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Sample records for human neutral ceramidase

  1. Discovery and evaluation of inhibitors of human ceramidase.

    PubMed

    Draper, Jeremiah M; Xia, Zuping; Smith, Ryan A; Zhuang, Yan; Wang, Wenxue; Smith, Charles D

    2011-11-01

    The ceramide/sphingosine-1-phosphate (S1P) rheostat has been hypothesized to play a critical role in regulating tumor cell fate, with elevated levels of ceramide inducing death and elevated levels of S1P leading to survival and proliferation. Ceramidases are key enzymes that control this rheostat by hydrolyzing ceramide to produce sphingosine and may also confer resistance to drugs and radiation. Therefore, ceramidase inhibitors have excellent potential for development as new anticancer drugs. In this study, we identify a novel ceramidase inhibitor (Ceranib-1) by screening a small molecule library and describe the synthesis of a more potent analogue (Ceranib-2). In a cell-based assay, both compounds were found to inhibit cellular ceramidase activity toward an exogenous ceramide analogue, induce the accumulation of multiple ceramide species, decrease levels of sphingosine and S1P, inhibit the proliferation of cells alone and in combination with paclitaxel, and induce cell-cycle arrest and cell death. In vivo, Ceranib-2 was found to delay tumor growth in a syngeneic tumor model without hematologic suppression or overt signs of toxicity. These data support the selection of ceramidases as suitable targets for anticancer drug development and provide the first nonlipid inhibitors of human ceramidase activity.

  2. Molecular cloning and characterization of neutral ceramidase homologue from the red flour beetle, Tribolium castaneum.

    PubMed

    Zhou, Ying; Lin, Xian-Wen; Yang, Qiong; Zhang, Yan-Ru; Yuan, Jing-Qun; Lin, Xin-Da; Xu, Ruijuan; Cheng, Jiaan; Mao, Cungui; Zhu, Zeng-Rong

    2011-07-01

    Ceramidase plays an important role in regulating the metabolism of sphingolipids, such as ceramide, sphingosine (SPH), and sphingosine-1-phosphate (S1P), by controlling the hydrolysis of ceramide. Here we report the cloning and biochemical characterization of a neutral ceramidase from the red flour beetle Tribolium castaneum which is an important storage pest. The Tribolium castaneum neutral ceramidase (Tncer) is a protein of 696 amino acids. It shares a high degree of similarity in protein sequence to neutral ceramidases from various species. Tncer mRNA levels are higher in the adult stage than in pre-adult stages, and they are higher in the reproductive organs than in head, thorax, and midgut. The mature ovary has higher mRNA levels than the immature ovary. Tncer is localized to the plasma membrane. It uses various ceramides (D-erythro-C(6), C(12), C(16), C(18:1), and C(24:1)-ceramide) as substrates and has an abroad pH optimum for its in vitro activity. Tncer has an optimal temperature of 37 °C for its in vitro activity. Its activity is inhibited by Fe(2+). These results suggest that Tncer has distinct biochemical properties from neutral ceramidases from other species.

  3. A neutral ceramidase homologue from Dictyostelium discoideum exhibits an acidic pH optimum.

    PubMed Central

    Monjusho, Hatsumi; Okino, Nozomu; Tani, Motohiro; Maeda, Mineko; Yoshida, Motonobu; Ito, Makoto

    2003-01-01

    The nucleotide sequence reported for the Dictyostelium discoideum ceramidase is available on the DNA Data Bank of Japan (DDBJ). Ceramidases (CDases) are currently classified into three categories (acid, neutral and alkaline) based on their optimal pHs and primary structures. Here, we report the first exception to this rule. We cloned the CDase cDNA, consisting of 2142 nucleotides encoding 714 amino-acid residues, from the slime mould, Dictyostelium discoideum. The putative amino-acid sequence indicates 32-42% identity with various neutral CDases, but does not show any similarity to the acid and alkaline CDases, indicating the enzyme should be classified as a neutral CDase. However, overexpression of the cDNA in D. discoideum resulted in increased CDase activity at an acidic, but not a neutral pH range. Knockout of the gene in slime mould eliminated CDase activity at acidic pH. The recombinant enzyme expressed in the slime mould was purified and then characterized. Consequently, the purified CDase was found to exhibit the maximal activity at approx. pH 3.0. The singular pH dependency of slime mould CDase is not derived from the specific post-translational modification in the slime mould, because the enzyme showed an acidic pH optimum even when expressed in Chinese hamster ovary cells, whereas rat neutral-CDase exhibited a neutral pH optimum when expressed in slime mould. PMID:12943537

  4. Pseudomonas-Derived Ceramidase Induces Production of Inflammatory Mediators from Human Keratinocytes via Sphingosine-1-Phosphate

    PubMed Central

    Oizumi, Ami; Nakayama, Hitoshi; Okino, Nozomu; Iwahara, Chihiro; Kina, Katsunari; Matsumoto, Ryo; Ogawa, Hideoki; Takamori, Kenji; Ito, Makoto; Suga, Yasushi; Iwabuchi, Kazuhisa

    2014-01-01

    Ceramide is important for water retention and permeability barrier functions in the stratum corneum, and plays a key role in the pathogenesis of atopic dermatitis (AD). A Pseudomonas aeruginosa-derived neutral ceramidase (PaCDase) isolated from a patient with AD was shown to effectively degrade ceramide in the presence of Staphylococcus aureus-derived lipids or neutral detergents. However, the effect of ceramide metabolites on the functions of differentiating keratinocytes is poorly understood. We found that the ceramide metabolite sphingosine-1-phosphate (S1P) stimulated the production of inflammatory mediators such as TNF-α and IL-8 from three-dimensionally cultured human primary keratinocytes (termed “3D keratinocytes”), which form a stratum corneum. PaCDase alone did not affect TNF-α gene expression in 3D keratinocytes. In the presence of the detergent Triton X-100, which damages stratum corneum structure, PaCDase, but not heat-inactivated PaCDase or PaCDase-inactive mutant, induced the production of TNF-α, endothelin-1, and IL-8, indicating that this production was dependent on ceramidase activity. Among various ceramide metabolites, sphingosine and S1P enhanced the gene expression of TNF-α, endothelin-1, and IL-8. The PaCDase-enhanced expression of these genes was inhibited by a sphingosine kinase inhibitor and by an S1P receptor antagonist VPC 23019. The TNF-α-binding antibody infliximab suppressed the PaCDase-induced upregulation of IL-8, but not TNF-α, mRNA. PaCDase induced NF-κB p65 phosphorylation. The NF-κB inhibitor curcumin significantly inhibited PaCDase-induced expression of IL-8 and endothelin-1. VPC 23019 and infliximab inhibited PaCDase-induced NF-κB p65 phosphorylation and reduction in the protein level of the NF-κB inhibitor IκBα. Collectively, these findings suggest that (i) 3D keratinocytes produce S1P from sphingosine, which is produced through the hydrolysis of ceramide by PaCDase, (ii) S1P induces the production of TNF

  5. 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

  6. Acid ceramidase expression modulates the sensitivity of A375 melanoma cells to dacarbazine.

    PubMed

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

    2011-08-12

    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.

  7. Acid Ceramidase in Melanoma

    PubMed Central

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

    2016-01-01

    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

  8. Cell Non-autonomous Function of Ceramidase in Photoreceptor Homeostasis

    PubMed Central

    Acharya, Jairaj K.; Dasgupta, Ujjaini; Rawat, Satinder S.; Yuan, Changqing; Sanxaridis, Parthena D.; Yonamine, Ikuko; Karim, Pusha; Nagashima, Kunio; Brodsky, Michael H.; Tsunoda, Susan; Acharya, Usha

    2008-01-01

    SUMMARY Neutral Ceramidase, a key enzyme of sphingolipid metabolism, hydrolyzes ceramide to sphingosine. These sphingolipids are critical structural components of cell membranes and act as second messengers in diverse signal transduction cascades. Here, we have isolated and characterized functional null mutants of Drosophila Ceramidase. We show that secreted Ceramidase functions in a cell non-autonomous manner to maintain photoreceptor homeostasis. In the absence of Ceramidase, photoreceptors degenerate in a light-dependent manner, are defective in normal endocytic turnover of Rhodopsin, and do not respond to light stimulus. Consistent with a cell non-autonomous function, our studies show that over expression of Ceramidase in a tissue distant from the photoreceptors can suppress photoreceptor degeneration in an Arrestin mutant and facilitate membrane turnover in a Rhodopsin null mutant. Furthermore, our results show that secreted CDase is internalized and localizes to endosomes. Our findings are the first to establish a role for a secreted sphingolipid enzyme in the regulation of photoreceptor structure and function. PMID:18184565

  9. In vivo delivery of human acid ceramidase via cord blood transplantation and direct injection of lentivirus as novel treatment approaches for Farber disease

    PubMed Central

    Ramsubir, Shobha; Nonaka, Takahiro; Girbés, Carmen Bedia; Carpentier, Stéphane; Levade, Thierry; Medin, Jeffrey A.

    2008-01-01

    Farber disease is a rare lysosomal storage disorder (LSD) caused by a deficiency of acid ceramidase (AC) activity and subsequent accumulation of ceramide. Currently, there is no treatment for Farber disease beyond palliative care and most patients succumb to the disorder at a very young age. Previously, our group showed that gene therapy using oncoretroviral vectors (RV) could restore enzyme activity in Farber patient cells. The studies described here employ novel RV and lentiviral (LV) vectors that engineer co-expression of AC and a cell surface marking transgene product, human CD25 (huCD25). Transduction of Farber patient fibroblasts and B cells with these vectors resulted in overexpression of AC and led to a 90% and 50% reduction in the accumulation of ceramide, respectively. Vectors were also evaluated in human hematopoietic stem/progenitor cells (HSPCs) and by direct in vivo delivery in mouse models. In a xenotransplantation model using NOD/SCID mice, we found that transduced CD34+ cells could repopulate irradiated recipient animals, as measured by CD25 expression. When virus was injected intravenously into mice, soluble CD25 was detected in the plasma and increased AC activity was present in the liver up to 14 weeks post-injection. These findings suggest that vector and transgene expression can persist long-term and offer the potential of a lasting cure. To our knowledge, this is the first report of in vivo testing of direct gene therapy strategies for Farber disease. PMID:18805722

  10. Drug targeting of sphingolipid metabolism: sphingomyelinases and ceramidases

    PubMed Central

    Canals, Daniel; Perry, David M; Jenkins, Russell W; Hannun, Yusuf A

    2011-01-01

    Sphingolipids represent a class of diverse bioactive lipid molecules that are increasingly appreciated as key modulators of diverse physiologic and pathophysiologic processes that include cell growth, cell death, autophagy, angiogenesis, and stress and inflammatory responses. Sphingomyelinases and ceramidases are key enzymes of sphingolipid metabolism that regulate the formation and degradation of ceramide, one of the most intensely studied classes of sphingolipids. Improved understanding of these enzymes that control not only the levels of ceramide but also the complex interconversion of sphingolipid metabolites has provided the foundation for the functional analysis of the roles of sphingolipids. Our current understanding of the roles of various sphingolipids in the regulation of different cellular processes has come from loss-of-function/gain-of-function studies utilizing genetic deletion/downregulation/overexpression of enzymes of sphingolipid metabolism (e.g. knockout animals, RNA interference) and from the use of pharmacologic inhibitors of these same enzymes. While genetic approaches to evaluate the functional roles of sphingolipid enzymes have been instrumental in advancing the field, the use of pharmacologic inhibitors has been equally important in identifying new roles for sphingolipids in important cellular processes.The latter also promises the development of novel therapeutic targets with implications for cancer therapy, inflammation, diabetes, and neurodegeneration. In this review, we focus on the status and use of pharmacologic compounds that inhibit sphingomyelinases and ceramidases, and we will review the history, current uses and future directions for various small molecule inhibitors, and will highlight studies in which inhibitors of sphingolipid metabolizing enzymes have been used to effectively treat models of human disease. PMID:21615386

  11. Ceramidase Regulates Synaptic Vesicle Exocytosis and Trafficking

    PubMed Central

    Rohrbough, Jeffrey; Rushton, Emma; Palanker, Laura; Woodruff, Elvin; Matthies, Heinrich J. G.; Acharya, Usha; Acharya, Jairaj K.; Broadie, Kendal

    2009-01-01

    A screen for Drosophila synaptic dysfunction mutants identified slug-a-bed (slab). The slab gene encodes ceramidase, a central enzyme in sphingolipid metabolism and regulation. Sphingolipids are major constituents of lipid rafts, membrane domains with roles in vesicle trafficking, and signaling pathways. Null slab mutants arrest as fully developed embryos with severely reduced movement. The SLAB protein is widely expressed in different tissues but enriched in neurons at all stages of development. Targeted neuronal expression of slab rescues mutant lethality, demonstrating the essential neuronal function of the protein. C5-ceramide applied to living preparations is rapidly accumulated at neuromuscular junction (NMJ) synapses dependent on the SLAB expression level, indicating that synaptic sphingolipid trafficking and distribution is regulated by SLAB function. Evoked synaptic currents at slab mutant NMJs are reduced by 50–70%, whereas postsynaptic glutamate-gated currents are normal, demonstrating a specific presynaptic impairment. Hypertonic saline-evoked synaptic vesicle fusion is similarly impaired by 50–70%, demonstrating a loss of readily releasable vesicles. In addition, FM1-43 dye uptake is reduced in slab mutant presynaptic terminals, indicating a smaller cycling vesicle pool. Ultrastructural analyses of mutants reveal a normal vesicle distribution clustered and docked at active zones, but fewer vesicles in reserve regions, and a twofold to threefold increased incidence of vesicles linked together and tethered at the plasma membrane. These results indicate that SLAB ceramidase function controls presynaptic terminal sphingolipid composition to regulate vesicle fusion and trafficking, and thus the strength and reliability of synaptic transmission. PMID:15356190

  12. 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.

  13. Molecular mechanism for sphingosine-induced Pseudomonas ceramidase expression through the transcriptional regulator SphR

    PubMed Central

    Okino, Nozomu; Ito, Makoto

    2016-01-01

    Pseudomonas aeruginosa, an opportunistic, but serious multidrug-resistant pathogen, secretes a ceramidase capable of cleaving the N-acyl linkage of ceramide to generate fatty acids and sphingosine. We previously reported that the secretion of P. aeruginosa ceramidase was induced by host-derived sphingolipids, through which phospholipase C-induced hemolysis was significantly enhanced. We herein investigated the gene(s) regulating sphingolipid-induced ceramidase expression and identified SphR, which encodes a putative AraC family transcriptional regulator. Disruption of the sphR gene in P. aeruginosa markedly decreased the sphingomyelin-induced secretion of ceramidase, reduced hemolytic activity, and resulted in the loss of sphingomyelin-induced ceramidase expression. A microarray analysis confirmed that sphingomyelin significantly induced ceramidase expression in P. aeruginosa. Furthermore, an electrophoretic mobility shift assay revealed that SphR specifically bound free sphingoid bases such as sphingosine, dihydrosphingosine, and phytosphingosine, but not sphingomyelin or ceramide. A β-galactosidase-assisted promoter assay showed that sphingosine activated ceramidase expression through SphR at a concentration of 100 nM. Collectively, these results demonstrated that sphingosine induces the secretion of ceramidase by promoting the mRNA expression of ceramidase through SphR, thereby enhancing hemolytic phospholipase C-induced cytotoxicity. These results facilitate understanding of the physiological role of bacterial ceramidase in host cells. PMID:27941831

  14. 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

  15. 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

  16. 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.

  17. Mechanism of human antibody-mediated neutralization of Marburg virus.

    PubMed

    Flyak, Andrew I; Ilinykh, Philipp A; Murin, Charles D; Garron, Tania; Shen, Xiaoli; Fusco, Marnie L; Hashiguchi, Takao; Bornholdt, Zachary A; Slaughter, James C; Sapparapu, Gopal; Klages, Curtis; Ksiazek, Thomas G; Ward, Andrew B; Saphire, Erica Ollmann; Bukreyev, Alexander; Crowe, James E

    2015-02-26

    The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably, several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of antibody-GP complexes reveal that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP, but not to MARV GP. The data suggest that MARV-neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site, revealing a mechanism of filovirus inhibition.

  18. Mechanism of Human Antibody-Mediated Neutralization of Marburg Virus

    PubMed Central

    Flyak, Andrew I.; Ilinykh, Philipp A.; Murin, Charles D.; Garron, Tania; Shen, Xiaoli; Fusco, Marnie L.; Hashiguchi, Takao; Bornholdt, Zachary A.; Slaughter, James C.; Sapparapu, Gopal; Klages, Curtis; Ksiazek, Thomas G.; Ward, Andrew B.; Saphire, Erica Ollmann; Bukreyev, Alexander; Crowe, James E.

    2015-01-01

    Summary The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably, several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of Antibody-GP complexes reveals that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP but not to MARV GP. The data suggest that MARV neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site, revealing a mechanism of filovirus inhibition. PMID:25723164

  19. 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

  20. Transcriptomic insights into human brain evolution: acceleration, neutrality, heterochrony.

    PubMed

    Somel, Mehmet; Rohlfs, Rori; Liu, Xiling

    2014-12-01

    Primate brain transcriptome comparisons within the last 12 years have yielded interesting but contradictory observations on how the transcriptome evolves, and its adaptive role in human cognitive evolution. Since the human-chimpanzee common ancestor, the human prefrontal cortex transcriptome seems to have evolved more than that of the chimpanzee. But at the same time, most expression differences among species, especially those observed in adults, appear as consequences of neutral evolution at cis-regulatory sites. Adaptive expression changes in the human brain may be rare events involving timing shifts, or heterochrony, in specific neurodevelopmental processes. Disentangling adaptive and neutral expression changes, and associating these with human-specific features of the brain require improved methods, comparisons across more species, and further work on comparative development.

  1. Influenza virus resistance to human neutralizing antibodies.

    PubMed

    Crowe, James E

    2012-01-01

    The human antibody repertoire has an exceptionally large capacity to recognize new or changing antigens through combinatorial and junctional diversity established at the time of V(D)J recombination and through somatic hypermutation. Influenza viruses exhibit a relentless capacity to escape the human antibody response by altering the amino acids of their surface proteins in hypervariable domains that exhibit a high level of structural plasticity. Both parties in this high-stakes game of shape shifting drive structural evolution of their functional proteins (the B cell receptor/antibody on one side and the viral hemagglutinin and neuraminidase proteins on the other) using error-prone polymerase systems. It is likely that most of the genetic mutations that occur in these systems are deleterious, resulting in the failure of the B cell or virus with mutations to propagate in the immune repertoire or viral quasispecies. A subset of mutations is tolerated in functional surface proteins that enter the B cell or virus progeny pool. In both cases, selection occurs in the population of mutated and unmutated species. In cases where the functional avidity of the B cell receptor is increased significantly, that clone may be selected for preferential expansion. In contrast, an influenza virus that "escapes" the inhibitory effect of secreted antibodies may represent a high proportion of the progeny virus in that host. The recent paper by O'Donnell et al. [C. D. O'Donnell et al., mBio 3(3):e00120-12, 2012] identifies a mechanism for antibody resistance that does not require escape from binding but rather achieves a greater efficiency in replication.

  2. 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

  3. Neutralization of Epstein-Barr Virus by Nonimmune Human Serum

    PubMed Central

    Nemerow, Glen R.; Jensen, Fred C.; Cooper, Neil R.

    1982-01-01

    These studies were carried out to investigate the mechanism of neutralization of purified Epstein-Barr virus (EBV) by fresh human serum from normal individuals lacking antibody to the EBV viral capsid (VCA) and nuclear antigens (EBNA). Such individuals thus lack serological evidence of immunity to EBV. Although an enzyme-linked immunosorbent assay (ELISA) with highly purified immobilized EBV detected low levels of IgG antibody reactive with EBV in these normal nonimmune sera, this antibody failed to neutralize EBV in the absence of complement. Studies with depleted sera and mixtures of purified complement proteins at physiologic concentrations showed that the IgG antibody and C1, C4, C2, and C3 of the classical pathway were able to fully neutralize EBV. Mixtures of the purified components of the alternative pathway at physiologic concentrations failed to neutralize purified EBV in the presence or absence of the antibody and the alternative pathway did not potentiate classical pathway-mediated neutralization. No evidence for a requirement for C8 was obtained, precluding lysis as the mechanism of neutralization. Since C3 deposition on the viral surface accompanied classical pathway activation, viral neutralization is most likely secondary to the accumulation of complement protein on the viral surface. A coating of protein on the virus could interfere with attachment to, or penetration of potentially susceptible cells. Experiments were undertaken to determine the specificity of the IgG antibody in the sera of EBV nonimmune individuals which, together with complement, neutralized EBV. Both purified EBV and herpes simplex I (HSV-1) absorbed the EBV ELISA reactivity and EBV-neutralizing activity of nonimmune sera, whereas another member of the herpesvirus group, cytomegalovirus, was inactive in this regard. HSV-1 was quantitatively more efficient than EBV in absorbing reactivity, a finding that indicates that the antibody has a higher affinity for HSV-1 than for EBV

  4. 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

    2016-09-12

    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.

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

    PubMed Central

    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. IRF8 Regulates Acid Ceramidase Expression to Mediate Apoptosis and Suppresses Myelogeneous Leukemia

    PubMed Central

    Hu, Xiaolin; Yang, Dafeng; Zimmerman, Mary; Liu, Feiyan; Yang, Jine; Kannan, Swati; Burchert, Andreas; Szulc, Zdzislaw; Bielawska, Alicja; Ozato, Keiko; Bhalla, Kapil; Liu, Kebin

    2011-01-01

    IFN regulatory factor 8 (IRF8) is a key transcription factor for myeloid cell differentiation and its expression is frequently lost in hematopoietic cells of human myeloid leukemia patients. IRF8-deficient mice exhibit uncontrolled clonal expansion of undifferentiated myeloid cells that can progress to a fatal blast crisis, thereby resembling human chronic myelogeneous leukemia (CML). Therefore, IRF8 is a myeloid leukemia suppressor. While the understanding of IRF8 function in CML has recently improved, the molecular mechanisms underlying IRF8 function in CML is still largely unknown. In this study, we identified acid ceramidase (A-CDase) as a general transcription target of IRF8. We demonstrated that IRF8 expression is regulated by IRF8 promoter DNA methylation in myeloid leukemia cells. Restoration of IRF8 expression repressed A-CDase expression, resulting in C16 ceramide accumulation and increased sensitivity of CML cells to FasL-induced apoptosis. In myeloid cells derived from IRF8-deficient mice, A-CDase protein level was dramatically increased. Furthermore, we demonstrated that IRF8 directly bind to the A-CDase promoter. At the functional level, inhibition of A-CDase activity, silencing A-CDase expression or application of exogenous C16 ceramide sensitized CML cells to FasL-induced apoptosis, whereas, overexpression of A-CDase decreased CML cells sensitivity to FasL-induced apoptosis. Consequently, restoration of IRF8 expression suppressed CML development in vivo at least partially through a Fas-dependent mechanism. In summary, our findings determine the mechanism of IRF8 downregulation in CML cells and they determine a primary pathway of resistance to Fas-mediated apoptosis and disease progression. PMID:21487040

  7. 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

    2015-04-02

    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.

  8. Recombinant human Fab fragments neutralize human type 1 immunodeficiency virus in vitro.

    PubMed Central

    Barbas, C F; Björling, E; Chiodi, F; Dunlop, N; Cababa, D; Jones, T M; Zebedee, S L; Persson, M A; Nara, P L; Norrby, E

    1992-01-01

    A panel of 20 recombinant Fab fragments reactive with the surface glycoprotein gp120 of human type 1 immunodeficiency virus (HIV-1) were examined for their ability to neutralize MN and IIIB strains of the virus. Neutralization was determined as the ability of the Fab fragments to inhibit infection as measured in both a p24 ELISA and a syncytium-formation assay. One group of closely sequence-related Fab fragments was found to neutralize virus in both assays with a 50% neutralization titer at approximately 1 micrograms/ml. Another Fab neutralized in the p24 ELISA but not in the syncytium assay. The other Fab fragments showed weak or no neutralizing ability. The results imply that virion aggregation or crosslinking of gp120 molecules on the virion surface is not an absolute requirement for HIV-1 neutralization. Further, all of the Fab fragments were shown to be competitive with soluble CD4 for binding to gp120 and yet few neutralized the virus effectively, implying that the mechanism of neutralization in this case may not involve receptor blocking. The observation of a preponderance of high-affinity Fab fragments with poor or no neutralizing ability could have implications for vaccine strategies. PMID:1384050

  9. Interfacial regulation of acid ceramidase activity. Stimulation of ceramide degradation by lysosomal lipids and sphingolipid activator proteins.

    PubMed

    Linke, T; Wilkening, G; Sadeghlar, F; Mozcall, H; Bernardo, K; Schuchman, E; Sandhoff, K

    2001-02-23

    The lysosomal degradation of ceramide is catalyzed by acid ceramidase and requires sphingolipid activator proteins (SAP) as cofactors in vivo. The aim of this study was to investigate how ceramide is hydrolyzed by acid ceramidase at the water-membrane interface in the presence of sphingolipid activator proteins in a liposomal assay system. The degradation of membrane-bound ceramide was significantly increased both in the absence and presence of SAP-D when anionic lysosomal phospholipids such as bis(monoacylglycero)phosphate, phosphatidylinositol, and dolichol phosphate were incorporated into substrate-bearing liposomes. Higher ceramide degradation rates were observed in vesicles with increased membrane curvature. Dilution assays indicated that acid ceramidase remained bound to the liposomal surface during catalysis. Not only SAP-D, but also SAP-C and SAP-A, were found to be stimulators of ceramide hydrolysis in the presence of anionic phospholipids. This finding was confirmed by cell culture studies, in which SAP-A, -C, and -D reduced the amount of ceramide storage observed in fibroblasts of a patient suffering from prosaposin deficiency. Strong protein-lipid interactions were observed for both SAP-D and acid ceramidase in surface plasmon resonance experiments. Maximum binding of SAP-D and acid ceramidase to lipid bilayers occurred at pH 4.0. Our results demonstrate that anionic, lysosomal lipids are required for efficient hydrolysis of ceramide by acid ceramidase.

  10. Human intestinal absorption--neutral molecules and ionic species.

    PubMed

    Abraham, Michael H

    2014-07-01

    Analysis of percentage human intestinal absorption (%HIA) for 280 drugs shows that an excellent fit can be obtained using only three descriptors for neutral molecules with a SD of 13.9%. Use of descriptors for individual cations and anions does not lead to any better goodness-of-fit. It is noted that diffusion coefficients in water for ionized molecules are almost identical to those for the corresponding neutral molecules. Comparison of equation coefficients for HIA with those for other processes shows that HIA resembles diffusion in water but does not resemble permeation through biological bilayers. It is shown that compound substituent effects on HIA are near those for diffusion but are far away from substituent effects on permeation through a typical bilayer. Calculations indicate that rates of permeation through an unstirred mucosal layer are of the same order as experimental rates of permeation in HIA. It is concluded that for the 280 compound set, diffusion through the unstirred mucosal layer is the rate determining step. The effect on pK(a) in transfer of acids and bases from water to another solvent, and of diffusion past a negative charge in a phase/bilayer is also considered.

  11. [Neutralizing Monoclonal and Chimeric Antibodies to Human IFN-γ].

    PubMed

    Larina, M V; Aliev, T K; Solopova, O N; Pozdnyakova, L P; Korobova, S V; Yakimov, S A; Sveshnikov, P G; Dolgikh, D A; Kirpichnikov, M P

    2015-01-01

    Autoiminune disorders are chronic diseases characterized by abnormal immune response directed against self-antigens that leads to tissue damage and violation of its normal functioning. Such diseases often result in disability or even death of patients. Nowadays a number of monoclonal antibodies to pro-inflammatory cytokines and their receptors are successfully used for the targeted treatment of autoimmune diseases. One of the perspective targets in autoimmune disease therapy is interferon gamma, a key cytokine in Th1 cells differentiation, activation of macrophages, and inflammation. In the present work, 5 monoclonal antibodies to human IFN-γ were obtained. For the development of potential therapeutic agent, we have performed neutralizing activity and affinity analysis of the antibodies. Based on the data obtained, the monoclonal antibody F1 was selected. This antibody has a dissociation constant 1.7 x 10(-9) M and IC90 = 8.9 ± 2.0 nM measured upon antibody inhibition of the IFN-γ-induced HLA-DR expression on the surface of U937 cells. We have constructed a bicistronic vector for the production of recombinant chimeric Fab fragment F1 chim in E. coli cells. The recombinant chimeric Fab fragment Fl chim neutralizes IFN-γ activity in vitro and has a dissociation constant 1.8 x 10(-9) M.

