobodies capable of binding to CyaA-Hly were selected from a humanized-camel VH/VHH phage-display library. Subsequently verified for binding activities by indirect ELISA and Western blotting, four CyaA-Hly-specific nanobodies were obtained and designated according to the presence/absence of VHH-hallmark amino acids as VHH2, VH5, VH18 and VHH37. In vitro neutralization assay revealed that all four ~17-kDa His-tagged VH/VHH nanobodies, in particular VHH37, which were over-expressed as inclusions and successfully unfolded-refolded, were able to effectively inhibit CyaA-Hly-mediated hemolysis. Phage-mimotope searching revealed that only peptides with sequence homologous to Linker 1 connecting Blocks I and II within the CyaA-RTX subdomain were able to bind to these four CyaA-Hly-specific nanobodies. Structural analysis of VHH37 via homology modeling and intermolecular docking confirmed that this humanized nanobody directly interacts with CyaA-RTX/Linker 1 through multiple hydrogen and ionic bonds. Altogether, our present data demonstrate that CyaA-RTX/Linker 1 could serve as a potential epitope of CyaA-protective antigen that may be useful for development of peptide-based pertussis vaccines. Additionally, such toxin-specific nanobodies have a potential for test-driven development of a ready-to-use therapeutic in passive immunization for mitigation of disease severity. PMID:27043627

  12. Ophiophagus hannah venom: proteome, components bound by Naja kaouthia antivenin and neutralization by N. kaouthia neurotoxin-specific human ScFv.

    PubMed

    Danpaiboon, Witchuda; Reamtong, Onrapak; Sookrung, Nitat; Seesuay, Watee; Sakolvaree, Yuwaporn; Thanongsaksrikul, Jeeraphong; Dong-din-on, Fonthip; Srimanote, Potjanee; Thueng-in, Kanyarat; Chaicumpa, Wanpen

    2014-05-01

    Venomous snakebites are an important health problem in tropical and subtropical countries. King cobra (Ophiophagus hannah) is the largest venomous snake found in South and Southeast Asia. In this study, the O. hannah venom proteome and the venom components cross-reactive to N. kaouthia monospecific antivenin were studied. O. hannah venom consisted of 14 different protein families, including three finger toxins, phospholipases, cysteine-rich secretory proteins, cobra venom factor, muscarinic toxin, L-amino acid oxidase, hypothetical proteins, low cysteine protein, phosphodiesterase, proteases, vespryn toxin, Kunitz, growth factor activators and others (coagulation factor, endonuclease, 5'-nucleotidase). N. kaouthia antivenin recognized several functionally different O. hannah venom proteins and mediated paratherapeutic efficacy by rescuing the O. hannah envenomed mice from lethality. An engineered human ScFv specific to N. kaouthia long neurotoxin (NkLN-HuScFv) cross-neutralized the O. hannah venom and extricated the O. hannah envenomed mice from death in a dose escalation manner. Homology modeling and molecular docking revealed that NkLN-HuScFv interacted with residues in loops 2 and 3 of the neurotoxins of both snake species, which are important for neuronal acetylcholine receptor binding. The data of this study are useful for snakebite treatment when and where the polyspecific antivenin is not available. Because the supply of horse-derived antivenin is limited and the preparation may cause some adverse effects in recipients, a cocktail of recombinant human ScFvs for various toxic venom components shared by different venomous snakes, exemplified by the in vitro produced NkLN-HuScFv in this study, should contribute to a possible future route for an improved alternative to the antivenins. PMID:24828754

  13. Ophiophagus hannah Venom: Proteome, Components Bound by Naja kaouthia Antivenin and Neutralization by N. kaouthia Neurotoxin-Specific Human ScFv

    PubMed Central

    Danpaiboon, Witchuda; Reamtong, Onrapak; Sookrung, Nitat; Seesuay, Watee; Sakolvaree, Yuwaporn; Thanongsaksrikul, Jeeraphong; Dong-din-on, Fonthip; Srimanote, Potjanee; Thueng-in, Kanyarat; Chaicumpa, Wanpen

    2014-01-01

    Venomous snakebites are an important health problem in tropical and subtropical countries. King cobra (Ophiophagus hannah) is the largest venomous snake found in South and Southeast Asia. In this study, the O. hannah venom proteome and the venom components cross-reactive to N. kaouthia monospecific antivenin were studied. O. hannah venom consisted of 14 different protein families, including three finger toxins, phospholipases, cysteine-rich secretory proteins, cobra venom factor, muscarinic toxin, L-amino acid oxidase, hypothetical proteins, low cysteine protein, phosphodiesterase, proteases, vespryn toxin, Kunitz, growth factor activators and others (coagulation factor, endonuclease, 5’-nucleotidase). N. kaouthia antivenin recognized several functionally different O. hannah venom proteins and mediated paratherapeutic efficacy by rescuing the O. hannah envenomed mice from lethality. An engineered human ScFv specific to N. kaouthia long neurotoxin (NkLN-HuScFv) cross-neutralized the O. hannah venom and extricated the O. hannah envenomed mice from death in a dose escalation manner. Homology modeling and molecular docking revealed that NkLN-HuScFv interacted with residues in loops 2 and 3 of the neurotoxins of both snake species, which are important for neuronal acetylcholine receptor binding. The data of this study are useful for snakebite treatment when and where the polyspecific antivenin is not available. Because the supply of horse-derived antivenin is limited and the preparation may cause some adverse effects in recipients, a cocktail of recombinant human ScFvs for various toxic venom components shared by different venomous snakes, exemplified by the in vitro produced NkLN-HuScFv in this study, should contribute to a possible future route for an improved alternative to the antivenins. PMID:24828754

  14. Structural Characterization of Humanized Nanobodies with Neutralizing Activity against the Bordetella pertussis CyaA-Hemolysin: Implications for a Potential Epitope of Toxin-Protective Antigen.

    PubMed

    Malik, Aijaz Ahmad; Imtong, Chompounoot; Sookrung, Nitat; Katzenmeier, Gerd; Chaicumpa, Wanpen; Angsuthanasombat, Chanan

    2016-04-01

    Previously, the 126-kDa CyaA-hemolysin (CyaA-Hly) fragment cloned from Bordetella pertussis--the causative agent of whooping cough--and functionally expressed in Escherichia coli was revealed as a key determinant for CyaA-mediated hemolysis against target erythrocytes. Here, phagemid-transfected E. coli clones producing nanobodies capable of binding to CyaA-Hly were selected from a humanized-camel VH/VHH phage-display library. Subsequently verified for binding activities by indirect ELISA and Western blotting, four CyaA-Hly-specific nanobodies were obtained and designated according to the presence/absence of VHH-hallmark amino acids as VHH2, VH5, VH18 and VHH37. In vitro neutralization assay revealed that all four ~17-kDa His-tagged VH/VHH nanobodies, in particular VHH37, which were over-expressed as inclusions and successfully unfolded-refolded, were able to effectively inhibit CyaA-Hly-mediated hemolysis. Phage-mimotope searching revealed that only peptides with sequence homologous to Linker 1 connecting Blocks I and II within the CyaA-RTX subdomain were able to bind to these four CyaA-Hly-specific nanobodies. Structural analysis of VHH37 via homology modeling and intermolecular docking confirmed that this humanized nanobody directly interacts with CyaA-RTX/Linker 1 through multiple hydrogen and ionic bonds. Altogether, our present data demonstrate that CyaA-RTX/Linker 1 could serve as a potential epitope of CyaA-protective antigen that may be useful for development of peptide-based pertussis vaccines. Additionally, such toxin-specific nanobodies have a potential for test-driven development of a ready-to-use therapeutic in passive immunization for mitigation of disease severity. PMID:27043627

  15. Structural Analysis of Human and Macaque mAbs 2909 and 2.5B: Implications for the Configuration of the Quaternary Neutralizing Epitope of HIV-1 gp120

    SciTech Connect

    Spurrier, Brett; Sampson, Jared M.; Totrov, Maxim; Li, Huiguang; O; Neal, Timothy; Williams, Constance; Robinson, James; Gorny, Miroslaw K.; Zolla-Pazner, Susan; Kong, Xiang-Peng

    2011-08-25

    The quaternary neutralizing epitope (QNE) of HIV-1 gp120 is preferentially expressed on the trimeric envelope spikes of intact HIV virions, and QNE-specific monoclonal antibodies (mAbs) potently neutralize HIV-1. Here, we present the crystal structures of the Fabs of human mAb 2909 and macaque mAb 2.5B. Both mAbs have long beta hairpin CDR H3 regions >20 {angstrom} in length that are each situated at the center of their respective antigen-binding sites. Computational analysis showed that the paratopes include the whole CDR H3, while additional CDR residues form shallow binding pockets. Structural modeling suggests a way to understand the configuration of QNEs and the antigen-antibody interaction for QNE mAbs. Our data will be useful in designing immunogens that may elicit potent neutralizing QNE Abs.

  16. Tailoring in vitro selection for a picomolar affinity human antibody directed against vascular endothelial growth factor receptor 2 for enhanced neutralizing activity.

    PubMed

    Lu, Dan; Shen, Juqun; Vil, Marie D; Zhang, Haifan; Jimenez, Xenia; Bohlen, Peter; Witte, Larry; Zhu, Zhenping

    2003-10-31

    Vascular endothelial growth factor (VEGF) and its receptors have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. We previously identified several fully human neutralizing anti-VEGF receptor 2 (or kinase inserting domain-containing receptor (KDR)) antibodies from a large antibody phage display library. These antibodies bind specifically to KDR, block VEGF/KDR interaction, and inhibit VEGF-induced proliferation of human endothelial cells and migration of KDR+ leukemia cells. Three of these antibodies, interestingly, share an identical heavy chain variable (VH) sequence. In this report, we constructed a new library comprising the single VH paired with the variable light chain (VL) repertoire obtained from the original naïve human library. Initial in vitro selection revealed that the single VH could pair with a number of different VL while retaining its specificity for KDR. However, a consensus VH/VL pair, clone 1121, was identified after three or four rounds of selection by tailoring the stringency of the panning conditions. Clone 1121 showed a >30-fold higher binding affinity to KDR (Kd, 100 pm) because of improvement on both association and dissociation constants and blocked VEGF/KDR interaction with an IC50 of approximately 1 nm, compared with that of 3-4 nm for the parent Fab fragments. Further, clone 1121 was more potent in inhibiting VEGF-stimulated KDR phosphorylation in endothelial cells. A binding epitope mapping study on clone 1121 and one of the parent clones, 2C6, demonstrated that both antibodies interacted with the third immunoglobulin domain within the extracellular region of KDR. Several peptide phage display libraries were utilized to further examine the fine binding specificities of the two antibodies. All of the 2C6-binding peptides are cysteine-constrained, whereas clone 1121 binds to both cysteine-constrained and linear peptides. It is noteworthy that most of the 2C6-binding peptides

  17. A Vaccine of L2 Epitope Repeats Fused with a Modified IgG1 Fc Induced Cross-Neutralizing Antibodies and Protective Immunity against Divergent Human Papillomavirus Types

    PubMed Central

    Zhang, Ting; Liu, Yanchun; Xie, Xixiu; Wang, Zhirong; Xu, Xuemei

    2014-01-01

    Current human papillomavirus (HPV) major capsid protein L1 virus-like particles (VLPs)-based vaccines in clinic induce strong HPV type-specific neutralizing antibody responses. To develop pan-HPV vaccines, here, we show that the fusion protein E3R4 consisting of three repeats of HPV16 L2 aa 17–36 epitope (E3) and a modified human IgG1 Fc scaffold (R4) induces cross-neutralizing antibodies and protective immunity against divergent HPV types. E3R4 was expressed as a secreted protein in baculovirus expression system and could be simply purified by one step Protein A affinity chromatography with the purity above 90%. Vaccination of E3R4 formulated with Freunds adjuvant not only induced cross-neutralizing antibodies against HPV pseudovirus types 16, 18, 45, 52, 58, 6, 11 and 5 in mice, but also protected mice against vaginal challenges with HPV pseudovirus types 16, 45, 52, 58, 11 and 5 for at least eleven months after the first immunization. Moreover, vaccination of E3R4 formulated with FDA approved adjuvant alum plus monophosphoryl lipid A also induced cross-neutralizing antibodies against HPV types 16, 18 and 6 in rabbits. Thus, our results demonstrate that delivery of L2 antigen as a modified Fc-fusion protein may facilitate pan-HPV vaccine development. PMID:24802101

  18. The Dubious Value of Value Neutrality

    ERIC Educational Resources Information Center

    Balch, Stephen H.

    2006-01-01

    Hard science is properly value neutral. But when that ideological neutrality extends to the whole university, the traditional foundation crumbles. Steve Balch laments the moral vacuum that now substitutes for fundamental principles, because it is impossible to frame a program of education--especially in the humanities and social sciences--without…

  19. Selection of affinity-improved neutralizing human scFv against HBV PreS1 from CDR3 VH/VL mutant library.

    PubMed

    Chen, YanMin; Bai, Yin; Guo, XiaoChen; Wang, WenFei; Zheng, Qi; Wang, FuXiang; Sun, Dejun; Li, DeShan; Ren, GuiPing; Yin, JieChao

    2016-07-01

    A CDR3 mutant library was constructed from a previously isolated anti-HBV neutralizing Homo sapiens scFv-31 template by random mutant primers PCR. Then the library was displayed on the inner membrane surface in Escherichia coli periplasmic space. Seven scFv clones were isolated from the mutant library through three rounds of screening by flow cytometry. Competition ELISA assay indicates that isolated scFv fragments show more efficient binding ability to HBV PreS1 compared with parental scFv-31. HBV neutralization assay indicated that two clones (scFv-3 and 59) show higher neutralizing activity by blocking the HBV infection to Chang liver cells. Our method provides a new strategy for rapid screening of mutant antibody library for affinity-enhanced scFv clones and the neutralizing scFvs obtained from this study provide a potential alternative of Hepatitis B immune globulin. PMID:27255707

  20. Determination of the Human Antibody Response to the Neutralization Epitopes Encompassing Amino Acids 313–327 and 432–443 of Hepatitis C Virus E1E2 Glycoproteins

    PubMed Central

    Liu, Ruyu; Rao, Huiying; Wang, Jianghua; Xie, Xingwang; Jiang, Dong; Pan, Xiaoben; Zhao, Ping; Zhang, Henghui; Wei, Lai

    2013-01-01

    It has been reported that monoclonal antibodies (MAbs) to the E1E2 glycoproteins may have the potential to prevent hepatitis C virus (HCV) infection. The protective epitopes targeted by these MAbs have been mapped to the regionsencompassing amino acids 313–327 and 432–443. In this study, we synthesized these two peptides and tested the reactivity of serum samples from 336 patients, 210 of whichwere from Chronic Hepatitis C (CHC) patients infected with diverse HCV genotypes.The remaining 126 samples were isolated from patients who had spontaneously clearedHCV infection.In the chronic HCV-infected group (CHC group), the prevalence of human serum antibodies reactive to epitopes 313–327 and 432–443was 24.29%(51 of 210) and4.76%(10 of 210),respectively. In thespontaneousclearance group (SC group),the prevalence was 0.79%(1 of 126) and 12.70%(16 of 126), respectively.The positive serum samples that contained antibodies reactive to epitope 313–327 neutralizedHCV pseudoparticles (HCVpp) bearing the envelope glycoproteins of genotypes 1a or 1b and/or 4, but genotypes 2a, 3a, 5 and 6 were not neutralized. The neutralizing activity of these serum samples could not be inhibited by peptide 313–327. Six samples (SC17, SC38, SC86, SC92, CHC75 and CHC198) containing antibodies reactive to epitope 432–443 had cross-genotype neutralizing activities. Theneutralizing activityof SC38, SC86, SC92 and CHC75waspartiallyinhibited by peptide 432–443. However,the neutralizing activity of sample SC17 for genotype 4HCVpp and sample CHC198 for genotype 1b HCVppwere notinhibited by the peptide.This study identifies the neutralizing ability of endogenous anti-HCV antibodies and warrants the exploration of antibodies reactive to epitope432–443as sources for future antibody therapies. PMID:23826163

  1. Automated facial coding software outperforms people in recognizing neutral faces as neutral from standardized datasets

    PubMed Central

    Lewinski, Peter

    2015-01-01

    Little is known about people’s accuracy of recognizing neutral faces as neutral. In this paper, I demonstrate the importance of knowing how well people recognize neutral faces. I contrasted human recognition scores of 100 typical, neutral front-up facial images with scores of an arguably objective judge – automated facial coding (AFC) software. I hypothesized that the software would outperform humans in recognizing neutral faces because of the inherently objective nature of computer algorithms. Results confirmed this hypothesis. I provided the first-ever evidence that computer software (90%) was more accurate in recognizing neutral faces than people were (59%). I posited two theoretical mechanisms, i.e., smile-as-a-baseline and false recognition of emotion, as possible explanations for my findings. PMID:26441761

  2. Neutral beam monitoring

    DOEpatents

    Fink, Joel H.

    1981-08-18

    Method and apparatus for monitoring characteristics of a high energy neutral beam. A neutral beam is generated by passing accelerated ions through a walled cell containing a low energy neutral gas, such that charge exchange neutralizes the high energy ion beam. The neutral beam is monitored by detecting the current flowing through the cell wall produced by low energy ions which drift to the wall after the charge exchange. By segmenting the wall into radial and longitudinal segments various beam conditions are further identified.

  3. Generation of Neutralizing Monoclonal Antibodies against a Conformational Epitope of Human Adenovirus Type 7 (HAdv-7) Incorporated in Capsid Encoded in a HAdv-3-Based Vector

    PubMed Central

    Li, Xiao; Zhou, Zhichao; Li, Chenyang; Zhou, Rong

    2014-01-01

    The generation of monoclonal antibodies (MAbs) by epitope-based immunization is difficult because the immunogenicity of simple peptides is poor and T cells must be potently stimulated and immunological memory elicited. A strategy in which antigen is incorporated into the adenoviral capsid protein has been used previously to develop antibody responses against several vaccine targets and may offer a solution to this problem. In this study, we used a similar strategy to develop HAdv-7-neutralizing MAbs using rAdMHE3 virions into which hexon hypervariable region 5 (HVR5) of adenovirus type 7 (HAdv-7) was incorporated. The epitope mutant rAdMHE3 was generated by replacing HVR5 of Ad3EGFP, a recombinant HAdv-3-based vector expressing enhanced green fluorescence protein, with HVR5 of HAdv-7. We immunized BALB/c mice with rAdMHE3 virions and produced 22 different MAbs against them, four of which showed neutralizing activity against HAdv-7 in vitro. Using an indirect enzyme-linked immunosorbent assay (ELISA) analysis and an antibody-binding-competition ELISA with Ad3EGFP, HAdv-7, and a series of chimeric adenoviral particles containing epitope mutants, we demonstrated that the four MAbs recognize the neutralization site within HVR5 of the HAdv-7 virion. Using an immunoblotting analysis and ELISA with HAdv-7, recombinant peptides, and a synthetic peptide, we also showed that the neutralizing epitope within HVR5 of the HAdv-7 virion is a conformational epitope. These findings suggest that it is feasible to use a strategy in which antigen is incorporated into the adenoviral capsid protein to generate neutralizing MAbs. This strategy may also be useful for developing therapeutic neutralizing MAbs and designing recombinant vector vaccines against HAdv-7, and in structural analysis of adenoviruses. PMID:25054273

  4. Molecular Features of the Broadly Neutralizing Immunoglobulin G1 b12 Required for Recognition of Human Immunodeficiency Virus Type 1 gp120

    PubMed Central

    Zwick, Michael B.; Parren, Paul W. H. I.; Saphire, Erica O.; Church, Sarah; Wang, Meng; Scott, Jamie K.; Dawson, Philip E.; Wilson, Ian A.; Burton, Dennis R.

    2003-01-01

    IgG1 b12 is a broadly neutralizing antibody against human immunodeficiency virus type 1 (HIV-1). The epitope recognized by b12 overlaps the CD4 receptor-binding site (CD4bs) on gp120 and has been a target for vaccine design. Determination of the three-dimensional structure of immunoglobulin G1 (IgG1) b12 allowed modeling of the b12-gp120 interaction in which the protruding third complementarity-determining region (CDR) of the heavy chain (H3) was crucial for antibody binding. In the present study, extensive mutational analysis of the antigen-binding site of Fab b12 was carried out to investigate the validity of the model and to identify residues important for gp120 recognition and, by inference, key to the anti-HIV-1 activity of IgG1 b12. In all, 50 mutations were tested: 40 in H3, 4 each in H2 and L1, and 2 in L3. The results suggest that the interaction of gp120 with H3 of b12 is crucially dependent not only on a Trp residue at the apex of the H3 loop but also on a number of residues at the base of the loop. The arrangement of these residues, including aromatic side chains and side chains that hydrogen bond across the base of the loop, may rigidify H3 for penetration of the recessed CD4-binding cavity. The results further emphasize the importance to gp120 binding of a Tyr residue at the apex of the H2 loop that forms a second finger-like structure and a number of Arg residues in L1 that form a positively charged, shelf-like structure. In general, the data are consistent with the b12-gp120 interaction model previously proposed. At the gene level, somatic mutation is seen to be crucial for the generation of many of the structural features described. The Fab b12 mutants were also tested against the b12 epitope-mimic peptide B2.1, and the reactivity profile had many similarities but also significant differences from that observed for gp120. The paratope map of b12 may facilitate the design of molecules that are able to elicit b12-like activities. PMID:12719580

  5. Identifying possible sites for antibody neutralization escape: Implications for unique functional properties of the C-terminal tail of Human Immunodeficiency Virus Type 1 gp41.

    PubMed

    Lu, Zhifeng; Huang, Yushen; Tan, Yue; Yu, Yang; Wang, Junyi; Chen, Ying-Hua

    2016-07-01

    A previous amino acid sequence analyses from our laboratory reported nine potential sites in gp41 glycoprotein of HIV-1 that may contribute to virus escape from antibody neutralization. Besides four sites found outside the membrane of HIV-1 virus, five located in the C-terminal tail of gp41 specifically in the lentivirus lytic peptides motifs (LLPs). To further study the bioinformatical results, the virus infectivity assay and the standard neutralization assay were conducted on conservatively mutated virus. Two sites in the LLP3 domain stood out with the ability to alter the resistance of HIV-1 virus to certain broadly neutralizing antibodies (bNAbs). While the glycoprotein incorporation on the viral membrane and the interaction of the LLP3 domain with the lipid membrane remained unaltered, the increase in neutralization resistance of the mutant virus was associated with the changes on Env conformation. Our findings demonstrate different sensibility of bNAbs to mutations in the C-terminal tail and indicate an unrecognized potential role for even minor sequence variation in the C-terminal tail in modulating the antigenicity of the ectodomain of HIV-1 envelope glycoprotein complex. PMID:27157128

  6. Neutralizing activities of human immunoglobulin derived from donors in Japan against mosquito-borne flaviviruses, Japanese encephalitis virus, West Nile virus, and dengue virus

    PubMed Central

    Yunoki, Mikihiro; Kurosu, Takeshi; Koketsu, Ritsuko Kubota; Takahashi, Kazuo; Okuno, Yoshinobu; Ikuta, Kazuyoshi

    2016-01-01

    Japanese encephalitis virus (JEV), West Nile virus (WNV), and dengue virus (DenV) are causal agents of Japanese encephalitis, West Nile fever, and dengue fever, respectively. JEV is considered to be indigenized and widespread in Japan, whereas WNV and DenV are not indigenized in Japan. Globulin products seem to reflect the status of the donor population according to antivirus neutralization activity. However, the anti-JEV, -WNV, and -DenV neutralization activities of globulin products derived from donors in Japan have not been clarified. Furthermore, potential candidates for the development of an effective immunotherapeutic drug for encephalitis caused by JEV, WNV, or DenV have also not been identified. Therefore, the aim of this study was to determine the overall status of the donor population in Japan based on globulin products by evaluating anti-JEV, -WNV, and -DenV neutralizing activities of intravenous immunoglobulin. Overall, intravenous immunoglobulin products showed stable neutralizing activity against JEV but showed no or only weak activity against WNV or DenV. These results suggest that the epidemiological level against WNV and DenV in the donor population of Japan is still low, suggesting that these viruses are not yet indigenized. In addition, JEV vaccinations and/or infections in the donor population do not induce a cross-reactive antibody against WNV. PMID:27462140

  7. Neutralizing activities of human immunoglobulin derived from donors in Japan against mosquito-borne flaviviruses, Japanese encephalitis virus, West Nile virus, and dengue virus.