  12. Preclinical development of a humanized neutralizing antibody targeting HGF

    PubMed Central

    Kim, Hyori; Hong, Sung Hee; Kim, Jung Yong; Kim, In-Chull; Park, Young-Whan; Lee, Song-Jae; Song, Seong-Won; Kim, Jung Ju; Park, Gunwoo; Kim, Tae Min; Kim, Yun-Hee; Park, Jong Bae; Chung, Junho; Kim, In-Hoo

    2017-01-01

    Hepatocyte growth factor (HGF) and its receptor, cMET, play critical roles in cell proliferation, angiogenesis and invasion in a wide variety of cancers. We therefore examined the anti-tumor activity of the humanized monoclonal anti-HGF antibody, YYB-101, in nude mice bearing human glioblastoma xenografts as a single agent or in combination with temozolomide. HGF neutralization, The extracellular signal-related kinases 1 and 2 (ERK1/2) phosphorylation, and HGF-induced scattering were assessed in HGF-expressing cell lines treated with YYB-101. To support clinical development, we also evaluated the preclinical pharmacokinetics and toxicokinetics in cynomolgus monkeys, and human and cynomolgus monkey tissue was stained with YYB-101 to test tissue cross-reactivity. We found that YYB-101 inhibited cMET activation in vitro and suppressed tumor growth in the orthotopic mouse model of human glioblastoma. Combination treatment with YYB-101 and temozolomide decreased tumor growth and increased overall survival compared with the effects of either agent alone. Five cancer-related genes (TMEM119, FST, RSPO3, ROS1 and NBL1) were overexpressed in YYB-101-treated mice that showed tumor regrowth. In the tissue cross-reactivity assay, critical cross-reactivity was not observed. The terminal elimination half-life was 21.7 days. Taken together, the in vitro and in vivo data demonstrated the anti-tumor efficacy of YYB-101, which appeared to be mediated by blocking the HGF/cMET interaction. The preclinical pharmacokinetics, toxicokinetics and tissue cross-reactivity data support the clinical development of YYB-101 for advanced cancer. PMID:28336956

  13. 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

  14. Expression of Human Immunodeficiency Virus Type 1 Neutralizing Antibody Fragments Using Human Vaginal Lactobacillus

    PubMed Central

    Marcobal, Angela; Liu, Xiaowen; Zhang, Wenlei; Dimitrov, Antony S.; Jia, Letong; Lee, Peter P.; Fouts, Timothy R.; Parks, Thomas P.

    2016-01-01

    Abstract Eradication of human immunodeficiency virus type 1 (HIV-1) by vaccination with epitopes that produce broadly neutralizing antibodies is the ultimate goal for HIV prevention. However, generating appropriate immune responses has proven difficult. Expression of broadly neutralizing antibodies by vaginal colonizing lactobacilli provides an approach to passively target these antibodies to the mucosa. We tested the feasibility of expressing single-chain and single-domain antibodies (dAbs) in Lactobacillus to be used as a topical microbicide/live biotherapeutic. Lactobacilli provide an excellent platform to express anti-HIV proteins. Broadly neutralizing antibodies have been identified against epitopes on the HIV-1 envelope and have been made into active antibody fragments. We tested single-chain variable fragment m9 and dAb-m36 and its derivative m36.4 as prototype antibodies. We cloned and expressed the antibody fragments m9, m36, and m36.4 in Lactobacillus jensenii-1153 and tested the expression levels and functionality. We made a recombinant L. jensenii 1153-1128 that expresses dAb-m36.4. All antibody fragments m9, m36, and m36.4 were expressed by lactobacilli. However, we noted the smaller m36/m36.4 were expressed to higher levels, ≥3 μg/ml. All L. jensenii-expressed antibody fragments bound to gp120/CD4 complex; Lactobacillus-produced m36.4 inhibited HIV-1BaL in a neutralization assay. Using a TZM-bl assay, we characterized the breadth of neutralization of the m36.4. Delivery of dAbs by Lactobacillus could provide passive transfer of these antibodies to the mucosa and longevity at the site of HIV-1 transmission. PMID:26950606

  15. 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.

  16. Neutralization of diverse human immunodeficiency virus type 1 variants by an anti-V3 human monoclonal antibody.

    PubMed Central

    Gorny, M K; Conley, A J; Karwowska, S; Buchbinder, A; Xu, J Y; Emini, E A; Koenig, S; Zolla-Pazner, S

    1992-01-01

    The third variable region (V3) of the HIV-1 gp120 envelope glycoprotein is thought to induce potent neutralizing antibodies which are generally defined as type specific and reactive with individual viral isolates. In contrast, the CD4-binding domain is thought to induce neutralizing antibodies that are group specific and capable of neutralizing all isolates of HIV-1. However, in this study, we used a panel of human monoclonal antibodies to these regions of gp120 which displays specificities and neutralizing activities that challenge these tenets. In particular, we used a human monoclonal antibody to the V3 domain with exceptionally potent and broad neutralizing activity against many diverse HIV-1 isolates. The anti-CD4-binding domain antibodies, on the other hand, showed a more restricted pattern of activity. PMID:1433529

  17. Induction of apoptosis in prostate cancer cells by the novel ceramidase inhibitor ceranib-2.

    PubMed

    Kus, Gokhan; Kabadere, Selda; Uyar, Ruhi; Kutlu, Hatice Mehtap

    2015-11-01

    Ceramidases are key enzymes that decrease ceramide levels in cells. A reduction in ceramide concentration impairs ceramide signalling, and results in apoptosis resistance in cancer cells. This study investigates the potential for ceranib-2, a novel ceramidase inhibitor, to affect the survival and/or promote apoptosis of prostate cancer cells (LNCaP and DU145) in vitro. Cell viability was determined using MTT, and apoptosis assessed via flow cytometry. We examined structural changes with both confocal and transmission electron microscopes. Ceranib-2 concentrations of 0.1, 1, 5, 10, 25 and 50 μM were applied to LNCaP and DU145 cell lines. The corresponding reduction in LNCaP cell viability (against the control) was 84%, 80%, 64%, 56%, 40% and 15% after 24 h, and 81%, 74%, 60%, 55%, 27% and 11% after 48 h. For DU145 cells, viability was reduced to 84%, 82%, 63%, 50%, 41% and 18% after 24 h, and 64%, 42%, 30%, 20%, 8% and 5% after 48 h. Following treatment with 25 and 50 μM ceranib-2, the respective observed rates of early apoptosis in LNCaP cells were 23% and 36% after 24 h and 27% and 58% after 48 h. The morphological and ultrastructural signs of apoptosis detected were fragmented nuclei, chromatin condensations and cytoskeleton laceration. The inhibitory effects of ceranib-2 on prostate cancer cell survival are dose and time dependent. For LNCaP cells, ceranib-2 toxicity was predominately apoptotic in nature, while for DU145 cells, cell death may be related to non-apoptotic mechanisms.

  18. Human Memory B Cells Producing Potent Cross-Neutralizing Antibodies against Human Parechovirus: Implications for Prevalence, Treatment, and Diagnosis

    PubMed Central

    Benschop, K. S. M.; Koen, G.; Claassen, Y. B.; Wagner, K.; Bakker, A. Q.; Wolthers, K. C.

    2015-01-01

    ABSTRACT The family Picornaviridae is a large and diverse group of positive-sense RNA viruses, including human enteroviruses (EVs) and human parechoviruses (HPeVs). The human immune response against EVs and HPeVs is thought to be mainly humoral, and an insufficient neutralizing antibody (Ab) response during infection is a risk factor and can ultimately be life threatening. The accessibility of different antigenic sites and observed cross-reactivity make HPeVs a good target for development of therapeutic human monoclonal antibodies (MAbs). In this study, we generated two different human MAbs specific for HPeV by screening culture supernatants of Ab-producing human B cell cultures for direct neutralization of HPeV1. Both MAbs showed HPeV1-specific neutralization as well as neutralization of HPeV2. One antibody, AM18, cross-neutralized HPeV4, -5, and -6 and coxsackievirus A9 (CV-A9). VP1 capsid protein-specific assays confirmed that AM18 bound VP1 of HPeV1, -2, and -4 with high affinity (11.5 pM). In contrast, the HPeV1-specific MAb AM28, which neutralized HPeV1 even more efficiently than did AM18, showed no cross-reactivity with HPeV3 to -6 or other EVs and did not bind any of the capsid proteins, suggesting that AM28 is specific for a conformation-dependent, nonlinear epitope on the virus. The discovery of MAbs that are cross-reactive between HPeVs may help development of HPeV treatment options with antibodies and vaccine design based on epitopes recognized by these antibodies. IMPORTANCE HPeV infections are widespread among young children and adults, causing a broad range of disease. Infections can be severe and life threatening, while no antiviral treatment is available. Given that the absence of neutralizing Abs is a risk factor for severe disease in infants, treatment of picornavirus infections with MAbs would be a therapeutic option. To study antibody neutralization of HPeV in more detail, we generated two different HPeV1-specific human MAbs. Both MAbs show HPe

  19. Can artificial techniques supply morally neutral human embryos for research?

    PubMed

    Cheshire, William P; Jones, Nancy L

    2005-01-01

    Amidst controversy surrounding research on human embryos, biotechnology has conceived a substitute in the artificial human embryo. We examine the claim that novel embryos constructed artificially should be exempt from ethical restraints appropriate for research on embryos that come into being through natural processes. Morally relevant differences in intrinsic value depend on the sense in which the entity may be artificial, whether in regard to constituent matter, genetic or cellular form, generative means, or intended purpose. Considering each of these Aristotelian categories from a physicalist viewpoint, technology can achieve only limited degrees of artificiality because redesigned embryos still retain most of their natural features and relationships. From an essentialist viewpoint, the very limits of technology preclude the capability of manipulating the fundamental nature or essence of the individual who, even at the embryonic stage of life, cannot be made to be artificial through and through. A human may possess artificially contributed attributes but cannot be an artificial being. Classification of novel human organisms as artificial, therefore, is insufficient grounds by which to relinquish the principle that human moral status should be recognized for all living beings of human origin. In uncertain cases, at least the possibility of special human moral status should be considered present in organisms that are derived asexually, are developmentally defective, or are otherwise technologically altered.

  20. The neutralizing human recombinant antibodies to pathogenic Orthopoxviruses derived from a phage display immune library.

    PubMed

    Tikunova, Nina; Dubrovskaya, Viktoriya; Morozova, Vera; Yun, Tatiana; Khlusevich, Yana; Bormotov, Nikolai; Laman, Aleksandr; Brovko, Fedor; Shvalov, Aleksandr; Belanov, Eugeni

    2012-01-01

    A panel of recombinant human antibodies to orthopoxviruses was isolated from a combinatorial phage display library of human scFv antibodies constructed from the Vh and Vl genes cloned from the peripheral blood lymphocytes of Vaccinia virus (VACV) immune donors. Plaque-reduction neutralization tests showed that seven selected phage-displaying scFv antibodies (pdAbs) neutralized both CPXV and VACV, and five of them neutralized Monkeypox virus (MPXV). Western blot analysis of VACV and CPXV proteins demonstrated that seven neutralizing antibodies recognized a 35 kDa protein. To identify this target protein, we produced a recombinant J3L protein of CPXV and showed that all the selected neutralizing antibodies recognized this protein. Neutralizing pdAb b9 was converted into fully human mAb b9 (fh b9), and scFv b9 displayed high binding affinities (K(d) of 0.7 and 3.2 nM). The fh b9 reduced VACV plaque formation in a dose-dependent manner.

  1. Production of a neutralizing mouse-human chimeric antibody against botulinum neurotoxin serotype E.

    PubMed

    Mukamoto, Masafumi; Maeda, Hiroaki; Kohda, Tomoko; Nozaki, Chikateru; Takahashi, Motohide; Kozaki, Shunji

    2013-01-01

    A mouse-human chimeric antibody that can neutralize botulinum neurotoxin serotype E (BoNT/E) was developed. Variable regions of heavy and light chains obtained using a mouse hybridoma clone (E9-4) cDNA, which was selected on the basis of neutralizing activity against BoNT/E, were fused with the upstream regions of the constant counterparts of human kappa light and gamma 1 heavy chain genes, respectively. CHO-DG44 cells were transfected with these plasmids and a mouse-human chimeric antibody (EC94) was purified to examine binding and neutralizing activity against BoNT/E. EC94 exhibited the same levels of binding activities against BoNT/E as those of a parent mouse monoclonal antibody and neutralized more than 4,000 LD(50)/mg antibody. This chimeric antibody seems to be a useful candidate for infant botulism in which the use of passive immunotherapy is not planned so as to avoid serious events such as anaphylactic shock. We designed shuffling chimeric antibodies with replacement of V(H) or V(L) of EC94 with that of a chimeric antibody (AC24) that possessed neutralizing activity against BoNT/A. These shuffling antibodies did not exhibit neutralizing activity against either BoNT/E or BoNT/A.

  2. Neutralizing activities against seasonal influenza viruses in human intravenous immunoglobulin

    PubMed Central

    Onodera, Hiroyuki; Urayama, Takeru; Hirota, Kazue; Maeda, Kazuhiro; Kubota-Koketsu, Ritsuko; Takahashi, Kazuo; Hagiwara, Katsuro; Okuno, Yoshinobu; Ikuta, Kazuyoshi; Yunoki, Mikihiro

    2017-01-01

    Influenza viruses A/H1N1, A/H3N2, and B are known seasonal viruses that undergo annual mutation. Intravenous immunoglobulin (IVIG) contains anti-seasonal influenza virus globulins. Although the virus-neutralizing (VN) titer is an indicator of protective antibodies, changes in this titer over extended time periods have yet to be examined. In this study, variations in hemagglutination inhibition (HI) and VN titers against seasonal influenza viruses in IVIG lots over extended time periods were examined. In addition, the importance of monitoring the reactivity of IVIG against seasonal influenza viruses with varying antigenicity was evaluated. A/H1N1, A/H3N2, and B influenza virus strains and IVIG lots manufactured from 1999 to 2014 were examined. The HI titer was measured by standard methods. The VN titer was measured using a micro-focus method. IVIG exhibited significant HI and VN titers against all investigated strains. Our results suggest that the donor population maintains both specific and cross-reactive antibodies against seasonal influenza viruses, except in cases of pandemic viruses, despite major antigen changes. The titers against seasonal influenza vaccine strains, including past strains, were stable over short time periods but increased slowly over time. PMID:28331286

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

    PubMed Central

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

    2015-01-01

    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

  4. 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.

  5. 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.

  6. Preventive and therapeutic applications of neutralizing antibodies to Human Immunodeficiency Virus Type 1 (HIV-1)

    PubMed Central

    Ringe, Rajesh

    2013-01-01

    The development of a preventive vaccine to neutralize the highly variable and antigenically diverse human immunodeficiency virus type 1 (HIV-1) has been an indomitable goal. The recent discovery of a number of cross-neutralizing and potent monoclonal antibodies from elite neutralizers has provided important insights in this field. Neutralizing antibodies (NAbs) are useful in identifying neutralizing epitopes of vaccine utility and for understanding the mechanism of potent and broad cross-neutralization thus providing a modality of preventive and therapeutic value. In this article we review the current understanding on the potential use of broadly neutralizing antibodies (bNAbs) in their full-length IgG structure, engineered domain antibody or bispecific versions towards preventive and therapeutic applications. The potential implications of NAbs are discussed in the light of the recent developments as key components in vaccination against HIV-1. The development of a vaccine immunogen which elicits bNAbs and confers protective immunity remains a real challenge. PMID:24757516

  7. Characterization of a human papillomavirus type 16 variant-dependent neutralizing epitope.

    PubMed Central

    Roden, R B; Armstrong, A; Haderer, P; Christensen, N D; Hubbert, N L; Lowy, D R; Schiller, J T; Kirnbauer, R

    1997-01-01

    We have determined that three type-specific and conformationally dependent monoclonal antibodies, H16.E70, H16.U4, and H16.V5, neutralize pseudotype human papillomavirus type 16 (HPV16) virions in vitro. H16.U4 and H16.V5 neutralized pseudotype virions derived from the German HPV16 variant 114K and the Zairian variant Z-1194 with equal efficiency. In contrast, neutralization of Z-1194 pseudotype virions by H16.E70 was two orders of magnitude weaker than neutralization of 114K pseudotype virions. This difference correlated with enzyme-linked immunosorbent assay reactivity of H16.E70 to L1 virus-like particles of the two variants. A substitution at residue 282 of L1 was responsible for this differential reactivity, suggesting that this residue constitutes part of the H16.E70 epitope. PMID:9223527

  8. Neutralizing human antibodies prevent Zika virus replication and fetal disease in mice.

    PubMed

    Sapparapu, Gopal; Fernandez, Estefania; Kose, Nurgun; Bin Cao; Fox, Julie M; Bombardi, Robin G; Zhao, Haiyan; Nelson, Christopher A; Bryan, Aubrey L; Barnes, Trevor; Davidson, Edgar; Mysorekar, Indira U; Fremont, Daved H; Doranz, Benjamin J; Diamond, Michael S; Crowe, James E

    2016-12-15

    Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that can cause severe disease, including congenital birth defects during pregnancy. To develop candidate therapeutic agents against ZIKV, we isolated a panel of human monoclonal antibodies from subjects that were previously infected with ZIKV. We show that a subset of antibodies recognize diverse epitopes on the envelope (E) protein and exhibit potent neutralizing activity. One of the most inhibitory antibodies, ZIKV-117, broadly neutralized infection of ZIKV strains corresponding to African and Asian-American lineages. Epitope mapping studies revealed that ZIKV-117 recognized a unique quaternary epitope on the E protein dimer-dimer interface. We evaluated the therapeutic efficacy of ZIKV-117 in pregnant and non-pregnant mice. Monoclonal antibody treatment markedly reduced tissue pathology, placental and fetal infection, and mortality in mice. Thus, neutralizing human antibodies can protect against maternal-fetal transmission, infection and disease, and reveal important determinants for structure-based rational vaccine design efforts.

  9. 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

  10. Neutralizing antibody responses in acute human immunodeficiency virus type 1 subtype C infection.

    PubMed

    Gray, E S; Moore, P L; Choge, I A; Decker, J M; Bibollet-Ruche, F; Li, H; Leseka, N; Treurnicht, F; Mlisana, K; Shaw, G M; Karim, S S Abdool; Williamson, C; Morris, L

    2007-06-01

    The study of the evolution and specificities of neutralizing antibodies during the course of human immunodeficiency virus type 1 (HIV-1) infection may be important in the discovery of possible targets for vaccine design. In this study, we assessed the autologous and heterologous neutralization responses of 14 HIV-1 subtype C-infected individuals, using envelope clones obtained within the first 2 months postinfection. Our data show that potent but relatively strain-specific neutralizing antibodies develop within 3 to 12 months of HIV-1 infection. The magnitude of this response was associated with shorter V1-to-V5 envelope lengths and fewer glycosylation sites, particularly in the V1-V2 region. Anti-MPER antibodies were detected in 4 of 14 individuals within a year of infection, while antibodies to CD4-induced (CD4i) epitopes developed to high titers in 12 participants, in most cases before the development of autologous neutralizing antibodies. However, neither anti-MPER nor anti-CD4i antibody specificity conferred neutralization breadth. These data provide insights into the kinetics, potency, breadth, and epitope specificity of neutralizing antibody responses in acute HIV-1 subtype C infection.

  11. Autoreactivity of primary human immunoglobulins ancestral to hypermutated human antibodies that neutralize HCMV.

    PubMed

    McLean, Gary R; Cho, Chin-wen; Schrader, John W

    2006-05-01

    The human antibody response to the AD-2S1 epitope of glycoprotein B (gB) of human cytomegalovirus (HCMV) is dominated by a family of closely related somatically mutated antibodies. These antibodies neutralize viral infectivity and the genes encoding them are derived from two commonly used germ-line variable (V) region genes, IGHV3-30 and IGKV3-11. Recombination of these V genes with the appropriate junctional diversity generates genes that encode primary immunoglobulins that bind to AD-2S1. To further understand the initial primary immunoglobulin response to AD-2S1 we synthesized the germ-line-based ancestor of one such family of antibodies and showed that it bound gB at the AD-2S1 epitope. Here we show that the germ-line ancestor of a second family of antibodies likewise binds to gB. We further show that one of the ancestral primary immunoglobulins, but not the other, also recognized autoantigens. In contrast, the hypermutated derivatives did not demonstrate autoreactivity and minor structural changes in the primary immunoglobulin were sufficient to generate or abolish autoreactivity or to change specificity. Thus, our demonstration that the ancestor of a highly mutated, non-autoreactive antiviral IgG antibody binds nuclear and cell-surface autoantigens indicates for the first time that self-reactivity is not necessarily a barrier to development into a follicular B lymphocyte that undergoes antigen-initiated affinity maturation.

  12. 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 =…

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

    PubMed Central

    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

  14. 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

    2016-09-11

    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.

  15. Identification of human immunodeficiency virus subtypes with distinct patterns of sensitivity to serum neutralization.

    PubMed Central

    Cheng-Mayer, C; Homsy, J; Evans, L A; Levy, J A

    1988-01-01

    The human immunodeficiency virus (HIV) type 1 displays a high degree of genetic variation, especially in the glycoprotein (gp120) domain of the envelope gene. To determine whether this genomic heterogeneity leads to the expression of independent HIV subtypes, 12 sera from HIV type 1 antibody-positive individuals were tested for their ability to neutralize 20 HIV isolates of various origins. Four distinct HIV subtypes with different sensitivity to serum neutralization were identified. These results suggest that a finite number of HIV subtypes exist and that the combined use of selected HIV isolates representing several subtypes may be necessary for the development of an effective vaccine. Images PMID:3357892

  16. Generation and characterization of neutralizing human recombinant antibodies against antigenic site II of rabies virus glycoprotein.

    PubMed

    Sun, Lina; Chen, Zhe; Yu, Li; Wei, Jingshuang; Li, Chuan; Jin, Jing; Shen, Xinxin; Lv, Xinjun; Tang, Qing; Li, Dexin; Liang, Mifang

    2012-10-01

    The currently recommended treatment for individuals exposed to rabies virus (RV) is post-exposure prophylaxis (PEP) through the combined administration of rabies vaccine and rabies immune globulin (RIG). Human monoclonal antibodies (mAbs) that neutralize RV offer an opportunity to replace RIG for rabies PEP. Here, a combinatorial human Fab library was constructed using antibody genes derived from the blood of RV-vaccinated donors. Selections of this library against purified RV virions resulted in the identification of 11 unique Fab antibodies specific for RV glycoprotein. Of the Fab antibodies, five were converted to full human IgG1 format. The human IgG antibodies revealed high binding affinity and neutralizing activities against RV fixed strains through a rapid fluorescent focus inhibition test in vitro as well as the early stage protective function after exposure to RV infection in vivo. Furthermore, epitope mapping and binding competition analysis showed that all of obtained human neutralizing and protective antibodies were directed to the antigenic site II of RV glycoprotein. Our results provide not only important insight into the protective immune response to RV in humans, but also more candidates eligible for use in a mAb cocktail aimed at replacing RIG for rabies post-exposure prophylaxis.

  17. 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

    2016-05-10

    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.

  18. 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

  19. Cortisol variation in humans affects memory for emotionally laden and neutral information.

    PubMed

    Abercrombie, Heather C; Kalin, Ned H; Thurow, Marchell E; Rosenkranz, Melissa A; Davidson, Richard J

    2003-06-01

    In a test of the effects of cortisol on emotional memory, 90 men were orally administered placebo or 20 or 40 mg cortisol and presented with emotionally arousing and neutral stimuli. On memory tests administered within 1 hr of stimulus presentation, cortisol elevations caused a reduction in the number of errors committed on free-recall tasks. Two evenings later, when cortisol levels were no longer manipulated, inverted-U quadratic trends were found for recognition memory tasks, reflecting memory facilitation in the 20-mg group for both negative and neutral information. Results suggest that the effects of cortisol on memory do not differ substantially for emotional and neutral information. The study provides evidence of beneficial effects of acute cortisol elevations on explicit memory in humans.

  20. Fine mapping of sequential neutralization epitopes on the subunit protein VP8 of human rotavirus.

    PubMed Central

    Kovacs-Nolan, Jennifer; Yoo, Dongwan; Mine, Yoshinori

    2003-01-01

    The epitopes of the HRV (human rotavirus), especially those involved in virus neutralization, have not been determined in their entirety, and would have significant implications for HRV vaccine development. In the present study, we report on the epitope mapping and identification of sequential neutralization epitopes, on the Wa strain HRV subunit protein VP8, using synthetic overlapping peptides. Polyclonal antibodies against recombinant Wa VP8 were produced previously in chicken, and purified from egg yolk, which showed neutralizing activity against HRV in vitro. Overlapping VP8 peptide fragments were synthesized and probed with the anti-VP8 antibodies, revealing five sequential epitopes on VP8. Further analysis suggested that three of the five epitopes detected, M1-L10, I55-D66 and L223-P234, were involved in virus neutralization, indicating that sequential epitopes may also be important for the HRV neutralization. The interactions of the antibodies with the five epitopes were characterized by an examination of the critical amino acids involved in antibody binding. Epitopes comprised primarily of hydrophobic amino acid residues, followed by polar and charged residues. The more critical amino acids appeared to be located near the centre of the epitopes, with proline, isoleucine, serine, glutamine and arginine playing an important role in the binding of antibody to the VP8 epitopes. PMID:12901721

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

    PubMed Central

    Bangaru, Sandhya; Nieusma, Travis; Kose, Nurgun; Thornburg, Natalie J.; Kaplan, Bryan S.; King, Hannah G.; Singh, Vidisha; Lampley, Rebecca M.; Cisneros, Alberto; Edwards, Kathryn M.; 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

  2. 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

  3. Recombinant human monoclonal antibodies to human cytomegalovirus glycoprotein B neutralize virus in a complement-dependent manner.

    PubMed

    Ohta, Akane; Fujita, Ayano; Murayama, Tsugiya; Iba, Yoshitaka; Kurosawa, Yoshikazu; Yoshikawa, Tetsushi; Asano, Yoshizo

    2009-11-01

    Human antibodies specific for HCMV are currently considered as potential anti-HCMV therapeutic agents. In this study, we used a combinatorial human antibody library to isolate and characterize complete human monoclonal antibodies that effectively neutralize HCMV in a complement-dependent manner. One hundred and six clones were isolated in two independent screens using HCMV virions and recombinant glycoprotein B, gB654, as antigens. All of the clones recognized the same molecule gB and were classified into 14 groups based on the amino acid sequence of the V(H) region. Seven representative clones from these 14 groups had a strong gB654 binding affinity by surface plasmon resonance (SPR). A pairwise binding competition analysis suggested that there were three groups based on differences in the gB recognition sites. Although Fab fragments of the seven groups showed strong affinity for gB, none of the Fab fragments neutralized HCMV infectivity in vitro. In contrast, complete human IgG(1) antibodies of at least three groups neutralized HCMV in a complement-dependent manner. These data suggest that potent therapeutic antibodies can be obtained from a human antibody library, including most of the functional antibodies that mediate humoral immunity to the selected pathogen.

  4. Acid Ceramidase Treatment Enhances the Outcome of Autologous Chondrocyte Implantation in a Rat Osteochondral Defect Model

    PubMed Central

    Frohbergh, Michael E.; Guevara, Johana M.; Grelsamer, Ronald P.; Barbe, Mary F.; He, Xingxuan; Simonaro, Calogera M.; Schuchman, Edward H.

    2015-01-01

    Objective The overall aim of this study was to evaluate how supplementation of chondrocyte media with recombinant acid ceramidase (rhAC) influenced cartilage repair in a rat osteochondral defect model. Methods Primary chondrocytes were grown as monolayers in polystyrene culture dishes with and without rhAC (added once at the time of cell plating) for 7 days, and then seeded onto Bio-Gide® collagen scaffolds and grown for an additional 3 days. The scaffolds were then introduced into osteochondroal defects created in Sprague-Dawley rat trochlea by a micordrilling procedure. Analysis was performed 6 weeks post-surgery macroscopically, by micro-CT, histologically, and by immunohistochemistry. Results Treatment with rhAC led to increased cell numbers and glycosaminoglycan production (~2 and 3-fold, respectively) following 7 days of expansion in vitro. Gene expression of collagen 2, aggrecan and Sox-9 also was significantly elevated. After seeding onto Bio-Gide®, more rhAC treated cells were evident within 4 hours. At 6 weeks post-surgery, defects containing rhAC-treated cells exhibited more soft tissue formation at the articular surface, as evidenced by microCT, as well as histological evidence of enhanced cartilage repair. Notably, collagen 2 immunostaining revealed greater surface expression in animals receiving rhAC treated cells as well. Collagen 10 staining was not enhanced. Conclusion The results further demonstrate the positive effects of rhAC treatment on chondrocyte growth and phenotype in vitro, and reveal for the first time the in vivo effects of the treated cells on cartilage repair. PMID:26524412

  5. Role of Human Immunodeficiency Virus Type 1 Envelope Structure in the Induction of Broadly Neutralizing Antibodies

    PubMed Central

    Benjelloun, F.; Lawrence, P.; Verrier, B.; Genin, C.

    2012-01-01

    Very soon after the discovery of neutralizing antibodies (NAbs) toward human immunodeficiency virus type 1 (HIV-1) infection, it became apparent that characterization of these NAbs would be an important step in finding a cure for or a vaccine to eradicate HIV-1. Since the initial description of broadly cross-clade NAbs naturally produced in HIV-1 patients, numerous studies have described new viral targets for these antibodies. More recently, studies concerning new groups of patients able to control their viremia, such as long-term nonprogressors (LTNPs) or elite controllers, have described the generation of numerous envelope-targeted NAbs. Recent studies have marked a new stage in research on NAbs with the description of antibodies obtained from a worldwide screening of HIV-positive patients. These studies have permitted the discovery of NAb families with great potential for both neutralization and neutralization breadth, such as PG, PGT, CH, and highly active agonistic anti-CD4 binding site antibodies (HAADs), of which VRC01 and its variants are members. These antibodies are able to neutralize more than 80% of circulating strains without any autoreactivity and can be rapidly integrated into clinical trials in order to test their protective potential. In this review, we will focus on new insights into HIV-1 envelope structure and their implications for the generation of potent NAbs. PMID:23015715

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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

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

    PubMed

    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

    2015-09-14

    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.

  12. Mapping the Human Memory B Cell and Serum Neutralizing Antibody Responses to Dengue Virus Serotype 4 Infection and Vaccination.

    PubMed

    Nivarthi, Usha K; Kose, Nurgun; Sapparapu, Gopal; Widman, Douglas; Gallichotte, Emily; Pfaff, Jennifer M; Doranz, Benjamin J; Weiskopf, Daniela; Sette, Alessandro; Durbin, Anna P; Whitehead, Steve S; Baric, Ralph; Crowe, James E; de Silva, Aravinda M

    2017-03-01

    The four dengue virus (DENV) serotypes are mosquito-borne flaviviruses responsible for dengue fever and dengue hemorrhagic fever. People exposed to DENV develop antibodies (Abs) that strongly neutralize the serotype responsible for infection. Historically, infection with DENV serotype 4 (DENV4) has been less common and less studied than infections with the other three serotypes. However, DENV4 has been responsible for recent large and sustained epidemics in Asia and Latin America. The neutralizing antibody responses and the epitopes targeted against DENV4 have not been characterized in human infection. In this study, we mapped and characterized epitopes on DENV4 recognized by neutralizing antibodies in people previously exposed to DENV4 infections or to a live attenuated DENV4 vaccine. To study the fine specificity of DENV4 neutralizing human antibodies, B cells from two people exposed to DENV4 were immortalized and screened to identify DENV-specific clones. Two human monoclonal antibodies (MAbs) that neutralized DENV4 were isolated, and their epitopes were finely mapped using recombinant viruses and alanine scan mutation array techniques. Both antibodies bound to quaternary structure epitopes near the hinge region between envelope protein domain I (EDI) and EDII. In parallel, to characterize the serum neutralizing antibody responses, convalescence-phase serum samples from people previously exposed to primary DENV4 natural infections or a monovalent DENV4 vaccine were analyzed. Natural infection and vaccination also induced serum-neutralizing antibodies that targeted similar epitope domains at the EDI/II hinge region. These studies defined a target of neutralizing antigenic site on DENV4 targeted by human antibodies following natural infection or vaccination.IMPORTANCE The four serotypes of dengue virus are the causative agents of dengue fever and dengue hemorrhagic fever. People exposed to primary DENV infections develop long-term neutralizing antibody responses

  13. Partitioning of polar and non-polar neutral organic chemicals into human and cow milk.

    PubMed

    Geisler, Anett; Endo, Satoshi; Goss, Kai-Uwe

    2011-10-01

    The aim of this work was to develop a predictive model for milk/water partition coefficients of neutral organic compounds. Batch experiments were performed for 119 diverse organic chemicals in human milk and raw and processed cow milk at 37°C. No differences (<0.3 log units) in the partition coefficients of these types of milk were observed. The polyparameter linear free energy relationship model fit the calibration data well (SD=0.22 log units). An experimental validation data set including hormones and hormone active compounds was predicted satisfactorily by the model. An alternative modelling approach based on log K(ow) revealed a poorer performance. The model presented here provides a significant improvement in predicting enrichment of potentially hazardous chemicals in milk. In combination with physiologically based pharmacokinetic modelling this improvement in the estimation of milk/water partitioning coefficients may allow a better risk assessment for a wide range of neutral organic chemicals.

  14. Molecular determinants of human neutralizing antibodies isolated from a patient infected with Zika virus.

    PubMed

    Wang, Qihui; Yang, Huabing; Liu, Xiaoqing; Dai, Lianpan; Ma, Tong; Qi, Jianxun; Wong, Gary; Peng, Ruchao; Liu, Sheng; Li, Junfu; Li, Shihua; Song, Jian; Liu, Jianying; He, Jianhua; Yuan, Hui; Xiong, Ying; Liao, Yong; Li, Jianhua; Yang, Jianping; Tong, Zhou; Griffin, Bryan D; Bi, Yuhai; Liang, Mifang; Xu, Xiaoning; Qin, Chuan; Cheng, Gong; Zhang, Xinzheng; Wang, Peiyi; Qiu, Xiangguo; Kobinger, Gary; Shi, Yi; Yan, Jinghua; Gao, George F

    2016-12-14

    The 2015-2016 outbreak of Zika virus (ZIKV) disease has affected many countries and is a major public health concern. ZIKV is associated with fetal microcephaly and neurological complications, and countermeasures are needed to treat and prevent ZIKV infection. We report the isolation of 13 specific human monoclonal antibodies from a single patient infected with ZIKV. Two of the isolated antibodies (Z23 and Z3L1) demonstrated potent ZIKV-specific neutralization in vitro without binding or neutralizing activity against strains 1 to 4 of dengue virus, the closest relative to ZIKV. These two antibodies provided postexposure protection to mice in vivo. Structural studies revealed that Z23 and Z3L1 bound to tertiary epitopes in envelope protein domain I, II, or III, indicating potential targets for ZIKV-specific therapy. Our results suggest the potential of antibody-based therapeutics and provide a structure-based rationale for the design of future ZIKV-specific vaccines.

  15. Human TNF cytokine neutralization with a vNAR from Heterodontus francisci shark

    PubMed Central

    Camacho-Villegas, Tanya; Mata-Gonzalez, Teresa; Paniagua-Solis, Jorge; Sanchez, Edna; Licea, Alexei

    2013-01-01

    The therapeutic use of single domain antibodies (sdAbs) is a promising new approach because these small antibodies maintain antigen recognition and neutralization capacity, have thermal and chemical stability and have good solubility. In this study, using phage display technology, we isolated a variable domain of a IgNAR (vNAR) from a Heterodontus francisci shark immunized against the recombinant human cytokine TNFα (rhTNFα). One clone T43, which expresses the vNAR protein in the periplasmic space, was isolated from the fourth round of panning. T43 had the capacity to recognize rhTNF and neutralize it in vitro, indicating that T43 has potential as a therapeutic that can be used for diseases in which this pro-inflammatory cytokine needs to be controlled. PMID:23221782

  16. Cortisol has different effects on human memory for emotional and neutral stimuli.

    PubMed

    Rimmele, Ulrike; Domes, Gregor; Mathiak, Klaus; Hautzinger, Martin

    2003-12-19

    Adrenal stress hormones are considered to play a role in memory enhancement of emotionally arousing events. To investigate the effects of cortisol on human emotional memory, subjects were administered hydrocortisone (25 mg) or placebo and presented with either an emotionally arousing or a neutral story. Memory for the story was tested 1 week later. In all memory tests, subjects who viewed the emotional story scored better for the emotionally arousing story parts, indicating that arousal enhances memory. In memory of details, cortisol showed an interaction with story valence but no main effect: cortisol enhanced memory for details of the neutral story version, but impaired memory for details of the emotionally arousing version. We thus confirm a non-linear interaction between cortisol and arousal on memory formation.

  17. Human skin permeation of neutral species and ionic species: extended linear free-energy relationship analyses.

    PubMed

    Zhang, Keda; Chen, Ming; Scriba, Gerhard K E; Abraham, Michael H; Fahr, Alfred; Liu, Xiangli

    2012-06-01

    The permeability, K(p), of some ionized solutes (including nine acids and nine bases) through human epidermis membrane was measured in this work. Combined with the experimental K(p) data set for neutral species created by Abraham and Martins and reliable K(p) data for ionic species from the literature, a linear free-energy relationship (LFER) analysis was conducted. The values of log K(p) for 118 compounds have been correlated with solute descriptors to yield an LFER equation that incorporates neutral species and ionic species, with R(2) = 0.861 and SD = 0.462 log units. The equation can be used to predict K(p) for neutral species and ionic species, as well as partly ionized solutes. Predicted values for the passive permeation of the sodium ion and the tetraethylammonium ion are in good accord with the experimental values. It was observed that neutral acids and bases are more permeable than their ionized forms, and that the ratio depends on the actual structure. The correlation between human skin permeation and water-organic solvent/artificial membrane partitions was investigated by comparison of the coefficients in the LFER equations. Partition into cerasome is a reasonable model for partition into skin, and using cerasome as a surrogate for the partitioning process, we separate permeation into partition and diffusion processes. We show that the poor permeability of ionic species is largely due to slow diffusion through the stratum corneum. This is especially marked for a number of protonated base cations.

  18. Natural selection affects multiple aspects of genetic variation at putatively neutral sites across the human genome.

    PubMed

    Lohmueller, Kirk E; Albrechtsen, Anders; Li, Yingrui; Kim, Su Yeon; Korneliussen, Thorfinn; Vinckenbosch, Nicolas; Tian, Geng; Huerta-Sanchez, Emilia; Feder, Alison F; Grarup, Niels; Jørgensen, Torben; Jiang, Tao; Witte, Daniel R; Sandbæk, Annelli; Hellmann, Ines; Lauritzen, Torsten; Hansen, Torben; Pedersen, Oluf; Wang, Jun; Nielsen, Rasmus

    2011-10-01

    A major question in evolutionary biology is how natural selection has shaped patterns of genetic variation across the human genome. Previous work has documented a reduction in genetic diversity in regions of the genome with low recombination rates. However, it is unclear whether other summaries of genetic variation, like allele frequencies, are also correlated with recombination rate and whether these correlations can be explained solely by negative selection against deleterious mutations or whether positive selection acting on favorable alleles is also required. Here we attempt to address these questions by analyzing three different genome-wide resequencing datasets from European individuals. We document several significant correlations between different genomic features. In particular, we find that average minor allele frequency and diversity are reduced in regions of low recombination and that human diversity, human-chimp divergence, and average minor allele frequency are reduced near genes. Population genetic simulations show that either positive natural selection acting on favorable mutations or negative natural selection acting against deleterious mutations can explain these correlations. However, models with strong positive selection on nonsynonymous mutations and little negative selection predict a stronger negative correlation between neutral diversity and nonsynonymous divergence than observed in the actual data, supporting the importance of negative, rather than positive, selection throughout the genome. Further, we show that the widespread presence of weakly deleterious alleles, rather than a small number of strongly positively selected mutations, is responsible for the correlation between neutral genetic diversity and recombination rate. This work suggests that natural selection has affected multiple aspects of linked neutral variation throughout the human genome and that positive selection is not required to explain these observations.

  19. Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus.

    PubMed

    Giang, Erick; Dorner, Marcus; Prentoe, Jannick C; Dreux, Marlène; Evans, Matthew J; Bukh, Jens; Rice, Charles M; Ploss, Alexander; Burton, Dennis R; Law, Mansun

    2012-04-17

    Hepatitis C virus (HCV) infects ∼2% of the world's population. It is estimated that there are more than 500,000 new infections annually in Egypt, the country with the highest HCV prevalence. An effective vaccine would help control this expanding global health burden. HCV is highly variable, and an effective vaccine should target conserved T- and B-cell epitopes of the virus. Conserved B-cell epitopes overlapping the CD81 receptor-binding site (CD81bs) on the E2 viral envelope glycoprotein have been reported previously and provide promising vaccine targets. In this study, we isolated 73 human mAbs recognizing five distinct antigenic regions on the virus envelope glycoprotein complex E1E2 from an HCV-immune phage-display antibody library by using an exhaustive-panning strategy. Many of these mAbs were broadly neutralizing. In particular, the mAb AR4A, recognizing a discontinuous epitope outside the CD81bs on the E1E2 complex, has an exceptionally broad neutralizing activity toward diverse HCV genotypes and protects against heterologous HCV challenge in a small animal model. The mAb panel will be useful for the design and development of vaccine candidates to elicit broadly neutralizing antibodies to HCV.

  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. 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.

  2. Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses.

    PubMed

    Sui, Jianhua; Hwang, William C; Perez, Sandra; Wei, Ge; Aird, Daniel; Chen, Li-mei; Santelli, Eugenio; Stec, Boguslaw; Cadwell, Greg; Ali, Maryam; Wan, Hongquan; Murakami, Akikazu; Yammanuru, Anuradha; Han, Thomas; Cox, Nancy J; Bankston, Laurie A; Donis, Ruben O; Liddington, Robert C; Marasco, Wayne A

    2009-03-01

    Influenza virus remains a serious health threat, owing to its ability to evade immune surveillance through rapid genetic drift and reassortment. Here we used a human non-immune antibody phage-display library and the H5 hemagglutinin ectodomain to select ten neutralizing antibodies (nAbs) that were effective against all group 1 influenza viruses tested, including H5N1 'bird flu' and the H1N1 'Spanish flu'. The crystal structure of one such nAb bound to H5 shows that it blocks infection by inserting its heavy chain into a conserved pocket in the stem region, thus preventing membrane fusion. Nine of the nAbs employ the germline gene VH1-69, and all seem to use the same neutralizing mechanism. Our data further suggest that this region is recalcitrant to neutralization escape and that nAb-based immunotherapy is a promising strategy for broad-spectrum protection against seasonal and pandemic influenza viruses.

  3. Structure of a Human Astrovirus Capsid-Antibody Complex and Mechanistic Insights into Virus Neutralization

    SciTech Connect

    Bogdanoff, Walter A.; Campos, Jocelyn; Perez, Edmundo I.; Yin, Lu; Alexander, David L.; DuBois, Rebecca M.; López, Susana

    2016-11-02

    ABSTRACT

    Human astroviruses (HAstVs) are a leading cause of viral diarrhea in young children, the immunocompromised, and the elderly. There are no vaccines or antiviral therapies against HAstV disease. Several lines of evidence point to the presence of protective antibodies in healthy adults as a mechanism governing protection against reinfection by HAstV. However, development of anti-HAstV therapies is hampered by the gap in knowledge of protective antibody epitopes on the HAstV capsid surface. Here, we report the structure of the HAstV capsid spike domain bound to the neutralizing monoclonal antibody PL-2. The antibody uses all six complementarity-determining regions to bind to a quaternary epitope on each side of the dimeric capsid spike. We provide evidence that the HAstV capsid spike is a receptor-binding domain and that the antibody neutralizes HAstV by blocking virus attachment to cells. We identify patches of conserved amino acids that overlap the antibody epitope and may comprise a receptor-binding site. Our studies provide a foundation for the development of therapies to prevent and treat HAstV diarrheal disease.

    IMPORTANCEHuman astroviruses (HAstVs) infect nearly every person in the world during childhood and cause diarrhea, vomiting, and fever. Despite the prevalence of this virus, little is known about how antibodies in healthy adults protect them against reinfection. Here, we determined the crystal structure of a complex of the HAstV capsid protein and a virus-neutralizing antibody. We show that the antibody binds to the outermost spike domain of the capsid, and we provide evidence that the antibody blocks virus attachment to human cells. Importantly, our findings suggest that a subunit-based vaccine focusing the immune system on the HAstV capsid spike domain could be effective in protecting children against HAstV disease.

  4. Targeting human vasohibin-2 by a neutralizing monoclonal antibody for anti-cancer treatment.

    PubMed

    Koyanagi, Takahiro; Suzuki, Yasuhiro; Komori, Kazuki; Saga, Yasushi; Matsubara, Shigeki; Fujiwara, Hiroyuki; Sato, Yasufumi

    2016-12-29

    There are 2 members of the vasohibin (VASH) family, VASH1 and VASH2. VASH1 is expressed mainly in endothelial cells to inhibit angiogenesis, whereas VASH2 is expressed mainly in cancer cells to stimulate tumor growth. The aim of the present study was to establish neutralizing monoclonal antibody (mAb) against human VASH2 and apply it as an anti-cancer treatment. We previously raised mAbs against several synthetic peptides of hVASH1, and found that one of them exhibited neutralizing activity against hVASH1. Because of the similarity in the amino acid sequences between VASH1 and VASH2, we hypothesized that they shared the bioactive center. When we mutated 4 amino acids within the region, the mutant VASH2 lost its pro-angiogenic activity. We therefore raised mAb against a synthetic peptide overlapping the mutated amino acids of hVASH2, and isolated one clone (1760) that almost completely inhibited the stimulatory effect of hVASH2 on the migration of and tube formation by ECs. When we used this clone 1760 antibody for cancer treatment, the peritoneal injection of it inhibited both tumor growth and angiogenesis in a mouse xenograft model of human cancer cells. In terms of anti-tumor activity, 25 mg/kg of clone 1760 was equivalent to 5 mg/kg of bevacizmab. From these results, we propose the targeting of human VASH2 with neutralizing mAb as a new strategy for cancer treatment. This article is protected by copyright. All rights reserved.

  5. Structural basis for antibody cross-neutralization of respiratory syncytial virus and human metapneumovirus.

    PubMed

    Wen, Xiaolin; Mousa, Jarrod J; Bates, John T; Lamb, Robert A; Crowe, James E; Jardetzky, Theodore S

    2017-01-30

    Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are two closely related viruses that cause bronchiolitis and pneumonia in infants and the elderly(1), with a significant health burden(2-6). There are no licensed vaccines or small-molecule antiviral treatments specific to these two viruses at present. A humanized murine monoclonal antibody (palivizumab) is approved to treat high-risk infants for RSV infection(7,8), but other treatments, as well as vaccines, for both viruses are still in development. Recent epidemiological modelling suggests that cross-immunity between RSV, HMPV and human parainfluenzaviruses may contribute to their periodic outbreaks(9), suggesting that a deeper understanding of host immunity to these viruses may lead to enhanced strategies for their control. Cross-reactive neutralizing antibodies to the RSV and HMPV fusion (F) proteins have been identified(10,11). Here, we examine the structural basis for cross-reactive antibody binding to RSV and HMPV F protein by two related, independently isolated antibodies, MPE8 and 25P13. We solved the structure of the MPE8 antibody bound to RSV F protein and identified the 25P13 antibody from an independent blood donor. Our results indicate that both antibodies use germline residues to interact with a conserved surface on F protein that could guide the emergence of cross-reactivity. The induction of similar cross-reactive neutralizing antibodies using structural vaccinology approaches could enhance intrinsic cross-immunity to these paramyxoviruses and approaches to controlling recurring outbreaks.

  6. 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.

  7. 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

  8. 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

  9. 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

  10. Recombinant tandem multi-linear neutralizing epitopes of human enterovirus 71 elicited protective immunity in mice

    PubMed Central

    2014-01-01

    Background Human Enterovirus 71 (EV71) has emerged as the leading cause of viral encephalitis in children, especially in the Asia-Pacific regions. EV71 vaccine development is of high priority at present, and neutralization antibodies have been documented to play critical roles during in vitro and in vivo protection against EV71 infection. Results In this study, a novel strategy to produce EV71 vaccine candidate based on recombinant multiple tandem linear neutralizing epitopes (mTLNE) was proposed. The three well identified EV71 linear neutralizing epitopes in capsid proteins, VP1-SP55, VP1-SP70 and VP2-SP28, were sequentially linked by a Gly-Ser linker ((G4S)3), and expressed in E.coli in fusion with the Trx and His tag at either terminal. The recombinant protein mTLNE was soluble and could be purified by standard affinity chromatography. Following three dosage of immunization in adult mice, EV71-specific IgG and neutralization antibodies were readily induced by recombinant mTLNE. IgG subtyping demonstrated that lgG1 antibodies dominated the mTLNE-induced humoral immune response. Especially, cytokine profiling in spleen cells from the mTLNE-immunized mice revealed high production of IL-4 and IL-6. Finally, in vivo challenge experiments showed that passive transfer with anti-mTLNE sera conferred full protection against lethal EV71 challenge in neonatal mice. Conclusion Our results demonstrated that this rational designed recombinant mTLNE might have the potential to be further developed as an EV71 vaccine in the future. PMID:24885030

  11. Application of a Neutral Community Model To Assess Structuring of the Human Lung Microbiome

    PubMed Central

    Venkataraman, Arvind; Bassis, Christine M.; Beck, James M.; Young, Vincent B.; Curtis, Jeffrey L.; Huffnagle, Gary B.

    2015-01-01

    ABSTRACT  DNA from phylogenetically diverse microbes is routinely recovered from healthy human lungs and used to define the lung microbiome. The proportion of this DNA originating from microbes adapted to the lungs, as opposed to microbes dispersing to the lungs from other body sites and the atmosphere, is not known. We use a neutral model of community ecology to distinguish members of the lung microbiome whose presence is consistent with dispersal from other body sites and those that deviate from the model, suggesting a competitive advantage to these microbes in the lungs. We find that the composition of the healthy lung microbiome is consistent with predictions of the neutral model, reflecting the overriding role of dispersal of microbes from the oral cavity in shaping the microbial community in healthy lungs. In contrast, the microbiome of diseased lungs was readily distinguished as being under active selection. We also assessed the viability of microbes from lung samples by cultivation with a variety of media and incubation conditions. Bacteria recovered by cultivation from healthy lungs represented species that comprised 61% of the 16S rRNA-encoding gene sequences derived from bronchoalveolar lavage samples. Importance  Neutral distribution of microbes is a distinguishing feature of the microbiome in healthy lungs, wherein constant dispersal of bacteria from the oral cavity overrides differential growth of bacteria. No bacterial species consistently deviated from the model predictions in healthy lungs, although representatives of many of the dispersed species were readily cultivated. In contrast, bacterial populations in diseased lungs were identified as being under active selection. Quantification of the relative importance of selection and neutral processes such as dispersal in shaping the healthy lung microbiome is a first step toward understanding its impacts on host health. PMID:25604788

  12. Levels of homotypic neutralizing antibody in human poliomyelitis three years after infection.

    PubMed

    WINSSER, J; SABIN, A B

    1952-11-01

    Quantitative neutralization tests in monkeys were carried out on sera obtained from 7 patients, 3 months, and 3 years after an attack of poliomyelitis. The serum specimens were tested against 100 to 1000 PD(50) of the patient's own strain of virus, recovered during the acute phase of the illness; all the strains were Type 1. The 6 patients, aged 6 months to 13 years, who had a paralytic attack of the disease, all exhibited very high levels of neutralizing antibody at 3 years as well as at 3 months after onset. The 50 per cent serum dilution titers ranged from about 1:180 to at least 1:860. Since the maximum titers were not established, it is not known to what extent, if any, the level of antibody may have dropped over the 3 year period. One of the patients, with a diagnosis of non-paralytic poliomyelitis, had a negligible or questionable antibody response during convalescence and no demonstrable antibody at 3 years; there is justifiable doubt as to whether the Type 1 poliomyelitis virus recovered from this patient had actually caused infection. Tests for Lansing neutralizing antibody indicated that the 5 patients who had no evidence of previous infection with Type 2 poliomyelitis virus had not become infected with it during the 3 year period. This suggested that these patients did not live in an environment in which infection with poliomyelitis virus is frequent. It is concluded, therefore, that in human beings, paralytic infections due to Type 1 poliomyelitis virus produce large amounts of homotypic neutralizing antibody, which persists at high levels for a period of at least 3 years.

  13. 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.

  14. Structural and Molecular Basis for Ebola Virus Neutralization by Protective Human Antibodies

    PubMed Central

    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.

    2017-01-01

    Ebola virus causes hemorrhagic fever with a high mortality rate and for which there is no approved therapy. Two human monoclonal antibodies, mAb100 and mAb114, in combination protect non-human 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 following 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

  15. Adenovirus-Vectored Broadly Neutralizing Antibodies Directed Against gp120 Prevent Human Immunodeficiency Virus Type 1 Acquisition in Humanized Mice

    PubMed Central

    Liu, Shan; Jackson, Andrew; Beloor, Jagadish; Kumar, Priti; Sutton, Richard E.

    2015-01-01

    Despite nearly three decades of research, a safe and effective vaccine against human immunodeficiency virus type 1 (HIV-1) has yet to be achieved. More recently, the discovery of highly potent anti-gp160 broadly neutralizing antibodies (bNAbs) has garnered renewed interest in using antibody-based prophylactic and therapeutic approaches. Here, we encoded bNAbs in first-generation adenoviral (ADV) vectors, which have the distinctive features of a large coding capacity and ease of propagation. A single intramuscular injection of ADV-vectorized bNAbs in humanized mice generated high serum levels of bNAbs that provided protection against multiple repeated challenges with a high dose of HIV-1, prevented depletion of peripheral CD4+ T cells, and reduced plasma viral loads to below detection limits. Our results suggest that ADV vectors may be a viable option for the prophylactic and perhaps therapeutic use of bNAbs in humans. PMID:25953321

  16. 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

  17. Two amino acid residues confer type specificity to a neutralizing, conformationally dependent epitope on human papillomavirus type 11.

    PubMed Central

    Ludmerer, S W; Benincasa, D; Mark, G E

    1996-01-01

    Characterization of virus binding by neutralizing antibodies is important both in understanding early events in viral infectivity and in development of vaccines. Neutralizing monoclonal antibodies (MAbs) to human papillomavirus type 11 (HPV11) have been described, but mapping the binding site has been difficult because of the conformational nature of key type-specific neutralization epitopes on the L1 coat protein. We have determined those residues of the L1 protein of HPV11 which confer type specificity to the binding of HPV11-neutralizing MAbs. Binding of three HPV11-specific neutralizing MAbs could be redirected to HPV6 L1 virus-like particles in which as few as two substitutions of corresponding amino acid residues from HPV11 L1 have been made, thus demonstrating the importance of these residues to MAb binding through the transfer of a conformationally dependent epitope. In addition, a fourth neutralizing MAb could be distinguished from the other neutralizing MAbs in terms of the amino acid residues which affect binding, suggesting the possibility that it neutralizes HPV11 through a different mechanism. PMID:8676509

  18. Polycistronic expression of human platelet factor 4 with heparin-neutralizing activity in Escherichia coli.

    PubMed

    Duan, Yitao; Wang, Zhe; Wu, Wei; Fang, Zhenjiang; Huang, He

    2012-01-01

    Human platelet factor 4 (hPF4) was evaluated as a clinical alternative to protamine for heparin neutralization, a protector against radiation injury and an anti-neoplastic. To achieve high-level expression of hPF4, expression vectors pET-28a(+)-nf PF4 (n=4, 5, 6) containing n tandem repeats of PF4 were constructed and transformed into the Escherichia coli BL21(DE3) strain. A higher expression level, about 45% of the total proteins (TP), was obtained for E. coli BL21(DE3)/pET28a(+)-nf PF4 (n=4, 5, 6). The purified His-PF4 protein was further identified by cleavage with enterokinase and MS, and its heparin-neutralizing activity was determined by colony formation assay. This study represents a novel approach to large-scale production of PF4 in E. coli, one that might be applied to large-scale production of PF4 protein for possible clinical application. It also provides theoretical points for the expression and purification of other small-molecule peptides.

  19. CROSS-REACTIVE AND POTENT NEUTRALIZING ANTIBODY RESPONSES IN HUMAN SURVIVORS OF NATURAL EBOLAVIRUS INFECTION

    PubMed Central

    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.

    2015-01-01

    Summary 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

  20. Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire

    PubMed Central

    Yeung, Yik Andy; Foletti, Davide; Deng, Xiaodi; Abdiche, Yasmina; Strop, Pavel; Glanville, Jacob; Pitts, Steven; Lindquist, Kevin; Sundar, Purnima D.; Sirota, Marina; Hasa-Moreno, Adela; Pham, Amber; Melton Witt, Jody; Ni, Irene; Pons, Jaume; Shelton, David; Rajpal, Arvind; Chaparro-Riggers, Javier

    2016-01-01

    Staphylococcus aureus is both an important pathogen and a human commensal. To explore this ambivalent relationship between host and microbe, we analysed the memory humoral response against IsdB, a protein involved in iron acquisition, in four healthy donors. Here we show that in all donors a heavily biased use of two immunoglobulin heavy chain germlines generated high affinity (pM) antibodies that neutralize the two IsdB NEAT domains, IGHV4-39 for NEAT1 and IGHV1-69 for NEAT2. In contrast to the typical antibody/antigen interactions, the binding is primarily driven by the germline-encoded hydrophobic CDRH-2 motifs of IGHV1-69 and IGHV4-39, with a binding mechanism nearly identical for each antibody derived from different donors. Our results suggest that IGHV1-69 and IGHV4-39, while part of the adaptive immune system, may have evolved under selection pressure to encode a binding motif innately capable of recognizing and neutralizing a structurally conserved protein domain involved in pathogen iron acquisition. PMID:27857134

  1. Accumulated Bending Energy Elicits Neutral Sphingomyelinase Activity in Human Red Blood Cells

    PubMed Central

    López, David J.; Egido-Gabas, Meritxell; López-Montero, Iván; Busto, Jon V.; Casas, Josefina; Garnier, Marie; Monroy, Francisco; Larijani, Banafshé; Goñi, Félix M.; Alonso, Alicia

    2012-01-01

    We propose that accumulated membrane bending energy elicits a neutral sphingomyelinase (SMase) activity in human erythrocytes. Membrane bending was achieved by osmotic or chemical processes, and SMase activity was assessed by quantitative thin-layer chromatography, high-performance liquid chromatography, and electrospray ionization-mass spectrometry. The activity induced by hypotonic stress in erythrocyte membranes had the pH dependence, ion dependence, and inhibitor sensitivity of mammalian neutral SMases. The activity caused a decrease in SM contents, with a minimum at 6 min after onset of the hypotonic conditions, and then the SM contents were recovered. We also elicited SMase activity by adding lysophosphatidylcholine externally or by generating it with phospholipase A2. The same effect was observed upon addition of chlorpromazine or sodium deoxycholate at concentrations below the critical micellar concentration, and even under hypertonic conditions. A unifying factor of the various agents that elicit this SMase activity is the accumulated membrane bending energy. Both hypo-and hypertonic conditions impose an increased curvature, whereas the addition of surfactants or phospholipase A2 activation increases the outer monolayer area, thus leading to an increased bending energy. The fact that this latent SMase activity is tightly coupled to the membrane bending properties suggests that it may be related to the general phenomenon of stress-induced ceramide synthesis and apoptosis. PMID:22824271

  2. Accumulated bending energy elicits neutral sphingomyelinase activity in human red blood cells.

    PubMed

    López, David J; Egido-Gabas, Meritxell; López-Montero, Iván; Busto, Jon V; Casas, Josefina; Garnier, Marie; Monroy, Francisco; Larijani, Banafshé; Goñi, Félix M; Alonso, Alicia

    2012-05-02

    We propose that accumulated membrane bending energy elicits a neutral sphingomyelinase (SMase) activity in human erythrocytes. Membrane bending was achieved by osmotic or chemical processes, and SMase activity was assessed by quantitative thin-layer chromatography, high-performance liquid chromatography, and electrospray ionization-mass spectrometry. The activity induced by hypotonic stress in erythrocyte membranes had the pH dependence, ion dependence, and inhibitor sensitivity of mammalian neutral SMases. The activity caused a decrease in SM contents, with a minimum at 6 min after onset of the hypotonic conditions, and then the SM contents were recovered. We also elicited SMase activity by adding lysophosphatidylcholine externally or by generating it with phospholipase A(2). The same effect was observed upon addition of chlorpromazine or sodium deoxycholate at concentrations below the critical micellar concentration, and even under hypertonic conditions. A unifying factor of the various agents that elicit this SMase activity is the accumulated membrane bending energy. Both hypo-and hypertonic conditions impose an increased curvature, whereas the addition of surfactants or phospholipase A(2) activation increases the outer monolayer area, thus leading to an increased bending energy. The fact that this latent SMase activity is tightly coupled to the membrane bending properties suggests that it may be related to the general phenomenon of stress-induced ceramide synthesis and apoptosis.