    PubMed

    Yunoki, Mikihiro; Kurosu, Takeshi; Koketsu, Ritsuko Kubota; Takahashi, Kazuo; Okuno, Yoshinobu; Ikuta, Kazuyoshi

    2016-01-01

    Japanese encephalitis virus (JEV), West Nile virus (WNV), and dengue virus (DenV) are causal agents of Japanese encephalitis, West Nile fever, and dengue fever, respectively. JEV is considered to be indigenized and widespread in Japan, whereas WNV and DenV are not indigenized in Japan. Globulin products seem to reflect the status of the donor population according to antivirus neutralization activity. However, the anti-JEV, -WNV, and -DenV neutralization activities of globulin products derived from donors in Japan have not been clarified. Furthermore, potential candidates for the development of an effective immunotherapeutic drug for encephalitis caused by JEV, WNV, or DenV have also not been identified. Therefore, the aim of this study was to determine the overall status of the donor population in Japan based on globulin products by evaluating anti-JEV, -WNV, and -DenV neutralizing activities of intravenous immunoglobulin. Overall, intravenous immunoglobulin products showed stable neutralizing activity against JEV but showed no or only weak activity against WNV or DenV. These results suggest that the epidemiological level against WNV and DenV in the donor population of Japan is still low, suggesting that these viruses are not yet indigenized. In addition, JEV vaccinations and/or infections in the donor population do not induce a cross-reactive antibody against WNV. PMID:27462140

  8. Soluble Human Cytomegalovirus gH/gL/pUL128–131 Pentameric Complex, but Not gH/gL, Inhibits Viral Entry to Epithelial Cells and Presents Dominant Native Neutralizing Epitopes*

    PubMed Central

    Loughney, John W.; Rustandi, Richard R.; Wang, Dai; Troutman, Matthew C.; Dick, Lawrence W.; Li, Guanghua; Liu, Zhong; Li, Fengsheng; Freed, Daniel C.; Price, Colleen E.; Hoang, Van M.; Culp, Timothy D.; DePhillips, Pete A.; Fu, Tong-Ming; Ha, Sha

    2015-01-01

    Congenital infection of human cytomegalovirus (HCMV) is one of the leading causes of nongenetic birth defects, and development of a prophylactic vaccine against HCMV is of high priority for public health. The gH/gL/pUL128–131 pentameric complex mediates HCMV entry into endothelial and epithelial cells, and it is a major target for neutralizing antibody responses. To better understand the mechanism by which antibodies interact with the epitopes of the gH/gL/pUL128–131 pentameric complex resulting in viral neutralization, we expressed and purified soluble gH/gL/pUL128–131 pentameric complex and gH/gL from Chinese hamster ovary cells to >95% purity. The soluble gH/gL, which exists predominantly as (gH/gL)2 homodimer with a molecular mass of 220 kDa in solution, has a stoichiometry of 1:1 and a pI of 6.0–6.5. The pentameric complex has a molecular mass of 160 kDa, a stoichiometry of 1:1:1:1:1, and a pI of 7.4–8.1. The soluble pentameric complex, but not gH/gL, adsorbs 76% of neutralizing activities in HCMV human hyperimmune globulin, consistent with earlier reports that the most potent neutralizing epitopes for blocking epithelial infection are unique to the pentameric complex. Functionally, the soluble pentameric complex, but not gH/gL, blocks viral entry to epithelial cells in culture. Our results highlight the importance of the gH/gL/pUL128–131 pentameric complex in HCMV vaccine design and emphasize the necessity to monitor the integrity of the pentameric complex during the vaccine manufacturing process. PMID:25947373

  9. On neutral plasma oscillations

    SciTech Connect

    Shadwick, B.A.; Morrison, P.J.

    1993-06-01

    We examine the conditions for the existence of spectrally stable neutral modes in a Vlasov-Poisson plasma and show that for stable equilibria of systems that have unbounded spatial domain, the only possible neutral modes are those with phase velocities that correspond to stationary inflection points of the equilibrium distribution function. It is seen that these neutral modes can possess positive or negative free energy.

  10. ALEX neutral beam probe

    SciTech Connect

    Pourrezaei, K.

    1982-01-01

    A neutral beam probe capable of measuring plasma space potential in a fully 3-dimensional magnetic field geometry has been developed. This neutral beam was successfully used to measure an arc target plasma contained within the ALEX baseball magnetic coil. A computer simulation of the experiment was performed to refine the experimental design and to develop a numerical model for scaling the ALEX neutral beam probe to other cases of fully 3-dimensional magnetic field. Based on this scaling a 30 to 50 keV neutral cesium beam probe capable of measuring space potential in the thermal barrier region of TMX Upgrade was designed.

  11. Human Papillomavirus neutralizing and cross-reactive antibodies induced in HIV-positive subjects after vaccination with quadrivalent and bivalent HPV vaccines.

    PubMed

    Faust, Helena; Toft, Lars; Sehr, Peter; Müller, Martin; Bonde, Jesper; Forslund, Ola; Østergaard, Lars; Tolstrup, Martin; Dillner, Joakim

    2016-03-18

    Ninety-one HIV-infected individuals (61 men and 30 women) were randomized to vaccination either with quadrivalent (Gardasil™) or bivalent (Cervarix™) HPV vaccine. Neutralizing and specific HPV-binding serum antibodies were measured at baseline and 12 months after the first vaccine dose. Presence of neutralizing and binding antibodies had good agreement (average Kappa for HPV types 6, 11, 16, 18, 31, 33 and 45 was 0.65). At baseline, 88% of subjects had antibodies against at least one genital HPV. Following vaccination with Cervarix™, all subjects became seropositive for HPV16 and 18. After Gardasil™ vaccination, 96% of subjects seroconverted for HPV16 and 73% for HPV18. Levels of HPV16-specific antibodies were <1 international unit (IU) in 87% of study subjects before vaccination but >10IU in 85% of study subjects after vaccination. Antibodies against non-vaccine HPV types appeared after Gardasil™ vaccination for >50% of vaccinated females for HPV 31, 35 and 73 and for >50% of Cervarix™-vaccinated females for HPV 31, 33, 35, 45, 56 and 58. Cross-reactivity with non-genital HPV types was also detected. In conclusion, HIV-infected subjects responded to HPV vaccination with induction of neutralizing antibodies against both vaccine and non-vaccine types. PMID:26896686

  12. Novel Clostridium difficile Anti-Toxin (TcdA and TcdB) Humanized Monoclonal Antibodies Demonstrate In Vitro Neutralization across a Broad Spectrum of Clinical Strains and In Vivo Potency in a Hamster Spore Challenge Model

    PubMed Central

    Qiu, Hongyu; Cassan, Robyn; Johnstone, Darrell; Han, Xiaobing; Joyee, Antony George; McQuoid, Monica; Masi, Andrea; Merluza, John; Hrehorak, Bryce; Reid, Ross; Kennedy, Kieron; Tighe, Bonnie; Rak, Carla; Leonhardt, Melanie; Dupas, Brian; Saward, Laura; Berry, Jody D.; Nykiforuk, Cory L.

    2016-01-01

    Clostridium difficile (C. difficile) infection (CDI) is the main cause of nosocomial antibiotic-associated colitis and increased incidence of community-associated diarrhea in industrialized countries. At present, the primary treatment of CDI is antibiotic administration, which is effective but often associated with recurrence, especially in the elderly. Pathogenic strains produce enterotoxin, toxin A (TcdA), and cytotoxin, toxin B (TcdB), which are necessary for C. difficile induced diarrhea and gut pathological changes. Administration of anti-toxin antibodies provides an alternative approach to treat CDI, and has shown promising results in preclinical and clinical studies. In the current study, several humanized anti-TcdA and anti-TcdB monoclonal antibodies were generated and their protective potency was characterized in a hamster infection model. The humanized anti-TcdA (CANmAbA4) and anti-TcdB (CANmAbB4 and CANmAbB1) antibodies showed broad spectrum in vitro neutralization of toxins from clinical strains and neutralization in a mouse toxin challenge model. Moreover, co-administration of humanized antibodies (CANmAbA4 and CANmAbB4 cocktail) provided a high level of protection in a dose dependent manner (85% versus 57% survival at day 22 for 50 mg/kg and 20 mg/kg doses, respectively) in a hamster gastrointestinal infection (GI) model. This study describes the protective effects conferred by novel neutralizing anti-toxin monoclonal antibodies against C. difficile toxins and their potential as therapeutic agents in treating CDI. PMID:27336843

  13. Neutrality in Language Policy

    ERIC Educational Resources Information Center

    Wee, Lionel

    2010-01-01

    The unavoidability of language makes it critical that language policies appeal to some notion of language neutrality as part of their rationale, in order to assuage concerns that the policies might otherwise be unduly discriminatory. However, the idea of language neutrality is deeply ideological in nature, since it is not only an attempt to treat…

  14. Neutral Sphingomyelinase 2

    PubMed Central

    Filosto, Simone; Castillo, Sianna; Danielson, Aaron; Franzi, Lisa; Khan, Elaine; Kenyon, Nick; Last, Jerold; Pinkerton, Kent; Tuder, Rubin; Goldkorn, Tzipora

    2011-01-01

    Chronic obstructive pulmonary disease (COPD) is caused by exposure to cigarette smoke (CS). One mechanism of CS-induced lung injury is aberrant generation of ceramide, which leads to elevated apoptosis of epithelial and endothelial cells in the alveolar spaces. Recently, we discovered that CS-induced ceramide generation and apoptosis in pulmonary cells is governed by neutral sphingomyelinase (nSMase) 2. In the current experiments, we expanded our studies to investigate whether nSMase2 governs ceramide generation and apoptosis in vivo using rodent and human models of CS-induced lung injury. We found that exposure of mice or rats to CS leads to colocalizing elevations of ceramide levels and terminal deoxynucleotidyl transferase mediated X-dUTP nick end labeling–positive cells in lung tissues. These increases are nSMase2 dependent, and are abrogated by treatment with N-acetyl cysteine or anti-nSMase2 small interfering RNA (siRNA). We further showed that mice that are heterozygous for nSMase2 demonstrate significant decrease in ceramide generation after CS exposure, whereas acidic sphingomyelinase (aSMase) knockout mice maintain wild-type ceramide levels, confirming our previous findings (in human airway epithelial cells) that only nSMase2, and not aSMase, is activated by CS exposure. Lastly, we found that lung tissues from patients with emphysema (smokers) display significantly higher levels of nSMase2 expression compared with lung tissues from healthy control subjects. Taken together, these data establish the central in vivo role of nSMase2 in ceramide generation, aberrant apoptosis, and lung injury under CS exposure, underscoring its promise as a novel target for the prevention of CS-induced airspace destruction. PMID:20448054

  15. Prevalence and Risk Factors for Neutralizing Antibodies to Human Papillomavirus Types 16 and 18 in HIV-positive Men who have Sex with Men

    PubMed Central

    Sharma, Rachna; Efird, Jimmy T.; Chein, Aung; Holly, Elizabeth A.; Krajden, Mel; Berry, Michael J.; Darragh, Teresa M.; Jay, Naomi; Palefsky, Joel M.

    2013-01-01

    Objective HPV vaccination is routinely recommended in HIV-positive MSM ≤ 26 years old. Levels of prior HPV exposure in older HIV-positive MSM are assumed to be too high to warrant routine HPV vaccination. However, little is known about the prevalence of and risk factors for neutralizing antibody seropositivity to HPV-16 or HPV-18, a key measure of prior exposure to these types. Methods Cross-sectional analysis of baseline visit for 296 HIV-positive MSM participating in a prospective cohort study of anal squamous intraepithelial lesions (ASIL) at a university-based research clinic. Participants completed a questionnaire detailing behaviors and medical history. Phlebotomy, anal cytology, HPV DNA testing with quantitation, and high resolution anoscopy with biopsy were performed. A pseudovirion-based neutralizing antibody (PBNA) assay was used to measure HPV-16 and HPV-18 neutralizing antibodies. Results 132/296 (45%) men were HPV-16-seropositive and 141/296 (48%) were HPV-18-seropositive. 175/296 (59%) of the men were positive for HPV-16 antibodies or DNA, and 167/296 (56%) were positive for HPV-18 antibodies or DNA. In multivariable analysis, HPV-16 seropositivity did not correlate with age, years of HIV positivity, CD4+ level or HIV viral load. Significant risk factors included HPV-16 DNA positivity with higher DNA levels (ptrend<.001) and higher number of receptive sexual partners in the last year (ptrend=.012). Conclusions A high proportion of HIV-positive MSM >26 years are DNA-negative and seronegative to HPV-16 and HPV-18 even when using a sensitive PBNA assay. Prospective studies are needed to determine the clinical- and cost-effectiveness of HPV vaccination in HIV-positive MSM > 26 years old. PMID:24231786

  16. Neutralizing antibodies against West Nile virus identified directly from human B cells by single-cell analysis and next generation sequencing.

    PubMed

    Tsioris, Konstantinos; Gupta, Namita T; Ogunniyi, Adebola O; Zimnisky, Ross M; Qian, Feng; Yao, Yi; Wang, Xiaomei; Stern, Joel N H; Chari, Raj; Briggs, Adrian W; Clouser, Christopher R; Vigneault, Francois; Church, George M; Garcia, Melissa N; Murray, Kristy O; Montgomery, Ruth R; Kleinstein, Steven H; Love, J Christopher

    2015-12-01

    West Nile virus (WNV) infection is an emerging mosquito-borne disease that can lead to severe neurological illness and currently has no available treatment or vaccine. Using microengraving, an integrated single-cell analysis method, we analyzed a cohort of subjects infected with WNV - recently infected and post-convalescent subjects - and efficiently identified four novel WNV neutralizing antibodies. We also assessed the humoral response to WNV on a single-cell and repertoire level by integrating next generation sequencing (NGS) into our analysis. The results from single-cell analysis indicate persistence of WNV-specific memory B cells and antibody-secreting cells in post-convalescent subjects. These cells exhibited class-switched antibody isotypes. Furthermore, the results suggest that the antibody response itself does not predict the clinical severity of the disease (asymptomatic or symptomatic). Using the nucleotide coding sequences for WNV-specific antibodies derived from single cells, we revealed the ontogeny of expanded WNV-specific clones in the repertoires of recently infected subjects through NGS and bioinformatic analysis. This analysis also indicated that the humoral response to WNV did not depend on an anamnestic response, due to an unlikely previous exposure to the virus. The innovative and integrative approach presented here to analyze the evolution of neutralizing antibodies from natural infection on a single-cell and repertoire level can also be applied to vaccine studies, and could potentially aid the development of therapeutic antibodies and our basic understanding of other infectious diseases. PMID:26481611

  17. Scorpion-Toxin Mimics of CD4 in Complex with Human Immunodeficiency Virus gp120: Crystal Structures, Molecular Mimicry, and Neutralization Breadth

    SciTech Connect

    Huang, Chih-chin; Stricher, Francois; Martin, Loic; Decker, Julie M.; Majeed, Shahzad; Barthe, Phillippe; Hendrickson, Wayne A.; Robinson, James; Roumestand, Christian; Sodroski, Joseph; Wyatt, Richard; Shaw, George M.; Vita, Claudio; Kwong, Peter D.

    2010-07-19

    The binding surface on CD4 for the HIV-1 gp120 envelope glycoprotein has been transplanted previously onto a scorpion-toxin scaffold. Here, we use X-ray crystallography to characterize atomic-level details of gp120 with this transplant, CD4M33. Despite known envelope flexibility, the conformation of gp120 induced by CD4M33 was so similar to that induced by CD4 that localized measures were required to distinguish ligand-induced differences from lattice variation. To investigate relationships between structure, function, and mimicry, an F23 analog of CD4M33 was devised. Structural and thermodynamic analyses showed F23 to be a better molecular mimic of CD4 than CD4M33. F23 also showed increased neutralization breadth, against diverse isolates of HIV-1, HIV-2, and SIVcpz. Our results lend insight into the stability of the CD4 bound conformation of gp120, define measures that quantify molecular mimicry as a function of evolutionary distance, and suggest how such evaluations might be useful in developing mimetic antagonists with increased neutralization breadth.

  18. Equivalent Neutral Wind

    NASA Technical Reports Server (NTRS)

    Liu, W. Timothy; Tang, Wenqing

    1996-01-01

    The definition of equivalent neutral wind and the rationale for using it as the geophysical product of a spaceborne scatterometer are reviewed. The differences between equivalent neutral wind and actual wind, which are caused by atmospheric density stratification, are demonstrated with measurements at selected locations. A method of computing this parameter from ship and buoy measurements is described and some common fallacies in accounting for the effects of atmospheric stratification on wind shear are discussed. The computer code for the model to derive equivalent neutral wind is provided.

  19. Neutralization escape mutants define a dominant immunogenic neutralization site on hepatitis A virus

    SciTech Connect

    Stapleton, J.T.; Lemon, S.M.

    1987-02-01

    Hepatitis A virus is an hepatotrophic human picornavirus which demonstrates little antigenic variability. To topologically map immunogenic sites on hepatitis A virus which elicit neutralizing antibodies, eight neutralizing monoclonal antibodies were evaluated in competition immunoassays employing radiolabeled monoclonal antibodies and HM-175 virus. Whereas two antibodies (K3-4C8 and K3-2F2) bound to intimately overlapping epitopes, the epitope bound by a third antibody (B5-B3) was distinctly different as evidenced by a lack of competition between antibodies for binding to the virus. The other five antibodies variably blocked the binding of both K3-4C8-K3-2F2 and B5-B3, suggesting that these epitopes are closely spaced and perhaps part of a single neutralization immunogenic site. Several combinations of monoclonal antibodies blocked the binding of polyclonal human convalescent antibody by greater than 96%, indicating that the neutralization epitopes bound by these antibodies are immunodominant in humans. Spontaneously arising HM-175 mutants were selected for resistance to monoclonal antibody-mediated neutralization. Neutralization resistance was associated with reduced antibody binding. These results suggest that hepatitis A virus may differ from poliovirus in possessing a single, dominant neutralization immunogenic site and therefore may be a better candidate for synthetic peptide or antiidiotype vaccine development.

  20. Solar Neutral Particles

    NASA Video Gallery

    This animation shows a neutral solar particle's path leaving the sun, following the magnetic field lines out to the heliosheath. The solar particle hits a hydrogen atom, stealing its electron, and ...

  1. A niche for neutrality.

    PubMed

    Adler, Peter B; Hillerislambers, Janneke; Levine, Jonathan M

    2007-02-01

    Ecologists now recognize that controversy over the relative importance of niches and neutrality cannot be resolved by analyzing species abundance patterns. Here, we use classical coexistence theory to reframe the debate in terms of stabilizing mechanisms (niches) and fitness equivalence (neutrality). The neutral model is a special case where stabilizing mechanisms are absent and species have equivalent fitness. Instead of asking whether niches or neutral processes structure communities, we advocate determining the degree to which observed diversity reflects strong stabilizing mechanisms overcoming large fitness differences or weak stabilization operating on species of similar fitness. To answer this question, we propose combining data on per capita growth rates with models to: (i) quantify the strength of stabilizing processes; (ii) quantify fitness inequality and compare it with stabilization; and (iii) manipulate frequency dependence in growth to test the consequences of stabilization and fitness equivalence for coexistence. PMID:17257097

  2. Is dispersal neutral?

    PubMed

    Lowe, Winsor H; McPeek, Mark A

    2014-08-01

    Dispersal is difficult to quantify and often treated as purely stochastic and extrinsically controlled. Consequently, there remains uncertainty about how individual traits mediate dispersal and its ecological effects. Addressing this uncertainty is crucial for distinguishing neutral versus non-neutral drivers of community assembly. Neutral theory assumes that dispersal is stochastic and equivalent among species. This assumption can be rejected on principle, but common research approaches tacitly support the 'neutral dispersal' assumption. Theory and empirical evidence that dispersal traits are under selection should be broadly integrated in community-level research, stimulating greater scrutiny of this assumption. A tighter empirical connection between the ecological and evolutionary forces that shape dispersal will enable richer understanding of this fundamental process and its role in community assembly. PMID:24962790

  3. Env-2dCD4 S60C complexes act as super immunogens and elicit potent, broadly neutralizing antibodies against clinically relevant human immunodeficiency virus type 1 (HIV-1).

    PubMed

    Killick, Mark A; Grant, Michelle L; Cerutti, Nichole M; Capovilla, Alexio; Papathanasopoulos, Maria A

    2015-11-17

    The ability to induce a broadly neutralizing antibody (bNAb) response following vaccination is regarded as a crucial aspect in developing an effective vaccine against human immunodeficiency virus type 1 (HIV-1). The bNAbs target the HIV-1 envelope glycoprotein (Env) which is exposed on the virus surface, thereby preventing cell entry. To date, conventional vaccine approaches such as the use of Env-based immunogens have been unsuccessful. We expressed, purified, characterized and evaluated the immunogenicity of several unique HIV-1 subtype C Env immunogens in small animals. Here we report that vaccine immunogens based on Env liganded to a two domain CD4 variant, 2dCD4(S60C) are capable of consistently eliciting potent, broadly neutralizing antibody responses in New Zealand white rabbits against a panel of clinically relevant HIV-1 pseudoviruses. This was irrespective of the Env protein subtype and context. Importantly, depletion of the anti-CD4 antibodies appeared to abrogate the neutralization activity in the rabbit sera. Taken together, this data suggests that the Env-2dCD4(S60C) complexes described here are "super" immunogens, and potentially immunofocus antibody responses to a unique epitope spanning the 2dCD4(60C). Recent data from the two available anti-CD4 monoclonal antibodies, Ibalizumab and CD4-Ig (and bispecific variants thereof) have highlighted that the use of these broad and potent entry inhibitors could circumvent the need for a conventional vaccine targeting HIV-1. Overall, the ability of the unique Env-2dCD4(S60C) complexes to elicit potent bNAb responses has not been described previously, reinforcing that further investigation for their utility in preventing and controlling HIV-1/SIV infection is warranted. PMID:26432912

  4. Neutral endopeptidase modulates neurogenic inflammation.

    PubMed

    Nadel, J A

    1991-06-01

    A noncholinergic, nonadrenergic nervous system has been described, involving the sensory nerves in the airways. Chemicals, dusts and other irritants stimulate these sensory nerves to release substance P and related neuropeptides. These neuropeptides have the remarkable ability to affect multiple cells in the airways and to provoke many responses including cough, mucus secretion, smooth muscle contraction, plasma extravasation and neutrophil adhesion. This series of effects is termed "neurogenic inflammation." An enzyme exists on the surfaces of all lung cells that contain receptors for these neuropeptides. This enzyme, neutral endopeptidase (NEP), by cleaving and thus inactivating the neuropeptides, limits the concentration of the neuropeptide that reaches the receptor on the cell surface. Thus, neurogenic inflammatory responses are normally mild and presumably protective in nature. However, when NEP is inhibited pharmacologically (with NEP inhibitors) or by cigarette smoke, respiratory viral infection, or by inhalation of the industrial pollutant toluene diisocyanate, neurogenic inflammatory responses are exaggerated. Delivery of exogenous human recombinant NEP inhibits neurogenic inflammation. Finally, evidence is provided that corticosteroids suppress neurogenic plasma extravasation and that this drug can upregulate NEP in human airway tissue. Neutral endopeptidase cleaves multiple peptides. Thus, its selectivity resides, at least in part, on its fixed location on the surfaces of specific cells where it can modulate effects of peptides exposed to the cells' surfaces. PMID:1889501

  5. Rapid isolation of dengue-neutralizing antibodies from single cell-sorted human antigen-specific memory B-cell cultures

    PubMed Central

    Cox, Kara S.; Tang, Aimin; Chen, Zhifeng; Horton, Melanie S.; Yan, Hao; Wang, Xin-Min; Dubey, Sheri A.; DiStefano, Daniel J.; Ettenger, Andrew; Fong, Rachel H.; Doranz, Benjamin J.; Casimiro, Danilo R.; Vora, Kalpit A.

    2016-01-01

    Monitoring antigen-specific memory B cells and the antibodies they encode is important for understanding the specificity, breadth and duration of immune response to an infection or vaccination. The antibodies isolated could further help design vaccine antigens for raising relevant protective immune responses. However, developing assays to measure and isolate antigen-specific memory B cells is technically challenging due to the low frequencies of these cells that exist in the circulating blood. Here, we describe a flow cytometry method to identify and isolate dengue envelope-specific memory B cells using a labeled dengue envelope protein. We enumerated dengue-envelope specific memory B cells from a cohort of dengue seropositive donors using this direct flow cytometry assay. A more established and conventional assay, the cultured B ELISPOT, was used as a benchmark comparator. Furthermore, we were able to confirm the single-sorted memory B-cell specificity by culturing B cells and differentiating them into plasma cells using cell lines expressing CD40L. The culture supernatants were assayed for antigen binding and the ability of the antibodies to neutralize the cognate dengue virus. Moreover, we successfully isolated the heavy and light Ig sequences and expressed them as full-length recombinant antibodies to reproduce the activity seen in culture supernatants. Mapping of these antibodies revealed a novel epitope for dengue 2 virus serotype. In conclusion, we established a reproducible methodology to enumerate antigen-specific memory B cells and assay their encoded antibodies for functional characterization. PMID:26491897

  6. Neutral particle lithography

    NASA Astrophysics Data System (ADS)

    Craver, Barry Paul

    Neutral particle lithography (NPL) is a high resolution, proximity exposure technique where a broad beam of energetic neutral particles floods a stencil mask and transmitted beamlets transfer the mask pattern to resist on a substrate, such that each feature is printed in parallel, rather than in the serial manner of electron beam lithography. It preserves the advantages of ion beam lithography (IBL), including extremely large depth-of-field, sub-5 nm resist scattering, and the near absence of diffraction, yet is intrinsically immune to charge-related artifacts including line-edge roughness and pattern placement errors due to charge accumulation on the mask and substrate. In our experiments, a neutral particle beam is formed by passing an ion beam (e.g., 30 keV He+) through a high pressure helium gas cell (e.g., 100 mTorr) to convert the ions to energetic neutrals through charge transfer scattering. The resolution of NPL is generally superior to that of IBL for applications involving insulating substrates, large proximity gaps, and ultra-small features. High accuracy stepped exposures with energetic neutral particles, where magnetic or electrostatic deflection is impossible, have been obtained by clamping the mask to the wafer, setting the proximity gap with a suitable spacer, and mechanically inclining the mask/wafer stack relative to the beam. This approach is remarkably insensitive to vibration and thermal drift; nanometer scale image offsets have been obtained with +/-2 nm placement accuracy for experiments lasting over one hour. Using this nanostepping technique, linewidth versus dose curves were obtained, from which the NPL lithographic blur was determined as 4.4+/-1.4 nm (1sigma), which is 2-3 times smaller than the blur of electron beam lithography. Neutral particle lithography has the potential to form high density, periodic patterns with sub-10 nm resolution.

  7. Ultracold neutral plasmas.

    PubMed

    Killian, Thomas C

    2007-05-01

    Ultracold neutral plasmas occupy an exotic regime of plasma physics in which electrons form a swarming, neutralizing background for ions that sluggishly move in a correlated manner. Strong interactions between the charged particles give rise to surprising dynamics such as oscillations of the average kinetic energy during equilibration and extremely fast recombination. Such phenomena offer stimulating and challenging problems for computational scientists, and the physics can be applied to other environments, such as the interior of gas giant planets and plasmas created by short-pulse laser irradiation of solid, liquid, and cluster targets. PMID:17478712

  8. Neutral beam development plan

    SciTech Connect

    Staten, H S

    1980-08-01

    The national plan is presented for developing advanced injection systems for use on upgrades of existing experiments, and use on future facilities such as ETF, to be built in the late 1980's or early 90's where power production from magnetic fusion will move closer to a reality. Not only must higher power and longer pulse length systems be developed , but they must operate reliably; they must be a tool for the experimenter, not the experiment itself. Neutral beam systems handle large amounts of energy and as such, they often are as complicated as the plasma physics experiment itself. This presents a significant challenge to the neutral beam developer.

  9. Enhanced HIV-1 neutralization by antibody heteroligation

    PubMed Central

    Mouquet, Hugo; Warncke, Malte; Scheid, Johannes F.; Seaman, Michael S.; Nussenzweig, Michel C.

    2012-01-01

    Passive transfer of broadly neutralizing human antibodies against HIV-1 protects macaques against infection. However, HIV-1 uses several strategies to escape antibody neutralization, including mutation of the gp160 viral surface spike, a glycan shield to block antibody access to the spike, and expression of a limited number of viral surface spikes, which interferes with bivalent antibody binding. The latter is thought to decrease antibody apparent affinity or avidity, thereby interfering with neutralizing activity. To test the idea that increasing apparent affinity might enhance neutralizing activity, we engineered bispecific anti–HIV-1 antibodies (BiAbs) that can bind bivalently by virtue of one scFv arm that binds to gp120 and a second arm to the gp41 subunit of gp160. The individual arms of the BiAbs preserved the binding specificities of the original anti-HIV IgG antibodies and together bound simultaneously to gp120 and gp41. Heterotypic bivalent binding enhanced neutralization compared with the parental antibodies. We conclude that antibody recognition and viral neutralization of HIV can be improved by heteroligation. PMID:22219363

  10. Enhancement of alpha -helicity in the HIV-1 inhibitory peptide DP178 leads to an increased affinity for human monoclonal antibody 2F5 but does not elicit neutralizing responses in vitro. Implications for vaccine design.

    PubMed

    Joyce, Joseph G; Hurni, William M; Bogusky, Michael J; Garsky, Victor M; Liang, Xiaoping; Citron, Michael P; Danzeisen, Renee C; Miller, Michael D; Shiver, John W; Keller, Paul M

    2002-11-29

    The synthetic peptide DP178, derived from the carboxyl-terminal heptad repeat region of human immunodeficiency virus type 1 GP41 protein is a potent inhibitor of viral-mediated fusion and contains the sequence ELDKWA, which constitutes the recognition epitope for the broadly neutralizing human monoclonal antibody 2F5. Efforts at eliciting a 2F5-like immune response by immunization with peptides or fusion proteins containing this sequence have not met with success, possibly because of incorrect structural presentation of the epitope. Although the structure of the carboxyl-terminal heptad repeat on the virion is not known, several recent reports have suggested a propensity for alpha-helical conformation. We have examined DP178 in the context of a model for optimized alpha-helices and show that the native sequence conforms poorly to the model. Solution conformation of DP178 was studied by circular dichroism and NMR spectroscopy and found to be predominantly random, consistent with previous reports. NMR mapping was used to show that the low percentage of alpha-helix present was localized to residues Glu(662) through Asn(671), a region encompassing the 2F5 epitope. Using NH(2)-terminal extensions derived from either GP41 or the yeast GCN4 leucine zipper dimerization domain, we designed peptide analogs in which the average helicity is significantly increased compared with DP178 and show that these peptides exhibit both a modest increase in affinity for 2F5 using a novel competitive solution-based binding assay and an increased ability to inhibit viral entry in a single-cycle infectivity model. Selected peptides were conjugated to carrier protein and used for guinea pig immunizations. High peptide-specific titers were achieved using these immunogens, but the resulting sera were incapable of viral neutralization. We discuss these findings in terms of structural and immunological considerations as to the utility of a 2F5-like response. PMID:12237296

  11. Vaccination of Rhesus Macaques with Recombinant Mycobacterium bovis Bacillus Calmette-Guérin Env V3 Elicits Neutralizing Antibody-Mediated Protection against Simian-Human Immunodeficiency Virus with a Homologous but Not a Heterologous V3 Motif

    PubMed Central

    Someya, Kenji; Cecilia, Dayaraj; Ami, Yasushi; Nakasone, Tadashi; Matsuo, Kazuhiro; Burda, Sherri; Yamamoto, Hiroshi; Yoshino, Naoto; Kaizu, Masahiko; Ando, Shuji; Okuda, Kenji; Zolla-Pazner, Susan; Yamazaki, Shudo; Yamamoto, Naoki; Honda, Mitsuo

    2005-01-01

    Although the correlates of vaccine-induced protection against human immunodeficiency virus type 1 (HIV-1) are not fully known, it is presumed that neutralizing antibodies (NAb) play a role in controlling virus infection. In this study, we examined immune responses elicited in rhesus macaques following vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing an HIV-1 Env V3 antigen (rBCG Env V3). We also determined the effect of vaccination on protection against challenge with either a simian-human immunodeficiency virus (SHIV-MN) or a highly pathogenic SHIV strain (SHIV-89.6PD). Immunization with rBCG Env V3 elicited significant levels of NAb for the 24 weeks tested that were predominantly HIV-1 type specific. Sera from the immunized macaques neutralized primary HIV-1 isolates in vitro, including HIV-1BZ167/X4, HIV-1SF2/X4, HIV-1CI2/X4, and, to a lesser extent, HIV-1MNp/X4, all of which contain a V3 sequence homologous to that of rBCG Env V3. In contrast, neutralization was not observed against HIV-1SF33/X4, which has a heterologous V3 sequence, nor was it found against primary HIV-1 R5 isolates from either clade A or B. Furthermore, the viral load in the vaccinated macaques was significantly reduced following low-dose challenge with SHIV-MN, and early plasma viremia was markedly decreased after high-dose SHIV-MN challenge. In contrast, replication of pathogenic SHIV-89.6PD was not affected by vaccination in any of the macaques. Thus, we have shown that immunization with an rBCG Env V3 vaccine elicits a strong, type-specific V3 NAb response in rhesus macaques. While this response was not sufficient to provide protection against a pathogenic SHIV challenge, it was able to significantly reduce the viral load in macaques following challenge with a nonpathogenic SHIV. These observations suggest that rBCG vectors have the potential to deliver an appropriate virus immunogen for desirable immune elicitations. PMID:15650171

  12. A Potential of an Anti-HTLV-I gp46 Neutralizing Monoclonal Antibody (LAT-27) for Passive Immunization against Both Horizontal and Mother-to-Child Vertical Infection with Human T Cell Leukemia Virus Type-I

    PubMed Central

    Fujii, Hideki; Shimizu, Mamoru; Miyagi, Takuya; Kunihiro, Marie; Tanaka, Reiko; Takahashi, Yoshiaki; Tanaka, Yuetsu

    2016-01-01

    Although the number of human T-cell leukemia virus type-I (HTLV-I)-infected individuals in the world has been estimated at over 10 million, no prophylaxis vaccines against HTLV-I infection are available. In this study, we took a new approach for establishing the basis of protective vaccines against HTLV-I. We show here the potential of a passively administered HTLV-I neutralizing monoclonal antibody of rat origin (LAT-27) that recognizes epitopes consisting of the HTLV-I gp46 amino acids 191–196. LAT-27 completely blocked HTLV-I infection in vitro at a minimum concentration of 5 μg/mL. Neonatal rats born to mother rats pre-infused with LAT-27 were shown to have acquired a large quantity of LAT-27, and these newborns showed complete resistance against intraperitoneal infection with HTLV-I. On the other hand, when humanized immunodeficient mice were pre-infused intravenously with humanized LAT-27 (hu-LAT-27), all the mice completely resisted HTLV-I infection. These results indicate that hu-LAT-27 may have a potential for passive immunization against both horizontal and mother-to-child vertical infection with HTLV-I. PMID:26848684

  13. CO2-Neutral Fuels

    NASA Astrophysics Data System (ADS)

    Goede, Adelbert; van de Sanden, Richard

    2016-06-01

    Mimicking the biogeochemical cycle of System Earth, synthetic hydrocarbon fuels are produced from recycled CO2 and H2O powered by renewable energy. Recapturing CO2 after use closes the carbon cycle, rendering the fuel cycle CO2 neutral. Non-equilibrium molecular CO2 vibrations are key to high energy efficiency.

  14. Bleach Neutralizes Mold Allergens

    ERIC Educational Resources Information Center

    Science Teacher, 2005

    2005-01-01

    Researchers at National Jewish Medical and Research Center have demonstrated that dilute bleach not only kills common household mold, but may also neutralize the mold allergens that cause most mold-related health complaints. The study, published in the Journal of Allergy and Clinical Immunology, is the first to test the effect on allergic…

  15. Modeling thermospheric neutral density

    NASA Astrophysics Data System (ADS)

    Qian, Liying

    Satellite drag prediction requires determination of thermospheric neutral density. The NCAR Thermosphere-Ionosphere-Electrodynamics General Circulation Model (TIEGCM) and the global-mean Thermosphere-Ionosphere-Mesosphere-Electrodynamics General Circulation Model (TIMEGCM) were used to quantify thermospheric neutral density and its variations, focusing on annual/semiannual variation, the effect of using measured solar irradiance on model calculations of solar-cycle variation, and global change in the thermosphere. Satellite drag data and the MSIS00 empirical model were utilized to compare to the TIEGCM simulations. The TIEGCM simulations indicated that eddy diffusion and its annual/semiannual variation is a mechanism for annual/semiannual density variation in the thermosphere. It was found that eddy diffusion near the turbopause can effectively influence thermospheric neutral density. Eddy diffusion, together with annual insolation variation and large-scale circulation, generated global annual/semiannual density variation observed by satellite drag. Using measured solar irradiance as solar input for the TIEGCM improved the solar-cycle dependency of the density calculation shown in F10.7 -based thermospheric empirical models. It has been found that the empirical models overestimate density at low solar activity. The TIEGCM simulations did not show such solar-cycle dependency. Using historic measurements of CO2 and F 10.7, simulations of the global-mean TIMEGCM showed that thermospheric neutral density at 400 km had an average long-term decrease of 1.7% per decade from 1970 to 2000. A forecast of density decrease for solar cycle 24 suggested that thermospheric density will decrease at 400 km from present to the end of solar cycle 24 at a rate of 2.7% per decade. Reduction in thermospheric density causes less atmospheric drag on earth-orbiting space objects. The implication of this long-term decrease of thermospheric neutral density is that it will increase the

  16. 42 CFR 412.352 - Budget neutrality adjustment.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 2 2014-10-01 2014-10-01 false Budget neutrality adjustment. 412.352 Section 412.352 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES... § 412.352 Budget neutrality adjustment. For FY 1992 through FY 1995, CMS will determine an adjustment...

  17. 42 CFR 412.352 - Budget neutrality adjustment.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 2 2013-10-01 2013-10-01 false Budget neutrality adjustment. 412.352 Section 412.352 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES... § 412.352 Budget neutrality adjustment. For FY 1992 through FY 1995, CMS will determine an adjustment...

  18. 42 CFR 412.352 - Budget neutrality adjustment.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 2 2011-10-01 2011-10-01 false Budget neutrality adjustment. 412.352 Section 412.352 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES... § 412.352 Budget neutrality adjustment. For FY 1992 through FY 1995, CMS will determine an adjustment...

  19. 42 CFR 412.352 - Budget neutrality adjustment.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 2 2012-10-01 2012-10-01 false Budget neutrality adjustment. 412.352 Section 412.352 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES... § 412.352 Budget neutrality adjustment. For FY 1992 through FY 1995, CMS will determine an adjustment...

  20. Troubling Neutrality: Toward a Philosophy of Teacher Ambiguity

    ERIC Educational Resources Information Center

    Heybach, Jessica A.

    2014-01-01

    Who is keeping watch to warn when policies and practices become essentially the same as those used in previous eras to justify the destruction of human beings? This question is asked by author Jessica Heybach, as she describes the etymological roots of the word "neutrality," the social function of teacher as neutral, and its relationship…

  1. A Yeast Glycoprotein Shows High-Affinity Binding to the Broadly Neutralizing Human Immunodeficiency Virus Antibody 2G12 and Inhibits gp120 Interactions with 2G12 and DC-SIGN▿

    PubMed Central

    Luallen, Robert J.; Fu, Hu; Agrawal-Gamse, Caroline; Mboudjeka, Innocent; Huang, Wei; Lee, Fang-Hua; Wang, Lai-Xi; Doms, Robert W.; Geng, Yu

    2009-01-01

    The human immunodeficiency virus type 1 (HIV-1) envelope (Env) protein contains numerous N-linked carbohydrates that shield conserved peptide epitopes and promote trans infection by dendritic cells via binding to cell surface lectins. The potent and broadly neutralizing monoclonal antibody 2G12 binds a cluster of high-mannose-type oligosaccharides on the gp120 subunit of Env, revealing a conserved and highly exposed epitope on the glycan shield. To find an effective antigen for eliciting 2G12-like antibodies, we searched for endogenous yeast proteins that could bind to 2G12 in a panel of Saccharomyces cerevisiae glycosylation knockouts and discovered one protein that bound weakly in a Δpmr1 strain deficient in hyperglycosylation. 2G12 binding to this protein, identified as Pst1, was enhanced by adding the Δmnn1 deletion to the Δpmr1 background, ensuring the exposure of terminal α1,2-linked mannose residues on the D1 and D3 arms of high-mannose glycans. However, optimum 2G12 antigenicity was found when Pst1, a heavily N-glycosylated protein, was expressed with homogenous Man8GlcNAc2 structures in Δoch1 Δmnn1 Δmnn4 yeast. Surface plasmon resonance analysis of this form of Pst1 showed high affinity for 2G12, which translated into Pst1 efficiently inhibiting gp120 interactions with 2G12 and DC-SIGN and blocking 2G12-mediated neutralization of HIV-1 pseudoviruses. The high affinity of the yeast glycoprotein Pst1 for 2G12 highlights its potential as a novel antigen to induce 2G12-like antibodies. PMID:19264785

  2. Conformation-specific antibodies targeting the trimer-of-hairpins motif of the human T-cell leukemia virus type 1 transmembrane glycoprotein recognize the viral envelope but fail to neutralize viral entry.

    PubMed

    Mirsaliotis, Antonis; Nurkiyanova, Kulpash; Lamb, Daniel; Woof, Jenny M; Brighty, David W

    2007-06-01

    Human T-cell leukemia virus type 1 (HTLV-1) entry into cells is dependent upon the viral envelope glycoprotein-catalyzed fusion of the viral and cellular membranes. Following receptor activation of the envelope, the transmembrane glycoprotein (TM) is thought to undergo a series of fusogenic conformational transitions through a rod-like prehairpin intermediate to a compact trimer-of-hairpins structure. Importantly, synthetic peptides that interfere with the conformational changes of TM are potent inhibitors of membrane fusion and HTLV-1 entry, suggesting that TM is a valid target for antiviral therapy. To assess the utility of TM as a vaccine target and to explore further the function of TM in HTLV-1 pathogenesis, we have begun to examine the immunological properties of TM. Here we demonstrate that a recombinant trimer-of-hairpins form of the TM ectodomain is strongly immunogenic. Monoclonal antibodies raised against the TM immunogen specifically bind to trimeric forms of TM, including structures thought to be important for membrane fusion. Importantly, these antibodies recognize the envelope on virally infected cells but, surprisingly, fail to neutralize envelope-mediated membrane fusion or infection by pseudotyped viral particles. Our data imply that, even in the absence of overt membrane fusion, there are multiple forms of TM on virally infected cells and that some of these display fusion-associated structures. Finally, we demonstrate that many of the antibodies possess the ability to recruit complement to TM, suggesting that envelope-derived immunogens capable of eliciting a combination of neutralizing and complement-fixing antibodies would be of value as subunit vaccines for intervention in HTLV infections. PMID:17376912

  3. Epitope determinants of a chimpanzee dengue virus type 4 (DENV-4)-neutralizing antibody and protection against DENV-4 challenge in mice and rhesus monkeys by passively transferred humanized antibody.

    PubMed

    Lai, Ching-Juh; Goncalvez, Ana P; Men, Ruhe; Wernly, Claire; Donau, Olivia; Engle, Ronald E; Purcell, Robert H

    2007-12-01

    The chimpanzee monoclonal antibody (MAb) 5H2 is specific for dengue virus type 4 (DENV-4) and neutralizes the virus at a high titer in vitro. The epitope detected by the antibody was mapped by sequencing neutralization escape variants of the virus. One variant contained a Lys174-Glu substitution and another contained a Pro176-Leu substitution in domain I of the DENV-4 envelope protein (E). These mutations reduced binding affinity for the antibody 18- to >100-fold. Humanized immunoglobulin G (IgG) 5H2, originally produced from an expression vector, has been shown to be a variant containing a nine-amino-acid deletion in the Fc region which completely ablates antibody-dependent enhancement of DENV replication in vitro. The variant MAb, termed IgG 5H2 deltaD, is particularly attractive for exploring its protective capacity in vivo. Passive transfer of IgG 5H2 deltaD at 20 microg/mouse afforded 50% protection of suckling mice against challenge with 25 50% lethal doses of mouse neurovirulent DENV-4 strain H241. Passive transfer of antibody to monkeys was conducted to demonstrate proof of concept for protection against DENV challenge. Monkeys that received 2 mg/kg of body weight of IgG 5H2 deltaD were completely protected against 100 50% monkey infectious doses (MID50) of DENV-4, as indicated by the absence of viremia and seroconversion. A DENV-4 escape mutant that contained a Lys174-Glu substitution identical to that found in vitro was isolated from monkeys challenged with 10(6) MID50 of DENV-4. This substitution was also present in all naturally occurring isolates belonging to DENV-4 genotype III. These studies have important implications for possible antibody-mediated prevention of DENV infection. PMID:17881450

  4. Neutral particle beam intensity controller

    DOEpatents

    Dagenhart, William K.

    1986-01-01

    A neutral beam intensity controller is provided for a neutral beam generator in which a neutral beam is established by accelerating ions from an ion source into a gas neutralizer. An amplitude modulated, rotating magnetic field is applied to the accelerated ion beam in the gas neutralizer to defocus the resultant neutral beam in a controlled manner to achieve intensity control of the neutral beam along the beam axis at constant beam energy. The rotating magnetic field alters the orbits of ions in the gas neutralizer before they are neutralized, thereby controlling the fraction of neutral particles transmitted out of the neutralizer along the central beam axis to a fusion device or the like. The altered path or defocused neutral particles are sprayed onto an actively cooled beam dump disposed perpendicular to the neutral beam axis and having a central open for passage of the focused beam at the central axis of the beamline. Virtually zero therough 100% intensity control is achieved by varying the magnetic field strength without altering the ion source beam intensity or its species yield.

  5. Between detection and neutralization.

    SciTech Connect

    Snell, Mark Kamerer; Green, Mary Wilson; Adams, Douglas Glenn; Pritchard, Daniel Allison

    2005-08-01

    Security system analytical performance analysis is generally based on the probability of system effectiveness. The probability of effectiveness is a function of the probabilities of interruption and neutralization. Interruption occurs if the response forces are notified in sufficient time to engage the adversary. Neutralization occurs if the adversary attack is defeated after the security forces have actively engaged the adversary. Both depend upon communications of data. This paper explores details of embedded communications functions that are often assumed to be inconsequential. It is the intent of the authors to bring focus to an issue in security system modeling that, if not well understood, has the potential to be a deciding factor in the overall system failure or effectiveness.

  6. Neutral atom traps.

    SciTech Connect

    Pack, Michael Vern

    2008-12-01

    This report describes progress in designing a neutral atom trap capable of trapping sub millikelvin atom in a magnetic trap and shuttling the atoms across the atom chip from a collection area to an optical cavity. The numerical simulation and atom chip design are discussed. Also, discussed are preliminary calculations of quantum noise sources in Kerr nonlinear optics measurements based on electromagnetically induced transparency. These types of measurements may be important for quantum nondemolition measurements at the few photon limit.

  7. Antihypertensive neutral lipid

    DOEpatents

    Snyder, F.L.; Blank, M.L.

    1984-10-26

    The invention relates to the discovery of a class of neutral acetylated either-linked glycerolipids having the capacity to lower blood presure in warm-blooded animals. This physiological effect is structure sensitive requiring a long chain alkyl group at the sn-1 position and a short carbon chain acyl group (acetyl or propionyl) at the sn-2 position, and a hydroxyl group at the sn-3 position.

  8. Antihypertensive neutral lipid

    DOEpatents

    Snyder, Fred L.; Blank, Merle L.

    1986-01-01

    The invention relates to the discovery of a class of neutral acetylated ether-linked glycerolipids having the capacity to lower blood pressure in warm-blooded animals. This physiological effect is structure sensitive requiring a long chain alkyl group at the sn-1 position and a short carbon chain acyl group (acetyl or propionyl) at the sn-2 position, and a hydroxyl group at the sn-3 position.

  9. Strong ion difference excess and corrected strong ion difference model incorporating the variations in apparent dissociation constant for human blood plasma electro-neutrality equilibria.

    PubMed

    Rana, Amar P S; Rana, Sam Pal; Rana, Jay Pal; Singh, Sumarjeet

    2006-01-01

    We introduce computed value of the corrected Strong Ion Difference (SID) by including the large variability of the apparent dissociation constant pK' in non-logarithmic form on SID in Henderson-Hasselbach bicarbonate ion aqueous equilibria thereby resulting in a significant correction of up to 27% in SID. We further introduce a new concept of Strong Ion Difference Excess (SIDE) as the change in SID from the reference value at pH = 7.4, pCO2 = 5.33 Kpa (or 40 torr). The SIDE is a particularly useful quick measure when one can rule out the effects of hemoglobin, weak proteins and unidentified components for human blood plasma. PMID:17051730

  10. Neutral particle beam intensity controller

    DOEpatents

    Dagenhart, W.K.

    1984-05-29

    The neutral beam intensity controller is based on selected magnetic defocusing of the ion beam prior to neutralization. The defocused portion of the beam is dumped onto a beam dump disposed perpendicular to the beam axis. Selective defocusing is accomplished by means of a magnetic field generator disposed about the neutralizer so that the field is transverse to the beam axis. The magnetic field intensity is varied to provide the selected partial beam defocusing of the ions prior to neutralization. The desired focused neutral beam portion passes along the beam path through a defining aperture in the beam dump, thereby controlling the desired fraction of neutral particles transmitted to a utilization device without altering the kinetic energy level of the desired neutral particle fraction. By proper selection of the magnetic field intensity, virtually zero through 100% intensity control of the neutral beam is achieved.

  11. Neutrality between Government and Religion.

    ERIC Educational Resources Information Center

    Mawdsley, Ralph D.

    1996-01-01

    The overall guiding principle of neutrality between government and religion masks a tension that exists between free exercise of religion and establishment of religion. Reviews the development and current status of "Lemon" as a test for neutrality; proposes a new test for neutrality, evenhandedness, that is common to both the Free Exercise and…

  12. Pulsed field sample neutralization

    DOEpatents

    Appelhans, Anthony D.; Dahl, David A.; Delmore, James E.