  3. 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.

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

    PubMed

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

    2013-09-03

    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.

  5. A human/murine chimeric fab antibody neutralizes anthrax lethal toxin in vitro.

    PubMed

    Ding, Guipeng; Chen, Ximin; Zhu, Jin; Duesbery, Nicholas S; Cheng, Xunjia; Cao, Brian

    2013-01-01

    Human anthrax infection caused by exposure to Bacillus anthracis cannot always be treated by antibiotics. This is mostly because of the effect of the remaining anthrax toxin in the body. Lethal factor (LF) is a component of lethal toxin (LeTx), which is the major virulence of anthrax toxin. A murine IgG monoclonal antibody (mAb) against LF with blocking activity (coded LF8) was produced in a previous study. In this report, a human/murine chimeric Fab mAb (coded LF8-Fab) was developed from LF8 by inserting murine variable regions into human constant regions using antibody engineering to reduce the incompatibility of the murine antibody for human use. The LF8-Fab expressed in Escherichia coli could specifically identify LF with an affinity of 3.46 × 10(7) L/mol and could neutralize LeTx with an EC50 of 85  μ g/mL. Even after LeTx challenge at various time points, the LF8-Fab demonstrated protection of J774A.1 cells in vitro. The results suggest that the LF8-Fab might be further characterized and potentially be used for clinical applications against anthrax infection.

  6. Autologous and heterologous neutralizing antibody responses following initial seroconversion in human immunodeficiency virus type 1-infected individuals.

    PubMed Central

    Moog, C; Fleury, H J; Pellegrin, I; Kirn, A; Aubertin, A M

    1997-01-01

    In the course of human immunodeficiency virus type 1 (HIV-1) infection, patients develop a strong and persistent immune response characterized by the production of HIV-specific antibodies. The aim of our study was to analyze the appearance of autologous and heterologous neutralizing antibodies in the sera of HIV-infected individuals. For this purpose, primary strains have been isolated from 18 HIV-1-infected subjects prior to seroconversion (in one case) or within 1 to 8 months after seroconversion. Sera, collected at the same time as the virus was isolated and at various times after isolation, have been analyzed for their ability to neutralize the autologous primary strains isolated early after infection, heterologous primary isolates, and cell-line adapted strains. Our neutralization assay, which combines serial dilutions of virus and serial dilutions of sera, is based on the determination of the serum dilution at which a fixed reduction in virus titer (90%) occurs. We have shown that (i) we could not detect autologous neutralizing antibodies in sera collected at the same time as we isolated viruses; (ii) we detected neutralizing antibodies against the autologous strains about 1 year after seroconversion, occasionally after 8 months, but sera were not always available to exclude the presence of neutralizing antibodies at earlier times; (iii) after 1 year, the neutralization response was highly specific to virus present during the early phase of HIV infection; and (iv) heterologous neutralization of primary isolates was detected later (after about 2 years). These results reveal the enormous diversity of neutralization determinants on primary isolates as well as a temporal evolution of the humoral response generating cross-reactive neutralizing antibodies. PMID:9094648

  7. 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

  8. 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

  9. KTP laser and neutral red phototherapy of human squamous cell carcinoma.

    PubMed

    VanderWerf, Q M; Castro, D J; Nguyen, R D; Paiva, M B; Chao, K H; Santillanes, M E; Saxton, R E

    1997-03-01

    Neutral red (NR) is a cationic, nontoxic vital dye employed as a histologic stain for proliferating cells; it has been used clinically for photodynamic treatment of herpes simplex virus lesions. NR is selectively taken up and concentrated by mitotic cells, an important characteristic for more effective antineoplastic agents. In the present study, UCLA-SO-P3 human squamous carcinoma cells displayed minimal toxicity when incubated with up to 50 microg/ml NR in the absence of light. However, cells incubated with greater than 0.5 microg/ml NR followed by exposure to KTP laser light at 532 nm exhibited nearly 100% tumor cell death. The degree of cell toxicity was proportional to NR dose and laser light fluence. This study demonstrates that NR is an excellent cancer cell photosensitizer in vitro, and, after adding additional in vivo preclinical testing, may prove to be a useful agent in photodynamic destruction of head and neck tumors.

  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. An equation for the prediction of human skin permeability of neutral molecules, ions and ionic species.

    PubMed

    Zhang, Keda; Abraham, Michael H; Liu, Xiangli

    2017-04-15

    Experimental values of permeability coefficients, as log Kp, of chemical compounds across human skin were collected by carefully screening the literature, and adjusted to 37°C for the effect of temperature. The values of log Kp for partially ionized acids and bases were separated into those for their neutral and ionic species, forming a total data set of 247 compounds and species (including 35 ionic species). The obtained log Kp values have been regressed against Abraham solute descriptors to yield a correlation equation with R(2)=0.866 and SD=0.432 log units. The equation can provide valid predictions for log Kp of neutral molecules, ions and ionic species, with predictive R(2)=0.858 and predictive SD=0.445 log units calculated by the leave-one-out statistics. The predicted log Kp values for Na(+) and Et4N(+) are in good agreement with the observed values. We calculated the values of log Kp of ketoprofen as a function of the pH of the donor solution, and found that log Kp markedly varies only when ketoprofen is largely ionized. This explains why models that neglect ionization of permeants still yield reasonable statistical results. The effect of skin thickness on log Kp was investigated by inclusion of two indicator variables, one for intermediate thickness skin and one for full thickness skin, into the above equation. The newly obtained equations were found to be statistically very close to the above equation. Therefore, the thickness of human skin used makes little difference to the experimental values of log Kp.

  12. 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.

  13. Viraemia suppressed in HIV-1-infected humans by broadly neutralizing antibody 3BNC117.

    PubMed

    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, 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

    2015-06-25

    HIV-1 immunotherapy with a combination of first generation monoclonal antibodies was largely ineffective in pre-clinical and clinical settings and was therefore abandoned. However, recently developed single-cell-based antibody cloning methods have uncovered a new generation of far more potent broadly neutralizing antibodies to HIV-1 (refs 4, 5). These antibodies can prevent infection and suppress viraemia in humanized mice and nonhuman primates, but their potential for human HIV-1 immunotherapy has not been evaluated. Here we report the results of a first-in-man dose escalation phase 1 clinical trial of 3BNC117, a potent human CD4 binding site antibody, in uninfected and HIV-1-infected individuals. 3BNC117 infusion was well tolerated and demonstrated favourable pharmacokinetics. A single 30 mg kg(-1) infusion of 3BNC117 reduced the viral load in HIV-1-infected individuals by 0.8-2.5 log10 and viraemia 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 viraemia, and that immunotherapy should be explored as a new modality for HIV-1 prevention, therapy and cure.

  14. 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

  15. 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.

  16. THE INCIDENCE OF NEUTRALIZING ANTIBODIES FOR SWINE INFLUENZA VIRUS IN THE SERA OF HUMAN BEINGS OF DIFFERENT AGES

    PubMed Central

    Shope, Richard E.

    1936-01-01

    Sera from a very high proportion of the human adults and new-born infants studied neutralized swine influenza virus; sera from children below the age of 12 years seldom exerted such an effect. The results of neutralization experiments with human sera and the virus of swine influenza have been compared with the outcome of similar tests with the virus of human influenza, and it seems evident that the presence of antibodies neutralizing swine influenza virus cannot be deemed the result of repeated exposures to the current human type of virus. From the known history of swine influenza and the similarity of its etiologic virus to that obtained from man it seems likely that the virus of swine influenza is the surviving prototype of the agent primarily responsible for the great human pandemic of 1918, as Laidlaw has already suggested. The presence in human sera of antibodies neutralizing swine influenza virus is believed to indicate a previous immunizing exposure to, or infection with, an influenza virus of the 1918 type. PMID:19870496

  17. Neutralization serotypes of human immunodeficiency virus type 1 field isolates are not predicted by genetic subtype. The WHO Network for HIV Isolation and Characterization.

    PubMed Central

    Weber, J; Fenyö, E M; Beddows, S; Kaleebu, P; Björndal, A

    1996-01-01

    Human immunodeficiency virus type 1 (HIV-1) primary isolates from four geographical locations in Thailand, Brazil, Rwanda, and Uganda, representing genetic subtypes A, B, C, D, and E, were examined for autologous and heterologous neutralization by panels of human HIV+ polyclonal plasma. In independent linked experiments in three laboratories using diverse methodologies and common reagents, no defined pattern of genetic subtype-specific neutralization was observed. Most plasma tested were broadly cross-neutralizing across two or more genetic subtypes, although the titer of neutralization varied across a wide range. We conclude that the genetic subtypes of HIV-1 are not classical neutralization serotypes. PMID:8892904

  18. In vitro generation and type-specific neutralization of a human papillomavirus type 16 virion pseudotype.

    PubMed Central

    Roden, R B; Greenstone, H L; Kirnbauer, R; Booy, F P; Jessie, J; Lowy, D R; Schiller, J T

    1996-01-01

    We report a system for generating infectious papillomaviruses in vitro that facilitates the analysis of papillomavirus assembly, infectivity, and serologic relatedness. Cultured hamster BPHE-1 cells harboring autonomously replicating bovine papillomavirus type 1 (BPV1) genomes were infected with recombinant Semliki Forest viruses that express the structural proteins of BPV1. When plated on C127 cells, extracts from cells expressing L1 and L2 together induced numerous transformed foci that could be specifically prevented by BPV neutralizing antibodies, demonstrating that BPV infection was responsible for the focal transformation. Extracts from BPHE-1 cells expressing L1 or L2 separately were not infectious. Although Semliki Forest virus-expressed L1 self-assembled into virus-like particles (VLPs), viral DNA was detected in particles only when L2 was coexpressed with L1, indicating that genome encapsidation requires L2. Expression of human papillomavirus type 16 (HPV16) L1 and L2 together in BPHE-1 cells also yielded infectious virus. These pseudotyped virions were neutralized by antiserum to HPV16 VLPs derived from European (114/K) or African (Z-1194) HPV16 variants but not by antisera to BPV VLPs, to a poorly assembling mutant HPV16 L1 protein, or to VLPs of closely related genital HPV types. Extracts from BPHE-1 cells coexpressing BPV L1 and HPV16 L2 or HPV16 L1 and BPV L2 were not infectious. We conclude that (i) mouse C127 cells express the cell surface receptor for HPV16 and are able to uncoat HPV16 capsids; (ii) if a papillomavirus DNA packaging signal exists, then it is conserved between the BPV and HPV16 genomes; (iii) functional L1-L2 interaction exhibits type specificity; and (iv) protection by HPV virus-like particle vaccines is likely to be type specific. PMID:8709207

  19. Evaluation of the suitability of chromatographic systems to predict human skin permeation of neutral compounds.

    PubMed

    Hidalgo-Rodríguez, Marta; Soriano-Meseguer, Sara; Fuguet, Elisabet; Ràfols, Clara; Rosés, Martí

    2013-12-18

    Several chromatographic systems (three systems of high-performance liquid chromatography and two micellar electrokinetic chromatography systems) besides the reference octanol-water partition system are evaluated by a systematic procedure previously proposed in order to know their ability to model human skin permeation. The precision achieved when skin-water permeability coefficients are correlated against chromatographic retention factors is predicted within the framework of the solvation parameter model. It consists in estimating the contribution of error due to the biological and chromatographic data, as well as the error coming from the dissimilarity between the human skin permeation and the chromatographic systems. Both predictions and experimental tests show that all correlations are greatly affected by the considerable uncertainty of the skin permeability data and the error associated to the dissimilarity between the systems. Correlations with much better predictive abilities are achieved when the volume of the solute is used as additional variable, which illustrates the main roles of both lipophilicity and size of the solute to penetrate through the skin. In this way, the considered systems are able to give precise estimations of human skin permeability coefficients. In particular, the HPLC systems with common C18 columns provide the best performances in emulating the permeation of neutral compounds from aqueous solution through the human skin. As a result, a methodology based on easy, fast, and economical HPLC measurements in a common C18 column has been developed. After a validation based on training and test sets, the method has been applied with good results to the estimation of skin permeation of several hormones and pesticides.

  20. 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.

  1. A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

    PubMed

    Scalley-Kim, Michelle L; Hess, Bruce W; Kelly, Ryan L; Krostag, Anne-Rachel F; Lustig, Kurt H; Marken, John S; Ovendale, Pamela J; Posey, Aaron R; Smolak, Pamela J; Taylor, Janelle D L; Wood, C L; Bienvenue, David L; Probst, Peter; Salmon, Ruth A; Allison, Daniel S; Foy, Teresa M; Raport, Carol J

    2012-01-01

    Chemokines play a key role in leukocyte recruitment during inflammation and are implicated in the pathogenesis of a number of autoimmune diseases. As such, inhibiting chemokine signaling has been of keen interest for the development of therapeutic agents. This endeavor, however, has been hampered due to complexities in the chemokine system. Many chemokines have been shown to signal through multiple receptors and, conversely, most chemokine receptors bind to more than one chemokine. One approach to overcoming this complexity is to develop a single therapeutic agent that binds and inactivates multiple chemokines, similar to an immune evasion strategy utilized by a number of viruses. Here, we describe the development and characterization of a novel therapeutic antibody that targets a subset of human CC chemokines, specifically CCL3, CCL4, and CCL5, involved in chronic inflammatory diseases. Using a sequential immunization approach, followed by humanization and phage display affinity maturation, a therapeutic antibody was developed that displays high binding affinity towards the three targeted chemokines. In vitro, this antibody potently inhibits chemotaxis and chemokine-mediated signaling through CCR1 and CCR5, primary chemokine receptors for the targeted chemokines. Furthermore, we have demonstrated in vivo efficacy of the antibody in a SCID-hu mouse model of skin leukocyte migration, thus confirming its potential as a novel therapeutic chemokine antagonist. We anticipate that this antibody will have broad therapeutic utility in the treatment of a number of autoimmune diseases due to its ability to simultaneously neutralize multiple chemokines implicated in disease pathogenesis.

  2. Three amino acid residues in the envelope of human immunodeficiency virus type 1 CRF07_BC regulate viral neutralization susceptibility to the human monoclonal neutralizing antibody IgG1b12.

    PubMed

    Nie, Jianhui; Zhao, Juan; Chen, Qingqing; Huang, Weijin; Wang, Youchun

    2014-10-01

    The CD4 binding site (CD4bs) of envelope glycoprotein (Env) is an important conserved target for anti-human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12 (b12) could recognize conformational epitopes that overlap the CD4bs of Env. Different virus strains, even derived from the same individual, showed distinct neutralization susceptibility to b12. We examined the key amino acid residues affecting b12 neutralization susceptibility using single genome amplification and pseudovirus neutralization assay. Eleven amino acid residues were identified that affect the sensitivity of Env to b12. Through site-directed mutagenesis, an amino acid substitution at position 182 in the V2 region of Env was confirmed to play a key role in regulating the b12 neutralization susceptibility. The introduction of V182L to a resistant strain enhanced its sensitivity to b12 more than twofold. Correspondingly, the introduction of L182V to a sensitive strain reduced its sensitivity to b12 more than tenfold. Amino acid substitution at positions 267 and 346 could both enhance the sensitivity to b12 more than twofold. However, no additive effect was observed when the three site mutageneses were introduced into the same strain, and the sensitivity was equivalent to the single V182L mutation. CRF07_BC is a major circulating recombinant form of HIV-1 prevalent in China. Our data may provide important information for understanding the molecular mechanism regulating the neutralization susceptibility of CRF07_BC viruses to b12 and may be helpful for a vaccine design targeting the CD4bs epitopes.

  3. Signature Biochemical Properties of Broadly Cross-Reactive HIV-1 Neutralizing Antibodies in Human Plasma

    PubMed Central

    Sajadi, Mohammad M.; Lewis, George K.; Seaman, Michael S.; Guan, Yongjun; Redfield, Robert R.

    2012-01-01

    The common properties of broadly cross-reactive HIV-1 neutralization antibodies found in certain HIV-1-infected individuals holds significant value for understanding natural and vaccine-mediated anti-HIV immunity. Recent efforts have addressed this question by deriving neutralizing monoclonal anti-envelope antibodies from memory B cell pools of selected subjects. However, it has been more difficult to identify whether broadly neutralizing antibodies circulating in plasma possess shared characteristics among individuals. To address this question, we used affinity chromatography and isoelectric focusing to fractionate plasma immunoglobulin from 10 HIV-1-infected subjects (5 subjects with broad HIV-1 neutralizing activity and 5 controls). We find that plasma neutralizing activity typically partitions into at least two subsets of antibodies. Antibodies with restricted neutralization breadth have relatively neutral isoelectric points and preferentially bind to envelope monomers and trimers versus core antigens from which variable loops and other domains have been deleted. In comparison, broadly neutralizing antibodies account for a minor fraction of the total anti-envelope response. They are consistently distinguished by more basic isoelectric points and specificity for epitopes shared by monomeric gp120, gp120 core, or CD4-induced structures. Such biochemical properties might be exploited to reliably predict or produce broad anti-HIV immunity. PMID:22379105

  4. Signature biochemical properties of broadly cross-reactive HIV-1 neutralizing antibodies in human plasma.

    PubMed

    Sajadi, Mohammad M; Lewis, George K; Seaman, Michael S; Guan, Yongjun; Redfield, Robert R; DeVico, Anthony L

    2012-05-01

    The common properties of broadly cross-reactive HIV-1 neutralization antibodies found in certain HIV-1-infected individuals holds significant value for understanding natural and vaccine-mediated anti-HIV immunity. Recent efforts have addressed this question by deriving neutralizing monoclonal anti-envelope antibodies from memory B cell pools of selected subjects. However, it has been more difficult to identify whether broadly neutralizing antibodies circulating in plasma possess shared characteristics among individuals. To address this question, we used affinity chromatography and isoelectric focusing to fractionate plasma immunoglobulin from 10 HIV-1-infected subjects (5 subjects with broad HIV-1 neutralizing activity and 5 controls). We find that plasma neutralizing activity typically partitions into at least two subsets of antibodies. Antibodies with restricted neutralization breadth have relatively neutral isoelectric points and preferentially bind to envelope monomers and trimers versus core antigens from which variable loops and other domains have been deleted. In comparison, broadly neutralizing antibodies account for a minor fraction of the total anti-envelope response. They are consistently distinguished by more basic isoelectric points and specificity for epitopes shared by monomeric gp120, gp120 core, or CD4-induced structures. Such biochemical properties might be exploited to reliably predict or produce broad anti-HIV immunity.

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

    PubMed Central

    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.

    2016-01-01

    Prevention of mother to child transmission (MTCT) of HIV remains a major objective where antenatal care is not readily accessible. We tested anti-HIV-1 human neutralizing monoclonal antibodies (NmAb) as post-exposure therapy in an infant macaque model for intrapartum MTCT. One-month-old rhesus macaques were inoculated orally with 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 following administration. Replicating virus was found in multiple tissues by day 1 in animals without treatment. All NmAb-treated macaques were free of virus in blood and tissues at 6 months post-exposure. We detected no anti-SHIV T cell responses in blood or tissues at necropsy, and no virus emerged following CD8+ T cell depletion. These results suggest early passive immunotherapy can eliminate early viral foci and thereby prevent the establishment of viral reservoirs. PMID:26998834

  6. 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

  7. A novel human recombinant antibody fragment capable of neutralizing Mexican scorpion toxins.

    PubMed

    Riaño-Umbarila, Lidia; Olamendi-Portugal, Timoteo; Morelos-Juárez, Citlalli; Gurrola, Georgina B; Possani, Lourival D; Becerril, Baltazar

    2013-12-15

    Using phage display and directed evolution, our group has progressed in the construction of a second family of human single chain variable fragments (scFv) which bind to scorpion toxins dangerous to mammals. It was observed that scFv C1 only bound initially to toxin Cn2, which constitutes 6.8% of whole venom from the scorpion Centruroides noxius Hoffman. Only a few amino acid changes were necessary to extend its recognition to other similar toxins and without affecting the recognition for its primary antigen (Cn2 toxin). One variant of scFv C1 (scFv 202F) was selected after two cycles of directed evolution against Cll1 toxin, the second major toxic component from the venom of the Mexican scorpion Centruroides limpidus limpidus Karsh (0.5% of the whole venom). scFv 202F is also capable of recognizing Cn2 toxin. Despite not having the highest affinity for toxins Cll1 (KD = 25.1 × 10(-9) M) or Cn2 (KD = 8.1 × 10(-9) M), this antibody fragment neutralized one LD50 of each one of these toxins. Additionally, scFv 202F moderately recognized Cll2 toxin which constitutes 1.5% of the venom from C. limpidus. Based on our previous experience, we consider that these results are promising; consequently, we continue working on generating new optimized variants from scFv C1 that could be part of a recombinant scorpion anti-venom from human origin, that might reach the market in the near future.

  8. 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.

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

    PubMed Central

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

    2015-01-01

    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

  10. Efficient neutralizing activity of cocktailed recombinant human antibodies against hepatitis A virus infection in vitro and in vivo.

    PubMed

    Cao, Jingyuan; Meng, Shufang; Li, Chuan; Ji, Yan; Meng, Qingling; Zhang, Quanfu; Liu, Feng; Li, Jiandong; Bi, Shengli; Li, Dexin; Liang, Mifang

    2008-07-01

    Hepatitis A virus (HAV) is the major pathogen responsible for acute infectious hepatitis A, a disease that is prevalent worldwide. Although HAV immunization effectively prevents infection, primary immunizations must be administered at least 2 weeks prior to HAV exposure. In contrast, passive immunization with pooled human immunoglobulin (Ig) can provide immediate and rapid protection from HAV infection. Because the use of human sera-derived Igs carries the risk of contamination, we sought to develop recombinant HAV-neutralizing human antibodies. We prepared a combinatorial phage display library of recombinant human anti-HAV antibodies from RNA extracted from the blood lymphocytes of a convalescent hepatitis A patient. Two recombinant human IgG antibodies, HAIgG16 and HAIgG78, were screened from the antibody library by their ability to bind with high affinity to purified, inactivated HAV virions. These antibodies recognized different epitopes of the HAV virion capsid, and competed with both patient sera and well-characterized neutralizing mouse monoclonal antibodies. A cocktailed mixture of HAIgG16 and HAIgG78 at a 3:1 ratio was prepared to compare its combined biological activity with that conferred by each antibody individually. The cocktailed antibodies displayed a stronger neutralizing activity in vitro than that observed with either HAIgG16 and HAIgG78 alone. To determine the in vivo neutralizing abilities of these antibodies, rhesus monkeys were inoculated with cocktailed antibodies and challenged with HAV. Whereas control animals developed hepatitis A and seroconverted to the HAV antibody, animals receiving cocktailed antibodies were protected either from viral infection or from developing clinical hepatitis. These results demonstrate that recombinant human antibody preparations could be used to prevent or treat early-stage HAV infection.

  11. A human monoclonal IgG1 potently neutralizing the pro-inflammatory cytokine GM-CSF.

    PubMed

    Krinner, Eva-Maria; Raum, Tobias; Petsch, Silke; Bruckmaier, Sandra; Schuster, Ioana; Petersen, Laetitia; Cierpka, Ronny; Abebe, Derege; Mølhøj, Michael; Wolf, Andreas; Sørensen, Poul; Locher, Mathias; Baeuerle, Patrick A; Hepp, Julia

    2007-02-01

    The pro-inflammatory cytokine GM-CSF is aberrantly produced in many autoimmune and chronic inflammatory human diseases. GM-CSF neutralization by antibodies has been shown to have a profound therapeutic effect in animal models of rheumatoid arthritis, inflammatory lung diseases, psoriasis and multiple sclerosis. Moreover, the absence of GM-CSF in null mutant mice ameliorates or prevents certain of these diseases. Here we describe the biophysical and biological properties of a human anti-GM-CSF IgG1 antibody designated MT203, which was derived by phage display guided selection. MT203 bound with picomolar affinity to an epitope on human and macaque GM-CSF involved in high-affinity receptor interaction. As a consequence, the antibody potently prevented both GM-CSF-induced proliferation of TF-1 cells with a sub-nanomolar IC50 value and the production of the chemokine IL-8 by U937 cells. MT203 neutralized equally well recombinant (r) human (h) GM-CSF from Escherichia coli and yeast, and also normally glycosylated GM-CSF secreted by human lung epithelial cells in response to IL-1beta stimulation. Furthermore, MT203 significantly reduced both survival and activation of peripheral human eosinophils as may be required for effective treatment of inflammatory lung diseases. The antibody did not show a detectable loss of neutralizing activity after 5 days in human serum at 37 degrees C. Based on its favorable properties, MT203 has been selected for development as a novel anti-inflammatory human monoclonal antibody with therapeutic potential in a multitude of human autoimmune and inflammatory diseases.

  12. 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.

  13. Neutralizing antibody responses against autologous and heterologous viruses in acute versus chronic human immunodeficiency virus (HIV) infection: evidence for a constraint on the ability of HIV to completely evade neutralizing antibody responses.

    PubMed

    Deeks, Steven G; Schweighardt, Becky; Wrin, Terri; Galovich, Justin; Hoh, Rebecca; Sinclair, Elizabeth; Hunt, Peter; McCune, Joseph M; Martin, Jeffrey N; Petropoulos, Christos J; Hecht, Frederick M

    2006-06-01

    Acute human immunodeficiency virus (HIV) infection is associated with the rapid development of neutralization escape mutations. The degree to which viral evolution persists in chronic infection has not been well characterized, nor is it clear if all patients develop high-level neutralization antibody escape. We therefore measured neutralizing antibody responses against autologous and heterologous viruses in a cohort of acutely and chronically infected subjects (n = 65). Neutralizing antibody responses against both autologous virus and heterologous viruses were lower among individuals with acute infection than among those with chronic infection. Among chronically infected individuals, there was a negative correlation between the level of neutralizing antibodies against autologous virus and the level of viremia. In contrast, there was a positive correlation between the level of neutralizing antibodies against a panel of heterologous viruses and the level of viremia. Viral evolution, as defined by the presence of higher neutralizing titers directed against earlier viruses than against contemporaneous viruses, was evident for subjects with recent infection but absent for those with chronic infection. In summary, neutralizing antibody responses against contemporaneous autologous viruses are absent in early HIV infection but can be detected at low levels in chronic infection, particularly among those controlling HIV in the absence of therapy. HIV replication either directly or indirectly drives the production of increasing levels of antibodies that cross-neutralize heterologous primary isolates. Collectively, these observations indicate that although HIV continuously drives the production of neutralizing antibodies, there may be limits to the capacity of the virus to evolve continuously in response to these antibodies. These observations also suggest that the neutralizing antibody response may contribute to the long-term control of HIV in some patients while protecting

  14. Human Ig knockin mice to study the development and regulation of HIV-1 broadly neutralizing antibodies.

    PubMed

    Verkoczy, Laurent; Alt, Frederick W; Tian, Ming

    2017-01-01

    A major challenge for HIV-1 vaccine research is developing a successful immunization approach for inducing broadly neutralizing antibodies (bnAbs). A key shortcoming in meeting this challenge has been the lack of animal models capable of identifying impediments limiting bnAb induction and ranking vaccine strategies for their ability to promote bnAb development. Since 2010, immunoglobulin knockin (KI) technology, involving inserting functional rearranged human variable exons into the mouse IgH and IgL loci has been used to express bnAbs in mice. This approach has allowed immune tolerance mechanisms limiting bnAb production to be elucidated and strategies to overcome such limitations to be evaluated. From these studies, along with the wealth of knowledge afforded by analyses of recombinant Ig-based bnAb structures, it became apparent that key functional features of bnAbs often are problematic for their elicitation in mice by classic vaccine paradigms, necessitating more iterative testing of new vaccine concepts. In this regard, bnAb KI models expressing deduced precursor V(D)J rearrangements of mature bnAbs or unrearranged germline V, D, J segments (that can be assembled into variable region exons that encode bnAb precursors), have been engineered to evaluate novel immunogens/regimens for effectiveness in driving bnAb responses. One promising approach emerging from such studies is the ability of sequentially administered, modified immunogens (designed to bind progressively more mature bnAb precursors) to initiate affinity maturation. Here, we review insights gained from bnAb KI studies regarding the regulation and induction of bnAbs, and discuss new Ig KI methodologies to manipulate the production and/or expression of bnAbs in vivo, to further facilitate vaccine-guided bnAb induction studies.