    1990-01-01

    An apparatus and method for alternating voltage and for varying the rate of extraction during the extraction of secondary particles, resulting in periods when either positive ions, or negative ions and electrons are extracted at varying rates. Using voltage with alternating charge during successive periods to extract particles from materials which accumulate charge opposite that being extracted causes accumulation of surface charge of opposite sign. Charge accumulation can then be adjusted to a ratio which maintains a balance of positive and negative charge emission, thus maintaining the charge neutrality of the sample.

  13. Producing human ceramide-NS by metabolic engineering using yeast Saccharomyces cerevisiae

    PubMed Central

    Murakami, Suguru; Shimamoto, Toshi; Nagano, Hideaki; Tsuruno, Masahiro; Okuhara, Hiroaki; Hatanaka, Haruyo; Tojo, Hiromasa; Kodama, Yukiko; Funato, Kouichi

    2015-01-01

    Ceramide is one of the most important intercellular components responsible for the barrier and moisture retention functions of the skin. Because of the risks involved with using products of animal origin and the low productivity of plants, the availability of ceramides is currently limited. In this study, we successfully developed a system that produces sphingosine-containing human ceramide-NS in the yeast Saccharomyces cerevisiae by eliminating the genes for yeast sphingolipid hydroxylases (encoded by SUR2 and SCS7) and introducing the gene for a human sphingolipid desaturase (encoded by DES1). The inactivation of the ceramidase gene YDC1, overexpression of the inositol phosphosphingolipid phospholipase C gene ISC1, and endoplasmic reticulum localization of the DES1 gene product resulted in enhanced production of ceramide-NS. The engineered yeast strains can serve as hosts not only for providing a sustainable source of ceramide-NS but also for developing further systems to produce sphingosine-containing sphingolipids. PMID:26573460

  14. Producing human ceramide-NS by metabolic engineering using yeast Saccharomyces cerevisiae.

    PubMed

    Murakami, Suguru; Shimamoto, Toshi; Nagano, Hideaki; Tsuruno, Masahiro; Okuhara, Hiroaki; Hatanaka, Haruyo; Tojo, Hiromasa; Kodama, Yukiko; Funato, Kouichi

    2015-01-01

    Ceramide is one of the most important intercellular components responsible for the barrier and moisture retention functions of the skin. Because of the risks involved with using products of animal origin and the low productivity of plants, the availability of ceramides is currently limited. In this study, we successfully developed a system that produces sphingosine-containing human ceramide-NS in the yeast Saccharomyces cerevisiae by eliminating the genes for yeast sphingolipid hydroxylases (encoded by SUR2 and SCS7) and introducing the gene for a human sphingolipid desaturase (encoded by DES1). The inactivation of the ceramidase gene YDC1, overexpression of the inositol phosphosphingolipid phospholipase C gene ISC1, and endoplasmic reticulum localization of the DES1 gene product resulted in enhanced production of ceramide-NS. The engineered yeast strains can serve as hosts not only for providing a sustainable source of ceramide-NS but also for developing further systems to produce sphingosine-containing sphingolipids. PMID:26573460

  15. Metabolism and excretion of the once-daily human glucagon-like peptide-1 analog liraglutide in healthy male subjects and its in vitro degradation by dipeptidyl peptidase IV and neutral endopeptidase.

    PubMed

    Malm-Erjefält, Monika; Bjørnsdottir, Inga; Vanggaard, Jan; Helleberg, Hans; Larsen, Uffe; Oosterhuis, Berend; van Lier, Jan Jaap; Zdravkovic, Milan; Olsen, Anette K

    2010-11-01

    Liraglutide is a novel once-daily human glucagon-like peptide (GLP)-1 analog in clinical use for the treatment of type 2 diabetes. To study metabolism and excretion of [(3)H]liraglutide, a single subcutaneous dose of 0.75 mg/14.2 MBq was given to healthy males. The recovered radioactivity in blood, urine, and feces was measured, and metabolites were profiled. In addition, [(3)H]liraglutide and [(3)H]GLP-1(7-37) were incubated in vitro with dipeptidyl peptidase-IV (DPP-IV) and neutral endopeptidase (NEP) to compare the metabolite profiles and characterize the degradation products of liraglutide. The exposure of radioactivity in plasma (area under the concentration-time curve from 2 to 24 h) was represented by liraglutide (≥89%) and two minor metabolites (totaling ≤11%). Similarly to GLP-1, liraglutide was cleaved in vitro by DPP-IV in the Ala8-Glu9 position of the N terminus and degraded by NEP into several metabolites. The chromatographic retention time of DPP-IV-truncated liraglutide correlated well with the primary human plasma metabolite [GLP-1(9-37)], and some of the NEP degradation products eluted very close to both plasma metabolites. Three minor metabolites totaling 6 and 5% of the administered radioactivity were excreted in urine and feces, respectively, but no liraglutide was detected. In conclusion, liraglutide is metabolized in vitro by DPP-IV and NEP in a manner similar to that of native GLP-1, although at a much slower rate. The metabolite profiles suggest that both DPP-IV and NEP are also involved in the in vivo degradation of liraglutide. The lack of intact liraglutide excreted in urine and feces and the low levels of metabolites in plasma indicate that liraglutide is completely degraded within the body. PMID:20709939

  16. Antibody neutralization of retargeted measles viruses.

    PubMed

    Lech, Patrycja J; Pappoe, Roland; Nakamura, Takafumi; Tobin, Gregory J; Nara, Peter L; Russell, Stephen J

    2014-04-01

    The measles virus (MV) vaccine lineage is a promising oncolytic but prior exposure to the measles vaccine or wild-type MV strains limits treatment utility due to the presence of anti-measles antibodies. MV entry can be redirected by displaying a polypeptide ligand on the Hemagglutinin (H) C-terminus. We hypothesized that retargeted MV would escape neutralization by monoclonal antibodies (mAbs) recognizing the H receptor-binding surface and be less susceptible to neutralization by human antisera. Using chimeric H proteins, with and without mutations that ablate MV receptor binding, we show that retargeted MVs escape mAbs that target the H receptor-binding surface by virtue of mutations that ablate infection via SLAM and CD46. However, C-terminally displayed domains do not mediate virus entry in the presence of human antibodies that bind to the underlying H domain. In conclusion, utility of retargeted oncolytic measles viruses does not extend to evasion of human serum neutralization. PMID:24725950

  17. Cryoprotectant toxicity neutralization.

    PubMed

    Fahy, Gregory M

    2010-07-01

    Cryoprotectant toxicity is a fundamental limiting factor for the successful cryopreservation of living systems by both freezing and vitrification, and the ability to negate it would be attractive. Past attempts to demonstrate "cryoprotectant toxicity neutralization" (CTN) have had many ups and downs. First convincingly introduced by Baxter and Lathe in 1971, the concept that certain amides can block toxic effects of dimethyl sulfoxide (Me(2)SO) was contradicted by direct experiments in 1990. But in 1995, the opposite mode of CTN, in which Me(2)SO blocked the damaging effects of formamide, was robustly demonstrated. Recent experiments have verified the original 1995 results and extended them to urea and acetamide, but no CTN was detected for N-methylamides (N-methylformamide, N,N-dimethylformamide, and N-methylacetamide). On the theory that the latter amides and acetamide might serve as low-toxicity structural analogs of formamide, urea, or Me(2)SO, competition experiments were carried out between them and formamide or urea, but CTN was not observed for these amide-amide systems. The idea that the N-methylamides might have non-specific rather than specific toxicity was supported by the fact that the concentrations of these amides that cause toxicity are similar to the concentrations that denature model proteins. Clear examples of neutralization of the toxicity of glycerol, propylene glycol, ethylene glycol, or Me(2)SO are presently lacking, but effects of the latter that depend on sulfhydryl oxidation have been reversed with reducing agents. In summary, CTN is a useful phenomenon with significant theoretical and practical implications. PMID:19501081

  18. Tissue dissociation enzyme neutral protease assessment.

    PubMed

    Breite, A G; Dwulet, F E; McCarthy, R C

    2010-01-01

    Neutral proteases, essential components of purified tissue dissociation enzymes required for successful human islet isolation, show variable activities and effects of substrate on their activities. Initially we used a spectrophotometric endpoint assay with azocasein substrate to measure neutral protease activity. After critical review of the results, we observed these data to be inconsistent and not correlating expected differences in specific activities between thermolysin and Bacillus polymyxa proteases. This observation led to the development of a fluorescent microplate assay using fluorescein isothyocyanate-conjugated bovine serum albumin (FITC-BSA) as the substrate. This simpler, more flexible method offered a homogeneous, kinetic enzyme assay allowing determination of steady state reaction rates of sample replicates at various dilutions. The assay had a linear range of 4- to 8-fold and interassay coefficients of variation for B polymyxa protease and thermolysin of <9% and <15%, respectively, which were lower than those using the spectrophotometric endpoint assay, namely, 54% and 36%, respectively. This format allowed for incorporation of enzyme inhibitors, as illustrated by addition of sulfhydryl protease inhibitors, which, consistent with earlier reports, strongly indicated that the main contaminant in purified collagenase preparations was clostripain. Determination of the specific activities for several purified neutral proteases showed that the B polymyxa and Clostridium histolyticum proteases had approximately 40% and 15% specific activities, respectively, of those obtained with purified thermolysin, indicating the different characteristics of neutral protease enzymes for cell isolation procedures. PMID:20692405

  19. Transient ion neutralization by electrons.

    NASA Technical Reports Server (NTRS)

    Wilhelm, H. E.

    1973-01-01

    The nonlinear initial-boundary-value problems describing the lateral neutralization of ion beams for the cases that (1) an auxiliary electric field accelerates the electrons into the ion space, and (2) the electrons are injected into the ion space at a prescribed current density are treated. Analytical solutions are derived which give the position and speed of the neutralization front as a function of time, and the temporal development of the electron density, velocity, and electric fields during the neutralization process.

  20. A proposed neutral line signature

    NASA Technical Reports Server (NTRS)

    Doxas, I.; Speiser, T. W.; Dusenbery, P. B.; Horton, W.

    1992-01-01

    An identifying signature is proposed for the existence and location of the neutral line in the magnetotail. The signature, abrupt density, and temperature changes in the Earthtail direction, was first discovered in test particle simulations. Such temperature variations have been observed in ISEE data (Huang et. al. 1992), but their connection to the possible existence of a neutral line in the tail has not yet been established. The proposed signature develops earlier than the ion velocity space ridge of Martin and Speiser (1988), but can only be seen by spacecraft in the vicinity of the neutral line, while the latter can locate a neutral line remotely.

  1. Chemistry of carotenoid neutral radicals.

    PubMed

    Ligia Focsan, A; Magyar, Adam; Kispert, Lowell D

    2015-04-15

    Proton loss from the carotenoid radical cations (Car(+)) to form neutral radicals (#Car) was investigated by numerous electrochemical, EPR, ENDOR and DFT studies described herein. The radical cation and neutral radicals were formed in solution electrochemically and stabilized on solid silica-alumina and MCM-41 matrices. Carotenoid neutral radicals were recently identified in Arabidopsis thaliana plant and photosystem II samples. Deprotonation at the terminal ends of a zeaxanthin radical cation could provide a secondary photoprotection pathway which involves quenching excited state chlorophyll by the long-lived zeaxanthin neutral radicals formed. PMID:25687648

  2. Emotionally Neutral Stimuli Are Not Neutral in Schizophrenia: A Mini Review of Functional Neuroimaging Studies.

    PubMed

    Potvin, Stéphane; Tikàsz, Andràs; Mendrek, Adrianna

    2016-01-01

    Reliable evidence shows that schizophrenia patients tend to experience negative emotions when presented with emotionally neutral stimuli. Similarly, several functional neuroimaging studies show that schizophrenia patients have increased activations in response to neutral material. However, results are heterogeneous. Here, we review the functional neuroimaging studies that have addressed this research question. Based on the 36 functional neuroimaging studies that we retrieved, it seems that the increased brain reactivity to neutral stimuli is fairly common in schizophrenia, but that the regions involved vary considerably, apart from the amygdala. Prefrontal and cingulate sub-regions and the hippocampus may also be involved. By contrasts, results in individuals at risk for psychosis are less consistent. In schizophrenia patients, results are less consistent in the case of studies using non-facial stimuli, explicit processing paradigms, and/or event-related designs. This means that human faces may convey subtle information (e.g., trustworthiness) other than basic emotional expressions. It also means that the aberrant brain reactivity to neutral stimuli is less likely to occur when experimental paradigms are too cognitively demanding as well as in studies lacking statistical power. The main hypothesis proposed to account for this increased brain reactivity to neutral stimuli is the aberrant salience hypothesis of psychosis. Other investigators propose that the aberrant brain reactivity to neutral stimuli in schizophrenia results from abnormal associative learning, untrustworthiness judgments, priming effects, and/or reduced habituation to neutral stimuli. In the future, the effects of antipsychotics on this aberrant brain reactivity will need to be determined, as well as the potential implication of sex/gender. PMID:27445871

  3. Emotionally Neutral Stimuli Are Not Neutral in Schizophrenia: A Mini Review of Functional Neuroimaging Studies

    PubMed Central

    Potvin, Stéphane; Tikàsz, Andràs; Mendrek, Adrianna

    2016-01-01

    Reliable evidence shows that schizophrenia patients tend to experience negative emotions when presented with emotionally neutral stimuli. Similarly, several functional neuroimaging studies show that schizophrenia patients have increased activations in response to neutral material. However, results are heterogeneous. Here, we review the functional neuroimaging studies that have addressed this research question. Based on the 36 functional neuroimaging studies that we retrieved, it seems that the increased brain reactivity to neutral stimuli is fairly common in schizophrenia, but that the regions involved vary considerably, apart from the amygdala. Prefrontal and cingulate sub-regions and the hippocampus may also be involved. By contrasts, results in individuals at risk for psychosis are less consistent. In schizophrenia patients, results are less consistent in the case of studies using non-facial stimuli, explicit processing paradigms, and/or event-related designs. This means that human faces may convey subtle information (e.g., trustworthiness) other than basic emotional expressions. It also means that the aberrant brain reactivity to neutral stimuli is less likely to occur when experimental paradigms are too cognitively demanding as well as in studies lacking statistical power. The main hypothesis proposed to account for this increased brain reactivity to neutral stimuli is the aberrant salience hypothesis of psychosis. Other investigators propose that the aberrant brain reactivity to neutral stimuli in schizophrenia results from abnormal associative learning, untrustworthiness judgments, priming effects, and/or reduced habituation to neutral stimuli. In the future, the effects of antipsychotics on this aberrant brain reactivity will need to be determined, as well as the potential implication of sex/gender. PMID:27445871

  4. CO2-neutral fuels

    NASA Astrophysics Data System (ADS)

    Goede, A. P. H.

    2015-08-01

    The need for storage of renewable energy (RE) generated by photovoltaic, concentrated solar and wind arises from the fact that supply and demand are ill-matched both geographically and temporarily. This already causes problems of overcapacity and grid congestion in countries where the fraction of RE exceeds the 20% level. A system approach is needed, which focusses not only on the energy source, but includes conversion, storage, transport, distribution, use and, last but not least, the recycling of waste. Furthermore, there is a need for more flexibility in the energy system, rather than relying on electrification, integration with other energy systems, for example the gas network, would yield a system less vulnerable to failure and better adapted to requirements. For example, long-term large-scale storage of electrical energy is limited by capacity, yet needed to cover weekly to seasonal demand. This limitation can be overcome by coupling the electricity net to the gas system, considering the fact that the Dutch gas network alone has a storage capacity of 552 TWh, sufficient to cover the entire EU energy demand for over a month. This lecture explores energy storage in chemicals bonds. The focus is on chemicals other than hydrogen, taking advantage of the higher volumetric energy density of hydrocarbons, in this case methane, which has an approximate 3.5 times higher volumetric energy density. More importantly, it allows the ready use of existing gas infrastructure for energy storage, transport and distribution. Intermittent wind electricity generated is converted into synthetic methane, the Power to Gas (P2G) scheme, by splitting feedstock CO2 and H2O into synthesis gas, a mixture of CO and H2. Syngas plays a central role in the synthesis of a range of hydrocarbon products, including methane, diesel and dimethyl ether. The splitting is accomplished by innovative means; plasmolysis and high-temperature solid oxygen electrolysis. A CO2-neutral fuel cycle is

  5. NEUTRAL-BEAM INJECTION

    SciTech Connect

    Kunkel, W.B.

    1980-06-01

    The emphasis in the preceding chapters has been on magnetic confinement of high temperature plasmas. The question of production and heating of such plasmas has been dealt with relatively more briefly. It should not be inferred, however, that these matters must therefore be either trivial or unimportant. A review of the history reveals that in the early days all these aspects of the controlled fusion problem were considered to be on a par, and were tackled simultaneously and with equal vigor. Only the confinement problem turned out to be much more complex than initially anticipated, and richer in challenge to the plasma physicist than the questions of plasma production and heating. On the other hand, the properties of high-temperature plasmas and plasma confinement can only be studied experimentally after the problems of production and of heating to adequate temperatures are solved. It is the purpose of this and the next chapter to supplement the preceding discussions with more detail on two important subjects: neutral-beam injection and radio-frequency heating. These are the major contenders for heating in present and future tokamak and mirror fusion experiments, and even in several proposed reactors. For neutral beams we emphasize here the technology involved, which has undergone a rather remarkable development. The physics of particle and energy deposition in the plasma, and the discussion of the resulting effects on the confined plasma, have been included in previous chapters, and some experimental results are quoted there. Other heating processes of relevance to fusion are mentioned elsewhere in this book, in connection with the experiments where they are used: i.e. ohmic heating, adiabatic compression heating, and alpha-particle heating in Chapter 3 by H.P. Furth; more ohmic heating in Chapter 7, and shock-implosion heating, laser heating, and relativistic-electron beam heating in Chapter 8, both by W. E. Quinn. These methods are relatively straightforward in

  6. Neutral Models of Microbiome Evolution

    PubMed Central

    Zeng, Qinglong; Sukumaran, Jeet; Wu, Steven; Rodrigo, Allen

    2015-01-01

    There has been an explosion of research on host-associated microbial communities (i.e.,microbiomes). Much of this research has focused on surveys of microbial diversities across a variety of host species, including humans, with a view to understanding how these microbiomes are distributed across space and time, and how they correlate with host health, disease, phenotype, physiology and ecology. Fewer studies have focused on how these microbiomes may have evolved. In this paper, we develop an agent-based framework to study the dynamics of microbiome evolution. Our framework incorporates neutral models of how hosts acquire their microbiomes, and how the environmental microbial community that is available to the hosts is assembled. Most importantly, our framework also incorporates a Wright-Fisher genealogical model of hosts, so that the dynamics of microbiome evolution is studied on an evolutionary timescale. Our results indicate that the extent of parental contribution to microbial availability from one generation to the next significantly impacts the diversity of microbiomes: the greater the parental contribution, the less diverse the microbiomes. In contrast, even when there is only a very small contribution from a constant environmental pool, microbial communities can remain highly diverse. Finally, we show that our models may be used to construct hypotheses about the types of processes that operate to assemble microbiomes over evolutionary time. PMID:26200800

  7. Variation and infectivity neutralization in influenza

    PubMed Central

    Knossow, Marcel; Skehel, John J

    2006-01-01

    Worldwide epidemics of influenza are caused by viruses that normally infect other species, particularly waterfowl, and that contain haemagglutinin membrane glycoproteins (HAs) to which the human population has no immunity. Anti-HA immunoglobulins neutralize influenza virus infectivity. In this review we outline structural differences that distinguish the HAs of the 16 antigenic subtypes (H1–16) found in viruses from avian species. We also describe structural changes in HA required for the effective transfer to humans of viruses containing three of them, H1, H2 and H3, in the 1918 (Spanish), the 1957 (Asian) and the 1968 (Hong Kong) pandemics, respectively. In addition, we consider changes that may be required before the current avian H5 viruses could pass from human to human. PMID:16925526

  8. Neutral Buoyancy Simulator (NBS)

    NASA Technical Reports Server (NTRS)

    1972-01-01

    This is a cutaway illustration of the Neutral Buoyancy Simulator (NBS) at the Marshall Space Flight Center (MSFC ). The MSFC NBS provided an excellent environment for testing hardware to examine how it would operate in space and for evaluating techniques for space construction and spacecraft servicing. Here, engineers, designers, and astronauts performed various tests to develop basic concepts, preliminary designs, final designs, and crew procedures. The NBS was constructed of welded steel with polyester-resin coating. The water tank was 75-feet (22.9- meters) in diameter, 40-feet (12.2-meters) deep, and held 1.32 million gallons of water. Since it opened for operation in 1968, the NBS had supported a number of successful space missions, such as the Skylab, Solar Maximum Mission Satellite, Marned Maneuvering Unit, Experimental Assembly of Structures in Extravehicular Activity/Assembly Concept for Construction of Erectable Space Structures (EASE/ACCESS), the Hubble Space Telescope, and the Space Station. The function of the MSFC NBS was moved to the larger simulator at the Johnson Space Center and is no longer operational.

  9. Is science metaphysically neutral?

    PubMed

    Fry, Iris

    2012-09-01

    This paper challenges the claim that science is metaphysically neutral upheld by contenders of the separation of peacefully co-existent science and religion and by evolutionary theists. True, naturalistic metaphysical claims can neither be refuted nor proved and are thus distinct from empirical hypotheses. However, metaphysical assumptions not only regulate the theoretical and empirical study of nature, but are increasingly supported by the growing empirical body of science. This historically evolving interaction has contributed to the development of a naturalistic worldview that renounces the necessity of a transcendent god and of purposeful design. The thesis presented here differs not only from the claims of the "separatists" and of evolutionary theists. In pointing to the metaphysical aspects of science, I also criticize the failure of some evolutionary naturalists to distinguish between empirical and metaphysical contentions. Most important, based on the examination of science suggested here, creationists' false accusation that science is only a naturalistic dogma is refuted. Finally, the difficulties involved in the position endorsed here for the public support of evolution are acknowledged, taking into account the high religious profile of the American society and the social and political context in the US and in other countries. PMID:22771725

  10. Neutralization tests on the SERT 2 spacecraft

    NASA Technical Reports Server (NTRS)

    Kerslake, W. R.; Domitz, S.

    1979-01-01

    Neutralization test data obtained on the SERT 2 spacecraft are presented. Tests included ion beam neutralization of a thruster by a close (normal design) neutralizer as well as by a distant (1 meter) neutralizer. Parameters affecting neutralization, such as neutralizer bias voltage, neutralizer anode voltage, local spacecraft plasma density, and solar array voltage configuration were varied and changes in plasma potentials were measured. A plasma model is presented as an approximation of observed results.

  11. Cross-clade neutralizing antibodies against HIV-1 induced in rabbits by focusing the immune response on a neutralizing epitope

    SciTech Connect

    Zolla-Pazner, Susan; Cohen, Sandra; Pinter, Abraham; Krachmarov, Chavdar; Wrin, Terri; Wang Shixia; Lu Shan

    2009-09-15

    Studies were performed to induce cross-clade neutralizing antibodies (Abs) by testing various combinations of prime and boost constructs that focus the immune response on structurally-conserved epitopes in the V3 loop of HIV-1 gp120. Rabbits were immunized with gp120 DNA containing a V3 loop characterized by the GPGR motif at its tip, and/or with gp120 DNA with a V3 loop carrying the GPGQ motif. Priming was followed by boosts with V3-fusion proteins (V3-FPs) carrying the V3 sequence from a subtype B virus (GPGR motif), and/or with V3 sequences from subtypes A and C (GPGQ motif). The broadest and most consistent neutralizing responses were generated when using a clade C gp120 DNA prime and with the V3{sub B}-FP boost. Immune sera displayed neutralizing activity in three assays against pseudoviruses and primary isolates from subtypes A, AG, B, C, and D. Polyclonal Abs in the immune rabbit sera neutralized viruses that were not neutralized by pools of human anti-V3 monoclonal Abs. Greater than 80% of the neutralizing Abs were specific for V3, showing that the immune response could be focused on a neutralizing epitope and that vaccine-induced anti-V3 Abs have cross-clade neutralizing activity.

  12. Polyvalent HIV-1 Env vaccine formulations delivered by the DNA priming plus protein boosting approach are effective in generating neutralizing antibodies against primary human immunodeficiency virus type 1 isolates from subtypes A, B, C, D and E.