  15. Detection of Neutralizing Antibodies against Human Papillomaviruses (HPV) by Inhibition of Gene Transfer Mediated by HPV Pseudovirions

    PubMed Central

    Bousarghin, Latifa; Combita-Rojas, Alba-Lucia; Touzé, Antoine; Mehdaoui, Slimane El; Sizaret, Pierre-Yves; Bravo, Maria-Mercedes; Coursaget, Pierre

    2002-01-01

    The goal of this study was to develop a human papillomavirus (HPV) neutralization assay using HPV pseudovirions generated in vitro. For this purpose, gene transfer efficiency of HPV virus-like particles (VLPs) was improved by using direct interaction between a reporter plasmid and the VLPs. Electron microscopic observation of the interaction between DNA molecules and VLPs revealed that VLPs always interact with a single DNA molecule and that VLPs bind to the end of linearized DNA molecules. An 100-fold improvement in the gene transfer was obtained by simple interaction between a linearized DNA molecule and VLPs. Moreover, direct interaction methods offer the possibility of transferring plasmids a size higher than that of the papillomavirus genome. The approach that we developed to generate HPV-16 and HPV-31 pseudovirions proved to be suitable for testing neutralizing antibodies in human sera both after immunization and after natural infection. PMID:11880418

  16. [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.

  17. 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

  18. Affinity maturation of a broadly neutralizing human monoclonal antibody that prevents acute hepatitis C virus infection in mice.

    PubMed

    Keck, Zhen-Yong; Wang, Yong; Lau, Patrick; Lund, Garry; Rangarajan, Sneha; Fauvelle, Catherine; Liao, Grant C; Holtsberg, Frederick W; Warfield, Kelly L; Aman, M Javad; Pierce, Brian G; Fuerst, Thomas R; Bailey, Justin R; Baumert, Thomas F; Mariuzza, Roy A; Kneteman, Norman M; Foung, Steven K H

    2016-12-01

    Direct-acting antivirals (DAAs) have led to a high cure rate in treated patients with chronic hepatitis C virus (HCV) infection, but this still leaves a large number of treatment failures secondary to the emergence of resistance-associated variants (RAVs). To increase the barrier to resistance, a complementary strategy is to use neutralizing human monoclonal antibodies (HMAbs) to prevent acute infection. However, earlier efforts with the selected antibodies led to RAVs in animal and clinical studies. Therefore, we identified an HMAb that is less likely to elicit RAVs for affinity maturation to increase potency and, more important, breadth of protection. Selected matured antibodies show improved affinity and neutralization against a panel of diverse HCV isolates. Structural and modeling studies reveal that the affinity-matured HMAb mediates virus neutralization, in part, by inducing conformational change to the targeted epitope, and that the maturated light chain is responsible for the improved affinity and breadth of protection. A matured HMAb protected humanized mice when challenged with an infectious HCV human serum inoculum for a prolonged period. However, a single mouse experienced breakthrough infection after 63 days when the serum HMAb concentration dropped by several logs; sequence analysis revealed no viral escape mutation.

  19. Deleterious alleles in the human genome are on average younger than neutral alleles of the same frequency.

    PubMed

    Kiezun, Adam; Pulit, Sara L; Francioli, Laurent C; van Dijk, Freerk; Swertz, Morris; Boomsma, Dorret I; van Duijn, Cornelia M; Slagboom, P Eline; van Ommen, G J B; Wijmenga, Cisca; de Bakker, Paul I W; Sunyaev, Shamil R

    2013-01-01

    Large-scale population sequencing studies provide a complete picture of human genetic variation within the studied populations. A key challenge is to identify, among the myriad alleles, those variants that have an effect on molecular function, phenotypes, and reproductive fitness. Most non-neutral variation consists of deleterious alleles segregating at low population frequency due to incessant mutation. To date, studies characterizing selection against deleterious alleles have been based on allele frequency (testing for a relative excess of rare alleles) or ratio of polymorphism to divergence (testing for a relative increase in the number of polymorphic alleles). Here, starting from Maruyama's theoretical prediction (Maruyama T (1974), Am J Hum Genet USA 6:669-673) that a (slightly) deleterious allele is, on average, younger than a neutral allele segregating at the same frequency, we devised an approach to characterize selection based on allelic age. Unlike existing methods, it compares sets of neutral and deleterious sequence variants at the same allele frequency. When applied to human sequence data from the Genome of the Netherlands Project, our approach distinguishes low-frequency coding non-synonymous variants from synonymous and non-coding variants at the same allele frequency and discriminates between sets of variants independently predicted to be benign or damaging for protein structure and function. The results confirm the abundance of slightly deleterious coding variation in humans.

  20. The human alpha defensin HD5 neutralizes JC polyomavirus infection by reducing endoplasmic reticulum traffic and stabilizing the viral capsid.

    PubMed

    Zins, Stephen R; Nelson, Christian D S; Maginnis, Melissa S; Banerjee, Rahul; O'Hara, Bethany A; Atwood, Walter J

    2014-01-01

    Progressive multifocal leukoencephalopathy (PML) is a fatal disease with limited treatment options, both clinically and in the research pipeline. Potential therapies would target and neutralize its etiologic agent, JC polyomavirus (JCPyV). The innate immune response to JCPyV infection has not been studied, and little is known about the initial host response to polyomavirus infection. This study examined the ability of a human alpha defensin, HD5, to neutralize JCPyV infection in human fetal glial cells. We show that HD5, by binding to the virion, blocks infection. The JCPyV-HD5 complexes bind to and enter host cells but are reduced in their ability to reach the endoplasmic reticulum (ER), where virions are normally uncoated. Furthermore, HD5 binding to the virion stabilizes the capsid and prevents genome release. Our results show that HD5 neutralizes JCPyV infection at an early postentry step in the viral life cycle by stabilizing the viral capsid and disrupting JCPyV trafficking. This study provides a naturally occurring platform for developing antivirals to treat PML and also expands on the known capabilities of human defensins.

  1. The Human Alpha Defensin HD5 Neutralizes JC Polyomavirus Infection by Reducing Endoplasmic Reticulum Traffic and Stabilizing the Viral Capsid

    PubMed Central

    Zins, Stephen R.; Nelson, Christian D. S.; Maginnis, Melissa S.; Banerjee, Rahul; O'Hara, Bethany A.

    2014-01-01

    Progressive multifocal leukoencephalopathy (PML) is a fatal disease with limited treatment options, both clinically and in the research pipeline. Potential therapies would target and neutralize its etiologic agent, JC polyomavirus (JCPyV). The innate immune response to JCPyV infection has not been studied, and little is known about the initial host response to polyomavirus infection. This study examined the ability of a human alpha defensin, HD5, to neutralize JCPyV infection in human fetal glial cells. We show that HD5, by binding to the virion, blocks infection. The JCPyV-HD5 complexes bind to and enter host cells but are reduced in their ability to reach the endoplasmic reticulum (ER), where virions are normally uncoated. Furthermore, HD5 binding to the virion stabilizes the capsid and prevents genome release. Our results show that HD5 neutralizes JCPyV infection at an early postentry step in the viral life cycle by stabilizing the viral capsid and disrupting JCPyV trafficking. This study provides a naturally occurring platform for developing antivirals to treat PML and also expands on the known capabilities of human defensins. PMID:24198413

  2. 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.

  3. Potent Intratype Neutralizing Activity Distinguishes Human Immunodeficiency Virus Type 2 (HIV-2) from HIV-1

    PubMed Central

    Özkaya Şahin, Gülşen; Holmgren, Birgitta; da Silva, Zacarias; Nielsen, Jens; Nowroozalizadeh, Salma; Esbjörnsson, Joakim; Månsson, Fredrik; Andersson, Sören; Norrgren, Hans; Aaby, Peter

    2012-01-01

    HIV-2 has a lower pathogenicity and transmission rate than HIV-1. Neutralizing antibodies could be contributing to these observations. Here we explored side by side the potency and breadth of intratype and intertype neutralizing activity (NAc) in plasma of 20 HIV-1-, 20 HIV-2-, and 11 dually HIV-1/2 (HIV-D)-seropositive individuals from Guinea-Bissau, West Africa. Panels of primary isolates, five HIV-1 and five HIV-2 isolates, were tested in a plaque reduction assay using U87.CD4-CCR5 cells as targets. Intratype NAc in HIV-2 plasma was found to be considerably more potent and also broader than intratype NAc in HIV-1 plasma. This indicates that HIV-2-infected individuals display potent type-specific neutralizing antibodies, whereas such strong type-specific antibodies are absent in HIV-1 infection. Furthermore, the potency of intratype NAc was positively associated with the viral load of HIV-1 but not HIV-2, suggesting that NAc in HIV-1 infection is more antigen stimulation dependent than in HIV-2 infection, where plasma viral loads typically are at least 10-fold lower than in HIV-1 infection. Intertype NAc of both HIV-1 and HIV-2 infections was, instead, of low potency. HIV-D subjects had NAc to HIV-2 with similar high potency as singly HIV-2-infected individuals, whereas neutralization of HIV-1 remained poor, indicating that the difference in NAc between HIV-1 and HIV-2 infections depends on the virus itself. We suggest that immunogenicity and/or antigenicity, meaning the neutralization phenotype, of HIV-2 is distinct from that of HIV-1 and that HIV-2 may display structures that favor triggering of potent neutralizing antibody responses. PMID:22072782

  4. Human health risks in an old gold mining area with circum-neutral drainage, central Portugal.

    PubMed

    Carvalho, P C S; Neiva, A M R; Silva, M M V G; Santos, A C T

    2017-02-01

    The former mine of Escádia Grande was active at the middle of 1900 and was exploited for Au and Ag. The mineralized quartz veins consist mainly of quartz, arsenopyrite, pyrite, rare chalcopyrite, galena, sphalerite, gold and argentite. The mine dumps and tailings were deposited close to a stream, and there is a river beach downstream used for recreational proposes. Two villages are also located close to the old mining area. Mine wastes contained up to 8090 mg/kg of As and 70.1 mg/kg of Sb. The waters of the stream that cross the mining area have circum-neutral pH values and contained elevated concentrations of As reaching up to 284 µg/L. However, geochemical speciation modeling (Phreeq C) revealed that As was mainly present as As (V). Arsenic concentrations in waters are attenuated throughout the stream, mainly by the iron-(hydro)-oxides adsorption upstream. However, at 2 km downstream of mine wastes in the river beach, the waters still exceeded 10 µg/L of As, the drinking water limit. The waters also have NO2(-), Cu and Cd concentrations higher than drinking water limit. The stream sediments have As concentrations up to 45 times higher (3140 mg/kg) than the limit of the sediment guideline values of NWQMS (2000). The maximum arsenic concentrations in soils are also up to 27 times higher (5940 mg/kg) than the maximum concentrations in streams from FOREGS Geochemical Atlas of Europe. The use of river beach for recreational purposes causes cancer risk (4.48 × 10(-6)) higher than USEPA limit, mainly due to the arsenic exposure. Even for recreational purposes, stream sediments and soils in the old mining area have high non-carcinogenic effects (2.76 and 4.78, respectively) for children, also related to the arsenic exposure mainly by the ingestion pathway, and the risk is unacceptable according to the limits of USEPA. Moreover, the cancer risk resulting from exposure of adults to arsenic in soils also has unacceptable non-cancer risk (1.13). Arsenic is the

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

    PubMed

    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.

  6. 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

  7. 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

  8. 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

  9. 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.

  10. Human Monoclonal Antibody 81.39a Effectively Neutralizes Emerging Influenza A Viruses of Group 1 and 2 Hemagglutinins

    PubMed Central

    Marjuki, Henju; Mishin, Vasiliy P.; Chai, Ning; Tan, Man-Wah; Newton, Elizabeth M.; Tegeris, John; Erlandson, Karl; Willis, Melissa; Jones, Joyce; Davis, Todd; Stevens, James

    2016-01-01

    ABSTRACT The pandemic threat posed by emerging zoonotic influenza A viruses necessitates development of antiviral agents effective against various antigenic subtypes. Human monoclonal antibody (hMAb) targeting the hemagglutinin (HA) stalk offers a promising approach to control influenza virus infections. Here, we investigated the ability of the hMAb 81.39a to inhibit in vitro replication of human and zoonotic viruses, representing 16 HA subtypes. The majority of viruses were effectively neutralized by 81.39a at a 50% effective concentration (EC50) of <0.01 to 4.9 μg/ml. Among group 2 HA viruses tested, a single A(H7N9) virus was not neutralized at 50 μg/ml; it contained HA2-Asp19Gly, an amino acid position previously associated with resistance to neutralization by the group 2 HA-neutralizing MAb CR8020. Notably, among group 1 HA viruses, H11-H13 and H16 subtypes were not neutralized at 50 μg/ml; they shared the substitution HA2-Asp19Asn/Ala. Conversely, H9 viruses harboring HA2-Asp19Ala were fully susceptible to neutralization. Therefore, amino acid variance at HA2-Asp19 has subtype-specific adverse effects on in vitro neutralization. Mice given a single injection (15 or 45 mg/kg of body weight) at 24 or 48 h after infection with recently emerged A(H5N2), A(H5N8), A(H6N1), or A(H7N9) viruses were protected from mortality and showed drastically reduced lung viral titers. Furthermore, 81.39a protected mice infected with A(H7N9) harboring HA2-Asp19Gly, although the antiviral effect was lessened. A(H1N1)pdm09-infected ferrets receiving a single dose (25 mg/kg) had reduced viral titers and showed less lung tissue injury, despite 24- to 72-h-delayed treatment. Taken together, this study provides experimental evidence for the therapeutic potential of 81.39a against diverse influenza A viruses. IMPORTANCE Zoonotic influenza viruses, such as A(H5N1) and A(H7N9) subtypes, have caused severe disease and deaths in humans, raising public health concerns. Development of novel

  11. Human TNF cytokine neutralization with a vNAR from Heterodontus francisci shark: a potential therapeutic use.

    PubMed

    Camacho-Villegas, Tanya; Mata-Gonzalez, Teresa; Paniagua-Solis, Jorge; Sanchez, Edna; Licea, Alexei

    2013-01-01

    The therapeutic use of single domain antibodies (sdAbs) is a promising new approach because these small antibodies maintain antigen recognition and neutralization capacity, have thermal and chemical stability and have good solubility. In this study, using phage display technology, we isolated a variable domain of a IgNAR (vNAR) from a Heterodontus francisci shark immunized against the recombinant human cytokine TNFα (rhTNFα). One clone T43, which expresses the vNAR protein in the periplasmic space, was isolated from the fourth round of panning. T43 had the capacity to recognize rhTNF and neutralize it in vitro, indicating that T43 has potential as a therapeutic that can be used for diseases in which this pro-inflammatory cytokine needs to be controlled.

  12. V3 variability can influence the ability of an antibody to neutralize or enhance infection by diverse strains of human immunodeficiency virus type 1.

    PubMed Central

    Kliks, S C; Shioda, T; Haigwood, N L; Levy, J A

    1993-01-01

    Human monoclonal antibodies (mAbs) to two contiguous epitopes in the V3 loop of the human immunodeficiency virus type 1 (HIV-1) envelope have shown different effects on three distinct strains of the virus: neutralization, enhancement, or resistance to both processes. Only one amino acid in the mAb epitopes proximal to the crown of the V3 loop was different among these three strains. Substitution of this amino acid in the neutralizable strain with the amino acid of the neutralization-resistant strain or the enhanceable strain resulted in loss of both activities. The conversion of this single amino acid in the neutralization-resistant strain to that of the amino acid found in the neutralization-sensitive strain did not confer the ability for the virus to be neutralized. However, additional changes in neighboring amino acids in the V3 loop succeeded in conferring the neutralization capability. These observations indicate that one antibody species can exert three different effects on various HIV-1 strains. They could explain the emergence of neutralization "escape" variants in the presence of the neutralizing antibodies. Moreover, the results suggest caution in immunization of individuals with the envelope region from one strain since the antibodies induced may show a neutralizing effect against the homologous strain but enhancing effects against other unrelated strains. Images Fig. 4 PMID:7505441

  13. 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.

  14. Crystal Structure of the Hendra Virus Attachment G Glycoprotein Bound to a Potent Cross-Reactive Neutralizing Human Monoclonal Antibody

    PubMed Central

    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

  15. A humanized neutralizing antibody against MERS-CoV targeting the receptor-binding domain of the spike protein.

    PubMed

    Li, Yan; Wan, Yuhua; Liu, Peipei; Zhao, Jincun; Lu, Guangwen; Qi, Jianxun; Wang, Qihui; Lu, Xuancheng; Wu, Ying; Liu, Wenjun; Zhang, Buchang; Yuen, Kwok-Yung; Perlman, Stanley; Gao, George F; Yan, Jinghua

    2015-11-01

    The newly-emerging Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe and fatal acute respiratory disease in humans. Despite global efforts, the potential for an associated pandemic in the future cannot be excluded. The development of effective counter-measures is urgent. MERS-CoV-specific anti-viral drugs or vaccines are not yet available. Using the spike receptor-binding domain of MERS-CoV (MERS-RBD) to immunize mice, we identified two neutralizing monoclonal antibodies (mAbs) 4C2 and 2E6. Both mAbs potently bind to MERS-RBD and block virus entry in vitro with high efficacy. We further investigated their mechanisms of neutralization by crystallizing the complex between the Fab fragments and the RBD, and solved the structure of the 4C2 Fab/MERS-RBD complex. The structure showed that 4C2 recognizes an epitope that partially overlaps the receptor-binding footprint in MERS-RBD, thereby interfering with the virus/receptor interactions by both steric hindrance and interface-residue competition. 2E6 also blocks receptor binding, and competes with 4C2 for binding to MERS-RBD. Based on the structure, we further humanized 4C2 by preserving only the paratope residues and substituting the remaining amino acids with the counterparts from human immunoglobulins. The humanized 4C2 (4C2h) antibody sustained similar neutralizing activity and biochemical characteristics to the parental mouse antibody. Finally, we showed that 4C2h can significantly abate the virus titers in lungs of Ad5-hCD26-transduced mice infected with MERS-CoV, therefore representing a promising agent for prophylaxis and therapy in clinical settings.

  16. Evaluation of three different formats of a neutralizing single chain human antibody against toxin Cn2: neutralization capacity versus thermodynamic stability.

    PubMed

    Quintero-Hernández, Veronica; Del Pozo-Yauner, Luis; Pedraza-Escalona, Martha; Juárez-González, Victor R; Alcántara-Recillas, Israel; Possani, Lourival D; Becerril, Baltazar

    2012-04-30

    The single-chain antibody fragment (scFv) 6009F, obtained by directed evolution, neutralizes the effects of the Cn2 toxin, which is the major toxic component of Centruroides noxius scorpion venom. In this work we compared the neutralization capacity and the thermodynamic stability of scFv 6009F with those of two other derived formats: Fab 6009F and diabody 6009F. Additionally, the affinity constants to Cn2 toxin of the three recombinant antibody fragments were determined by means of BIAcore. We found a correlation between the thermodynamic stability of these antibody fragments with their neutralization capacity. The order of thermodynamic stability determined was Fab≫scFv>diabody. The Fab and scFv were capable of neutralizing the toxic effects of Cn2 and whole venom but the diabody was unable to fully neutralize intoxication. In silico analysis of the diabody format indicates that the reduction of stability and neutralization capacity could be explained by a less cooperative interface between the heavy and the light variable domains.

  17. Molecular cloning of the first human monoclonal antibodies neutralizing with high potency swine-origin influenza A pandemic virus (S-OIV).

    PubMed

    Burioni, Roberto; Canducci, Filippo; Mancini, Nicasio; Clementi, Nicola; Sassi, Monica; De Marco, Donata; Saita, Diego; Diotti, Roberta Antonia; Sautto, Giuseppe; Sampaolo, Michela; Clementi, Massimo

    2009-10-01

    The pandemic caused by the new H1N1 swine-origin influenza virus (S-OIV) strain is a worldwide health emergency and alternative therapeutic and prophylactic options are greatly needed. Two human monoclonal antibody Fab fragments (HMab) neutralizing the novel H1N1 influenza strain at very low concentrations were cloned from a patient who had a broad-range anti-H1N1 serum neutralizing activity. The two HMabs neutralized S-OIV with an IC50 of 2.8 and 4 microg/mL. The genes coding for the neutralizing HMabs could be used for generating full human monoclonal IgGs that can be safely administered with the potentially of representing a novel drug to be used in the prophylaxis and the treatment of this human infection. This is the first report of molecular cloning of human monoclonal antibodies against the new pandemic swine-origin influenza virus.

  18. 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

  19. 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.

  20. 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

  1. Broad-range neutralizing anti-influenza A human monoclonal antibodies: new perspectives in therapy and prophylaxis.

    PubMed

    Clementi, Nicola; Criscuolo, Elena; Castelli, Matteo; Clementi, Massimo

    2012-10-01

    Broadly neutralizing monoclonal antibodies (mAbs) directed against different subtypes of influenza A viruses are novel tools for the potential development of effective anti-influenza prophylactic and therapeutic strategies. In both cases, the main candidates for passive transfer and new vaccine development are represented by protective mAbs directed against influenza hemagglutinin (HA). A large number of mAbs directed against influenza HA has been developed to date. However, even if they can be useful and contribute to develop new vaccinal strategies, only few of them can be a good candidate for human administration. In this review, we will describe the most relevant human mAb directed against influenza HA able to recognize highly divergent influenza isolates and possibly useful for human therapy and prophylaxis.

  2. 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

  3. Protection of Macaques against pathogenic simian/human immunodeficiency virus 89.6PD by passive transfer of neutralizing antibodies.

    PubMed

    Mascola, J R; Lewis, M G; Stiegler, G; Harris, D; VanCott, T C; Hayes, D; Louder, M K; Brown, C R; Sapan, C V; Frankel, S S; Lu, Y; Robb, M L; Katinger, H; Birx, D L

    1999-05-01

    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.

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

    SciTech Connect

    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.

    2015-10-26

    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. In this paper, 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. Finally, 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.

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

    DOE PAGES

    Long, Feng; Fong, Rachel H.; Austin, Stephen K.; ...

    2015-10-26

    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. In this paper, 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 ofmore » 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. Finally, 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.« less

  6. International technology transfer of a GCLP-compliant HIV-1 neutralizing antibody assay for human clinical trials.

    PubMed

    Ozaki, Daniel A; Gao, Hongmei; 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.

  7. 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

  8. Neutral red uptake inhibition in adhered and adhering rat hepatoma-derived Fa32 cells to predict human toxicity.

    PubMed

    Dierickx, Paul J; Scheers, Ellen M

    2002-01-01

    The cytotoxicity of the MEIC (Multicentre Evaluation of In vitro Cytotoxicity) reference chemicals was investigated by measuring the neutral red uptake inhibition in adhered and adhering rat hepatoma-derived Fa32 cells. The adhered cells were seeded and then treated and the adhering cells were treated simultaneously upon seeding. Five of the 44 test chemicals were twofold more toxic in adhering cells; ethylene glycol was 28-fold more toxic and mercuric chloride was 5.2-fold more toxic than in adhered cells. The cytotoxicity of dithiothreitol was altered in the same way as that of ethylene glycol, probably by interacting with calcium. When the neutral red uptake inhibition was compared with human toxicity, the correlation coefficient for adhering cells was almost identical to that obtained previously in human hepatoma-derived Hep G2 cells and slightly higher for adhered cells. The Hep G2 assay was the best acute in vitro assay for the prediction of human toxicity within the MEIC study. An obviously better correlation was obtained when the strong intoxicant mercuric chloride was withdrawn from the comparison, both for the adhered and the adhering cells. Altogether, the results can be integrated very well with the basal cytotoxicity concept.

  9. 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

  10. 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.

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

    PubMed

    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

    2016-03-03

    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 C(18:1)-ceramide, a substrate of Acer3, in murine immune cells or CECs. Knocking out Acer3 enhanced the elevation of C(18:1)-ceramide and the expression of pro-inflammatory cytokines in immune cells and CECs in response to LPS challenge. Similar to Acer3 knockout, treatment with C(18:1)-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 C(18:1)-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 C(18

  12. Identification of cross-reactive and serotype 2-specific neutralization epitopes on VP3 of human rotavirus.

    PubMed Central

    Taniguchi, K; Maloy, W L; Nishikawa, K; Green, K Y; Hoshino, Y; Urasawa, S; Kapikian, A Z; Chanock, R M; Gorziglia, M

    1988-01-01

    The group A rotaviruses are composed of at least seven serotypes. Serotype specificity is defined mainly by an outer capsid protein, VP7. In contrast, the other surface protein, VP3 (775 amino acids), appears to be associated with both serotype-specific and heterotypic immunity. To identify the cross-reactive and serotype-specific neutralization epitopes on VP3 of human rotavirus, we sequenced the VP3 gene of antigenic mutants resistant to each of seven anti-VP3 neutralizing monoclonal antibodies (N-MAbs) which exhibited heterotypic or serotype 2-specific reactivity, and we defined three distinct neutralization epitopes on VP3. The mutants sustained single amino acid substitutions at position 305, 392, 433, or 439. Amino acid position 305 was critical to epitope I, whereas amino acid position 433 was critical to epitope III. In contrast, epitope II appeared to be more dependent upon conformation and protein folding because both amino acid positions 392 and 439 appeared to be critical. These four positions clustered in a relatively limited area of VP5, the larger of the two cleavage products of VP3. At the positions where amino acid substitutions occurred, there was a correlation between amino acid sequence homology among different serotypes and the reactivity patterns of various viruses with the N-MAbs used for selection of mutants. A synthetic peptide (amino acids 296 to 313) which included the sequence of epitope I reacted with its corresponding N-MAb, suggesting that the region contains a sequential antigenic determinant. These data may prove useful in current efforts to develop vaccines against human rotavirus infection. PMID:2453680

  13. Structural basis of clade-specific HIV-1 neutralization by humanized anti-V3 monoclonal antibody KD-247

    PubMed Central

    Kirby, Karen A.; Ong, Yee Tsuey; Hachiya, Atsuko; Laughlin, Thomas G.; Chiang, Leslie A.; Pan, Yun; Moran, Jennifer L.; Marchand, Bruno; Singh, Kamalendra; Gallazzi, Fabio; Quinn, Thomas P.; Yoshimura, Kazuhisa; Murakami, Toshio; Matsushita, Shuzo; Sarafianos, Stefan G.

    2015-01-01

    Humanized monoclonal antibody KD-247 targets the Gly312-Pro313-Gly314-Arg315 arch of the third hypervariable (V3) loop of the HIV-1 surface glycoprotein. It potently neutralizes many HIV-1 clade B isolates, but not of other clades. To understand the molecular basis of this specificity, we solved a high-resolution (1.55 Å) crystal structure of the KD-247 antigen binding fragment and examined the potential interactions with various V3 loop targets. Unlike most antibodies, KD-247 appears to interact with its target primarily through light chain residues. Several of these interactions involve Arg315 of the V3 loop. To evaluate the role of light chain residues in the recognition of the V3 loop, we generated 20 variants of KD-247 single-chain variable fragments with mutations in the antigen-binding site. Purified proteins were assessed for V3 loop binding using AlphaScreen technology and for HIV-1 neutralization. Our data revealed that recognition of the clade-specificity defining residue Arg315 of the V3 loop is based on a network of interactions that involve TyrL32, TyrL92, and AsnL27d that directly interact with Arg315, thus elucidating the molecular interactions of KD-247 with its V3 loop target.—Kirby, K. A., Ong, Y. T., Hachiya, A., Laughlin, T. G., Chiang, L. A., Pan, Y., Moran, J. L., Marchand, B., Singh, K., Gallazzi, F., Quinn, T. P., Yoshimura, K., Murakami, T., Matsushita, S., Sarafianos, S. G. Structural basis of clade-specific HIV-1 neutralization by humanized anti-V3 monoclonal antibody KD-247. PMID:25351987

  14. 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.

  15. Human Immunodeficiency Virus Type 1 Neutralization Measured by Flow Cytometric Quantitation of Single-Round Infection of Primary Human T Cells

    PubMed Central

    Mascola, John R.; Louder, Mark K.; Winter, Christine; Prabhakara, Ranjani; De Rosa, Stephen C.; Douek, Daniel C.; Hill, Brenna J.; Gabuzda, Dana; Roederer, Mario

    2002-01-01

    There is currently intensive research on the design of novel human immunodeficiency virus type 1 (HIV-1) vaccine immunogens that can elicit potent neutralizing antibodies. A prerequisite for comparing and optimizing these strategies is the ability to precisely measure neutralizing antibody responses. To this end, we sought to develop an assay that directly quantifies single-round HIV-1 infection of peripheral blood mononuclear cells (PBMC). Initial experiments demonstrated that essentially all productively infected PBMC could be identified by flow cytometric detection of intracellular p24 antigen (p24-Ag). After infection of PBMC with HIV-1, p24+ lymphocytes could be distinguished beginning 1 day postinfection, and the majority of CD8− T cells were p24-Ag positive by 3 to 4 days postinfection. To directly quantify first-round infection, we included a protease inhibitor in PBMC cultures. The resulting 2-day assay was highly sensitive and specific for the detection of HIV-1-infected PBMC. Serial dilutions of virus stocks demonstrated that the number of target cells infected was directly related to the amount of infectious virus input into the assay. In neutralization assays, the flow cytometric enumeration of first-round infection of PBMC provided quantitative data on the number of target cells infected and on the inactivation of infectious virus due to reaction with antibody. We also used this single-round assay to compare the percentage of cells expressing p24-Ag to the number of copies of HIV-1 gag per 100 PBMC. The precision and reproducibility of this assay will facilitate the measurement of HIV-1 neutralization, particularly incrementally improved neutralizing antibody responses generated by new candidate vaccines. PMID:11967298

  16. Neutralizing IgG at the portal of infection mediates protection against vaginal simian/human immunodeficiency virus challenge.