    PubMed

    Wang, Shixia; Pal, Ranajit; Mascola, John R; Chou, Te-Hui W; Mboudjeka, Innocent; Shen, Siyuan; Liu, Qin; Whitney, Stephen; Keen, Timothy; Nair, B C; Kalyanaraman, V S; Markham, Philip; Lu, Shan

    2006-06-20

    A major challenge in developing an HIV-1 vaccine is to identify immunogens and their delivery methods that can elicit broad neutralizing antibodies against primary isolates of different genetic subtypes. Recently, we demonstrated that priming with DNA vaccines expressing primary HIV-1 envelope glycoprotein (Env) followed by recombinant Env protein boosting was successful in generating positive neutralizing antibody responses against a clade B primary HIV-1 isolate, JR-FL, that was not easily neutralized. In the current study, we examined whether the DNA priming plus recombinant protein boosting approach delivering a polyvalent primary Env formulation was able to generate neutralizing antibodies against primary HIV-1 viral isolates from various genetic subtypes. New Zealand White rabbits were first immunized with DNA vaccines expressing one, three or eight primary HIV-1 gp120 antigens delivered by a gene gun followed by recombinant gp120 protein boosting. Neutralizing antibody responses were examined by two independently executed neutralization assays: the first one was a single round infection neutralization assay against a panel of 10 primary HIV-1 isolates of subtypes A, B, C and E and the second one used the PhenoSense assay against a panel of 12 pseudovirues expressing primary HIV-1 Env antigens from subtypes A, B, C, D and E as well as 2 pseudoviruses expressing the Env antigens from MN and NL4-3 viruses. Rabbit sera immunized with the DNA priming plus protein boosting approach, but not DNA vaccine alone or Env protein alone, were capable of neutralizing 7 of 10 viruses in the first assay and 12 of 14 viruses in the second assay. More importantly, sera immunized with the polyvalent Env antigens were able to neutralize a significantly higher percentage of viruses than the sera immunized with the monovalent antigens. Our results suggest that DNA priming followed by recombinant Env protein boosting can be used to deliver polyvalent Env-antigen-based HIV-1

  13. Characterization of neutralization epitopes in the V2 region of human immunodeficiency virus type 1 gp120: role of glycosylation in the correct folding of the V1/V2 domain.

    PubMed Central

    Wu, Z; Kayman, S C; Honnen, W; Revesz, K; Chen, H; Vijh-Warrier, S; Tilley, S A; McKeating, J; Shotton, C; Pinter, A

    1995-01-01

    A number of monoclonal antibodies (MAbs) with various levels of neutralizing activity that recognize epitopes in the V1/V2 domain of LAI-related gp120s have been described. These include rodent antibodies directed against linear and conformational epitopes and a chimpanzee MAb, C108G, with extremely potent neutralizing activity directed against a glycan-dependent epitope. A fusion glycoprotein expression system that expressed the isolated V1/V2 domain of gp120 in native form was used to analyze the structural characteristics of these epitopes. A number of MAbs (C108G, G3-4, 684-238, SC258, 11/68b, 38/66a, 38/66c, 38/62c, and CRA3) that did not bind with high affinity to peptides immunoprecipitated a fusion glycoprotein expressing the V1/V2 domain of HXB2 gp120 in the absence of other human immunodeficiency virus sequences, establishing that their epitopes were fully specified within this region. Biochemical analyses indicated that in the majority of V1/V2 fusion molecules only five of the six glycosylation signals in the V1/V2 domain were utilized, and the glycoforms were found to be differentially recognized by particular MAbs. Both C108G and MAbs directed against conformational epitopes reacted with large fractions of the fully glycosylated molecules but with only small fractions of the incompletely glycosylated molecules. Mutational analysis of the V1 and V2 glycosylation signals indicated that in most cases the unutilized site was located either at position 156 or at position 160, suggesting the occurrence of competition for glycan addition at these neighboring positions. Mutation of glycosylation site 160 destroyed the C108G epitope but increased the fraction of the molecules that presented the conformational epitopes, while mutation of the highly conserved glycosylation site at position 156 greatly diminished the expression of the conformational epitopes and increased expression of the C108G epitope. Similar heterogeneity in glycosylation was also observed

  14. Ceramide stimulates ABCA12 expression via peroxisome proliferator-activated receptor {delta} in human keratinocytes.

    PubMed

    Jiang, Yan J; Uchida, Yoshikazu; Lu, Biao; Kim, Peggy; Mao, Cungui; Akiyama, Masashi; Elias, Peter M; Holleran, Walter M; Grunfeld, Carl; Feingold, Kenneth R

    2009-07-10

    ABCA12 (ATP binding cassette transporter, family 12) is a cellular membrane transporter that facilitates the delivery of glucosylceramides to epidermal lamellar bodies in keratinocytes, a process that is critical for permeability barrier formation. Following secretion of lamellar bodies into the stratum corneum, glucosylceramides are metabolized to ceramides, which comprise approximately 50% of the lipid in stratum corneum. Gene mutations of ABCA12 underlie harlequin ichthyosis, a devastating skin disorder characterized by abnormal lamellar bodies and a severe barrier abnormality. Recently we reported that peroxisome proliferator-activated receptor (PPAR) and liver X receptor activators increase ABCA12 expression in human keratinocytes. Here we demonstrate that ceramide (C(2)-Cer and C(6)-Cer), but not C(8)-glucosylceramides, sphingosine, or ceramide 1-phosphate, increases ABCA12 mRNA expression in a dose- and time-dependent manner. Inhibitors of glucosylceramide synthase, sphingomyelin synthase, and ceramidase and small interfering RNA knockdown of human alkaline ceramidase, which all increase endogenous ceramide levels, also increased ABCA12 mRNA levels. Moreover, simultaneous treatment with C(6)-Cer and each of these same inhibitors additively increased ABCA12 expression, indicating that ceramide is an important inducer of ABCA12 expression and that the conversion of ceramide to other sphingolipids or metabolites is not required. Finally, both exogenous and endogenous ceramides preferentially stimulate PPARdelta expression (but not other PPARs or liver X receptors), whereas PPARdelta knockdown by siRNA transfection specifically diminished the ceramide-induced increase in ABCA12 mRNA levels, indicating that PPARdelta is a mediator of the ceramide effect. Together, these results show that ceramide, an important lipid component of epidermis, up-regulates ABCA12 expression via the PPARdelta-mediated signaling pathway, providing a substrate-driven, feed

  15. Environmental neutralization of polonium-218

    SciTech Connect

    Goldstein, S.D.; Hopke, P.K.

    1985-01-01

    Previous work has indicated that two mechanisms of neutralization of the singly charged polonium ion exist. Charged Polonium-218 can be neutralized by reacting with oxygen to form a polonium oxide ion with a higher ionization potential than that of the polonium metal and then accepting an electron transferred from a lower ionization potential gas. In this present work, this mechanism has been verified by determining that the polonium oxide has an ionization potential in the range 10.35-10.53 eV. It was also previously reported that /sup 218/Po can be neutralized, in the absence of oxygen, by the scavenging of electrons by a trace gas such as water or nitrogen dioxide and their diffusion to the polonium ion. To verify this second neutralization mechanism, concentrations of nitrogen dioxide in nitrogen in the range of 50 ppb-1 ppm were examined for their ability to neutralize the polonium ion. Complete neutralization of /sup 218/Po was observed at nitrogen dioxide concentrations greater than 700 ppb. For concentrations below 700 ppb, the degree of neutralization was found to increase smoothly with the nitrogen dioxide concentration.

  16. [Neutral Medical Claim Management Committee].

    PubMed

    Komatsu, Mitsuru

    2013-03-01

    The Ibaraki Medical Association established the Committee for Alternative Dispute Resolution called the Neutral Medical Claim Management Committee in 2006. Among 64 claims presented to the committee, 29 were settled through mediation or consultation. Patients were generally satisfied that their claims were considered fairly by the committee and that they were able to talk directly with healthcare professionals. However, some did not consider the committee to be completely neutral. The healthcare professionals involved rated the committee highly because they felt that the processes were neutral and no emotional aspects were involved. PMID:23617190

  17. Mechanism of Neutralization by the Broadly Neutralizing HIV-1 Monoclonal Antibody VRC01▿†

    PubMed Central

    Li, Yuxing; O'Dell, Sijy; Walker, Laura M.; Wu, Xueling; Guenaga, Javier; Feng, Yu; Schmidt, Stephen D.; McKee, Krisha; Louder, Mark K.; Ledgerwood, Julie E.; Graham, Barney S.; Haynes, Barton F.; Burton, Dennis R.; Wyatt, Richard T.; Mascola, John R.

    2011-01-01

    The structure of VRC01 in complex with the HIV-1 gp120 core reveals that this broadly neutralizing CD4 binding site (CD4bs) antibody partially mimics the interaction of the primary virus receptor, CD4, with gp120. Here, we extended the investigation of the VRC01-gp120 core interaction to the biologically relevant viral spike to better understand the mechanism of VRC01-mediated neutralization and to define viral elements associated with neutralization resistance. In contrast to the interaction of CD4 or the CD4bs monoclonal antibody (MAb) b12 with the HIV-1 envelope glycoprotein (Env), occlusion of the VRC01 epitope by quaternary constraints was not a major factor limiting neutralization. Mutagenesis studies indicated that VRC01 contacts within the gp120 loop D, the CD4 binding loop, and the V5 region were necessary for optimal VRC01 neutralization, as suggested by the crystal structure. In contrast to interactions with the soluble gp120 monomer, VRC01 interaction with the native viral spike did not occur in a CD4-like manner; VRC01 did not induce gp120 shedding from the Env spike or enhance gp41 membrane proximal external region (MPER)-directed antibody binding to the Env spike. Finally, VRC01 did not display significant reactivity with human antigens, boding well for potential in vivo applications. The data indicate that VRC01 interacts with gp120 in the context of the functional spike in a manner distinct from that of CD4. It achieves potent neutralization by precisely targeting the CD4bs without requiring alterations of Env spike configuration and by avoiding steric constraints imposed by the quaternary structure of the functional Env spike. PMID:21715490

  18. Higher Throughput Quantification of Neutralizing Antibody to Herpes Simplex Viruses

    PubMed Central

    Blevins, Tamara P.; Mitchell, Michelle C.; Korom, Maria; Wang, Hong; Yu, Yinyi; Morrison, Lynda A.; Belshe, Robert B.

    2015-01-01

    We report a rapid, higher throughput method for measuring neutralizing antibody to herpes simplex virus (HSV) in human sera. Clinical isolates and sera from the Herpevac Trial for Women were used in a colorimetric assay in which infection of tissue culture (lack of neutralization) was indicated by substrate metabolism by beta-galactosidase induced in the ELVIS cell line. The neutralization assay was optimized by addition of guinea pig complement, which particularly enhanced neutralizing antibody titers to HSV-2. Higher neutralizing antibody titers were also achieved using virus particles isolated from the supernatant of infected cells rather than lysate of infected cells as the source of virus. The effect of assay incubation time and incubation time with substrate were also optimized. We found that incubating with substrate until a standard optical density of 1.0 was reached permitted a better comparison among virus isolates, and achieved reliable measurement of neutralizing antibody activity. Interestingly, in contrast to results in the absence of complement, addition of complement allowed sera from HSV-2 gD-vaccinated subjects to neutralize HSV-1 and HSV-2 clinical and laboratory isolates with equal potency. PMID:26658766

  19. Higher Throughput Quantification of Neutralizing Antibody to Herpes Simplex Viruses.

    PubMed

    Blevins, Tamara P; Mitchell, Michelle C; Korom, Maria; Wang, Hong; Yu, Yinyi; Morrison, Lynda A; Belshe, Robert B

    2015-01-01

    We report a rapid, higher throughput method for measuring neutralizing antibody to herpes simplex virus (HSV) in human sera. Clinical isolates and sera from the Herpevac Trial for Women were used in a colorimetric assay in which infection of tissue culture (lack of neutralization) was indicated by substrate metabolism by beta-galactosidase induced in the ELVIS cell line. The neutralization assay was optimized by addition of guinea pig complement, which particularly enhanced neutralizing antibody titers to HSV-2. Higher neutralizing antibody titers were also achieved using virus particles isolated from the supernatant of infected cells rather than lysate of infected cells as the source of virus. The effect of assay incubation time and incubation time with substrate were also optimized. We found that incubating with substrate until a standard optical density of 1.0 was reached permitted a better comparison among virus isolates, and achieved reliable measurement of neutralizing antibody activity. Interestingly, in contrast to results in the absence of complement, addition of complement allowed sera from HSV-2 gD-vaccinated subjects to neutralize HSV-1 and HSV-2 clinical and laboratory isolates with equal potency. PMID:26658766

  20. Incomplete Neutralization and Deviation from Sigmoidal Neutralization Curves for HIV Broadly Neutralizing Monoclonal Antibodies

    PubMed Central

    McCoy, Laura E.; Falkowska, Emilia; Doores, Katie J.; Le, Khoa; Sok, Devin; van Gils, Marit J.; Euler, Zelda; Burger, Judith A.; Seaman, Michael S.; Sanders, Rogier W.; Schuitemaker, Hanneke; Poignard, Pascal; Wrin, Terri; Burton, Dennis R.

    2015-01-01

    The broadly neutralizing HIV monoclonal antibodies (bnMAbs) PG9, PG16, PGT151, and PGT152 have been shown earlier to occasionally display an unusual virus neutralization profile with a non-sigmoidal slope and a plateau at <100% neutralization. In the current study, we were interested in determining the extent of non-sigmoidal slopes and plateaus at <100% for HIV bnMAbs more generally. Using both a 278 panel of pseudoviruses in a CD4 T-cell (U87.CCR5.CXCR4) assay and a panel of 117 viruses in the TZM-bl assay, we found that bnMAbs targeting many neutralizing epitopes of the spike had neutralization profiles for at least one virus that plateaued at <90%. Across both panels the bnMAbs targeting the V2 apex of Env and gp41 were most likely to show neutralization curves that plateaued <100%. Conversely, bnMAbs targeting the high-mannose patch epitopes were less likely to show such behavior. Two CD4 binding site (CD4bs) Abs also showed this behavior relatively infrequently. The phenomenon of incomplete neutralization was also observed in a large peripheral blood mononuclear cells (PBMC)-grown molecular virus clone panel derived from patient viral swarms. In addition, five bnMAbs were compared against an 18-virus panel of molecular clones produced in 293T cells and PBMCs and assayed in TZM-bl cells. Examples of plateaus <90% were seen with both types of virus production with no consistent patterns observed. In conclusion, incomplete neutralization and non-sigmoidal neutralization curves are possible for all HIV bnMAbs against a wide range of viruses produced and assayed in both cell lines and primary cells with implications for the use of antibodies in therapy and as tools for vaccine design. PMID:26267277

  1. Incomplete Neutralization and Deviation from Sigmoidal Neutralization Curves for HIV Broadly Neutralizing Monoclonal Antibodies.

    PubMed

    McCoy, Laura E; Falkowska, Emilia; Doores, Katie J; Le, Khoa; Sok, Devin; van Gils, Marit J; Euler, Zelda; Burger, Judith A; Seaman, Michael S; Sanders, Rogier W; Schuitemaker, Hanneke; Poignard, Pascal; Wrin, Terri; Burton, Dennis R

    2015-08-01

    The broadly neutralizing HIV monoclonal antibodies (bnMAbs) PG9, PG16, PGT151, and PGT152 have been shown earlier to occasionally display an unusual virus neutralization profile with a non-sigmoidal slope and a plateau at <100% neutralization. In the current study, we were interested in determining the extent of non-sigmoidal slopes and plateaus at <100% for HIV bnMAbs more generally. Using both a 278 panel of pseudoviruses in a CD4 T-cell (U87.CCR5.CXCR4) assay and a panel of 117 viruses in the TZM-bl assay, we found that bnMAbs targeting many neutralizing epitopes of the spike had neutralization profiles for at least one virus that plateaued at <90%. Across both panels the bnMAbs targeting the V2 apex of Env and gp41 were most likely to show neutralization curves that plateaued <100%. Conversely, bnMAbs targeting the high-mannose patch epitopes were less likely to show such behavior. Two CD4 binding site (CD4bs) Abs also showed this behavior relatively infrequently. The phenomenon of incomplete neutralization was also observed in a large peripheral blood mononuclear cells (PBMC)-grown molecular virus clone panel derived from patient viral swarms. In addition, five bnMAbs were compared against an 18-virus panel of molecular clones produced in 293T cells and PBMCs and assayed in TZM-bl cells. Examples of plateaus <90% were seen with both types of virus production with no consistent patterns observed. In conclusion, incomplete neutralization and non-sigmoidal neutralization curves are possible for all HIV bnMAbs against a wide range of viruses produced and assayed in both cell lines and primary cells with implications for the use of antibodies in therapy and as tools for vaccine design. PMID:26267277

  2. Neutral changes during divergent evolution of hemoglobins

    NASA Technical Reports Server (NTRS)

    Jukes, T. H.

    1978-01-01

    A comparison of the mRNAs for rabbit and human beta-hemoglobins shows that synonymous changes in codons have accumulated three times as rapidly as nucleotide replacements that produced changes in amino acids. This agrees with predictions based on the so-called neutral theory. In addition, seven codon changes that appear to be single-base changes (according to maximum parsimony) are actually two-base changes. This indicates that the construction of primordial sequences is of limited significance when based on inferences that assume minimum base changes for amino acid replacements.

  3. Structural basis of hepatitis C virus neutralization by broadly neutralizing antibody HCV1

    SciTech Connect

    Kong, Leopold; Giang, Erick; Robbins, Justin B.; Stanfield, Robyn L.; Burton, Dennis R.; Wilson, Ian A.; Law, Mansun

    2012-10-29

    Hepatitis C virus (HCV) infects more than 2% of the global population and is a leading cause of liver cirrhosis, hepatocellular carcinoma, and end-stage liver diseases. Circulating HCV is genetically diverse, and therefore a broadly effective vaccine must target conserved T- and B-cell epitopes of the virus. Human mAb HCV1 has broad neutralizing activity against HCV isolates from at least four major genotypes and protects in the chimpanzee model from primary HCV challenge. The antibody targets a conserved antigenic site (residues 412-423) on the virus E2 envelope glycoprotein. Two crystal structures of HCV1 Fab in complex with an epitope peptide at 1.8-{angstrom} resolution reveal that the epitope is a {beta}-hairpin displaying a hydrophilic face and a hydrophobic face on opposing sides of the hairpin. The antibody predominantly interacts with E2 residues Leu{sup 413} and Trp{sup 420} on the hydrophobic face of the epitope, thus providing an explanation for how HCV isolates bearing mutations at Asn{sup 415} on the same binding face escape neutralization by this antibody. The results provide structural information for a neutralizing epitope on the HCV E2 glycoprotein and should help guide rational design of HCV immunogens to elicit similar broadly neutralizing antibodies through vaccination.

  4. Glucose and palmitate uncouple AMPK from autophagy in human aortic endothelial cells

    PubMed Central

    Cacicedo, José M.; Ruderman, Neil B.; Ido, Yasuo

    2014-01-01

    Dysregulated autophagy and decreased AMP-activated protein kinase (AMPK) activity are each associated with atherogenesis. Atherogenesis is preceded by high circulating concentrations of glucose and fatty acids, yet the mechanism by which these nutrients regulate autophagy in human aortic endothelial cells (HAECs) is not known. Furthermore, whereas AMPK is recognized as an activator of autophagy in cells with few nutrients, its effects on autophagy in nutrient-rich HAECs has not been investigated. We maintained and passaged primary HAECs in media containing 25 mM glucose and incubated them subsequently with 0.4 mM palmitate. These conditions impaired basal autophagy and rendered HAECs more susceptible to apoptosis and adhesion of monocytes, outcomes attenuated by the autophagy activator rapamycin. Glucose and palmitate diminished AMPK activity and phosphorylation of the uncoordinated-51-like kinase 1 (ULK1) at Ser555, an autophagy-activating site targeted by AMPK. 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR)-mediated activation of AMPK phosphorylated acetyl-CoA carboxylase, but treatment with AICAR or other AMPK activators (A769662, phenformin) did not restore ULK1 phosphorylation or autophagosome formation. To determine whether palmitate-induced ceramide accumulation contributed to this finding, we overexpressed a ceramide-metabolizing enzyme, acid ceramidase. The increase in acid ceramidase expression ameliorated the effects of excess nutrients on ULK1 phosphorylation, without altering the effects of the AMPK activators. Thus, unlike low nutrient conditions, AMPK becomes uncoupled from autophagy in HAECs in a nutrient-rich environment, such as that found in patients with increased cardiovascular risk. These findings suggest that combinations of AMPK-independent and AMPK-dependent therapies may be more effective alternatives than either therapy alone for treating nutrient-induced cellular dysfunction. PMID:25354528

  5. Neutral and Non-Neutral Evolution of Drosophila Mitochondrial DNA

    PubMed Central

    Rand, D. M.; Dorfsman, M.; Kann, L. M.

    1994-01-01

    To test hypotheses of neutral evolution of mitochondrial DNA (mtDNA), nucleotide sequences were determined for 1515 base pairs of the NADH dehydrogenase subunit 5 (ND5) gene in the mitochondrial DNA of 29 lines of Drosophila melanogaster and 9 lines of its sibling species Drosophila simulans. In contrast to the patterns for nuclear genes, where D. melanogaster generally exhibits much less nucleotide polymorphism, the number of segregating sites was slightly higher in a global sample of nine ND5 sequences in D. melanogaster (s = 8) than in the nine lines of D. simulans (s = 6). When compared to variation at nuclear loci, the mtDNA variation in D. melanogaster does not depart from neutral expectations. The ND5 sequences in D. simulans, however, show fewer than half the number of variable sites expected under neutrality when compared to sequences from the period locus. While this reduction in variation is not significant at the 5% level, HKA tests with published restriction data for mtDNA in D. simulans do show a significant reduction of variation suggesting a selective sweep of variation in the mtDNA in this species. Tests of neutral evolution based on the ratios of synonymous and replacement polymorphism and divergence are generally consistent with neutral expectations, although a significant excess of amino acid polymorphism within both species is localized in one region of the protein. The rate of mtDNA evolution has been faster in D. melanogaster than in D. simulans and the population structure of mtDNA is distinct in these species. The data reveal how different rates of mtDNA evolution between species and different histories of neutral and adaptive evolution within species can compromise historical inferences in population and evolutionary biology. PMID:7851771

  6. Gas cell neutralizers (Fundamental principles)

    SciTech Connect

    Fuehrer, B.

    1985-06-01

    Neutralizing an ion-beam of the size and energy levels involved in the neutral-particle-beam program represents a considerable extension of the state-of-the-art of neutralizer technology. Many different mediums (e.g., solid, liquid, gas, plasma, photons) can be used to strip the hydrogen ion of its extra electron. A large, multidisciplinary R and D effort will no doubt be required to sort out all of the ''pros and cons'' of these various techniques. The purpose of this particular presentation is to discuss some basic configurations and fundamental principles of the gas type of neutralizer cell. Particular emphasis is placed on the ''Gasdynamic Free-Jet'' neutralizer since this configuration has the potential of being much shorter than other type of gas cells (in the beam direction) and it could operate in nearly a continuous mode (CW) if necessary. These were important considerations in the ATSU design which is discussed in some detail in the second presentation entitled ''ATSU Point Design''.

  7. A Re-Examiniation of Phonological Neutralization.

    ERIC Educational Resources Information Center

    Dinnsen, D.

    1985-01-01

    Reviews research studies that raise serious questions about phonological neutralization, that is, the merger of a contrast in certain contexts. Some findings cast doubt on the very existence of neutralization and the correctness of the theoretical principles that make assumptions based on neutralization. Reanalyzes neutralization in light of these…

  8. Simulations of neutralized final focus

    SciTech Connect

    Welch, D.R.; Rose, D.V.; Genoni, T.C.; Yu, S.S.; Barnard, J.J.

    2005-01-18

    In order to drive an inertial fusion target or study high energy density physics with heavy ion beams, the beam radius must be focused to < 3 mm and the pulse length must be compressed to < 10 ns. The conventional scheme for temporal pulse compression makes use of an increasing ion velocity to compress the beam as it drifts and beam space charge to stagnate the compression before final focus. Beam compression in a neutralizing plasma does not require stagnation of the compression, enabling a more robust method. The final pulse shape at the target can be programmed by an applied velocity tilt. In this paper, neutralized drift compression is investigated. The sensitivity of the compression and focusing to beam momentum spread, plasma, and magnetic field conditions is studied with realistic driver examples. Using the 3D particle-in-cell code, we examine issues associated with self-field generation, stability, and vacuum-neutralized transport transition and focusing.

  9. Conserved Role of an N-Linked Glycan on the Surface Antigen of Human Immunodeficiency Virus Type 1 Modulating Virus Sensitivity to Broadly Neutralizing Antibodies against the Receptor and Coreceptor Binding Sites

    PubMed Central

    Townsley, Samantha; Li, Yun; Kozyrev, Yury; Cleveland, Brad

    2015-01-01

    ABSTRACT HIV-1 establishes persistent infection in part due to its ability to evade host immune responses. Occlusion by glycans contributes to masking conserved sites that are targets for some broadly neutralizing antibodies (bNAbs). Previous work has shown that removal of a highly conserved potential N-linked glycan (PNLG) site at amino acid residue 197 (N7) on the surface antigen gp120 of HIV-1 increases neutralization sensitivity of the mutant virus to CD4 binding site (CD4bs)-directed antibodies compared to its wild-type (WT) counterpart. However, it is not clear if the role of the N7 glycan is conserved among diverse HIV-1 isolates and if other glycans in the conserved regions of HIV-1 Env display similar functions. In this work, we examined the role of PNLGs in the conserved region of HIV-1 Env, particularly the role of the N7 glycan in a panel of HIV-1 strains representing different clades, tissue origins, coreceptor usages, and neutralization sensitivities. We demonstrate that the absence of the N7 glycan increases the sensitivity of diverse HIV-1 isolates to CD4bs- and V3 loop-directed antibodies, indicating that the N7 glycan plays a conserved role masking these conserved epitopes. However, the effect of the N7 glycan on virus sensitivity to neutralizing antibodies directed against the V2 loop epitope is isolate dependent. These findings indicate that the N7 glycan plays an important and conserved role modulating the structure, stability, or accessibility of bNAb epitopes in the CD4bs and coreceptor binding region, thus representing a potential target for the design of immunogens and therapeutics. IMPORTANCE N-linked glycans on the HIV-1 envelope protein have been postulated to contribute to viral escape from host immune responses. However, the role of specific glycans in the conserved regions of HIV-1 Env in modulating epitope recognition by broadly neutralizing antibodies has not been well defined. We show here that a single N-linked glycan plays a

  10. RE: Pedagogy--After Neutrality

    ERIC Educational Resources Information Center

    I'Anson, John

    2010-01-01

    Within the UK and in many parts of the world, official accounts of what it is to make sense of religion are framed within a rhetorics of neutrality in which such study is premised upon the possibility of dispassionate engagement and analysis. This paper, which is largely theoretical in scope, explores both the affordances and the costs of such an…

  11. Neutralization kinetics for polonium-218

    SciTech Connect

    Chu, K.D.; Hopke, P.K.

    1988-06-01

    In a well-defined experimental system the neutralization of polonium-218 ions was investigated as a function of the physical and chemical properties of the controlled composition atmosphere. The diffusion coefficient of polonium-218 under various concentrations of trace gas NO/sub 2/ in nitrogen was measured. The mobilities of Po/sup +/ and PoO/sub 2//sup +/ are determined by combining experimental results with a computer model of the system. Three neutralization mechanisms were individually studied. The small-ion recombination rate has been found to be proportional to the square root of radon concentration. The electron-scavenging mechanism is responsible for the neutralization of Po/sup +/ in NO/sub 2/ or H/sub 2/O in nitrogen. When PoO/sub 2//sup +/ is formed, the electron-transfer mechanism dominates the neutralization process. The electron is transferred to PoO/sub 2//sup +/ from molecules with lower ionization potentials. The ionization molecules with lower ionization potentials. The ionization potential of PoO/sub 2//sup +/ is also determined to be 10.44 +/- 0.05 eV.

  12. Photodetachment process for beam neutralization

    DOEpatents

    Fink, Joel H. [Livermore, CA; Frank, Alan M. [Livermore, CA

    1979-02-20

    A process for neutralization of accelerated ions employing photo-induced charge detachment. The process involves directing a laser beam across the path of a negative ion beam such as to effect photodetachment of electrons from the beam ions. The frequency of the laser beam employed is selected to provide the maximum cross-section for the photodetachment process.

  13. Photodetachment process for beam neutralization

    DOEpatents

    Fink, J.H.; Frank, A.M.

    1979-02-20

    A process for neutralization of accelerated ions employing photo-induced charge detachment is disclosed. The process involves directing a laser beam across the path of a negative ion beam such as to effect photodetachment of electrons from the beam ions. The frequency of the laser beam employed is selected to provide the maximum cross-section for the photodetachment process. 2 figs.

  14. MSFC Skylab neutral buoyancy simulator

    NASA Technical Reports Server (NTRS)

    1974-01-01

    The use of a neutral buoyancy simulator for developing extravehicular activity systems and for training astronauts in weightless activities is discussed. The construction of the facility and the operations are described. The types of tests and the training activities conducted in the simulator are reported. Photographs of the components of the simulator and actual training exercises are included.

  15. Electron-electron interactions in fast neutral-neutral collisions

    SciTech Connect

    DuBois, R.D. ); Manson, S.T. . Dept. of Physics and Astronomy)

    1992-11-01

    Differential electron emission is studied for 50--500 keV H[sup +] and H atom impact on helium. Using the first Born formulation, it is shown that projectile electron-target electron interactions are expected to dominate the differential cross sections for low energy target electron emission induced by fast neutral projectile impact on any target. Measurements of the 15[degrees] electron emission were made in order to investigate this prediction. For low impact energies, a constant ratio between the hydrogen atom and proton impact cross sections was found for emitted electron velocities less than half the projectile velocity, V[sub p] But as the collision energy increased, for electron velocities less than 0.25 V[sub p], the cross section ratio increased as the emitted electron velocity decreased. This is interpreted as a signature of projectile electron-target electron interactions becoming dominant for distant collisions between neutral particles.

  16. Electron-electron interactions in fast neutral-neutral collisions

    SciTech Connect

    DuBois, R.D.; Manson, S.T.

    1992-11-01

    Differential electron emission is studied for 50--500 keV H{sup +} and H atom impact on helium. Using the first Born formulation, it is shown that projectile electron-target electron interactions are expected to dominate the differential cross sections for low energy target electron emission induced by fast neutral projectile impact on any target. Measurements of the 15{degrees} electron emission were made in order to investigate this prediction. For low impact energies, a constant ratio between the hydrogen atom and proton impact cross sections was found for emitted electron velocities less than half the projectile velocity, V{sub p} But as the collision energy increased, for electron velocities less than 0.25 V{sub p}, the cross section ratio increased as the emitted electron velocity decreased. This is interpreted as a signature of projectile electron-target electron interactions becoming dominant for distant collisions between neutral particles.

  17. Envelope Variants Circulating as Initial Neutralization Breadth Developed in Two HIV-Infected Subjects Stimulate Multiclade Neutralizing Antibodies in Rabbits

    PubMed Central

    Malherbe, Delphine C.; Pissani, Franco; Sather, D. Noah; Guo, Biwei; Pandey, Shilpi; Sutton, William F.; Stuart, Andrew B.; Robins, Harlan; Park, Byung; Krebs, Shelly J.; Schuman, Jason T.; Kalams, Spyros; Hessell, Ann J.

    2014-01-01

    ABSTRACT Identifying characteristics of the human immunodeficiency virus type 1 (HIV-1) envelope that are effective in generating broad, protective antibodies remains a hurdle to HIV vaccine design. Emerging evidence of the development of broad and potent neutralizing antibodies in HIV-infected subjects suggests that founder and subsequent progeny viruses may express unique antigenic motifs that contribute to this developmental pathway. We hypothesize that over the course of natural infection, B cells are programmed to develop broad antibodies by exposure to select populations of emerging envelope quasispecies variants. To test this hypothesis, we identified two unrelated subjects whose antibodies demonstrated increasing neutralization breadth against a panel of HIV-1 isolates over time. Full-length functional env genes were cloned longitudinally from these subjects from months after infection through 2.6 to 5.8 years of infection. Motifs associated with the development of breadth in published, cross-sectional studies were found in both subjects. We compared the immunogenicity of envelope vaccines derived from time points obtained during and after broadening of neutralization activity within these subjects. Rabbits were coimmunized four times with selected multiple gp160 DNAs and gp140-trimeric envelope proteins. The affinity of the polyclonal response increased as a function of boosting. The most rapid and persistent neutralization of multiclade tier 1 viruses was elicited by envelopes that were circulating in plasma at time points prior to the development of 50% neutralization breadth in both human subjects. The breadth elicited in rabbits was not improved by exposure to later envelope variants. These data have implications for vaccine development in describing a target time point to identify optimal envelope immunogens. IMPORTANCE Vaccine protection against viral infections correlates with the presence of neutralizing antibodies; thus, vaccine components capable

  18. Arenavirus Glycan Shield Promotes Neutralizing Antibody Evasion and Protracted Infection

    PubMed Central

    Malinge, Pauline; Magistrelli, Giovanni; Fischer, Nicolas; Sahin, Mehmet; Bergthaler, Andreas; Igonet, Sebastien; ter Meulen, Jan; Rigo, Dorothée; Meda, Paolo; Rabah, Nadia; Coutard, Bruno; Bowden, Thomas A.; Lambert, Paul-Henri; Siegrist, Claire-Anne; Pinschewer, Daniel D.

    2015-01-01

    Arenaviruses such as Lassa virus (LASV) can cause severe hemorrhagic fever in humans. As a major impediment to vaccine development, delayed and weak neutralizing antibody (nAb) responses represent a unifying characteristic of both natural infection and all vaccine candidates tested to date. To investigate the mechanisms underlying arenavirus nAb evasion we engineered several arenavirus envelope-chimeric viruses and glycan-deficient variants thereof. We performed neutralization tests with sera from experimentally infected mice and from LASV-convalescent human patients. NAb response kinetics in mice correlated inversely with the N-linked glycan density in the arenavirus envelope protein’s globular head. Additionally and most intriguingly, infection with fully glycosylated viruses elicited antibodies, which neutralized predominantly their glycan-deficient variants, both in mice and humans. Binding studies with monoclonal antibodies indicated that envelope glycans reduced nAb on-rate, occupancy and thereby counteracted virus neutralization. In infected mice, the envelope glycan shield promoted protracted viral infection by preventing its timely elimination by the ensuing antibody response. Thus, arenavirus envelope glycosylation impairs the protective efficacy rather than the induction of nAbs, and thereby prevents efficient antibody-mediated virus control. This immune evasion mechanism imposes limitations on antibody-based vaccination and convalescent serum therapy. PMID:26587982

  19. Arenavirus Glycan Shield Promotes Neutralizing Antibody Evasion and Protracted Infection.

    PubMed

    Sommerstein, Rami; Flatz, Lukas; Remy, Melissa M; Malinge, Pauline; Magistrelli, Giovanni; Fischer, Nicolas; Sahin, Mehmet; Bergthaler, Andreas; Igonet, Sebastien; Ter Meulen, Jan; Rigo, Dorothée; Meda, Paolo; Rabah, Nadia; Coutard, Bruno; Bowden, Thomas A; Lambert, Paul-Henri; Siegrist, Claire-Anne; Pinschewer, Daniel D

    2015-11-01

    Arenaviruses such as Lassa virus (LASV) can cause severe hemorrhagic fever in humans. As a major impediment to vaccine development, delayed and weak neutralizing antibody (nAb) responses represent a unifying characteristic of both natural infection and all vaccine candidates tested to date. To investigate the mechanisms underlying arenavirus nAb evasion we engineered several arenavirus envelope-chimeric viruses and glycan-deficient variants thereof. We performed neutralization tests with sera from experimentally infected mice and from LASV-convalescent human patients. NAb response kinetics in mice correlated inversely with the N-linked glycan density in the arenavirus envelope protein's globular head. Additionally and most intriguingly, infection with fully glycosylated viruses elicited antibodies, which neutralized predominantly their glycan-deficient variants, both in mice and humans. Binding studies with monoclonal antibodies indicated that envelope glycans reduced nAb on-rate, occupancy and thereby counteracted virus neutralization. In infected mice, the envelope glycan shield promoted protracted viral infection by preventing its timely elimination by the ensuing antibody response. Thus, arenavirus envelope glycosylation impairs the protective efficacy rather than the induction of nAbs, and thereby prevents efficient antibody-mediated virus control. This immune evasion mechanism imposes limitations on antibody-based vaccination and convalescent serum therapy. PMID:26587982

  20. Passive neutralizing antibody controls SHIV viremia and enhances B cell responses in infant macaques

    PubMed Central

    Ng, Cherie T.; Jaworski, J. Pablo; Jayaraman, Pushpa; Sutton, William F.; Delio, Patrick; Kuller, LaRene; Anderson, David; Landucci, Gary; Richardson, Barbra A.; Burton, Dennis R.; Forthal, Donald N.; Haigwood, Nancy L.

    2010-01-01

    Maternal HIV-1-specific antibodies are efficiently transferred to newborns; their role in disease control is unknown. We administered non-sterilizing levels of neutralizing IgG, including the human neutralizing monoclonal IgG1b12, to six newborn macaques before oral challenge with SHIVSF612P3. All rapidly developed neutralizing antibodies and had significantly reduced plasma viremia for 6 months. These studies support the use of neutralizing antibodies in enhancing B cell responses and viral control in perinatal settings. PMID:20890292

  1. Coulomb blockade with neutral modes.

    PubMed

    Kamenev, Alex; Gefen, Yuval

    2015-04-17

    We study transport through a quantum dot in the fractional quantum Hall regime with filling factors ν=2/3 and ν=5/2, weakly coupled to the leads. We account for both injection of electrons to or from the leads, and quasiparticle rearrangement processes between the edge and the bulk of the quantum dot. The presence of neutral modes introduces topological constraints that modify qualitatively the features of the Coulomb blockade (CB). The periodicity of CB peak spacings doubles and the ratio of spacing between adjacent peaks approaches (in the low temperature and large dot limit) a universal value: 2∶1 for ν=2/3 and 3∶1 for ν=5/2. The corresponding CB diamonds alternate their width in the direction of the bias voltage and allow for the determination of the neutral mode velocity, and of the topological numbers associated with it. PMID:25933323

  2. HIV Neutralizing Antibodies Induced by Native-like Envelope Trimers

    PubMed Central

    Sanders, Rogier W.; van Gils, Marit J.; Derking, Ronald; Sok, Devin; Ketas, Thomas J.; Burger, Judith A.; Ozorowski, Gabriel; Cupo, Albert; Simonich, Cassandra; Goo, Leslie; Arendt, Heather; Kim, Helen J.; Lee, Jeong Hyun; Pugach, Pavel; Williams, Melissa; Debnath, Gargi; Moldt, Brian; van Breemen, Mariëlle J.; Isik, Gözde; Medina-Ramírez, Max; Back, Jaap Willem; Koff, Wayne; Julien, Jean-Philippe; Rakasz, Eva G.; Seaman, Michael S.; Guttman, Miklos; Lee, Kelly K.; Klasse, Per Johan; LaBranche, Celia; Schief, William R.; Wilson, Ian A.; Overbaugh, Julie; Burton, Dennis R.; Ward, Andrew B.; Montefiori, David C.; Dean, Hansi; Moore, John P.

    2015-01-01

    A challenge for HIV-1 immunogen design is inducing neutralizing antibodies (NAbs) against neutralization-resistant (Tier-2) viruses that dominate human transmissions. We show that a soluble recombinant HIV-1 envelope glycoprotein trimer that adopts a native conformation (BG505 SOSIP.664) induced NAbs potently against the sequence-matched Tier-2 virus in rabbits and similar but weaker responses in macaques. The trimer also consistently induced cross-reactive NAbs against more sensitive (Tier-1) viruses. Tier-2 NAbs recognized conformational epitopes that differed between animals and in some cases overlapped with those recognized by broadly neutralizing antibodies (bNAbs), whereas Tier-1 responses targeted linear V3 epitopes. A second trimer, B41 SOSIP.664, also induced a strong autologous Tier-2 NAb response in rabbits. Thus, native-like trimers represent a promising starting point for developing HIV-1 vaccines aimed at inducing bNAbs. PMID:26089353

  3. EVA Development and Verification Testing at NASA's Neutral Buoyancy Laboratory

    NASA Technical Reports Server (NTRS)

    Jairala, Juniper; Durkin, Robert

    2012-01-01

    As an early step in preparing for future EVAs, astronauts perform neutral buoyancy testing to develop and verify EVA hardware and operations. To date, neutral buoyancy demonstrations at NASA JSC’s Sonny Carter Training Facility have primarily evaluated assembly and maintenance tasks associated with several elements of the ISS. With the retirement of the Space Shuttle, completion of ISS assembly, and introduction of commercial participants for human transportation into space, evaluations at the NBL will take on a new focus. In this session, Juniper Jairala briefly discussed the design of the NBL and, in more detail, described the requirements and process for performing a neutral buoyancy test, including typical hardware and support equipment requirements, personnel and administrative resource requirements, examples of ISS systems and operations that are evaluated, and typical operational objectives that are evaluated. Robert Durkin discussed the new and potential types of uses for the NBL, including those by non-NASA external customers.

  4. EVA Development and Verification Testing at NASA's Neutral Buoyancy Laboratory

    NASA Technical Reports Server (NTRS)

    Jairala, Juniper; Durkin, Robert

    2012-01-01

    As an early step in preparing for future EVAs, astronauts perform neutral buoyancy testing to develop and verify EVA hardware and operations. To date, neutral buoyancy demonstrations at NASA JSC's Sonny Carter Training Facility have primarily evaluated assembly and maintenance tasks associated with several elements of the ISS. With the retirement of the Space Shuttle, completion of ISS assembly, and introduction of commercial participants for human transportation into space, evaluations at the NBL will take on a new focus. In this session, Juniper Jairala briefly discussed the design of the NBL and, in more detail, described the requirements and process for performing a neutral buoyancy test, including typical hardware and support equipment requirements, personnel and administrative resource requirements, examples of ISS systems and operations that are evaluated, and typical operational objectives that are evaluated. Robert Durkin discussed the new and potential types of uses for the NBL, including those by non-NASA external customers.

  5. Neutral Buoyancy Simulator - Hubble Space Telescope Training

    NASA Technical Reports Server (NTRS)

    1993-01-01

    This photograph shows STS-61 crewmemmbers training for the Hubble Space Telescope (HST) servicing mission in the Marshall Space Flight Center's (MSFC's) Neutral Buoyancy Simulator (NBS). Two months after its deployment in space, scientists detected a 2-micron spherical aberration in the primary mirror of the HST that affected the telescope's ability to focus faint light sources into a precise point. This imperfection was very slight, one-fiftieth of the width of a human hair. A scheduled Space Service servicing mission (STS-61) in 1993 permitted scientists to correct the problem. The MSFC NBS provided an excellent environment for testing hardware to examine how it would operate in space and for evaluating techniques for space construction and spacecraft servicing.

  6. Astronaut Training in the Neutral Buoyancy Simulator

    NASA Technical Reports Server (NTRS)

    1993-01-01

    This photograph shows an STS-61 astronaut training for the Hubble Space Telescope (HST) servicing mission (STS-61) in the Marshall Space Flight Center's (MSFC's) Neutral Buoyancy Simulator (NBS). Two months after its deployment in space, scientists detected a 2-micron spherical aberration in the primary mirror of the HST that affected the telescope's ability to focus faint light sources into a precise point. This imperfection was very slight, one-fiftieth of the width of a human hair. A scheduled Space Service servicing mission (STS-61) in 1993 permitted scientists to correct the problem. The MSFC NBS provided an excellent environment for testing hardware to examine how it would operate in space and for evaluating techniques for space construction and spacecraft servicing.

  7. Optimization of neutral atom imagers

    NASA Astrophysics Data System (ADS)

    Shappirio, M.; Coplan, M.; Balsamo, E.; Chornay, D.; Collier, M.; Hughes, P.; Keller, J.; Ogilvie, K.

    2008-12-01

    : The interactions between plasma structures and neutral atom populations in interplanetary space can be effectively studied with energetic neutral atom imagers. For neutral atoms with energies less than 1 keV, the most efficient detection method that preserves direction and energy information is conversion to negative ions on surfaces. We have examined a variety of surface materials and conversion geometries in order to identify the factors that determine conversion efficiency. For chemically and physically stable surfaces smoothness is of primary importance while properties such as work function have no obvious correlation to conversion efficiency. For the noble metals, tungsten, silicon, and graphite with comparable smoothness, conversion efficiency varies by a factor of two to three. We have also examined the way in which surface conversion efficiency varies with the angle of incidence of the neutral atom and have found that the highest efficiencies are obtained at angles of incidence greater then 80°. The conversion efficiency of silicon, tungsten and graphite were examined most closely and the energy dependent variation of conversion efficiency measured over a range of incident angles. We have also developed methods for micromachining silicon in order to reduce the volume to surface area over that of a single flat surface and have been able to reduce volume to surface area ratios by up to a factor of 60. With smooth micro-machined surfaces of the optimum geometry, conversion efficiencies can be increased by an order of magnitude over instruments like LENA on the IMAGE spacecraft without increase the instruments mass or volume. Work was supported by grant ACT-05-40 from the ESTO office of NASA

  8. Optimization of Neutral Atom Imagers

    NASA Technical Reports Server (NTRS)

    Shappirio, M.; Coplan, M.; Balsamo, E.; Chornay, D.; Collier, M.; Hughes, P.; Keller, J.; Ogilvie, K.; Williams, E.

    2008-01-01

    The interactions between plasma structures and neutral atom populations in interplanetary space can be effectively studied with energetic neutral atom imagers. For neutral atoms with energies less than 1 keV, the most efficient detection method that preserves direction and energy information is conversion to negative ions on surfaces. We have examined a variety of surface materials and conversion geometries in order to identify the factors that determine conversion efficiency. For chemically and physically stable surfaces smoothness is of primary importance while properties such as work function have no obvious correlation to conversion efficiency. For the noble metals, tungsten, silicon, and graphite with comparable smoothness, conversion efficiency varies by a factor of two to three. We have also examined the way in which surface conversion efficiency varies with the angle of incidence of the neutral atom and have found that the highest efficiencies are obtained at angles of incidence greater then 80deg. The conversion efficiency of silicon, tungsten and graphite were examined most closely and the energy dependent variation of conversion efficiency measured over a range of incident angles. We have also developed methods for micromachining silicon in order to reduce the volume to surface area over that of a single flat surface and have been able to reduce volume to surface area ratios by up to a factor of 60. With smooth micro-machined surfaces of the optimum geometry, conversion efficiencies can be increased by an order of magnitude over instruments like LENA on the IMAGE spacecraft without increase the instruments mass or volume.

  9. Plasma sources for spacecraft neutralization

    NASA Technical Reports Server (NTRS)

    Davis, V. A.; Katz, I.; Mandell, M. J.

    1990-01-01

    The principles of the operation of plasma sources for the neutralization of the surface of a spacecraft traveling in the presence of hot plasma are discussed with special attention given to the hollow-cathode-based plasma contactors. Techiques are developed that allow the calculation of the potentials and particle densities in the near environment of a hollow cathode plasma contactor in both the test tank and the LEO environment. The techniques and codes were validated by comparison of calculated and measured results.

  10. Broadly Neutralizing Activity of Zika Virus-Immune Sera Identifies a Single Viral Serotype.

    PubMed

    Dowd, Kimberly A; DeMaso, Christina R; Pelc, Rebecca S; Speer, Scott D; Smith, Alexander R Y; Goo, Leslie; Platt, Derek J; Mascola, John R; Graham, Barney S; Mulligan, Mark J; Diamond, Michael S; Ledgerwood, Julie E; Pierson, Theodore C

    2016-08-01

    Recent epidemics of Zika virus (ZIKV) have been associated with congenital malformation during pregnancy and Guillain-Barré syndrome. There are two ZIKV lineages (African and Asian) that share >95% amino acid identity. Little is known regarding the ability of neutralizing antibodies elicited against one lineage to protect against the other. We investigated the breadth of the neutralizing antibody response following ZIKV infection by measuring the sensitivity of six ZIKV strains to neutralization by ZIKV-confirmed convalescent human serum or plasma samples. Contemporary Asian and early African ZIKV strains were similarly sensitive to neutralization regardless of the cellular source of virus. Furthermore, mouse immune serum generated after infection with African or Asian ZIKV strains was capable of neutralizing homologous and heterologous ZIKV strains equivalently. Because our study only defines a single ZIKV serotype, vaccine candidates eliciting robust neutralizing antibody responses should inhibit infection of both ZIKV lineages, including strains circulating in the Americas. PMID:27481466

  11. Neutral-current x-distributions

    DOE R&D Accomplishments Database

    Friedman, J. I.; Kendall, H. W.; Bogert, D.; Burnstein, R.; Fisk, R.; Fuess, S.; Bofill, J.; Busza, W.; Eldridge, T.; Abolins, M.; Brock, R.; et al.

    1984-06-01

    The role of the semi leptonic neutral current interaction as a probe of nucleon structure is examined. Previous measurements of neutral current x-distributions are reviewed, and new results from the Fermilab - MIT - MSU collaboration are presented.

  12. A novel linear neutralizing epitope of hepatitis E virus.

    PubMed

    Tang, Zi-Min; Tang, Ming; Zhao, Min; Wen, Gui-Ping; Yang, Fan; Cai, Wei; Wang, Si-Ling; Zheng, Zi-Zheng; Xia, Ning-Shao

    2015-07-01

    Hepatitis E virus (HEV) is a serious public health problem that causes acute hepatitis in humans and is primarily transmitted through fecal and oral routes. The major anti-HEV antibody responses are against conformational epitopes located in a.a. 459-606 of HEV pORF2. All reported neutralization epitopes are present on the dimer domain constructed by this peptide. While looking for a neutralizing monoclonal antibody (MAb)-recognized linear epitope, we found a novel neutralizing linear epitope (L2) located in a.a. 423-437 of pORF2. Moreover, epitope L2 is proved non-immunodominant in the HEV-infection process. Using the hepatitis B virus core protein (HBc) as a carrier to display this novel linear epitope, we show herein that this epitope could induce a neutralizing antibody response against HEV in mice and could protect rhesus monkeys from HEV infection. Collectively, our results showed a novel non-immunodominant linear neutralizing epitope of hepatitis E virus, which provided additional insight of HEV vaccine. PMID:26051517

  13. Plasma/Neutral-Beam Etching Apparatus

    NASA Technical Reports Server (NTRS)

    Langer, William; Cohen, Samuel; Cuthbertson, John; Manos, Dennis; Motley, Robert

    1989-01-01

    Energies of neutral particles controllable. Apparatus developed to produce intense beams of reactant atoms for simulating low-Earth-orbit oxygen erosion, for studying beam-gas collisions, and for etching semiconductor substrates. Neutral beam formed by neutralization and reflection of accelerated plasma on metal plate. Plasma ejected from coaxial plasma gun toward neutralizing plate, where turned into beam of atoms or molecules and aimed at substrate to be etched.