    PubMed

    Klein, Katja; Veazey, Ronald S; Warrier, Ranjit; Hraber, Peter; Doyle-Meyers, Lara A; Buffa, Viviana; Liao, Hua-Xin; Haynes, Barton F; Shaw, George M; Shattock, Robin J

    2013-11-01

    Neutralizing antibodies may have critical importance in immunity against human immunodeficiency virus type 1 (HIV-1) infection. However, the amount of protective antibody needed at mucosal surfaces has not been fully established. Here, we evaluated systemic and mucosal pharmacokinetics (PK) and pharmacodynamics (PD) of 2F5 IgG and 2F5 Fab fragments with respect to protection against vaginal challenge with simian-human immunodeficiency virus-BaL in macaques. Antibody assessment demonstrated that 2F5 IgG was more potent than polymeric forms (IgM and IgA) across a range of cellular and tissue models. Vaginal challenge studies demonstrated a dose-dependent protection for 2F5 IgG and no protection with 2F5 Fab despite higher vaginal Fab levels at the time of challenge. Animals receiving 50 or 25 mg/kg of body weight 2F5 IgG were completely protected, while 3/5 animals receiving 5 mg/kg were protected. In the control animals, infection was established by a minimum of 1 to 4 transmitted/founder (T/F) variants, similar to natural human infection by this mucosal route; in the two infected animals that had received 5 mg 2F5 IgG, infection was established by a single T/F variant. Serum levels of 2F5 IgG were more predictive of sterilizing protection than measured vaginal levels. Fc-mediated antiviral activity did not appear to influence infection of primary target cells in cervical explants. However, PK studies highlighted the importance of the Fc portion in tissue biodistribution. Data presented in this study may be important in modeling serum levels of neutralizing antibodies that need to be achieved by either vaccination or passive infusion to prevent mucosal acquisition of HIV-1 infection in humans.

  17. Resistance to neutralization by broadly reactive antibodies to the human immunodeficiency virus type 1 gp120 glycoprotein conferred by a gp41 amino acid change.

    PubMed Central

    Thali, M; Charles, M; Furman, C; Cavacini, L; Posner, M; Robinson, J; Sodroski, J

    1994-01-01

    A neutralization-resistant variant of human immunodeficiency virus type 1 (HIV-1) that emerged during in vitro propagation of the virus in the presence of neutralizing serum from an infected individual has been described. A threonine-for-alanine substitution at position 582 in the gp41 transmembrane envelope glycoprotein of the variant virus was responsible for the neutralization-resistant phenotype (M.S. Reitz, Jr., C. Wilson, C. Naugle, R. C. Gallo, and M. Robert-Guroff, Cell 54:57-63, 1988). The mutant virus also exhibited reduced sensitivity to neutralization by 30% of HIV-1-positive sera that neutralized the parental virus, suggesting that a significant fraction of the neutralizing activity within these sera can be affected by the amino acid change in gp41 (C. Wilson, M. S. Reitz, Jr., K. Aldrich, P. J. Klasse, J. Blomberg, R. C. Gallo, and M. Robert-Guroff, J. Virol. 64:3240-3248, 1990). It is shown here that the change of alanine 582 to threonine specifically confers resistance to neutralizing by antibodies directed against both groups of discontinuous, conserved epitopes related to the CD4 binding site on the gp120 exterior envelope glycoprotein. Only minor differences in binding of these antibodies to wild-type and mutant envelope glycoproteins were observed. Thus, the antigenic structure of gp120 can be subtly affected by an amino acid change in gp41, with important consequences for sensitivity to neutralization. Images PMID:7507184

  18. An investigation of the genetic basis of increased susceptibility to neutralization by anti-fusion glycoprotein antibody arising on passage of human respiratory syncytial virus in cell culture.

    PubMed

    Hiriote, W; Gias, E L Michael; Welsh, S H; Toms, G L

    2015-01-01

    Human respiratory syncytial virus isolates have previously been shown to exhibit resistance to neutralization by anti-fusion glycoprotein antibodies that is lost on passage in cell culture. Early passage resistant and late passage susceptible stocks of two virus isolates from different epidemics were cloned by plaque purification. Early passage stocks of both isolates yielded predominantly neutralization resistant clones while late passage stocks yielded predominantly susceptible clones. On further characterization of resistant and susceptible clones, resistant virus yields were lower and they were relatively resistant to both neutralization and fusion inhibition by anti-F murine monoclonal antibodies and were also resistant to neutralization by human sera and by Palivizumab. The full genome of resistant and susceptible clones from one of the isolates was sequenced. Four differences, confirmed by sequencing sister clones, were found between resistant and susceptible clones, one in each of the SH, G, F, and L genes.

  19. Plasma pharmacokinetics and biological activity of a human immunodeficiency virus type 1 neutralizing human monoclonal antibody, F105, in cynomolgus monkeys.

    PubMed

    Cavacini, L A; Power, J; Emes, C L; Mace, K; Treacy, G; Posner, M R

    1994-05-01

    The IgG1 kappa human monoclonal antibody (HMab), F105 reacts with a discontinuous epitope on the CD4 binding site (CD4BS) of human immunodeficiency virus type 1 (HIV-1)/gp120 and has broad neutralizing activity. F105 HMab (60 mg/kg bolus) was administered intravenously to four monkeys and serum was collected at intervals to determine pharmacokinetics in a primate model. Average serum F105 concentrations, as determined by enzyme-linked immunosorbent assay, were analyzed with MINSQ software using a two-compartment, first-order model. The half-life for the alpha phase of the distribution curve is 6.7 h and for the beta elimination phase, 9.6 days. The volume of distribution is 0.65 L/kg and the rate of clearance 2 ml/kg/h. Serum levels of 1.3-1.6 mg/ml of F105 were maintained for 24 h. When monkey serum from day 15 postdose was tested, total serum F105 was 230 +/- 79 micrograms/ml and was immunoreactive with cells infected with the MN and IIIB strains of HIV-1 as determined by flow cytometry. Binding activity was identical to that obtained with stock F105 HMab. Identical neutralizing activity between the injected and uninjected antibody was also observed. Thus, serum neutralizing titers (90%) of 1:2000 at peak and 1:30 at day 15 postdose for MN virus were observed. These data indicate that high in vivo levels of HMab F105 can be attained by single bolus administration with full retention of biological activity. Of importance, levels of antibody necessary for effective neutralization can be achieved and maintained.

  20. In vivo effects of antirheumatic drugs on neutral collagenolytic proteases in human rheumatoid arthritis cartilage and synovium.

    PubMed

    Martel-Pelletier, J; Cloutier, J M; Pelletier, J P

    1988-08-01

    The destruction of joints in rheumatoid arthritis (RA) is thought to be related in part to an increased synthesis of proteolytic enzymes. We have determined neutral collagenolytic protease activity levels in human RA synovia and articular cartilage and examined the in vivo effects of various therapeutic regimens on enzyme levels. Neutral metallocollagenolytic enzyme (NMCE) was measured in 29 RA cartilages and synovial membranes. In addition, synovial serine protease levels were determined. Specimens were divided into 4 groups according to prescribed medications: (1) nonsteroidal antiinflammatory drugs alone; (2) steroids alone; (3) steroids and gold; and (4) steroids and methotrexate (MTX). Ten normal specimens were used as controls. Total and active NMCE measured in both RA cartilage and synovial membrane specimens showed a significantly higher level of activity than in controls (p less than 0.0001, p less than 0.005; p less than 0.004, p less than 0.02). MTX was found to markedly decrease NMCE activity; cartilage NMCE level in patients with RA receiving MTX was reduced, compared to the other subgroups. This was particularly noted for the active form. Synovial NMCE levels from the MTX subgroup for both enzyme forms were much lower than in any other RA subgroup, significantly lower than in the RA group as a whole (p less than 0.05), and similar to controls. RA synovial membrane serine protease activity showed an increase compared to controls. Again, MTX markedly decreased the activity of this class of enzyme. Our data strongly support the role of neutral proteases in the destruction of RA joints. MTX was the only drug to consistently decrease these enzyme levels in joint tissues.

  1. Crystal structure of human antibody 2909 reveals conserved features of quaternary structure-specific antibodies that potently neutralize HIV-1.

    PubMed

    Changela, Anita; Wu, Xueling; Yang, Yongping; Zhang, Baoshan; Zhu, Jiang; Nardone, Glenn A; O'Dell, Sijy; Pancera, Marie; Gorny, Miroslaw K; Phogat, Sanjay; Robinson, James E; Stamatatos, Leonidas; Zolla-Pazner, Susan; Mascola, John R; Kwong, Peter D

    2011-03-01

    Monoclonal antibody 2909 belongs to a class of potently neutralizing antibodies that recognize quaternary epitopes on HIV-1. Some members of this class, such as 2909, are strain specific, while others, such as antibody PG16, are broadly neutralizing; all, however, recognize a region on the gp120 envelope glycoprotein that includes two loops (V2 and V3) and forms appropriately only in the oligomeric HIV-1 spike (gp120(3)/gp41(3)). Here we present the crystal structure of 2909 and report structure-function analysis with antibody chimeras composed of 2909 and other members of this antibody class. The 2909 structure was dominated by a heavy-chain third-complementarity-determining region (CDR H3) of 21 residues, which comprised 36% of the combining surface and formed a β-hairpin club extending ∼20 Å beyond the rest of the antibody. Sequence analysis and mass spectrometry identified sites of tyrosine sulfation at the middle and top of CDR H3; substitutions with phenylalanine either ablated (middle substitution) or substantially diminished (top substitution) neutralization. Chimeric antibodies composed of heavy and light chains, exchanged between 2909 and other members of the class, indicated a substantial lack of complementation. Comparison of 2909 to PG16 (which is tyrosine sulfated and the only other member of the class for which a structure has previously been reported) showed that both utilize protruding, anionic CDR H3s for recognition. Thus, despite some diversity, members of this class share structural and functional similarities, with conserved features of the CDR H3 subdomain likely reflecting prevalent solutions by the human immune system for recognition of a quaternary site of HIV-1 vulnerability.

  2. Generation and characterization of tabalumab, a human monoclonal antibody that neutralizes both soluble and membrane-bound B-cell activating factor

    PubMed Central

    Manetta, Joseph; Bina, Holly; Ryan, Paul; Fox, Niles; Witcher, Derrick R; Kikly, Kristine

    2014-01-01

    B-cell activating factor (BAFF) is a B-cell survival factor with a key role in B-cell homeostasis and tolerance. Dysregulated BAFF expression may contribute to autoimmune diseases or B-cell malignancies via effects on abnormal B-lymphocyte activation, proliferation, survival, and immunoglobulin secretion. Monoclonal antibodies were generated against human BAFF, characterized for species specificity and affinity, and screened for the ability to neutralize both membrane-bound and soluble BAFF. In addition, studies were undertaken to determine the relative potency of membrane-bound and soluble BAFF. Tabalumab has a high affinity for human, cynomolgus monkey, and rabbit BAFF. No binding to mouse BAFF was detected. Tabalumab was able to neutralize soluble human, cynomolgus monkey, or rabbit BAFF with equal potency. Our data demonstrate that membrane-bound BAFF can be a more potent stimulus for B-cells than soluble BAFF, and tabalumab also neutralized membrane-bound BAFF. Tabalumab prevented BAFF from binding to BAFF receptors and demonstrated pharmacodynamic effects in human BAFF transgenic mice. Tabalumab is a high-affinity human antibody with neutralizing activity against membrane-bound and soluble BAFF. Given our findings that membrane-bound BAFF can have greater in vitro potency than soluble BAFF, neutralization of both forms of BAFF is likely to be important for optimal therapeutic effect. PMID:25258549

  3. Involvement of ACSL in local synthesis of neutral lipids in cytoplasmic lipid droplets in human hepatocyte HuH7.

    PubMed

    Fujimoto, Yasuyuki; Itabe, Hiroyuki; Kinoshita, Tetsuaki; Homma, Koichi J; Onoduka, Jun; Mori, Masahiro; Yamaguchi, Shinji; Makita, Minoru; Higashi, Yusuke; Yamashita, Atsushi; Takano, Tatsuya

    2007-06-01

    Lipid droplets (LDs) function as intracellular storage depots of neutral lipids. Recently, we identified long-chain acyl-coenzyme A synthetase 3 (ACSL3) as a major LD-associated protein in the human hepatocyte cell line HuH7. In this study, we investigated whether droplet-associated ACSL is involved in lipid metabolism in LDs. Addition of oleic acid (OA) to culture medium was shown to enhance the intracellular accumulation of LDs in the cells, which was accompanied by an increase of droplet ACSL3. When LD-enriched cells induced by OA were further incubated without OA for 3 days, approximately 80% of LDs were retained in the cells. Conversely, cellular LD content was greatly decreased after the addition of an ACSL inhibitor, triacsin C. This was accompanied by a concomitant decrease of the droplet ACSL3. Incubation of isolated LD fractions with (14)C-labeled OA or palmitic acid resulted in [(14)C]acyl-CoA generation in vitro, indicating the presence of ACSL activity in LDs. The droplet ACSL activity varied according to the quantity of LDs in their emergence and disappearance in cells. Incubation of the LD fraction with [(14)C]oleoyl-CoA resulted in radioactive triacylglycerol and cholesteryl esters. These results suggest that LD ACSL activity is involved in local synthesis of neutral lipids and LD formation.

  4. 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

  5. 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.

  6. A neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection.

    PubMed

    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

    2009-10-01

    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 TCID(50) 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.

  7. Quantitative model of antibody- and soluble CD4-mediated neutralization of primary isolates and T-cell line-adapted strains of human immunodeficiency virus type 1.

    PubMed Central

    Klasse, P J; Moore, J P

    1996-01-01

    Primary isolates (PI) of human immunodeficiency virus type 1 (HIV-1) are considerably less sensitive than T-cell line-adapted strains to neutralization by soluble CD4 and by most cross-reactive monoclonal antibodies to the viral envelope (Env) glycoprotein, as well as by postinfection and postvaccination sera (J. P. Moore and D. D. Ho, AIDS 9 [suppl. A]:5117-5136, 1995). We developed a quantitative model to explain the neutralization resistance of PI. The factors incorporated into the model are the dissociation constants for the binding of the neutralizing agent to native Env oligomers, the number of outer Env molecules on the viral surface (which decreases by shedding), and the minimum number of Env molecules required for attachment and fusion. We conclude that modest differences in all these factors can, when combined, explain a relative neutralization resistance of PI versus T-cell line-adapted strains that sometimes amounts to several orders of magnitude. The hypothesis that neutralization of HIV is due to the reduction below a minimum number of the Env molecules on a virion available for attachment and fusion is at odds with single- and few-hit neutralization theories. Our analysis of these ideas favors the hypothesis that neutralization of HIV is instead a competitive blocking of interactions with cellular factors, including adsorption receptors. PMID:8648701

  8. Presence of neutralizing antibodies to heterologous human immunodeficiency virus type 1 isolates in sera of infected individuals is not predictive of rate of disease progression.

    PubMed Central

    Warren, R Q; Wong, M T; Melcher, G P; Blatt, S P; Zapiola, I; Bouzas, M B; Muchinik, G; Anderson, S A; Kennedy, R C

    1995-01-01

    These studies were undertaken to examine whether the presence of human immunodeficiency virus type 1 (HIV-1)-neutralizing antibodies in sera of infected individuals would alter the rate of disease progression. HIV-1-infected individuals (n = 87) were initially examined for neutralizing activity in vitro against both laboratory and tissue culture-adapted clinical heterologous HIV-1 isolates. The neutralizing activities of sera were determined by a 90% or greater reduction in HIV-1 p24 levels in vitro. In a cross-sectional analysis of all infected individuals, we observed that sera from asymptomatic individuals neutralized a significantly greater number of heterologous HIV-1 isolates than sera from symptomatic patients. Patients who could be followed up longitudinally (n = 24) were then studied to determine the impact of neutralizing antibodies on the rate of disease progression. We observed no significant difference between the numbers of HIV-1 isolates neutralized in vitro by sera from patients who remained clinically stable and by those from patients who progressed rapidly. Our data indicated that the presence or absence of neutralizing antibodies to heterologous HIV-1 isolates was not associated with the rate of disease progression. PMID:7583913

  9. Antibodies Elicited by Multiple Envelope Glycoprotein Immunogens in Primates Neutralize Primary Human Immunodeficiency Viruses (HIV-1) Sensitized by CD4-Mimetic Compounds

    PubMed Central

    Madani, Navid; Princiotto, Amy M.; Easterhoff, David; Bradley, Todd; Luo, Kan; Williams, Wilton B.; Liao, Hua-Xin; Moody, M. Anthony; Phad, Ganesh E.; Vázquez Bernat, Néstor; Melillo, Bruno; Santra, Sampa; Smith, Amos B.; Karlsson Hedestam, Gunilla B.; Haynes, Barton

    2016-01-01

    ABSTRACT The human immunodeficiency virus (HIV-1) envelope glycoproteins (Env) mediate virus entry through a series of complex conformational changes triggered by binding to the receptors CD4 and CCR5/CXCR4. Broadly neutralizing antibodies that recognize conserved Env epitopes are thought to be an important component of a protective immune response. However, to date, HIV-1 Env immunogens that elicit broadly neutralizing antibodies have not been identified, creating hurdles for vaccine development. Small-molecule CD4-mimetic compounds engage the CD4-binding pocket on the gp120 exterior Env and induce Env conformations that are highly sensitive to neutralization by antibodies, including antibodies directed against the conserved Env region that interacts with CCR5/CXCR4. Here, we show that CD4-mimetic compounds sensitize primary HIV-1 to neutralization by antibodies that can be elicited in monkeys and humans within 6 months by several Env vaccine candidates, including gp120 monomers. Monoclonal antibodies directed against the gp120 V2 and V3 variable regions were isolated from the immunized monkeys and humans; these monoclonal antibodies neutralized a primary HIV-1 only when the virus was sensitized by a CD4-mimetic compound. Thus, in addition to their direct antiviral effect, CD4-mimetic compounds dramatically enhance the HIV-1-neutralizing activity of antibodies that can be elicited with currently available immunogens. Used as components of microbicides, the CD4-mimetic compounds might increase the protective efficacy of HIV-1 vaccines. IMPORTANCE Preventing HIV-1 transmission is a high priority for global health. Eliciting antibodies that can neutralize transmitted strains of HIV-1 is difficult, creating problems for the development of an effective vaccine. We found that small-molecule CD4-mimetic compounds sensitize HIV-1 to antibodies that can be elicited in vaccinated humans and monkeys. These results suggest an approach to prevent HIV-1 sexual transmission in

  10. 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

  11. Neutralizing Antibodies from the Sera of Human Immunodeficiency Virus Type 1-Infected Individuals Bind to Monomeric gp120 and Oligomeric gp140

    PubMed Central

    Stamatos, Nicholas M.; Mascola, John R.; Kalyanaraman, Vaniambadi S.; Louder, Mark K.; Frampton, Lynn M.; Birx, Deborah L.; VanCott, Thomas C.

    1998-01-01

    Antibodies that neutralize primary isolates of human immunodeficiency virus type 1 (HIV-1) appear during HIV-1 infection but are difficult to elicit by immunization with current vaccine products comprised of monomeric forms of HIV-1 envelope glycoprotein gp120. The limited neutralizing antibody response generated by gp120 vaccine products could be due to the absence or inaccessibility of the relevant epitopes. To determine whether neutralizing antibodies from HIV-1-infected patients bind to epitopes accessible on monomeric gp120 and/or oligomeric gp140 (ogp140), purified total immunoglobulin from the sera of two HIV-1-infected patients as well as pooled HIV immune globulin were selectively depleted of antibodies which bound to immobilized gp120 or ogp140. After passage of each immunoglobulin preparation through the respective columns, antibody titers against gp120 and ogp140 were specifically reduced at least 128-fold. The gp120- and gp140-depleted antibody fraction from each serum displayed reduced neutralization activity against three primary and two T-cell line-adapted (TCLA) HIV-1 isolates. Significant residual neutralizing activity, however, persisted in the depleted sera, indicating additional neutralizing antibody specificities. gp120- and ogp140-specific antibodies eluted from each column neutralized both primary and TCLA viruses. These data demonstrate the presence and accessibility of epitopes on both monomeric gp120 and ogp140 that are specific for antibodies that are capable of neutralizing primary isolates of HIV-1. Thus, the difficulties associated with eliciting neutralizing antibodies by using current monomeric gp120 subunit vaccines may be related less to improper protein structure and more to ineffective immunogen formulation and/or presentation. PMID:9811699

  12. Human Immunodeficiency Virus Type 1 gp120 Induces Apoptosis in Human Primary Neurons through Redox-Regulated Activation of Neutral Sphingomyelinase

    PubMed Central

    Jana, Arundhati; Pahan, Kalipada

    2007-01-01

    Human immunodeficiency virus type 1 (HIV-1) infection is known to cause disorders of the CNS, including HIV-associated dementia (HAD). HIV-1 coat protein gp120 (glycoprotein 120) induces neuronal apoptosis and has been implicated in the pathogenesis of HAD. However, the mechanism by which gp120 causes neuronal apoptosis is poorly understood. The present study underlines the importance of gp120 in inducing the production of ceramide, an important inducer of apoptosis, in human primary neurons. gp120 induced the activation of sphingomyelinases (primarily the neutral one) and the production of ceramide in primary neurons. Antisense knockdown of neutral (NSMase) but not acidic (ASMase) sphingomyelinase markedly inhibited gp120-mediated apoptosis and cell death of primary neurons, suggesting that the activation of NSMase but not ASMase plays an important role in gp120-mediated neuronal apoptosis. Similarly, the HIV-1 regulatory protein Tat also induced neuronal cell death via NSMase. Furthermore, gp120-induced production of ceramide was redox sensitive, because reactive oxygen species were involved in the activation of NSMase but not ASMase. gp120 coupled CXCR4 (CXC chemokine receptor 4) to induce NADPH oxidase-mediated production of superoxide radicals in neurons, which was involved in the activation of NSMase but not ASMase. These studies suggest that gp120 may induce neuronal apoptosis in the CNS of HAD patients through the CXCR4–NADPH oxidase–superoxide–NSMase–ceramide pathway. PMID:15509740

  13. Neutral metoclopramide sensitizes cytotoxicity induced by ionizing radiation in SCID mice xenografted with a human brain astrocytoma.

    PubMed

    Hua, J; Olsson, A R; Pero, R W

    1997-12-10

    A formulation of metoclopramide (MCA) conformationally altered by neutralization of pH (nMCA, Neu-Sensamide) has been shown to have the same efficacy of enhancing the cytotoxicity of a single dose of 1 Gy radiation as acidic formulations (e.g., Primperan, Sensamide) in a human lung adenocarcinoma (H2981) xenografted into SCID mice. In the present study, 2 x 1 Gy radiation was combined with 2 x 2 mg nMCA/kg body weight injected 2 hr before radiation treatment for evaluation of radiosensitization in SCID mice xenografted with a human brain astrocytoma (T24). Given in this treatment schedule, nMCA alone at 2 mg/kg showed no cytotoxic effect on tumor growth in vivo. When combined with 2 x 1 Gy of radiation, however, the cytotoxicity was significantly increased as measured by tumor growth delay over the radiation-only-treated group. Furthermore, nMCA was absorbed into brains of mice and rats as efficiently as acidic MCA (aMCA) when analyzed 45 min after i.m. injection by high-performance liquid chromatography.

  14. 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.

  15. Enhanced humanization and affinity maturation of neutralizing anti-hepatitis B virus preS1 antibody based on antigen-antibody complex structure.

    PubMed

    Kim, Jin Hong; Gripon, Philippe; Bouezzedine, Fidaa; Jeong, Mun Sik; Chi, Seung-Wook; Ryu, Seong-Eon; Hong, Hyo Jeong

    2015-01-16

    To improve a previously constructed broadly neutralizing hepatitis B virus (HBV)-specific preS1 humanized antibody (HzKR127), we further humanized it through specificity-determining residue (SDR) grafting. Moreover, we improved affinity by mutating two residues in heavy-chain complementarity-determining regions (CDR), on the basis of the crystal structure of the antigen-antibody complex. HzKR127-3.2 exhibited 2.5-fold higher affinity and enhanced virus-neutralizing activity compared to the original KR127 antibody and showed less immunogenic potential than HzKR127. Enhanced virus-neutralizing activity was achieved by the increased association rate, providing insights into engineering potent antibody therapeutics for HBV immunoprophylaxis. HzKR127-3.2 may be a good candidate for HBV immunoprophylaxis.

  16. 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.

  17. Structural basis for Marburg virus neutralization by a cross-reactive human antibody.

    PubMed

    Hashiguchi, Takao; Fusco, Marnie L; Bornholdt, Zachary A; Lee, Jeffrey E; Flyak, Andrew I; Matsuoka, Rei; Kohda, Daisuke; Yanagi, Yusuke; Hammel, Michal; Crowe, James E; Saphire, Erica Ollmann

    2015-02-26

    The filoviruses, including Marburg and Ebola, express a single glycoprotein on their surface, termed GP, which is responsible for attachment and entry of target cells. Filovirus GPs differ by up to 70% in protein sequence, and no antibodies are yet described that cross-react among them. Here, we present the 3.6 Å crystal structure of Marburg virus GP in complex with a cross-reactive antibody from a human survivor, and a lower resolution structure of the antibody bound to Ebola virus GP. The antibody, MR78, recognizes a GP1 epitope conserved across the filovirus family, which likely represents the binding site of their NPC1 receptor. Indeed, MR78 blocks binding of the essential NPC1 domain C. These structures and additional small-angle X-ray scattering of mucin-containing MARV and EBOV GPs suggest why such antibodies were not previously elicited in studies of Ebola virus, and provide critical templates for development of immunotherapeutics and inhibitors of entry.

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

    PubMed

    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; Mascola, John R; Nabel, Gary J

    2015-08-01

    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 g/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.

  19. Administration of nucleoside-modified mRNA encoding broadly neutralizing antibody protects humanized mice from HIV-1 challenge

    PubMed Central

    Pardi, Norbert; Secreto, Anthony J.; Shan, Xiaochuan; Debonera, Fotini; Glover, Joshua; Yi, Yanjie; Muramatsu, Hiromi; Ni, Houping; Mui, Barbara L.; Tam, Ying K.; Shaheen, Farida; Collman, Ronald G.; Karikó, Katalin; Danet-Desnoyers, Gwenn A.; Madden, Thomas D.; Hope, Michael J.; Weissman, Drew

    2017-01-01

    Monoclonal antibodies are one of the fastest growing classes of pharmaceutical products, however, their potential is limited by the high cost of development and manufacturing. Here we present a safe and cost-effective platform for in vivo expression of therapeutic antibodies using nucleoside-modified mRNA. To demonstrate feasibility and protective efficacy, nucleoside-modified mRNAs encoding the light and heavy chains of the broadly neutralizing anti-HIV-1 antibody VRC01 are generated and encapsulated into lipid nanoparticles. Systemic administration of 1.4 mg kg−1 of mRNA into mice results in ∼170 μg ml−1 VRC01 antibody concentrations in the plasma 24 h post injection. Weekly injections of 1 mg kg−1 of mRNA into immunodeficient mice maintain trough VRC01 levels above 40 μg ml−1. Most importantly, the translated antibody from a single injection of VRC01 mRNA protects humanized mice from intravenous HIV-1 challenge, demonstrating that nucleoside-modified mRNA represents a viable delivery platform for passive immunotherapy against HIV-1 with expansion to a variety of diseases. PMID:28251988

  20. 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

  1. Highly efficient neutralization by plasma antibodies from human immunodeficiency virus type-1 infected individuals on antiretroviral drug therapy.

    PubMed

    Andrabi, Raiees; Makhdoomi, M A; Kumar, Rajesh; Bala, Manju; Parray, Hilal; Gupta, Arjun; Kotnala, Ankita; Thirumurthy, Velpandian; Luthra, Kalpana

    2014-05-01

    Little is known about the neutralizing antibodies induced in HIV-1 patients on antiretroviral treatment, which constitute an interesting group of individuals with improved B cell profile. Plasma samples from 34 HIV-1 seropositive antiretroviral drug treated (ART) patients were tested for neutralization against a panel of 14 subtype-A, B and C tier 1 and tier 2 viruses in TZM-bl assay. Of the 34 plasma samples, remarkably all the plasma samples were able to neutralize at least one virus while 32 (94 %) were found to neutralize ≥50 % viruses tested. In terms of overall neutralization frequency, approximately 86 %, 68 % and 17 % of the virus/plasma combinations showed 50 % neutralizing activity at 1 > 60, 1 ≥ 200 and 1 ≥ 2000 dilutions respectively. The improvement in neutralizing activity was shown to be associated with ART in two follow up patients. The neutralization of viruses by two representative plasma samples, AIIMS221 and AIIMS265, was exclusively mediated by immunoglobulin G fractions independent of ART drugs and IgG retained cross-reactive binding to recombinant gp120 proteins. We observed a positive trend of neutralization with duration of ART (p = 0.06), however no such correlation was found with clinical and immunological variables like CD4 count (p = 0.35), viral load (p = 0.09) and plasma total IgG (p = 0.46). Our study suggests that the plasma antibodies from ART patients display high neutralizing activity most likely due to an improved B cell function induced by ART despite low antigenic stimulation.

  2. The relationship between environmental exposures to phthalates and DNA damage in human sperm using the neutral comet assay.

    PubMed Central

    Duty, Susan M; Singh, Narendra P; Silva, Manori J; Barr, Dana B; Brock, John W; Ryan, Louise; Herrick, Robert F; Christiani, David C; Hauser, Russ

    2003-01-01

    Phthalates are industrial chemicals widely used in many commercial applications. The general population is exposed to phthalates through consumer products as well as through diet and medical treatments. To determine whether environmental levels of phthalates are associated with altered DNA integrity in human sperm, we selected a population without identified sources of exposure to phthalates. One hundred sixty-eight subjects recruited from the Massachusetts General Hospital Andrology Laboratory provided a semen and a urine sample. Eight phthalate metabolites were measured in urine by using high-performance liquid chromatography and tandem mass spectrometry; data were corrected for urine dilution by adjusting for specific gravity. The neutral single-cell microgel electrophoresis assay (comet assay) was used to measure DNA integrity in sperm. VisComet image analysis software was used to measure comet extent, a measure of total comet length (micrometers); percent DNA in tail (tail%), a measure of the proportion of total DNA present in the comet tail; and tail distributed moment (TDM), an integrated measure of length and intensity (micrometers). For an interquartile range increase in specific gravity-adjusted monoethyl phthalate (MEP) level, the comet extent increased significantly by 3.6 micro m [95% confidence interval (95% CI), 0.74-6.47]; the TDM also increased 1.2 micro m (95% CI, -0.05 to 2.38) but was of borderline significance. Monobutyl, monobenzyl, monomethyl, and mono-2-ethylhexyl phthalates were not significantly associated with comet assay parameters. In conclusion, this study represents the first human data to demonstrate that urinary MEP, at environmental levels, is associated with increased DNA damage in sperm. PMID:12842768

  3. 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.

  4. 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.

  5. Neutralization of Diverse Human Cytomegalovirus Strains Conferred by Antibodies Targeting Viral gH/gL/pUL128-131 Pentameric Complex

    PubMed Central

    Ha, Sha; Li, Fengsheng; Troutman, Matthew C.; Freed, Daniel C.; Tang, Aimin; Loughney, John W.; Wang, I-Ming; Vlasak, Josef; Nickle, David C.; Rustandi, Richard R.; Hamm, Melissa; DePhillips, Pete A.; Zhang, Ningyan; McLellan, Jason S.; Zhu, Hua; Adler, Stuart P.; McVoy, Michael A.; An, Zhiqiang

    2017-01-01

    ABSTRACT Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection, and developing a prophylactic vaccine is of high priority to public health. We recently reported a replication-defective human cytomegalovirus with restored pentameric complex glycoprotein H (gH)/gL/pUL128-131 for prevention of congenital HCMV infection. While the quantity of vaccine-induced antibody responses can be measured in a viral neutralization assay, assessing the quality of such responses, including the ability of vaccine-induced antibodies to cross-neutralize the field strains of HCMV, remains a challenge. In this study, with a panel of neutralizing antibodies from three healthy human donors with natural HCMV infection or a vaccinated animal, we mapped eight sites on the dominant virus-neutralizing antigen—the pentameric complex of glycoprotein H (gH), gL, and pUL128, pUL130, and pUL131. By evaluating the site-specific antibodies in vaccine immune sera, we demonstrated that vaccination elicited functional antiviral antibodies to multiple neutralizing sites in rhesus macaques, with quality attributes comparable to those of CMV hyperimmune globulin. Furthermore, these immune sera showed antiviral activities against a panel of genetically distinct HCMV clinical isolates. These results highlighted the importance of understanding the quality of vaccine-induced antibody responses, which includes not only the neutralizing potency in key cell types but also the ability to protect against the genetically diverse field strains. IMPORTANCE HCMV is the leading cause of congenital viral infection, and development of a preventive vaccine is a high public health priority. To understand the strain coverage of vaccine-induced immune responses in comparison with natural immunity, we used a panel of broadly neutralizing antibodies to identify the immunogenic sites of a dominant viral antigen—the pentameric complex. We further demonstrated that following vaccination of a replication

  6. 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.

  7. Trivalency of a Nanobody Specific for the Human Respiratory Syncytial Virus Fusion Glycoprotein Drastically Enhances Virus Neutralization and Impacts Escape Mutant Selection.

    PubMed

    Palomo, Concepción; Mas, Vicente; Detalle, Laurent; Depla, Erik; Cano, Olga; Vázquez, Mónica; Stortelers, Catelijne; Melero, José A

    2016-11-01

    ALX-0171 is a trivalent Nanobody derived from monovalent Nb017 that binds to antigenic site II of the human respiratory syncytial virus (hRSV) fusion (F) glycoprotein. ALX-0171 is about 6,000 to 10,000 times more potent than Nb017 in neutralization tests with strains of hRSV antigenic groups A and B. To explore the effect of this enhanced neutralization on escape mutant selection, viruses resistant to either ALX-0171 or Nb017 were isolated after serial passage of the hRSV Long strain in the presence of suboptimal concentrations of the respective Nanobodies. Resistant viruses emerged notably faster with Nb017 than with ALX-0171 and in both cases contained amino acid changes in antigenic site II of hRSV F. Detailed binding and neutralization analyses of these escape mutants as well as previously described mutants resistant to certain monoclonal antibodies (MAbs) offered a comprehensive description of site II mutations which are relevant for neutralization by MAbs and Nanobodies. Notably, ALX-0171 showed a sizeable neutralization potency with most escape mutants, even with some of those selected with the Nanobody, and these findings make ALX-0171 an attractive antiviral for treatment of hRSV infections.

  8. Neutralization of multiple laboratory and clinical isolates of human immunodeficiency virus type 1 (HIV-1) by antisera raised against gp120 from the MN isolate of HIV-1.

    PubMed Central

    Berman, P W; Matthews, T J; Riddle, L; Champe, M; Hobbs, M R; Nakamura, G R; Mercer, J; Eastman, D J; Lucas, C; Langlois, A J

    1992-01-01

    Vaccines prepared from the envelope glycoprotein, gp120, of the common laboratory isolate of human immunodeficiency virus type 1 (HIV-1) (IIIB/LAV-1) elicit antibodies that neutralize the homologous virus but show little if any cross-neutralizing activity. This may be because the principal neutralizing determinant (PND) of gp120 is highly unusual in the IIIB/LAV-1 strain and is not representative of those found in the majority of field isolates. We have now examined the immunogenicity of recombinant gp120 prepared from the MN strain of HIV-1 (MN-rgp120), whose PND is thought to be representative of approximately 60% of the isolates in North America. Our results show that MN-rgp120 is a potent immunogen and elicits anti-gp120 titers comparable to those found in HIV-1-infected individuals. While both MN-rgp120 and IIIB-rgp120 induced antibodies able to block gp120 binding to CD4, strain-specific and type-common blocking antibodies were detected. Finally, antibodies to MN-rgp120 but not to IIIB-rgp120 were effective in neutralizing a broad range of laboratory and clinical isolates of HIV-1. These studies demonstrate that susceptibility or resistance to neutralization by antibodies to gp120 correlates with the PND sequence and suggest that the problem of antigenic variation may not be insurmountable in the development of an effective AIDS vaccine. PMID:1602554

  9. Neutralizing human recombinant antibodies against herpes simplex virus type 1 glycoproteins B from a phage-displayed scFv antibody library.

    PubMed

    Bagheri, Vahid; Nejatollahi, Foroogh; Esmaeili, Seyed Alireza; Momtazi, Amir Abbas; Motamedifar, Mohamad; Sahebkar, Amirhossein

    2017-01-15

    The HSV-1 envelope glycoprotein B (gB) plays a critical role in virus entry into host cells. Neutralizing antibodies can therefore potentially prevent virus entry into target cells and cell-to-cell spread of infection. Our present study focused on the selection of neutralizing single-chain Fv (scFv) antibodies of a phage-displayed nonimmune human scFv antibody library against gB of HSV-1. To enrich specific scFvs, two phage antibodies were isolated against amino acid residues 31-43 derived from the N-terminal part of gB using panning technique. Two scFvs, scFv-gB1 and scFv-gB2, with frequencies of 45% and 20% were obtained from scFv clones after performing PCR and MvaI fingerprinting. In phage ELISA analysis, both gB1 and gB2 scFvs demonstrated high reactivity with the gB peptide. In the neutralization assay, scFv-gB1 and scFv-gB2 represented neutralizing effects of 55% and 59%, respectively. Upon further enhancement of the neutralizing effects of these antibodies, they can be considered as new potential alternatives in the treatment and prophylaxis of HSV-1 infections.

  10. Novelty-seeking DRD4 polymorphisms are associated with human migration distance out-of-Africa after controlling for neutral population gene structure.

    PubMed

    Matthews, Luke J; Butler, Paul M

    2011-07-01

    Numerous lines of evidence suggest that Homo sapiens evolved as a distinct species in Africa by 150,000 years before the present (BP) and began major migrations out-of-Africa ∼50,000 BP. By 20,000 BP, our species had effectively colonized the entire Old World, and by 12,000 BP H. sapiens had a global distribution. We propose that this rapid migration into new habitats selected for individuals with low reactivity to novel stressors. Certain dopamine receptor D4 (DRD4) polymorphisms are associated with low neuronal reactivity and increased exploratory behavior, novelty seeking, and risk taking, collectively considered novelty-seeking trait (NS). One previous report (Chen et al.: Evol Hum Behav 20 (1999) 309-324) demonstrated a correlation between migratory distance and the seven-repeat (7R) VNTR DRD4 allele at exon 3 for human populations. This study, however, failed to account for neutral genetic processes (drift and admixture) that might create such a correlation in the absence of natural selection. Furthermore, additional loci surrounding DRD4 are now recognized to influence NS. Herein we account for neutral genetic structure by modeling the nonindependence of neutral allele frequencies between human populations. We retest the DRD4 exon 3 alleles, and also test two other loci near DRD4 that are associated with NS. We conclude there is an association between migratory distance and DRD4 exon 3 2R and 7R alleles that cannot be accounted for by neutral genetic processes alone.

  11. 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.

  12. Analysis of cross-reactive neutralizing antibodies in human HFMD serum with an EV71 pseudovirus-based assay.

    PubMed

    Zhang, Huafei; An, Dong; Liu, Wei; Mao, Qunying; 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.

  13. 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

  14. 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.

  15. Characterization of a plant-produced recombinant human secretory IgA with broad neutralizing activity against HIV

    PubMed Central

    Paul, Matthew; Reljic, Rajko; Klein, Katja; Drake, Pascal MW; van Dolleweerd, Craig; Pabst, Martin; Windwarder, Markus; Arcalis, Elsa; Stoger, Eva; Altmann, Friedrich; Cosgrove, Catherine; Bartolf, Angela; Baden, Susan; Ma, Julian K-C

    2014-01-01

    Recombinant Secretory IgA (SIgA) complexes have the potential to improve antibody-based passive immunotherapeutic approaches to combat many mucosal pathogens. In this report, we describe the expression, purification and characterization of a human SIgA format of the broadly neutralizing anti-HIV monoclonal antibody (mAb) 2G12, using both transgenic tobacco plants and transient expression in Nicotiana benthamiana as expression hosts (P2G12 SIgA). The resulting heterodecameric complexes accumulated in intracellular compartments in leaf tissue, including the vacuole. SIgA complexes could not be detected in the apoplast. Maximum yields of antibody were 15.2 μg/g leaf fresh mass (LFM) in transgenic tobacco and 25 μg/g LFM after transient expression, and assembly of SIgA complexes was superior in transgenic tobacco. Protein L purified antibody specifically bound HIV gp140 and neutralised tier 2 and tier 3 HIV isolates. Glycoanalysis revealed predominantly high mannose structures present on most N-glycosylation sites, with limited evidence for complex glycosylation or processing to paucimannosidic forms. O-glycan structures were not identified. Functionally, P2G12 SIgA, but not IgG, effectively aggregated HIV virions. Binding of P2G12 SIgA was observed to CD209 / DC-SIGN, but not to CD89 / FcalphaR on a monocyte cell line. Furthermore, P2G12 SIgA demonstrated enhanced stability in mucosal secretions in comparison to P2G12 IgG mAb. PMID:25484063

  16. 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

  17. Cloning of the first human anti-JCPyV/VP1 neutralizing monoclonal antibody: epitope definition and implications in risk stratification of patients under natalizumab therapy.

    PubMed

    Diotti, Roberta Antonia; Mancini, Nicasio; Clementi, Nicola; Sautto, Giuseppe; Moreno, Guisella Janett; Criscuolo, Elena; Cappelletti, Francesca; Man, Petr; Forest, Eric; Remy, Louise; Giannecchini, Simone; Clementi, Massimo; Burioni, Roberto

    2014-08-01

    JC virus (JCPyV) has gained novel clinical importance as cause of progressive multifocal leukoencephalopathy (PML), a rare demyelinating disease recently associated to immunomodulatory drugs, such as natalizumab used in multiple sclerosis (MS) cases. Little is known about the mechanisms leading to PML, and this makes the need of PML risk stratification among natalizumab-treated patients very compelling. Clinical and laboratory-based risk-stratification markers have been proposed, one of these is represented by the JCPyV-seropositive status, which includes about 54% of MS patients. We recently proposed to investigate the possible protective role of neutralizing humoral immune response in preventing JCPyV reactivation. In this proof-of-concept study, by cloning the first human monoclonal antibody (GRE1) directed against a neutralizing epitope on JCPyV/VP1, we optimized a robust anti-JCPyV neutralization assay. This allowed us to evaluate the neutralizing activity in JCPyV-positive sera from MS patients, demonstrating the lack of correlation between the level of anti-JCPyV antibody and anti-JCPyV neutralizing activity. Relevant consequences may derive from future clinical studies induced by these findings; indeed the study of the serum anti-JCPyV neutralizing activity could allow not only a better risk stratification of the patients during natalizumab treatment, but also a better understanding of the pathophysiological mechanisms leading to PML, highlighting the contribution of peripheral versus central nervous system JCPyV reactivation. Noteworthy, the availability of GRE1 could allow the design of novel immunoprophylactic strategies during the immunomodulatory treatment.

  18. Antibody neutralization poses a barrier to intravitreal adeno-associated viral vector gene delivery to non-human primates.

    PubMed

    Kotterman, M A; Yin, L; Strazzeri, J M; Flannery, J G; Merigan, W H; Schaffer, D V

    2015-02-01

    Gene delivery vectors based on adeno-associated viruses (AAV) have exhibited promise in both preclinical disease models and human clinical trials for numerous disease targets, including the retinal degenerative disorders Leber's congenital amaurosis and choroideremia. One general challenge for AAV is that preexisting immunity, as well as subsequent development of immunity following vector administration, can severely inhibit systemic AAV vector gene delivery. However, the role of neutralizing antibodies (NABs) in AAV transduction of tissues considered to be immune privileged, such as the eye, is unclear in large animals. Intravitreal AAV administration allows for broad retinal delivery, but is more susceptible to interactions with the immune system than subretinal administration. To assess the effects of systemic anti-AAV antibody levels on intravitreal gene delivery, we quantified the anti-AAV antibodies present in sera from non-human primates before and after intravitreal injections with various AAV capsids. Analysis showed that intravitreal administration resulted in an increase in anti-AAV antibodies regardless of the capsid serotype, transgene or dosage of virus injected. For monkeys injected with wild-type AAV2 and/or an AAV2 mutant, the variable that most significantly affected the production of anti-AAV2 antibodies was the amount of virus delivered. In addition, post-injection antibody titers were highest against the serotype administered, but the antibodies were also cross-reactive against other AAV serotypes. Furthermore, NAB levels in serum correlated with those in vitreal fluid, demonstrating both that this route of administration exposes AAV capsid epitopes to the adaptive immune system and that serum measurements are predictive of vitreous fluid NAB titers. Moreover, the presence of preexisting NAB titers in the serum of monkeys correlated strongly (R=0.76) with weak, decaying or no transgene expression following intravitreal administration of AAV

  19. Differential regulation of cellular tropism and sensitivity to soluble CD4 neutralization by the envelope gp120 of human immunodeficiency virus type 1.

    PubMed Central

    Stamatatos, L; Werner, A; Cheng-Mayer, C

    1994-01-01

    Using recombinant and mutant viruses generated between two human immunodeficiency virus type 1 isolates that display differences in cell tropism and sensitivity to soluble CD4 neutralization, we show that these two properties of the virus are regulated by different mechanisms. Whereas there is an association between V3 loop conformation and a particular cellular tropism, soluble CD4 neutralization sensitivity appears to be determined by amino acid differences in the C2 domain of the envelope gp120 that modulate the stability of gp120-gp41 association. Our findings further illustrate the importance of functional interactions among different regions of the envelope gp120 in regulating the biological phenotypes of human immunodeficiency virus and suggest that additional probing of the V3 loop with monoclonal antibodies may identify specific structural features of this loop that determine cell tropism. Images PMID:8035496

  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.

  1. Uridine uptake inhibition as a cytotoxicity test for a human hepatoma cell line (HepG2 cells): comparison with the neutral red assay.

    PubMed

    Valentin, I; Philippe, M; Lhuguenot, J; Chagnon, M

    2001-02-14

    This study describes a sensitive microassay for measuring cytotoxicity based on the degree of inhibition of RNA synthesis in HepG2 cells. RNA synthesis is measured by the kinetic uptake of radiolabeled uridine. A large number of compounds were tested in a wide range of concentrations. The concentration required to induce 50% inhibition of HepG2 uridine uptake rates (IC(50)) was determined for each compound and used to rank its potency. These IC(50)s were compared with IC(50)s measured with the neutral red assay. 2-acetylaminofluorene, benzo[a]pyrene and methylnitrosourea were not cytotoxic in the neutral red assay. Uridine uptake was always inhibited at lower concentrations than those required in the neutral red assay, suggesting that the uridine uptake assay is a more sensitive indicator of toxic action than the neutral red inclusion. Uridine uptake assay provides a rapid and quantitative method for assessing toxicity in a human cell line. Application of this method to bottled spring waters are described. Due to its high sensitivity and reproducibility, this method provides a suitable tool for screening a great number of samples and will be a helpful test for evaluating food safety and controlling the recycling process of wrapping materials.

  2. Hyperimmune antisera against synthetic peptides representing the glycoprotein of human immunodeficiency virus type 2 can mediate neutralization and antibody-dependent cytotoxic activity.

    PubMed Central

    Björling, E; Broliden, K; Bernardi, D; Utter, G; Thorstensson, R; Chiodi, F; Norrby, E

    1991-01-01

    Twenty-five 13- to 35-amino-acid-long peptides representing regions of human immunodeficiency virus type 2 (HIV-2), strain SBL6669, envelope proteins were evaluated for their immunogenic activity in guinea pigs. The peptides were selected to provide homologous representation of sites in the HIV-1 envelope proteins that were previously documented to have a particular immunogenic importance. A number of the HIV-2 peptides were found to be capable of inducing strain SBL6669 neutralizing and antibody-dependent cellular cytotoxicity (ADCC) antibodies. Two overlapping peptides covering amino acids 311-337 representing the central and C-terminal part of the variable third (V3) region, terminology according to Modrow et al. [Modrow, S., Hahn, B., Shaw, G. M., Gallo, R. C., Wong-Staal, F. & Wolf, H. (1987) J. Virol. 61, 570-578], showed the most pronounced capacity to induce neutralizing antibodies. One of the peptides (amino acids 318-337) also induced antibodies mediating ADCC. Two additional regions in the large glycoprotein, gp125, containing linear sites reacting with neutralizing antibodies were identified (amino acids, 119-137 and 472-509). The transmembrane protein, gp36, of HIV-2 harbored two regions of importance for induction of neutralizing antibodies (amino acids 595-614 and 714-729). ADCC activity was induced by two additional gp125-specific peptides (amino acids 291-311 and 446-461). Thus, except for the single V3-specific site there was no correlation between linear immunogenic sites stimulating neutralizing antibody and ADCC activity. These findings pave the way for development of synthetic vaccines against HIV-2 and possibly also simian immunodeficiency virus infections. The capacity of such a product to induce protective immunity can be evaluated in macaque monkeys. Images PMID:2068087

  3. Replication and neutralization of human immunodeficiency virus type 1 lacking the V1 and V2 variable loops of the gp120 envelope glycoprotein.

    PubMed Central

    Cao, J; Sullivan, N; Desjardin, E; Parolin, C; Robinson, J; Wyatt, R; Sodroski, J

    1997-01-01

    A human immunodeficiency virus type 1 (HIV-1) mutant lacking the V1 and V2 variable loops in the gp120 exterior envelope glycoprotein replicated in Jurkat lymphocytes with only modest delays compared with the wild-type virus. Revertants that replicated with wild-type efficiency rapidly emerged and contained only a few amino acid changes in the envelope glycoproteins compared with the parent virus. Both the parent and revertant viruses exhibited increased sensitivity to neutralization by antibodies directed against the V3 loop or a CD4-induced epitope on gp120 but not by soluble CD4 or an antibody against the CD4 binding site. This result demonstrates the role of the gp120 V1 and V2 loops in protecting HIV-1 from some subsets of neutralizing antibodies. PMID:9371651

  4. The membrane-proximal external region of the human immunodeficiency virus type 1 envelope: dominant site of antibody neutralization and target for vaccine design.

    PubMed

    Montero, Marinieve; van Houten, Nienke E; Wang, Xin; Scott, Jamie K

    2008-03-01

    Enormous efforts have been made to produce a protective vaccine against human immunodeficiency virus type 1; there has been little success. However, the identification of broadly neutralizing antibodies against epitopes on the highly conserved membrane-proximal external region (MPER) of the gp41 envelope protein has delineated this region as an attractive vaccine target. Furthermore, emerging structural information on the MPER has provided vaccine designers with new insights for building relevant immunogens. This review describes the current state of the field regarding (i) the structure and function of the gp41 MPER; (ii) the structure and binding mechanisms of the broadly neutralizing antibodies 2F5, 4E10, and Z13; and (iii) the development of an MPER-targeting vaccine. In addition, emerging approaches to vaccine design are presented.

  5. 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.

  6. The neutralization sensitivity of viruses representing human immunodeficiency virus type 1 variants of diverse subtypes from early in infection is dependent on producer cell, as well as characteristics of the specific antibody and envelope variant.

    PubMed

    Provine, Nicholas M; Cortez, Valerie; Chohan, Vrasha; Overbaugh, Julie

    2012-05-25

    Neutralization properties of human immunodeficiency virus (HIV-1) are often defined using pseudoviruses grown in transformed cells, which are not biologically relevant HIV-1 producer cells. Little information exists on how these viruses compare to viruses produced in primary lymphocytes, particularly for globally relevant HIV-1 strains. Therefore, replication-competent chimeras encoding envelope variants from the dominant HIV-1 subtypes (A, C, and D) obtained early after infection were generated and the neutralization properties explored. Pseudoviruses generated in 293T cells were the most sensitive to antibody neutralization. Replicating viruses generated in primary lymphocytes were most resistant to neutralization by plasma antibodies and most monoclonal antibodies (b12, 4E10, 2F5, VRC01). These differences were not associated with differences in envelope content. Surprisingly, the virus source did not impact neutralization sensitivity of most viruses to PG9. These findings suggest that producer cell type has a major effect on neutralization sensitivity, but in an antibody dependent manner.

  7. Generation and selection of novel fully human monoclonal antibodies that neutralize Dickkopf-1 (DKK1) inhibitory function in vitro and increase bone mass in vivo.

    PubMed

    Glantschnig, Helmut; Hampton, Richard A; Lu, Ping; Zhao, Jing Z; Vitelli, Salvatore; Huang, Lingyi; Haytko, Peter; Cusick, Tara; Ireland, Cheryl; Jarantow, Stephen W; Ernst, Robin; Wei, Nan; Nantermet, Pascale; Scott, Kevin R; Fisher, John E; Talamo, Fabio; Orsatti, Laura; Reszka, Alfred A; Sandhu, Punam; Kimmel, Donald; Flores, Osvaldo; Strohl, William; An, Zhiqiang; Wang, Fubao

    2010-12-17

    Wnt/LRP5 signaling is a central regulatory component of bone formative and resorptive activities, and the pathway inhibitor DKK1 is a suppressor of bone formation and bone mass accrual in mice. In addition, augmented DKK1 levels are associated with high bone turnover in diverse low bone mass states in rodent models and disease etiologies in human. However, examination of the precise role of DKK1 in the normal skeleton and in higher species requires the development of refined DKK1-specific pharmacological tools. Here, we report the strategy resulting in isolation of a panel of fully human anti-DKK1 antibodies applicable to studies interrogating the roles of mouse, rhesus, and human DKK1. Selected anti-DKK1 antibodies bind primate and human DKK-1 with picomolar affinities yet do not appreciably bind to DKK2 or DKK4. Epitopes mapped within the DKK1 C-terminal domain necessary for interaction with LRP5/6 and consequently effectively neutralized DKK1 function in vitro. When introduced into naïve normal growing female mice, IgGs significantly improved trabecular bone volume and structure and increased both trabecular and cortical bone mineral densities in a dose-related fashion. Furthermore, fully human DKK1-IgG displayed favorable pharmacokinetic parameters in non-human primates. In summary, we demonstrate here a rate-limiting function of physiologic DKK1 levels in the regulation of bone mass in intact female mice, amendable to specific pharmacologic neutralization by newly identified DKK1-IgGs. Importantly the fully human IgGs display a profile of attributes that recommends their testing in higher species and their use in evaluating DKK1 function in relevant disease models.

  8. 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

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

    PubMed Central

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

    2011-01-01

    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 Å 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 β-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. PMID:21489992

  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. Structural basis of neutralization of the major toxic component from the scorpion Centruroides noxius Hoffmann by a human-derived single-chain antibody fragment.

    PubMed

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

    2011-06-10

    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 Å 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 β-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.

  12. A neutralizing monoclonal antibody previously mapped exclusively on human immunodeficiency virus type 1 gp41 recognizes an epitope in p17 sharing the core sequence IEEE.

    PubMed Central

    Buratti, E; Tisminetzky, S G; D'Agaro, P; Baralle, F E

    1997-01-01

    We report here that a human immunodeficiency virus type 1 (HIV-1)-specific neutralizing monoclonal antibody (MAb 1575) mapped to the conserved putative intracellular region from amino acid residues 735 to 752 (735-752 region) of gp41 also recognizes a region in an extracellular portion of p17. Both epitopes have a core recognition sequence (IEEE) in a nonhomologous context. The IEEE motif found in HIV-1 p17 is located in a region known as HGP-30 (residues 86 to 115) which has been previously associated with virus neutralization, cytotoxic T lymphocyte activity, and mother-to-child transmission. An analysis of available gp41 and p17 sequences demonstrates that in these regions both IEEE sequences are highly conserved in different HIV-1 clades. The presence of the IEEE epitope in p17 allows us to explain some unexpected neutralizing characteristics of MAb 1575. In addition, the gp41 735-752 region has been previously reported both in intra- and extracellular locations. Our results suggest that the extracellular location was the result of cross-reactivity with p17. PMID:9032383

  13. Structural basis for penetration of the glycan shield of hepatitis C virus E2 glycoprotein by a broadly neutralizing human antibody.

    PubMed

    Li, Yili; Pierce, Brian G; Wang, Qian; Keck, Zhen-Yong; Fuerst, Thomas R; Foung, Steven K H; Mariuzza, Roy A

    2015-04-17

    Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. A challenge for HCV vaccine development is to identify conserved epitopes able to elicit protective antibodies against this highly diverse virus. Glycan shielding is a mechanism by which HCV masks such epitopes on its E2 envelope glycoprotein. Antibodies to the E2 region comprising residues 412-423 (E2(412-423)) have broadly neutralizing activities. However, an adaptive mutation in this linear epitope, N417S, is associated with a glycosylation shift from Asn-417 to Asn-415 that enables HCV to escape neutralization by mAbs such as HCV1 and AP33. By contrast, the human mAb HC33.1 can neutralize virus bearing the N417S mutation. To understand how HC33.1 penetrates the glycan shield created by the glycosylation shift to Asn-415, we determined the structure of this broadly neutralizing mAb in complex with its E2(412-423) epitope to 2.0 Å resolution. The conformation of E2(412-423) bound to HC33.1 is distinct from the β-hairpin conformation of this peptide bound to HCV1 or AP33, because of disruption of the β-hairpin through interactions with the unusually long complementarity-determining region 3 of the HC33.1 heavy chain. Whereas Asn-415 is buried by HCV1 and AP33, it is solvent-exposed in the HC33.1-E2(412-423) complex, such that glycosylation of Asn-415 would not prevent antibody binding. Furthermore, our results highlight the structural flexibility of the E2(412-423) epitope, which may serve as an immune evasion strategy to impede induction of antibodies targeting this site by reducing its antigenicity.