  14. The Net Neutrality Debate: The Basics

    ERIC Educational Resources Information Center

    Greenfield, Rich

    2006-01-01

    Rich Greenfield examines the basics of today's net neutrality debate that is likely to be an ongoing issue for society. Greenfield states the problems inherent in the definition of "net neutrality" used by Common Cause: "Network neutrality is the principle that Internet users should be able to access any web content they choose and use any…

  15. Neutral beamline with improved ion energy recovery

    DOEpatents

    Kim, Jinchoon

    1984-01-01

    A neutral beamline employing direct energy recovery of unneutralized residual ions is provided which enhances the energy recovery of the full energy ion component of the beam exiting the neutralizer cell, and thus improves the overall neutral beamline efficiency. The unneutralized full energy ions exiting the neutralizer are deflected from the beam path and the electrons in the cell are blocked by a magnetic field applied transverse to the beam direction in the neutral izer exit region. The ions which are generated at essentially ground potential and accelerated through the neutralizer cell by a negative acceleration voltage are collected at ground potential. A neutralizer cell exit end region is provided which allows the magnetic and electric fields acting on the exiting ions to be loosely coupled. As a result, the fractional energy ions exiting the cell are reflected onto and collected at an interior wall of the neutralizer formed by the modified end geometry, and thus do not detract from the energy recovery efficiency of full energy ions exiting the cell. Electrons within the neutralizer are prevented from exiting the neutralizer end opening by the action of crossed fields drift (ExB) and are terminated to a collector collar around the downstream opening of the neutralizer. The correct combination of the extended neutralizer end structure and the magnet region is designed so as to maximize the exit of full energy ions and to contain the fractional energy ions.

  16. Magnetostatic traps for charged and neutral particles

    NASA Astrophysics Data System (ADS)

    Gomer, V.; Harms, O.; Haubrich, D.; Schadwinkel, H.; Strauch, F.; Ueberholz, B.; Aus der Wiesche, S.; Meschede, D.

    1997-08-01

    We have constructed magnetostatic traps from permanent magnets for trapping charged and neutral atoms. Two storage experiments are presented: a compact Penning trap for light ions and magnetic trapping of single neutral atoms. The dynamics of cold neutral atoms and their loss mechanisms in a quadrupole magnetostatic trap are discussed.

  17. Ion-beam Plasma Neutralization Interaction Images

    SciTech Connect

    Igor D. Kaganovich; Edward Startsev; S. Klasky; Ronald C. Davidson

    2002-04-09

    Neutralization of the ion beam charge and current is an important scientific issue for many practical applications. The process of ion beam charge and current neutralization is complex because the excitation of nonlinear plasma waves may occur. Computer simulation images of plasma neutralization of the ion beam pulse are presented.

  18. 46 CFR 502.404 - Neutrals.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Dispute Resolution § 502.404 Neutrals. (a) A neutral may be a permanent or temporary officer or employee of the Federal Government or any other individual who is acceptable to the parties to a dispute... Maritime Commission Dispute Resolution Specialist will seek to provide a neutral in dispute...

  19. 46 CFR 502.404 - Neutrals.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Dispute Resolution § 502.404 Neutrals. (a) A neutral may be a permanent or temporary officer or employee of the Federal Government or any other individual who is acceptable to the parties to a dispute... Maritime Commission Dispute Resolution Specialist will seek to provide a neutral in dispute...

  20. Priming Effects for Affective vs. Neutral Faces

    ERIC Educational Resources Information Center

    Burton, Leslie A.; Rabin, Laura; Wyatt, Gwinne; Frohlich, Jonathan; Vardy, Susan B.; Dimitri, Diana

    2005-01-01

    Affective and Neutral Tasks (faces with negative or neutral content, with different lighting and orientation) requiring reaction time judgments of poser identity were administered to 32 participants. Speed and accuracy were better for the Affective than Neutral Task, consistent with literature suggesting facilitation of performance by affective…

  1. Gas Flow Measurements of a Novel Geometry for Neutral Beam Neutralizers.

    NASA Astrophysics Data System (ADS)

    Pirkle, David Ross

    The gas flow characteristics of a novel geometry (pumped neutralizer) for decreasing the flow of gas from neutral beam neutralizers were measured and compared with a conventional (passive) neutralizer. A passive neutralizer is typically a duct attached to the ion source. For the pumped neutralizer the top and bottom surfaces of the duct are replaced by a Venetian blind geometry which opens into ballast vacuum pumping volumes. With guidance from a Monte Carlo program which models gas flow at low pressure, a one-half scale model with pumped neutralizer geometry was built and compared to a passive neutralizer with comparable dimensions. With the vanes on the pumped neutralizer opened to 55 degrees, the line density of the pumped neutralizer was 1.6 times less than the passive neutralizer. The amount of gas flowing from the exit of the pumped neutralizer was from 2 to 5 times less than the amount flowing from the pumped neutralizer. Hence, the pumped neutralizer geometry appears to be a promising method of limiting the flow of gas from neutral beam gas cell neutralizers.

  2. Poliovirus neutralization epitopes: analysis and localization with neutralizing monoclonal antibodies.

    PubMed Central

    Emini, E A; Jameson, B A; Lewis, A J; Larsen, G R; Wimmer, E

    1982-01-01

    Two hybridomas (H3 and D3) secreting monoclonal neutralizing antibody to intact poliovirus type 1 (Mahoney strain) were established. Each antibody bound to a site qualitatively different from that to which the other antibody bound. The H3 site was located on intact virions and, to a lesser extent, on 80S naturally occurring empty capsids and 14S precursor subunits. The D3 site was found only on virions and empty capsids. Neither site was expressed on 80S heat-treated virions. The antibodies did not react with free denatured or undenatured viral structural proteins. Viral variants which were no longer capable of being neutralized by either one or the other antibody were obtained. Such variants arose during normal cell culture passage of wild-type virus and were present in the progeny viral population on the order of 10(-4) variant per wild-type virus PFU. Toluene-2,4-diisocyanate, a heterobifunctional covalent cross-linking reagent, was used to irreversibly bind the F(ab) fragments of the two antibodies to their respective binding sites. In this way, VP1 was identified as the structural protein containing both sites. PMID:6183443

  3. Kinetic Simulations of Ion Beam Neutralization

    SciTech Connect

    Wang, Joseph

    2010-05-21

    Ion beam emission/neutralization is one of the most fundamental problems in spacecraft plasma interactions and electric propulsion. Although ion beam neutralization is readily achieved in experiments, the understanding of the underlying physical process remains at a rather primitive level. No theoretical or simulation models have convincingly explained the detailed neutralization mechanism, and no conclusions have been reached. This paper presents a fully kinetic simulation of ion beam neutralization and plasma beam propagation and discusses the physics of electron-ion coupling and the resulting propagation of a neutralized mesothermal plasma.

  4. Neutralization of measles virus wild-type isolates after immunization with a synthetic peptide vaccine which is not recognized by neutralizing passive antibodies.

    PubMed

    El Kasmi, K C; Fillon, S; Theisen, D M; Hartter, H; Brons, N H; Muller, C P

    2000-03-01

    The sequence H379-410 of the measles virus haemagglutinin (MV-H) protein forms a surface-exposed loop and contains three cysteine residues (Cys-381, Cys-386 and Cys-394) which are conserved among all measles isolates. It comprises the minimal sequential B cell epitope (BCE) (H386-400) of the neutralizing and protective MAb BH6 that neutralizes all wild-type viruses tested. The aim of this study was to design synthetic peptides which induce neutralizing antibodies against MV wild-type isolates. Peptides containing one or two copies of T cell epitopes (TCE) and BCEs of different lengths (H386-400, B(CC); H379-400, B(CCC)), in different combinations and orientations were produced and iteratively optimized for inducing neutralizing antibodies. Peptides with the shorter BCE induced sera that cross-reacted with MV but did not neutralize. The longer BCE containing the three cysteines (B(CCC)) and two homologous TCE were required for neutralization activity. These sera neutralized wild-type strains of different clades and geographic origins. Neutralizing serum was also obtained after immunization with human promiscuous TCEs. Furthermore B(CCC)-based peptides were fully immunogenic even in the presence of pre-existing MV-specific antibodies. The results suggest that subunit vaccines based on such peptides could potentially be used to actively protect infants against wild-type viruses irrespective of persisting maternal antibodies. PMID:10675410

  5. Space station neutral external environment

    NASA Technical Reports Server (NTRS)

    Ehlers, H.; Leger, L.

    1988-01-01

    Molecular contamination levels arising from the external induced neutral environment of the Space Station (Phase 1 configuration) were calculated using the MOLFLUX model. Predicted molecular column densities and deposition rates generally meet the Space Station contamination requirements. In the doubtful cases of deposition due to materials outgassing, proper material selection, generally excluding organic products exposed to the external environment, must be considered to meet contamination requirements. It is important that the Space Station configuration, once defined, is not significantly modified to avoid introducing new unacceptable contamination sources.

  6. Neutral gas dynamics in fireballs

    SciTech Connect

    Stenzel, R. L.; Ionita, C.; Schrittwieser, R.

    2011-06-01

    Fireballs are local discharge phenomena on positively biased electrodes in partially ionized plasmas. Electrons, energized at a double layer, heat neutral gas which expands. The gas pressure exceeds the plasma pressure, hence becomes important to the stability and transport in fireballs. The flow of gas moves the electrode and sensors similar to a mica pendulum. Flow speed and directions are measured. A fireball gun has been developed to partially collimate the flow of hot gas and heat objects in its path. New applications of fireballs are suggested.

  7. Use of beta-maltosides (p-nitrophenyl-beta-D-maltoside, 2-chloro-4- nitrophenyl-beta-D-maltoside and 4-methylumbelliferyl-beta-D-maltoside) as substrates for the assay of neutral alpha-glucosidase from human kidney and urine.

    PubMed

    Lukomskaya, I S; Voznyi, Y V; Lanskaya, I M; Podkidisheva, E I

    1996-01-31

    The method of assay of neutral alpha-glucosidase from human kidney and urine using beta-maltosides (p-nitrophenyl-beta-D-maltoside [NP-beta-D-maltoside], 2-chloro-4-nitrophenyl-beta-D-maltoside]) [CNP-beta-D-maltoside] and 4-methylumbelliferyl-beta-D-maltosides ([MU-beta-D-maltoside]) as substrates and beta-glucosidase as an auxiliary enzyme is proposed. All three beta-maltosides are suitable substrates for the determination of neutral alpha-glucosidase activity but MU-beta-D-maltoside is the most sensitive due to its methylumbelliferyl moiety. The method is simple, convenient and 10-fold more sensitive than the commonly used alpha-glucosidase assay procedure with the corresponding synthetic alpha-glucosides, p-nitrophenyl- alpha-D-glucoside (NP-alpha-D-glucoside) and 4-methylumbelliferyl-alpha-D-glucoside (MU-alpha-D-glucoside). A modification of the method, with p-nitrophenyl-beta-D-maltoside as substrate, was applied to the semiautomatic assay of urinary alpha-glucosidase in 96-well microtitre plates. PMID:8714432

  8. Detailed Atomic Structure of Neutral and Near-Neutral Systems

    SciTech Connect

    Oliver, Paul; Hibbert, Alan

    2011-05-11

    This paper highlights the issues which need to be addressed in undertaking accurate calculations of multi-electron atoms and ions, particularly at or near the neutral end of an isoelectronic sequence. We illustrate the processes through two calculations--of transitions in Cl I and Sn II--and discuss the convergence of our results as well as updating previous work. In particular, in the case of Cl I, we propose new identifications of the levels involved in certain transitions which are important in determining the abundance of chlorine in the inter-stellar medium (ISM), while in singly ionised tin, our calculations suggest a re-evaluation of the the abundance of tin in the ISM. We also confirm recent identification of Sn II lines seen in tokamak plasmas.

  9. Site Neutral Payments: An Overview.

    PubMed

    Dresevic, Adrienne; Rojas, Leslie

    2015-01-01

    A site neutral payment policy would entail CMS paying the same rate for the same healthcare service regardless of the location in which the service is provided. From the government's perspective, the reason behind this policy is potentially billions of dollars in savings. The rationale for using various payment systems is that there are different costs associated with providing healthcare services in different locations. Each payment system has a separate methodology for determining rates for services based on these costs. Hospitals may choose to prepare early for the inevitable through accurate cost reporting, shifting certain ancillary services to more appropriate outpatient, off site locations, and participating in the Medicare Shared Savings Program. PMID:26571969

  10. High Resolution Neutral Atom Microscope

    NASA Astrophysics Data System (ADS)

    Bucay, Igal; Castillo-Garza, Rodrigo; Stratis, Georgios; Raizen, Mark

    2015-03-01

    We are developing a high resolution neutral atom microscope based on metastable atom electron spectroscopy (MAES). When a metastable atom of a noble gas is near a solid, a surface electron will tunnel to an empty energy level of the metastable atom, thereby ejecting the excited electron from the atom. The emitted electrons carry information regarding the local topography and electronic, magnetic, and chemical structures of most hard materials. Furthermore, using a chromatic aberration corrected magnetic hexapole lens we expect to attain a spatial resolution below 10 nm. We will use this microscope to investigate how local phenomena can give rise to macroscopic effects in materials that cannot be probed using a scanning tunneling microscope, namely insulating transition metal oxides.

  11. Neutral composition in the thermosphere.

    NASA Technical Reports Server (NTRS)

    Taeusch, D. R.; Carignan, G. R.

    1972-01-01

    Data obtained from the Ogo 6 neutral atmospheric composition (NAC) experiment for a period of more than 1 year are used to compare the average constituent composition at an altitude of 400 km with that predicted by the Jacchia 1971 and 1965 models. The comparison shows that the Jacchia 1971 model underestimates the molecular nitrogen densities at 400 km by a factor of 2. An atmosphere is constructed down to 120 km by means of the Stein and Walker technique. A fit is made with (1) the 400-km total densities from drag measurements and the composition from Ogo 6 NAC, (2) the 250-km measured molecular nitrogen densities, and (3) the 150-km total densities from drag. This fit shows that the Jacchia 1971 model overestimates the atomic oxygen content at 150 km.

  12. Ergonomically neutral arm support system

    DOEpatents

    Siminovitch, Michael J; Chung, Jeffrey Y; Dellinges, Steven; Lafever, Robin E

    2005-08-02

    An ergonomic arm support system maintains a neutral position for the forearm. A mechanical support structure attached to a chair or other mounting structure supports the arms of a sitting or standing person. The system includes moving elements and tensioning elements to provide a dynamic balancing force against the forearms. The support structure is not fixed or locked in a rigid position, but is an active dynamic system that is maintained in equipoise by the continuous operation of the opposing forces. The support structure includes an armrest connected to a flexible linkage or articulated or pivoting assembly, which includes a tensioning element such as a spring. The pivoting assembly moves up and down, with the tensioning element providing the upward force that balances the downward force of the arm.

  13. An accessible heavy neutral lepton

    NASA Astrophysics Data System (ADS)

    Chang, Chao-Hsi

    1982-09-01

    In the SUL(2) ⊗ SUR(2) ⊗ UB-L(1) model, an accessible heavy neutral lepton may exist owing to the mixing of generations. Based on a rough estimate, it is pointed out that the most hopeful experiments to observe this lepton are ν-production in an emulsion (because the track of a particle with lifetime 10-11-10-13 s could be seen) and e-production. The author thanks Professor J.D. Bjorken, Professor He Zuo-Xiu, Professor R.E. Mashark, Professor S.J. Chang, Professor Zhu Cong-Yuan and Professor M. Dine for helpful discussions and comments. He would like to thank the referee for valuable comments.

  14. After treatment ends: neutral time.

    PubMed

    Hurt, G J; McQuellon, R P; Barrett, R J

    1994-01-01

    For persons diagnosed with cancer, the remission period may be marked by increased anxiety and distress. While the medical team may view remission as an eagerly anticipated milestone, the decreased medical surveillance during this time can cause a heightened fear of recurrence for the patient. One author has called this period of remission "neutral time," a time characterized by uncertainty. The safety signal hypothesis, developed by Martin Seligman, may help to explain the anxiety experienced by some patients during the remission period. Because cancer is frequently a silent disease with no overt symptoms, patients in remission often have no safety signal to indicate that the disease will not return. A case study is presented and discussed in light of these two concepts. PMID:7697080

  15. BEAMS3D Neutral Beam Injection Model

    SciTech Connect

    Lazerson, Samuel

    2014-04-14

    With the advent of applied 3D fi elds in Tokamaks and modern high performance stellarators, a need has arisen to address non-axisymmetric effects on neutral beam heating and fueling. We report on the development of a fully 3D neutral beam injection (NBI) model, BEAMS3D, which addresses this need by coupling 3D equilibria to a guiding center code capable of modeling neutral and charged particle trajectories across the separatrix and into the plasma core. Ionization, neutralization, charge-exchange, viscous velocity reduction, and pitch angle scattering are modeled with the ADAS atomic physics database [1]. Benchmark calculations are presented to validate the collisionless particle orbits, neutral beam injection model, frictional drag, and pitch angle scattering effects. A calculation of neutral beam heating in the NCSX device is performed, highlighting the capability of the code to handle 3D magnetic fields.

  16. Three-Dimensional Normal Human Neutral Progenitor Tissue-Like Assemblies: A Model for Persistent Varicella-Zoster Virus Infection and Platform to Study Oxidate Stress and Damage in Multiple Hit Scenarios

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J.; McCarthy, M.; Osterrieder, N.; Cohrs, R. J.; Kaufer, B. B.

    2014-01-01

    The environment of space results in a multitude of challenges to the human physiology that present barriers to extended habitation and exploration. Over 40 years of investigation to define countermeasures to address space flight adaptation has left gaps in our knowledge regarding mitigation strategies partly due to the lack of investigative tools, monitoring strategies, and real time diagnostics to understand the central causative agent(s) responsible for physiologic adaptation and maintaining homeostasis. Spaceflight-adaptation syndrome is the combination of space environmental conditions and the synergistic reaction of the human physiology. Our work addresses the role of oxidative stress and damage (OSaD) as a negative and contributing Risk Factor (RF) in the following areas of combined spaceflight related dysregulation: i) radiation induced cellular damage [1], [2] ii) immune impacts and the inflammatory response [3], [4] and iii) varicella zoster virus (VZV) reactivation [5]. Varicella-zoster (VZV)/Chicken Pox virus is a neurotropic human alphaherpes virus resulting in varicella upon primary infection, suppressed by the immune system becomes latent in ganglionic neurons, and reactivates under stress events to re-express in zoster and possibly shingles. Our laboratory has developed a complex three-dimensional (3D) normal human neural tissue model that emulates several characteristics of the human trigeminal ganglia (TG) and allows the study of combinatorial experimentation which addresses, simultaneously, OSaD associated with Spaceflight adaptation and habitation [6]. By combining the RFs of microgravity, radiation, and viral infection we will demonstrate that living in the space environment leads to significant physiological consequences for the peripheral and subsequently the central nervous system (PNS, CNS) associated with OSaD generation and consequentially endangers long-duration and exploration-class missions.

  17. Structure and Design of Broadly-Neutralizing Antibodies Against HIV

    PubMed Central

    Ryu, Seong Eon; Hendrickson, Wayne A.

    2012-01-01

    Since the discovery more than 30 years ago of human immunodeficiency virus (HIV) as the causative agent of the deadly disease, acquired immune deficiency disease (AIDS), there have been no efficient vaccines against the virus. For the infection of the virus, the HIV surface glycoprotein gp120 first recognizes the CD4 receptor on the target helper T-cell, which initiates HIV fusion with the target cell and, if unchecked, leads to destruction of the patient’s immune system. Despite the difficulty of developing appropriate immune responses in HIV-infected individuals, patient sera often contain antibodies that have broad neutralization activity, indicating the possibility of immunological treatment and prevention. Recently, through extensive structural studies of neutralizing antibodies of HIV in complex with gp120, the critical mechanisms of broad neutralization against HIV have been elucidated. Based on these discoveries, the structure-aided designs of antibodies and novel scaffolds were performed to create extremely potent neutralizing antibodies against HIV. These new discoveries and advances shed light on the road to development of efficient immunological therapies against AIDS. PMID:22736269

  18. An ultrasensitive universal detector based on neutralizer displacement

    NASA Astrophysics Data System (ADS)

    Das, Jagotamoy; Cederquist, Kristin B.; Zaragoza, Alexandre A.; Lee, Paul E.; Sargent, Edward H.; Kelley, Shana O.

    2012-08-01

    Diagnostic technologies that can provide the simultaneous detection of nucleic acids for gene expression, proteins for host response and small molecules for profiling the human metabolome will have a significant advantage in providing comprehensive patient monitoring. Molecular sensors that report changes in the electrostatics of a sensor's surface on analyte binding have shown unprecedented sensitivity in the detection of charged biomolecules, but do not lend themselves to the detection of small molecules, which do not carry significant charge. Here, we introduce the neutralizer displacement assay that allows charge-based sensing to be applied to any class of molecule irrespective of the analyte charge. The neutralizer displacement assay starts with an aptamer probe bound to a neutralizer. When analyte binding occurs the neutralizer is displaced, which results in a dramatic change in the surface charge for all types of analytes. We have tested the sensitivity, speed and specificity of this system in the detection of a panel of molecules: (deoxy)ribonucleic acid, ribonucleic acid, cocaine, adenosine triphosphate and thrombin.

  19. The edge of neutral evolution in social dilemmas

    NASA Astrophysics Data System (ADS)

    Cremer, Jonas; Reichenbach, Tobias; Frey, Erwin

    2009-09-01

    The functioning of animal as well as human societies fundamentally relies on cooperation. Yet, defection is often favorable for the selfish individual, and social dilemmas arise. Selection by individuals' fitness, usually the basic driving force of evolution, quickly eliminates cooperators. However, evolution is also governed by fluctuations that can be of greater importance than fitness differences, and can render evolution effectively neutral. Here, we investigate the effects of selection versus fluctuations in social dilemmas. By studying the mean extinction times of cooperators and defectors, a variable sensitive to fluctuations, we are able to identify and quantify an emerging 'edge of neutral evolution' that delineates regimes of neutral and Darwinian evolution. Our results reveal that cooperation is significantly maintained in the neutral regimes. In contrast, the classical predictions of evolutionary game theory, where defectors beat cooperators, are recovered in the Darwinian regimes. Our studies demonstrate that fluctuations can provide a surprisingly simple way to partly resolve social dilemmas. Our methods are generally applicable to estimate the role of random drift in evolutionary dynamics.

  20. Engineered Bispecific Antibodies with Exquisite HIV-1-Neutralizing Activity.

    PubMed

    Huang, Yaoxing; Yu, Jian; Lanzi, Anastasia; Yao, Xin; Andrews, Chasity D; Tsai, Lily; Gajjar, Mili R; Sun, Ming; Seaman, Michael S; Padte, Neal N; Ho, David D

    2016-06-16

    While the search for an efficacious HIV-1 vaccine remains elusive, emergence of a new generation of virus-neutralizing monoclonal antibodies (mAbs) has re-ignited the field of passive immunization for HIV-1 prevention. However, the plasticity of HIV-1 demands additional improvements to these mAbs to better ensure their clinical utility. Here, we report engineered bispecific antibodies that are the most potent and broad HIV-neutralizing antibodies to date. One bispecific antibody, 10E8V2.0/iMab, neutralized 118 HIV-1 pseudotyped viruses tested with a mean 50% inhibitory concentration (IC50) of 0.002 μg/mL. 10E8V2.0/iMab also potently neutralized 99% of viruses in a second panel of 200 HIV-1 isolates belonging to clade C, the dominant subtype accounting for ∼50% of new infections worldwide. Importantly, 10E8V2.0/iMab reduced virus load substantially in HIV-1-infected humanized mice and also provided complete protection when administered prior to virus challenge. These bispecific antibodies hold promise as novel prophylactic and/or therapeutic agents in the fight against HIV-1. PMID:27315479

  1. EVA Development and Verification Testing at NASA's Neutral Buoyancy Laboratory

    NASA Technical Reports Server (NTRS)

    Jairala, Juniper C.; Durkin, Robert; Marak, Ralph J.; Sipila, Stepahnie A.; Ney, Zane A.; Parazynski, Scott E.; Thomason, Arthur H.

    2012-01-01

    As an early step in the preparation for future Extravehicular Activities (EVAs), astronauts perform neutral buoyancy testing to develop and verify EVA hardware and operations. Neutral buoyancy demonstrations at NASA Johnson Space Center's Sonny Carter Training Facility to date have primarily evaluated assembly and maintenance tasks associated with several elements of the International Space Station (ISS). With the retirement of the Shuttle, completion of ISS assembly, and introduction of commercial players for human transportation to space, evaluations at the Neutral Buoyancy Laboratory (NBL) will take on a new focus. Test objectives are selected for their criticality, lack of previous testing, or design changes that justify retesting. Assembly tasks investigated are performed using procedures developed by the flight hardware providers and the Mission Operations Directorate (MOD). Orbital Replacement Unit (ORU) maintenance tasks are performed using a more systematic set of procedures, EVA Concept of Operations for the International Space Station (JSC-33408), also developed by the MOD. This paper describes the requirements and process for performing a neutral buoyancy test, including typical hardware and support equipment requirements, personnel and administrative resource requirements, examples of ISS systems and operations that are evaluated, and typical operational objectives that are evaluated.

  2. Neutralizing and non-neutralizing monoclonal antibodies against dengue virus E protein derived from a naturally infected patient

    PubMed Central

    2010-01-01

    Background Antibodies produced in response to infection with any of the four serotypes of dengue virus generally provide homotypic immunity. However, prior infection or circulating maternal antibodies can also mediate a non-protective antibody response that can enhance the course of disease in a subsequent heterotypic infection. Naturally occurring human monoclonal antibodies can help us understand the protective and pathogenic roles of the humoral immune system in dengue virus infection. Results Epstein-Barr Virus (EBV) transformation of B cells isolated from the peripheral blood of a human subject with previous dengue infection was performed. B cell cultures were screened by ELISA for antibodies to dengue (DENV) envelope (E) protein. ELISA positive cultures were cloned by limiting dilution. Three IgG1 human monoclonal antibodies (HMAbs) were purified and their binding specificity to E protein was verified by ELISA and biolayer interferometry. Neutralization and enhancement assays were conducted in epithelial and macrophage-like cell lines, respectively. All three HMAbs bound to E from at least two of the four DENV serotypes, one of the HMAbs was neutralizing, and all were able to enhance DENV infection. Conclusions HMAbs against DENV can be successfully generated by EBV transformation of B cells from patients at least two years after naturally acquired DENV infections. These antibodies show different patterns of cross-reactivity, neutralizing, and enhancement activity. PMID:20132551

  3. Neutral Buoyancy Test - NB23 - Space Telescope

    NASA Technical Reports Server (NTRS)

    1979-01-01

    Once the United States' space program had progressed from Earth's orbit into outerspace, the prospect of building and maintaining a permanent presence in space was realized. To accomplish this feat, NASA launched a temporary workstation, Skylab, to discover the effects of low gravity and weightlessness on the human body, and also to develop tools and equipment that would be needed in the future to build and maintain a more permanent space station. The structures, techniques, and work schedules had to be carefully designed to fit this unique construction site. The components had to be lightweight for transport into orbit, yet durable. The station also had to be made with removable parts for easy servicing and repairs by astronauts. All of the tools necessary for service and repairs had to be designed for easy manipulation by a suited astronaut. And construction methods had to be efficient due to limited time the astronauts could remain outside their controlled environment. In lieu of all the specific needs for this project, an environment on Earth had to be developed that could simulate a low gravity atmosphere. A Neutral Buoyancy Simulator (NBS) was constructed by NASA Marshall Space Flight Center (MSFC) in 1968. Since then, NASA scientists have used this facility to understand how humans work best in low gravity and also provide information about the different kinds of structures that can be built. Included in the plans for the space station was a space telescope. This telescope would be attached to the space station and directed towards outerspace. Astronomers hoped that the space telescope would provide a look at space that is impossible to see from Earth because of Earth's atmosphere and other man made influences. In an effort to make replacement and repairs easier on astronauts the space telescope was designed to be modular. Practice makes perfect as demonstrated in this photo: an astronaut practices moving modular pieces of the space telescope in the Neutral

  4. ION SOURCE WITH SPACE CHARGE NEUTRALIZATION

    DOEpatents

    Flowers, J.W.; Luce, J.S.; Stirling, W.L.

    1963-01-22

    This patent relates to a space charge neutralized ion source in which a refluxing gas-fed arc discharge is provided between a cathode and a gas-fed anode to provide ions. An electron gun directs a controlled, monoenergetic electron beam through the discharge. A space charge neutralization is effected in the ion source and accelerating gap by oscillating low energy electrons, and a space charge neutralization of the source exit beam is effected by the monoenergetic electron beam beyond the source exit end. The neutralized beam may be accelerated to any desired energy at densities well above the limitation imposed by Langmuir-Child' s law. (AEC)

  5. Influenza virus antigenicity and broadly neutralizing epitopes

    PubMed Central

    Air, Gillian M.

    2015-01-01

    A vaccine formulation that would be effective against all strains of influenza virus has long been a goal of vaccine developers, but antibodies after infection or vaccination were seen to be strain specific and there was little evidence of cross-reactive antibodies that neutralized across subtypes. Recently a number of broadly neutralizing monoclonal antibodies have been characterized. This review describes the different classes of broadly neutralizing antibodies and discusses the potential of their therapeutic use or for design of immunogens that induce a high proportion of broadly neutralizing antibodies. PMID:25846699

  6. Neutral Vlasov kinetic theory of magnetized plasmas

    SciTech Connect

    Tronci, Cesare; Camporeale, Enrico

    2015-02-15

    The low-frequency limit of Maxwell equations is considered in the Maxwell-Vlasov system. This limit produces a neutral Vlasov system that captures essential features of plasma dynamics, while neglecting radiation effects. Euler-Poincaré reduction theory is used to show that the neutral Vlasov kinetic theory possesses a variational formulation in both Lagrangian and Eulerian coordinates. By construction, the new model recovers all collisionless neutral models employed in plasma simulations. Then, comparisons between the neutral Vlasov system and hybrid kinetic-fluid models are presented in the linear regime.

  7. Neutralization efficiency estimation in a neutral beam source based on inductively coupled plasma

    NASA Astrophysics Data System (ADS)

    Vozniy, O. V.; Yeom, G. Y.

    2009-01-01

    This study examined the optimal conditions of neutral beam generation to maintain a high degree of neutralization and focusing during beam energy variation for a neutral beam source based on inductively coupled plasma with a three-grid ion beam acceleration system. The neutral beam energy distribution was estimated by measuring the energy profiles of ions that "survived" the neutralization after reflection. The energy measurements of the primary and reflected ions showed narrow distribution functions, each with only one peak. At higher beam energies, both the ratio of the ion energy loss to the primary energy and the degree of energy divergence decreased, confirming the precise alignment of the neutral beam. The neutralization efficiency of the neutral beam source with a three-grid acceleration system was found to be affected mainly by the beam angle divergence rather than by the particle translation energy.

  8. Neutralization efficiency estimation in a neutral beam source based on inductively coupled plasma

    SciTech Connect

    Vozniy, O. V.; Yeom, G. Y.

    2009-01-01

    This study examined the optimal conditions of neutral beam generation to maintain a high degree of neutralization and focusing during beam energy variation for a neutral beam source based on inductively coupled plasma with a three-grid ion beam acceleration system. The neutral beam energy distribution was estimated by measuring the energy profiles of ions that 'survived' the neutralization after reflection. The energy measurements of the primary and reflected ions showed narrow distribution functions, each with only one peak. At higher beam energies, both the ratio of the ion energy loss to the primary energy and the degree of energy divergence decreased, confirming the precise alignment of the neutral beam. The neutralization efficiency of the neutral beam source with a three-grid acceleration system was found to be affected mainly by the beam angle divergence rather than by the particle translation energy.

  9. Modelling the Neutral Sodium Tails of Comets

    NASA Astrophysics Data System (ADS)

    Birkett, K. S.; Jones, G. H.; Coates, A. J.

    2014-12-01

    Neutral sodium is typically easy to detect in active comets around perihelion, due to the very high efficiency of the sodium D transition, and at some comets a distinct neutral sodium tail is observed. The first distinct neutral sodium tail images were apparent in comet Hale-Bopp (C/1995 O1) data taken using CoCam [Cremonese et al, 1997], but since this initial detection similar features have been observed at a number of near-Sun comets using the SOHO/LASCO coronagraph. An understanding of the distribution and evolution of neutral cometary sodium may best be developed using a combination of spectra and images in different filters at multiple times throughout a comet's orbit. At present the source of neutral sodium in comets is unknown, primarily because the evolution of neutral cometary sodium is difficult to intuitively predict due to the Swings and Greenstein effects. Several authors [review presented in Cremonese et al, 1999] have suggested various combinations of sources of neutral sodium in the nuclear region, near-nuclear region, dust tail and ion tail. In order to understand the wide variety of cometary observations of neutral sodium available we have developed the first fully three dimensional, heliocentric distance dependent, versatile Monte Carlo neutral sodium tail model (initially based on a model developed by [Brown et al, 1998]). Our model is known as COMPASS (Cometary Orbital Motion at Perihelion: an Adaptable Sodium Simulation), and incorporates the unintuitive variation in radiation pressure influences on sodium atoms with different heliocentric velocities. We present the initial results of a comparison between COMPASS and observational data. We have found good agreement between the overall morphology of the neutral sodium tail imaged at comet Hale-Bopp and COMPASS, and have begun to extend the study to other comets of interest. We also present a comparison between simulated COMPASS spectra and observations. The versatility of COMPASS allows it to

  10. SPARQL Assist language-neutral query composer

    PubMed Central

    2012-01-01

    Background SPARQL query composition is difficult for the lay-person, and even the experienced bioinformatician in cases where the data model is unfamiliar. Moreover, established best-practices and internationalization concerns dictate that the identifiers for ontological terms should be opaque rather than human-readable, which further complicates the task of synthesizing queries manually. Results We present SPARQL Assist: a Web application that addresses these issues by providing context-sensitive type-ahead completion during SPARQL query construction. Ontological terms are suggested using their multi-lingual labels and descriptions, leveraging existing support for internationalization and language-neutrality. Moreover, the system utilizes the semantics embedded in ontologies, and within the query itself, to help prioritize the most likely suggestions. Conclusions To ensure success, the Semantic Web must be easily available to all users, regardless of locale, training, or preferred language. By enhancing support for internationalization, and moreover by simplifying the manual construction of SPARQL queries through the use of controlled-natural-language interfaces, we believe we have made some early steps towards simplifying access to Semantic Web resources. PMID:22373327

  11. Targets for high power neutral beams

    SciTech Connect

    Kim, J.

    1980-01-01

    Stopping high-power, long-pulse beams is fast becoming an engineering challenge, particularly in neutral beam injectors for heating magnetically confined plasmas. A brief review of neutral beam target technology is presented along with heat transfer calculations for some selected target designs.

  12. 32 CFR 644.323 - Neutral language.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 32 National Defense 4 2014-07-01 2013-07-01 true Neutral language. 644.323 Section 644.323 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY (CONTINUED) REAL PROPERTY REAL ESTATE HANDBOOK Disposal § 644.323 Neutral language. Wherever the words “man”, “men”, or their...

  13. 32 CFR 644.323 - Neutral language.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 32 National Defense 4 2013-07-01 2013-07-01 false Neutral language. 644.323 Section 644.323 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY (CONTINUED) REAL PROPERTY REAL ESTATE HANDBOOK Disposal § 644.323 Neutral language. Wherever the words “man”, “men”, or their...

  14. 32 CFR 644.323 - Neutral language.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 32 National Defense 4 2012-07-01 2011-07-01 true Neutral language. 644.323 Section 644.323 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY (CONTINUED) REAL PROPERTY REAL ESTATE HANDBOOK Disposal § 644.323 Neutral language. Wherever the words “man”, “men”, or their...

  15. 32 CFR 644.323 - Neutral language.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 32 National Defense 4 2011-07-01 2011-07-01 false Neutral language. 644.323 Section 644.323 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY (CONTINUED) REAL PROPERTY REAL ESTATE HANDBOOK Disposal § 644.323 Neutral language. Wherever the words “man”, “men”, or their...

  16. Efficient laser production of energetic neutral beams

    NASA Astrophysics Data System (ADS)

    Mollica, F.; Antonelli, L.; Flacco, A.; Braenzel, J.; Vauzour, B.; Folpini, G.; Birindelli, G.; Schnuerer, M.; Batani, D.; Malka, V.

    2016-03-01

    Laser-driven ion acceleration by intense, ultra-short, laser pulse has received increasing attention in recent years, and the availability of much compact and versatile ions sources motivates the study of laser-driven sources of energetic neutral atoms. We demonstrate the production of a neutral and directional beam of hydrogen and carbon atoms up to 200 keV per nucleon, with a peak flow of 2.7× {{10}13} atom s-1. Laser accelerated ions are neutralized in a pulsed, supersonic argon jet with tunable density between 1.5× {{10}17} cm-3and 6× {{10}18} cm-3. The neutralization efficiency has been measured by a time-of-flight detector for different argon densities. An optimum is found, for which complete neutralization occurs. The neutralization rate can be explained only at high areal densities (>1× {{10}17} cm-2) by single electron charge transfer processes. These results suggest a new perspective for the study of neutral production by laser and open discussion of neutralization at a lower density.

  17. Controversial Issues: A Case for Neutrality?

    ERIC Educational Resources Information Center

    Cain, Paul

    1999-01-01

    Arguments against teachers asserting their own views about controversial issues say that neutrality enables students to develop autonomous reflection. Others claim that a nonneutral stance is morally preferable. There are some teaching situations in which a neutral stance may not be an option. (SK)

  18. Gender Neutrality: Women's Friend or Foe?

    ERIC Educational Resources Information Center

    Steuernagel, Trudy

    Gender neutral public policies are those that are either silent on the question of the existence of significant gender differences or incorporate a perspective which mandates that such differences be ignored. Prominent voices today contend that gender neutrality favors males and have held the male standard as the one for which women should aspire.…

  19. Ion-Neutral Coupling in Solar Prominences

    NASA Technical Reports Server (NTRS)

    Gilbert, Holly

    2011-01-01

    Interactions between ions and neutrals in a partially ionized plasma are important throughout heliophysics, including near the solar surface in prominences. Understanding how ion-neutral coupling affects formation, support, structure, and dynamics of prominences will advance our physical understanding of magnetized systems involving a transition from a weakly ionized dense gas to a fully ionized tenuous plasma. We address the fundamental physics of prominence support, which is normally described in terms of a magnetic force on the prominence plasma that balances the solar gravitational force, and the implications for observations. Because the prominence plasma is only partially ionized, it is necessary to consider the support of the both the ionized and neutral components. Support of the neutrals is accomplished through a frictional interaction between the neutral and ionized components of the plasma, and its efficacy depends strongly on the degree of ionization of the plasma. More specifically, the frictional force is proportional to the relative flow of neutral and ion species, and for a sufficiently weakly ionized plasma, this flow must be relatively large to produce a frictional force that balances gravity. A large relative flow, of course, implies significant draining of neutral particles from the prominence. We evaluate the importance of this draining effect for a hydrogen-helium plasma, and consider the observational evidence for cross-field diffusion of neutral prominence material.

  20. 6 CFR 27.305 - Neutral adjudications.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 6 Domestic Security 1 2012-01-01 2012-01-01 false Neutral adjudications. 27.305 Section 27.305 Domestic Security DEPARTMENT OF HOMELAND SECURITY, OFFICE OF THE SECRETARY CHEMICAL FACILITY ANTI-TERRORISM STANDARDS Orders and Adjudications § 27.305 Neutral adjudications. (a) Any facility or other person who...

  1. 6 CFR 27.305 - Neutral adjudications.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 6 Domestic Security 1 2013-01-01 2013-01-01 false Neutral adjudications. 27.305 Section 27.305 Domestic Security DEPARTMENT OF HOMELAND SECURITY, OFFICE OF THE SECRETARY CHEMICAL FACILITY ANTI-TERRORISM STANDARDS Orders and Adjudications § 27.305 Neutral adjudications. (a) Any facility or other person who...

  2. 6 CFR 27.305 - Neutral adjudications.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 6 Domestic Security 1 2014-01-01 2014-01-01 false Neutral adjudications. 27.305 Section 27.305 Domestic Security DEPARTMENT OF HOMELAND SECURITY, OFFICE OF THE SECRETARY CHEMICAL FACILITY ANTI-TERRORISM STANDARDS Orders and Adjudications § 27.305 Neutral adjudications. (a) Any facility or other person who...

  3. 6 CFR 27.305 - Neutral adjudications.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 6 Domestic Security 1 2011-01-01 2011-01-01 false Neutral adjudications. 27.305 Section 27.305 Domestic Security DEPARTMENT OF HOMELAND SECURITY, OFFICE OF THE SECRETARY CHEMICAL FACILITY ANTI-TERRORISM STANDARDS Orders and Adjudications § 27.305 Neutral adjudications. (a) Any facility or other person who...

  4. 6 CFR 27.305 - Neutral adjudications.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 6 Domestic Security 1 2010-01-01 2010-01-01 false Neutral adjudications. 27.305 Section 27.305 Domestic Security DEPARTMENT OF HOMELAND SECURITY, OFFICE OF THE SECRETARY CHEMICAL FACILITY ANTI-TERRORISM STANDARDS Orders and Adjudications § 27.305 Neutral adjudications. (a) Any facility or other person who...

  5. Types of Neutralization and Types of Delinquency.

    ERIC Educational Resources Information Center

    Mitchell, Jim; Dodder, Richard A.

    1983-01-01

    Neutralization theory was tested with questionnaires administered to a random sample of public high school students (N-298) and institutionalized male delinquents (N-53). Neutralization acceptance technique patterns were similar across subsamples; however, correlations between each technique and each type of delinquency were statistically…

  6. 32 CFR 644.323 - Neutral language.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 32 National Defense 4 2010-07-01 2010-07-01 true Neutral language. 644.323 Section 644.323 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY (CONTINUED) REAL PROPERTY REAL ESTATE HANDBOOK Disposal § 644.323 Neutral language. Wherever the words “man”, “men”, or their...

  7. Neutral Buoyancy Simulator - Space Station

    NASA Technical Reports Server (NTRS)

    1985-01-01

    Skylab's success proved that scientific experimentation in a low gravity environment was essential to scientific progress. A more permanent structure was needed to provide this space laboratory. President Ronald Reagan, on January 25, 1984, during his State of the Union address, claimed that the United States should exploit the new frontier of space, and directed NASA to build a permanent marned space station within a decade. The idea was that the space station would not only be used as a laboratory for the advancement of science and medicine, but would also provide a staging area for building a lunar base and manned expeditions to Mars and elsewhere in the solar system. President Reagan invited the international community to join with the United States in this endeavour. NASA and several countries moved forward with this concept. By December 1985, the first phase of the space station was well underway with the design concept for the crew compartments and laboratories. Pictured are two NASA astronauts, at Marshall Space Flight Center's (MSFC) Neutral Buoyancy Simulator (NBS), practicing construction techniques they later used to construct the space station after it was deployed.

  8. The status of neutral currents

    SciTech Connect

    Zwirner, F.

    1987-11-01

    The situation of particle physics today is quite puzzling. On the one hand, the Standard Model (SM) of strong and electroweak interactions is consistent with all confirmed experimental data but theoretically rather unsatisfactory. On the other hand, none of the many theoretical speculations which try to go beyond the SM has (yet) received the slightest experimental support. The solution to this dilemma can only come from new data: either from the detection of a new particle threshold at high energy colliders, or from the appearance of some small discrepancy in high-precision experiments. A crucial sector for testing the SM and its extensions is that of neutral currents (NC), where an impressive amount of data has been collected in recent years. While waiting for the next generation of experiments, it is certainly useful to take stock of our knowledge, determining the NC parameters as precisely as we can and putting limits on possible deviations from the SM. The present talk contains the results of a recent analysis along these lines: the first part illustrates how a set of 'model-independent' parameters can be extracted from the available NC data, the second part particularizes the analysis to the SM and to some superstring-inspired models with an additional Z' in their low-energy spectrum. 27 refs., 3 figs., 1 tab.

  9. Neutral Buoyancy underwater electrical cornector test

    NASA Technical Reports Server (NTRS)

    1978-01-01

    Once the United States' space program had progressed from Earth's orbit into outerspace, the prospect of building and maintaining a permanent presence in space was realized. To accomplish this feat, NASA launched a temporary workstation, Skylab, to discover the effects of low gravity and weightlessness on the human body, and also to develop tools and equipment that would be needed in the future to build and maintain a more permanent space station. The structures, techniques, and work schedules had to be carefully designed to fit this unique construction site. The components had to be lightweight for transport into orbit, yet durable. The station also had to be made with removable parts for easy servicing and repairs by astronauts. All of the tools necessary for service and repairs had to be designed for easy manipulation by a suited astronaut. And construction methods had to be efficient due to limited time the astronauts could remain outside their controlled environment. In lieu of all the specific needs for this project, an environment on Earth had to be developed that could simulate a low gravity atmosphere. A Neutral Buoyancy Simulator (NBS) was constructed by NASA Marshall Space Flight Center (MSFC) in 1968. Since then, NASA scientists have used this facility to understand how humans work best in low gravity and also provide information about the different kinds of structures that can be built. Included in the plans for the space station was a space telescope. This telescope would be attached to the space station and directed towards outerspace. Astronomers hoped that the space telescope would provide a look at space that is impossible to see from Earth because of Earth's atmosphere and other man made influences. Pictured is a large structure that is being used as the antenna base for the space telescope.

  10. Neutral Buoyancy Simulator - EASE Project (NB32)

    NASA Technical Reports Server (NTRS)

    1980-01-01

    Once the United States' space program had progressed from Earth's orbit into outerspace, the prospect of building and maintaining a permanent presence in space was realized. To accomplish this feat, NASA launched a temporary workstation, Skylab, to discover the effects of low gravity and weightlessness on the human body, and also to develop tools and equipment that would be needed in the future to build and maintain a more permanent space station. The structures, techniques, and work schedules had to be carefully designed to fit this unique construction site. The components had to be lightweight for transport into orbit, yet durable. The station also had to be made with removable parts for easy servicing and repairs by astronauts. All of the tools necessary for service and repairs had to be designed for easy manipulation by a suited astronaut. Construction methods had to be efficient due to the limited time the astronauts could remain outside their controlled environment. In lieu of all the specific needs for this project, an environment on Earth had to be developed that could simulate a low gravity atmosphere. A Neutral Buoyancy Simulator (NBS) was constructed by NASA's Marshall Space Flight Center (MSFC) in 1968. Since then, NASA scientists have used this facility to understand how humans work best in low gravity and also provide information about the different kinds of structures that can be built. Pictured is a Massachusetts Institute of Technology (MIT) student working in a spacesuit on the Experimental Assembly of Structures in Extravehicular Activity (EASE) project which was developed as a joint effort between MFSC and MIT. The EASE experiment required that crew members assemble small components to form larger components, working from the payload bay of the space shuttle. The MIT student in this photo is assembling two six-beam tetrahedrons.

  11. Neutral depletion and the helicon density limit

    SciTech Connect

    Magee, R. M.; Galante, M. E.; Carr, J. Jr.; Lusk, G.; McCarren, D. W.; Scime, E. E.

    2013-12-15

    It is straightforward to create fully ionized plasmas with modest rf power in a helicon. It is difficult, however, to create plasmas with density >10{sup 20} m{sup −3}, because neutral depletion leads to a lack of fuel. In order to address this density limit, we present fast (1 MHz), time-resolved measurements of the neutral density at and downstream from the rf antenna in krypton helicon plasmas. At the start of the discharge, the neutral density underneath the antenna is reduced to 1% of its initial value in 15 μs. The ionization rate inferred from these data implies that the electron temperature near the antenna is much higher than the electron temperature measured downstream. Neutral density measurements made downstream from the antenna show much slower depletion, requiring 14 ms to decrease by a factor of 1/e. Furthermore, the downstream depletion appears to be due to neutral pumping rather than ionization.

  12. Neutralization Properties of Simian Immunodeficiency Viruses Infecting Chimpanzees and Gorillas

    PubMed Central

    Barbian, Hannah J.; Decker, Julie M.; Bibollet-Ruche, Frederic; Galimidi, Rachel P.; West, Anthony P.; Learn, Gerald H.; Parrish, Nicholas F.; Iyer, Shilpa S.; Li, Yingying; Pace, Craig S.; Song, Ruijiang; Huang, Yaoxing; Denny, Thomas N.; Mouquet, Hugo; Martin, Loic; Acharya, Priyamvada; Zhang, Baoshan; Kwong, Peter D.; Mascola, John R.; Verrips, C. Theo; Strokappe, Nika M.; Rutten, Lucy; McCoy, Laura E.; Weiss, Robin A.; Brown, Corrine S.; Jackson, Raven; Silvestri, Guido; Connors, Mark; Burton, Dennis R.; Shaw, George M.; Nussenzweig, Michel C.; Bjorkman, Pamela J.; Ho, David D.; Farzan, Michael

    2015-01-01

    ABSTRACT Broadly cross-reactive neutralizing antibodies (bNabs) represent powerful tools to combat human immunodeficiency virus type 1 (HIV-1) infection. Here, we examined whether HIV-1-specific bNabs are capable of cro