  14. Neutralization of non-vaccine human papillomavirus pseudoviruses from the A7 and A9 species groups by bivalent HPV vaccine sera.

    PubMed

    Draper, Eve; Bissett, Sara L; Howell-Jones, Rebecca; Edwards, Debbie; Munslow, Graham; Soldan, Kate; Beddows, Simon

    2011-11-03

    The majority of cervical cancers are associated with infection by one or more Human Papillomavirus (HPV) types from just two distinct Alpha-Papillomavirus species groups, A7 and A9. The extent to which the current HPV16/18 vaccines will protect against other genetically related HPV types is of interest to inform vaccine implementation, cervical disease surveillance and the development of second generation HPV vaccines. The aim of this study was to determine the frequency and titer of neutralizing antibodies against a range of A7 (18, 39, 45, 59, 68) and A9 (16, 31, 33, 35, 52, 58) HPV types using sera from individuals immunized with the bivalent HPV vaccine within the school-based, UK national HPV immunization programme. Serum samples were collected from 69 girls aged 13-14 years, a median 5.9 months (inter-quartile range, IQR, 5.7-6.0) after their third vaccine dose. Cross-neutralizing antibodies against HPV31, HPV33, HPV35 and HPV45 were common and strongly associated with the titer for the related vaccine-type, but were considerably lower (<1%) than their related vaccine type-specific response. The low prevalence of these HPV types in the population and the ages within the study cohort suggest these responses are due to vaccination. It is unclear whether such low levels of neutralizing antibodies would be sufficient to protect at the site of infection in the absence of other immune effectors but the coincidence with HPV types reported from efficacy studies is intriguing. The utility of neutralizing antibodies as surrogate markers of protection remains to be determined.

  15. A human antibody to the CD4 binding site of gp120 capable of highly potent but sporadic cross clade neutralization of primary HIV-1.

    PubMed

    Gach, Johannes S; Quendler, Heribert; Tong, Tommy; Narayan, Kristin M; Du, Sean X; Whalen, Robert G; Binley, James M; Forthal, Donald N; Poignard, Pascal; Zwick, Michael B

    2013-01-01

    Primary isolates of HIV-1 resist neutralization by most antibodies to the CD4 binding site (CD4bs) on gp120 due to occlusion of this site on the trimeric spike. We describe 1F7, a human CD4bs monoclonal antibody that was found to be exceptionally potent against the HIV-1 primary isolate JR-FL. However, 1F7 failed to neutralize a patient-matched primary isolate, JR-CSF even though the two isolates differ by <10% in gp120 at the protein level. In an HIV-1 cross clade panel (n = 157), 1F7 exhibited moderate breadth, but occasionally achieved considerable potency. In binding experiments using monomeric gp120s of select resistant isolates and domain-swap chimeras between JR-FL and JR-CSF, recognition by 1F7 was limited by sequence polymorphisms involving at least the C2 region of Env. Putative N-linked glycosylation site (PNGS) mutations, notably at position 197, allowed 1F7 to neutralize JR-CSF potently without improving binding to the cognate, monomeric gp120. In contrast, flow cytometry experiments using the same PNGS mutants revealed that 1F7 binding is enhanced on cognate trimeric Env. BN-PAGE mobility shift experiments revealed that 1F7 is sensitive to the diagnostic mutation D368R in the CD4 binding loop of gp120. Our data on 1F7 reinforce how exquisitely targeted CD4bs antibodies must be to achieve cross neutralization of two closely related primary isolates. High-resolution analyses of trimeric Env that show the orientation of glycans and polymorphic elements of the CD4bs that affect binding to antibodies like 1F7 are desirable to understand how to promote immunogenicity of more conserved elements of the CD4bs.

  16. Insertion of primary syncytium-inducing (SI) and non-SI envelope V3 loops in human immunodeficiency virus type 1 (HIV-1) LAI reduces neutralization sensitivity to autologous, but not heterologous, HIV-1 antibodies.

    PubMed Central

    Hogervorst, E; de Jong, J; van Wijk, A; Bakker, M; Valk, M; Nara, P; Goudsmit, J

    1995-01-01

    The aim of the study was to investigate the influence of V3 loops from naturally occurring viruses on the neutralization sensitivity of a molecularly cloned virus. A selection of well-defined syncytium-inducing (SI) and non-SI V3 loops of a single human immunodeficiency virus type 1-infected individual (H594) and the V3 regions of two SI laboratory strains were inserted in an infectious molecular clone of human immunodeficiency type 1 LAI. Neutralization was performed with a heterologous serum pool and autologous patient serum, using the virus reduction neutralization assay and peripheral blood lymphocytes as target cells. High sensitivity of the chimeric viruses containing the laboratory strain V3 regions to neutralization by H594 sequential sera as well as the heterologous serum pool was found. A statistically significant correlation between the sensitivities of these viruses was seen. In contrast, insertion of the primary isolate NSI and SI envelope V3 loops significantly reduced the neutralization by autologous serum but not by the heterologous serum pool. No correlation was found between the neutralization of the viruses with laboratory strain-derived V3 regions and the viruses with primary isolate V3 domains. We conclude that heterologous antibodies are able to neutralize infectious molecular clones with V3 loops of both SI and NSI viruses, regardless of whether they originated from laboratory strains or primary isolates. However, serum of patient H594 discriminated between the two types of viruses and showed reduced neutralization of the viruses with the autologous NSI and SI primary isolate V3 loops. These results indicated that the neutralization sensitivity of the viruses depended on the capacity of the V3 region to influence the conformation of the virus envelope. These V3-dependent conformational changes partially explain the neutralization sensitivity of laboratory strains and the relative neutralization resistance of primary isolates. PMID:7666535

  17. Characterization of human single-chain antibodies against highly pathogenic avian influenza H5N1 viruses: mimotope and neutralizing activity.

    PubMed

    Yang, Jiupian; Yoshida, Reiko; Kariya, Yuki; Zhang, Xu; Hashiguchi, Shuhei; Nakashima, Toshihiro; Suda, Yasuo; Takada, Ayato; Ito, Yuji; Sugimura, Kazuhisa

    2010-10-01

    The development of new therapeutic targets and strategies to control highly pathogenic avian influenza (HPAI) H5N1 virus infection in humans is urgently needed. Neutralizing recombinant human antibodies would provide important agents for immunotherapy on human H5N1 virus infection and definition of the critical mimotope for vaccine development. In this study, we have characterized an anti-H5-specific scFv clone, 3D1 from the human-scFv-displaying phage library. 3D1 blocked the binding of H5-Fc to MDCK cells in flow cytometry and neutralized H5N1 subtype influenza A viruses in a microneutralization assay. Employing a peptide-displaying phage library, Ph.D-12, the mimotope was determined to be at #128-131 and #204-211 of H5, which are silic acid-binding regions. In consistency with this result, 3D1 binds the recombinant sugar-binding domain (#50G-#272E) produced by a baculovirus vector. The 3D1 antibody employs the germline gene VH1-23. As this antibody is the first human anti-H5 scFv clearly defined on the sugar-binding epitope, it allows us to investigate the influence of amino acid substitutions in this region on the determination of the binding specificity to either sialic acid α2,6-galactose (SA α2,6Gal) or sialic acid α2,3-galactose (SA α2,3Gal) providing new insight for the development of effective H5N1 pandemic vaccines.

  18. 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.

  19. Exercise Equipment: Neutral Buoyancy

    NASA Technical Reports Server (NTRS)

    Shackelford, Linda; Valle, Paul

    2016-01-01

    Load Bearing Equipment for Neutral Buoyancy (LBE-NB) is an exercise frame that holds two exercising subjects in position as they apply counter forces to each other for lower extremity and spine loading resistance exercises. Resistance exercise prevents bone loss on ISS, but the ISS equipment is too massive for use in exploration craft. Integrating the human into the load directing, load generating, and motion control functions of the exercise equipment generates safe exercise loads with less equipment mass and volume.

  20. 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.

  1. Serotype-specific neutralizing antibody epitopes of human adenovirus type 3 (HAdV-3) and HAdV-7 reside in multiple hexon hypervariable regions.

    PubMed

    Qiu, Hongling; Li, Xiao; Tian, Xingui; Zhou, Zhichao; Xing, Ke; Li, Haitao; Tang, Ni; Liu, Wenkuan; Bai, Peisheng; Zhou, Rong

    2012-08-01

    Human adenovirus types 3 and 7 (HAdV-3 and HAdV-7) occur epidemically and contribute greatly to respiratory diseases, but there is no currently available licensed recombinant HAdV-3/HAdV-7 bivalent vaccine. Identification of serotype-specific neutralizing antibody (NAb) epitopes for HAdV-3 and HAdV-7 will be beneficial for development of recombinant HAdV-3/HAdV-7 bivalent vaccines. In this study, four NAb epitopes within hexon hypervariable regions (HVRs) were predicted for HAdV-3 and HAdV-7, respectively, by using bioinformatics. Eight hexon chimeric adenovirus vectors with the alternation of only one predicted neutralizing epitope were constructed. Further in vitro and in vivo neutralization assays indicated that E2 (residing in HVR2) and E3 (residing in HVR5) are NAb epitopes for HAdV-7, and E3 plays a more important role in generating NAb responses. Cross-neutralization assays indicated that all four predicted epitopes, R1 to R4, are NAb epitopes for HAdV-3, and R1 (residing in HVR1) plays the most important role in generating NAb responses. Humoral immune responses elicited by the recombinant rAdH7R1 (containing the R1 epitope) were significantly and durably suppressed by HAdV-3-specific NAbs. Surprisingly, the rAdΔE3GFP-specific neutralizing epitope responses induced by rAdMHE3 (R3 replaced by E3) and rAdMHE4 (R4 replaced by E4) were weaker than those of rAdMHE1 (R1 replaced by E1) or rAdMHE2 (R2 relaced by E2) in vitro and in vivo. Furthermore, rAdMHE4 replicated more slowly in HEp-2 cells, and the final yield was about 10-fold lower than that of rAdΔE3GFP. The current findings contribute not only to the development of new adenovirus vaccine candidates, but also to the construction of new gene delivery vectors.

  2. Beyond Viral Neutralization.

    PubMed

    Lewis, George K; Pazgier, Marzena; Evans, David; Ferrari, Guido; Bournazos, Stylianos; Parsons, Matthew S; Bernard, Nicole F; Finzi, Andrés

    2017-01-13

    It has been known for more than 30 years that Human Immunodeficiency Virus 1 (HIV-1) infection drives a very potent B cell response resulting in the production of anti-HIV-1 antibodies targeting several viral proteins, particularly its envelope glycoproteins (Env). Env epitopes are exposed on the surfaces of viral particles and infected cells where they are targets of potentially protective antibodies. These antibodies can interdict infection by neutralization and there is strong evidence suggesting that Fc-mediated effector function can also contribute to protection. Current evidence suggests that Fc-mediated effector function plays a role in protection against infection by broadly neutralizing antibodies (bnAbs) and it might be important for protection by non-neutralizing antibodies. Fc-mediated effector function includes diverse mechanisms that include antibody-dependent cellular cytotoxicity (ADCC), antibody-mediated complement activation (ADC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cell-mediated virus inhibition (ADCVI), antibody-mediated trancytosis inhibition, and antibody-mediated virus opsonization. All these functions could be beneficial in fighting viral infections including HIV-1. In this perspective, we discuss the latest developments for ADCC responses discussed at the HIVR4P satellite session on non-neutralizing antibodies, with emphasis on the mechanisms of ADCC resistance employed by HIV-1, the structural basis of epitopes recognized by antibodies that mediate ADCC, NK-cell education and ADCC, and murine models to study ADCC against HIV-1.

  3. Characterization of the neutralizing activity of three anti-human TNF monoclonal antibodies and prediction of their TNF epitopes by molecular modeling and mutant protein approach.

    PubMed

    Zhu, Cansheng; Liu, Xuesong; Feng, Jiannan; Zhang, Wei; Shen, Beifen; Ou'yang, Weiming; Cao, Yunxin; Jin, Boquan

    2006-02-15

    The neutralizing activity of three anti-human TNF monoclonal antibodies, designated D2, E6, and F6 were investigated by three experimental systems. The results from the systems showed that all the three mAbs could neutralize TNF-mediated cytotoxicity in L929 cells, TNF-induced NF-kappaB activation in ECV304 cells, and TNF-upregulated ICAM-1 surface expression on ECV304 cells in dose-dependent manners. D2 had the highest neutralizing activity of the three mAbs, and F6 had higher level of neutralizing activity than E6. We also cloned the VH and VL cDNAs and obtained their cDNA sequences. The sequences were used in molecular modeling to establish the complex structures of TNF with variable regions of the three mAbs, respectively. In the structures, the TNF epitopes of D2, E6, and F6 were predicted at amino acids of (A109, A111-A112, C19, C21-C29, C44-C46, C66-C75, C77, C79, C90, C101, C103, C105, C114, C134-C148), (C18-C19, C21-C30, C32, C37, C43-C47, C67-C75, C83, C105-C106, C131, C135-C141), and (C21-C32, C45-C47, C65, C67-C72, C74, C81, C83, C90-C95, C105-C113, C133-C147), respectively, and the affinities of D2, E6, and F6 to TNF were predicted as -252.69, -232.83, and -299.92 kcal, respectively. Moreover, we proved the binding ability of F6 to the epitopes of amino acids of 141-146 in TNF molecule was better than that of E6, and that of D2 was the best of the three mAbs by Western blot and ELISA, in which the mutant TNF deleted the amino acids of 141-146 in TNF molecule was employed. These results make a basic foundation for selecting candidate mAbs for various purposes, such as construction of chimeric or humanized mAbs for therapeutic purpose, establishment of ELISA kits for determination of TNF, and production of affinity columns to purify TNF.

  4. Alkaline ceramidase 2 is a novel direct target of p53 and induces autophagy and apoptosis through ROS generation

    PubMed Central

    Wang, Yitao; Zhang, Chunxue; Jin, Yuelei; Wang; He, Qing; Liu, Zhu; Ai, Qing; Lei, Yunlong; Li, Yi; Song, Fangzhou; Bu, Youquan

    2017-01-01

    ACER2 is a critical sphingolipid metabolizing enzyme, and has been shown to be remarkably up-regulated following various stimuli such as DNA damage. However, the transcriptional regulatory mechanism of ACER2 gene and its potential role in the regulation of autophagy remain unknown. In this study, we have for the first time identified the human ACER2 gene promoter, and found that human ACER2 transcription is directly regulated by p53 and ACER2 is implicated in the induction of autophagy as well as apoptosis. A series of luciferase reporter assay demonstrated that ACER2 major promoter is located within its first intron where the consensus p53-binding sites exist. Consistently, forced expression of p53 significantly stimulated ACER2 transcription. Notably, p53-mediated autophagy and apoptosis were markedly enhanced by ACER2. Depletion of the essential autophagy gene ATG5 revealed that ACER2-induced autophagy facilitates its effect on apoptosis. Further studies clearly showed that ACER2-mediated autophagy and apoptosis are accompanied by ROS generation. In summary, our present study strongly suggests that ACER2 plays a pivotal role in p53-induced autophagy and apoptosis, and thus might serve as a novel and attractive molecular target for cancer treatment. PMID:28294157

  5. Alkaline ceramidase 2 is a novel direct target of p53 and induces autophagy and apoptosis through ROS generation.

    PubMed

    Wang, Yitao; Zhang, Chunxue; Jin, Yuelei; Wang; He, Qing; Liu, Zhu; Ai, Qing; Lei, Yunlong; Li, Yi; Song, Fangzhou; Bu, Youquan

    2017-03-15

    ACER2 is a critical sphingolipid metabolizing enzyme, and has been shown to be remarkably up-regulated following various stimuli such as DNA damage. However, the transcriptional regulatory mechanism of ACER2 gene and its potential role in the regulation of autophagy remain unknown. In this study, we have for the first time identified the human ACER2 gene promoter, and found that human ACER2 transcription is directly regulated by p53 and ACER2 is implicated in the induction of autophagy as well as apoptosis. A series of luciferase reporter assay demonstrated that ACER2 major promoter is located within its first intron where the consensus p53-binding sites exist. Consistently, forced expression of p53 significantly stimulated ACER2 transcription. Notably, p53-mediated autophagy and apoptosis were markedly enhanced by ACER2. Depletion of the essential autophagy gene ATG5 revealed that ACER2-induced autophagy facilitates its effect on apoptosis. Further studies clearly showed that ACER2-mediated autophagy and apoptosis are accompanied by ROS generation. In summary, our present study strongly suggests that ACER2 plays a pivotal role in p53-induced autophagy and apoptosis, and thus might serve as a novel and attractive molecular target for cancer treatment.

  6. Model for screening-level assessment of near-field human exposure to neutral organic chemicals released indoors.

    PubMed

    Zhang, Xianming; Arnot, Jon A; Wania, Frank

    2014-10-21

    Screening organic chemicals for hazard and risk to human health requires near-field human exposure models that can be readily parametrized with available data. The integration of a model of human exposure, uptake, and bioaccumulation into an indoor mass balance model provides a quantitative framework linking emissions in indoor environments with human intake rates (iRs), intake fractions (iFs) and steady-state concentrations in humans (C) through consideration of dermal permeation, inhalation, and nondietary ingestion exposure pathways. Parameterized based on representative indoor and adult human characteristics, the model is applied here to 40 chemicals of relevance in the context of human exposure assessment. Intake fractions and human concentrations (C(U)) calculated with the model based on a unit emission rate to air for these 40 chemicals span 2 and 5 orders of magnitude, respectively. Differences in priority ranking based on either iF or C(U) can be attributed to the absorption, biotransformation and elimination processes within the human body. The model is further applied to a large data set of hypothetical chemicals representative of many in-use chemicals to show how the dominant exposure pathways, iF and C(U) change as a function of chemical properties and to illustrate the capacity of the model for high-throughput screening. These simulations provide hypotheses for the combination of chemical properties that may result in high exposure and internal dose. The model is further exploited to highlight the role human contaminant uptake plays in the overall fate of certain chemicals indoors and consequently human exposure.

  7. Human anti-varicella-zoster virus (VZV) recombinant monoclonal antibody produced after Zostavax immunization recognizes the gH/gL complex and neutralizes VZV infection.

    PubMed

    Birlea, Marius; Owens, Gregory P; Eshleman, Emily M; Ritchie, Alanna; Traktinskiy, Igor; Bos, Nathan; Seitz, Scott; Azarkh, Yevgeniy; Mahalingam, Ravi; Gilden, Don; Cohrs, Randall J

    2013-01-01

    Varicella-zoster virus (VZV) is a ubiquitous, highly cell-associated, and exclusively human neurotropic alphaherpesvirus. VZV infection is initiated by membrane fusion, an event dependent in part on VZV glycoproteins gH and gL. Consistent with its location on the virus envelope, the gH/gL complex is a target of neutralizing antibodies produced after virus infection. One week after immunizing a 59-year-old VZV-seropositive man with Zostavax, we sorted his circulating blood plasma blasts and amplified expressed immunoglobulin variable domain sequences by single-cell PCR. Sequence analysis identified two plasma blast clones, one of which was used to construct a recombinant monoclonal antibody (rec-RC IgG). The rec-RC IgG colocalized with VZV gE on the membranes of VZV-infected cells and neutralized VZV infection in tissue culture. Mass spectrometric analysis of proteins immunoprecipitated by rec-RC IgG identified both VZV gH and gL. Transfection experiments showed that rec-RC IgG recognized a VZV gH/gL protein complex but not individual gH or gL proteins. Overall, our recombinant monoclonal anti-VZV antibody effectively neutralizes VZV and recognizes a conformational epitope within the VZV gH/L protein complex. An unlimited supply of this antibody provides the opportunity to analyze membrane fusion events that follow virus attachment and to identify multiple epitopes on VZV-specific proteins.

  8. A neutralizing epitope of human papillomavirus type 11 is principally described by a continuous set of residues which overlap a distinct linear, surface-exposed epitope.

    PubMed Central

    Ludmerer, S W; Benincasa, D; Mark, G E; Christensen, N D

    1997-01-01

    A panel of monoclonal antibodies (MAbs) which neutralize human papillomavirus type 11 (HPV11) in the athymic mouse xenograph neutralization assay and bind HPV11 virus-like particles (VLPs) has been described. We recently presented evidence that the Gly131-Tyr132 residues of the major capsid protein L1 confer type 11-specific binding. However, residues distally located on the primary L1 sequence also were shown to affect binding. This poses the question whether the epitope is principally centered in the region of Gly131-Tyr132 or, alternatively, is comprised of diversely located residues which come into proximity only upon proper assembly. We analyzed the result of numerous substitutions located between Tyr123 and Val142 of the HPV11 L1 sequence. We show that substitutions at five positions result in loss of binding for one or more of these MAbs by an enzyme-linked immunosorbent assay which measures antibody binding to VLPs. We demonstrate that binding of these MAbs is redirected to HPV16 VLPs which harbor eight type 11-like substitutions within the homologous region. Three of these substitutions did not affect binding when individually substituted in HPV11 but yet were still required to transfer binding to substituted HPV16 VLPs. The results demonstrate that the epitope for this class of neutralizing MAbs, although conformational and requiring VLP assembly for presentation, principally lies along a 20-residue stretch of the L1 major capsid protein. This targets the region for evaluation of the possibility of receptor binding and suggests possibilities for the design of peptide inhibitors of virus infectivity. PMID:9094659

  9. Protective efficacy of VP1-specific neutralizing antibody associated with a reduction of viral load and pro-inflammatory cytokines in human SCARB2-transgenic mice.

    PubMed

    Chang, Hsuen-Wen; Lin, Yi-Wen; Ho, Hui-Min; Lin, Min-Han; Liu, Chia-Chyi; Shao, Hsiao-Yun; Chong, Pele; Sia, Charles; Chow, Yen-Hung

    2013-01-01

    Hand-foot-mouth diseases (HFMD) caused by enterovirus 71 (EV71) and coxsackievirus 16 (CVA16) in children have now become a severe public health issue in the Asian-Pacific region. Recently we have successfully developed transgenic mice expressing human scavenger receptor class B member 2 (hSCARB2, a receptor of EV71 and CVA16) as an animal model for evaluating the pathogenesis of enterovirus infections. In this study, hSCARB2-transgenic mice were used to investigate the efficacy conferred by a previously described EV71 neutralizing antibody, N3. A single injection of N3 effectively inhibited the HFMD-like skin scurfs in mice pre-infected with clinical isolate of EV71 E59 (B4 genotype) or prevented severe limb paralysis and death in mice pre-inoculated with 5746 (C2 genotype). This protection was correlated with remarkable reduction of viral loads in the brain, spinal cord and limb muscles. Accumulated viral loads and the associated pro-inflammatory cytokines were all reduced. The protective efficacy of N3 was not observed in animals challenged with CVA16. This could be due to dissimilarity sequences of the neutralizing epitope found in CVA16. These results indicate N3 could be useful in treating severe EV71 infections and the hSCARB2-transgenic mouse could be used to evaluate the protective efficacy of potential anti-enterovirus agent candidates.

  10. Highly pathogenic avian influenza A(H5N1) mutants transmissible by air are susceptible to human and animal neutralizing antibodies.

    PubMed

    Du, Lanying; Li, Ye; Zhao, Guangyu; Wang, Lili; Zou, Peng; Lu, Lu; Zhou, Yusen; Jiang, Shibo

    2013-10-15

    A laboratory-generated reassortant H5 hemagglutinin (HA)/influenza A(H1N1) strain containing 4 mutations in influenza A(H5N1) HA has become transmissible by air among mammals. Here, we constructed 15 influenza A(H5N1) pseudoviruses containing a single mutation or a combination of mutations and showed that the pseudoviruses were susceptible to neutralizing antibodies from patients with influenza A(H5N1) infection and from mice immunized with a vaccine containing the conserved HA1 sequence of influenza A(H5N1). These results indicate that antibodies in patients currently infected by influenza A(H5N1) and antibodies induced by vaccines containing conserved sequences in HA1 of wild-type influenza A(H5N1) are highly effective in cross-neutralizing future influenza A(H5N1) mutants with airborne transmissibility, suggesting that human influenza pandemics caused by these influenza A(H5N1) variants can be prevented.

  11. 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

  12. 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

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

    PubMed

    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

    2015-08-01

    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.

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

    DOE PAGES

    Santra, Sampa; Tomaras, Georgia D.; Warrier, Ranjit; ...

    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

  15. 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.

  16. 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

    PubMed Central

    2013-01-01

    Background Differently from HIV-1, HIV-2 disease progression usually takes decades without antiretroviral 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. Results 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. Conclusion Our data suggests that the molecular and phenotypic evolution of the human immunodeficiency virus type 2 envelope are related with the dynamics of the

  17. 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 PAGES

    Rocha, Cheila; Calado, Rita; Borrego, Pedro; ...

    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

  18. Human Immunodeficiency Virus Type 1 Escape from Cyclotriazadisulfonamide-Induced CD4-Targeted Entry Inhibition Is Associated with Increased Neutralizing Antibody Susceptibility▿

    PubMed Central

    Vermeire, Kurt; Van Laethem, Kristel; Janssens, Wouter; Bell, Thomas W.; Schols, Dominique

    2009-01-01

    Continuous specific downmodulation of CD4 receptor expression in T lymphocytes by the small molecule cyclotriazadisulfonamide (CADA) selected for the CADA-resistant human immunodeficiency virus type 1 (HIV-1) NL4.3 virus containing unique mutations in the C4 and V5 regions of gp120, likely stabilizing the CD4-binding conformation. The amino acid changes in Env were associated with decreased susceptibility to anti-CD4 monoclonal antibody treatment of the cells and with higher susceptibility of the virus to soluble CD4. In addition, the acquired ability of a CADA-resistant virus to infect cells with low CD4 expression was associated with an increased susceptibility of the virus to neutralizing antibodies from sera of several HIV-1-infected patients. PMID:19570853

  19. Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1.

    PubMed Central

    Roben, P; Moore, J P; Thali, M; Sodroski, J; Barbas, C F; Burton, D R

    1994-01-01

    Six recombinant human Fab fragments that were derived from the same human immunodeficiency virus type 1 (HIV-1)-infected individual and are directed against the CD4 binding site (CD4bs) of the gp120 envelope glycoprotein were studied. A range of neutralizing activity against the HIV-1 (HXBc2) isolate was observed, with Fab b12 exhibiting the greatest potency among the Fabs tested. The neutralizing potency of Fab b12 was better than that of monoclonal whole antibodies directed against the third variable (V3) region of gp120. To explore the basis for the efficient neutralizing activity of b12, the recognition of a panel of HIV-1 gp120 mutants by the six Fabs was studied. The patterns of sensitivity to particular gp120 amino acid changes were similar for all six Fabs to those seen for anti-CD4bs monoclonal antibodies derived from HIV-1-infected individuals by conventional means. In addition, recognition by Fab b12 demonstrated an atypical sensitivity to changes in the V1 and V2 variable regions. Next, the binding of the Fabs to monomeric gp120 and to the envelope glycoprotein complex was examined. Neither the binding properties of the b12 Fab to monomeric gp120 nor the ability of the Fab to compete with soluble CD4 for monomeric gp120 binding appeared to account for the greater neutralizing potency. However, both quantitative and qualitative differences between the binding of b12 and that of less potent Fabs to the cell surface envelope glycoprotein complex were observed. Relative to less potently neutralizing Fabs, Fab b12 exhibited a higher affinity for a subpopulation of cell surface envelope glycoproteins, the conformation of which was best approximated by the mature gp120 glycoprotein. Apparently, subtle differences in the gp120 epitope recognized allow some members of the group of anti-CD4bs antibodies to bind to the functionally relevant envelope glycoprotein complex and to neutralize virus more efficiently. Images PMID:7518527

  20. Sub-inhibitory concentrations of human α-defensin potentiate neutralizing antibodies against HIV-1 gp41 pre-hairpin intermediates in the presence of serum.

    PubMed

    Demirkhanyan, Lusine; Marin, Mariana; Lu, Wuyuan; Melikyan, Gregory B

    2013-01-01

    Human defensins are at the forefront of the host responses to HIV and other pathogens in mucosal tissues. However, their ability to inactivate HIV in the bloodstream has been questioned due to the antagonistic effect of serum. In this study, we have examined the effect of sub-inhibitory concentrations of human α-defensin HNP-1 on the kinetics of early steps of fusion between HIV-1 and target cells in the presence of serum. Direct measurements of HIV-cell fusion using an enzymatic assay revealed that, in spite of the modest effect on the extent of fusion, HNP-1 prolonged the exposure of functionally important transitional epitopes of HIV-1 gp41 on the cell surface. The increased lifetime of gp41 intermediates in the presence of defensin was caused by a delay in the post-coreceptor binding steps of HIV-1 entry that correlated with the marked enhancement of the virus' sensitivity to neutralizing anti-gp41 antibodies. By contrast, the activity of antibodies to gp120 was not affected. HNP-1 appeared to specifically potentiate antibodies and peptides targeting the first heptad repeat domain of gp41, while its effect on inhibitors and antibodies to other gp41 domains was less prominent. Sub-inhibitory concentrations of HNP-1 also promoted inhibition of HIV-1 entry into peripheral blood mononuclear cells by antibodies and, more importantly, by HIV-1 immune serum. Our findings demonstrate that: (i) sub-inhibitory doses of HNP-1 potently enhance the activity of a number of anti-gp41 antibodies and peptide inhibitors, apparently by prolonging the lifetime of gp41 intermediates; and (ii) the efficiency of HIV-1 fusion inhibitors and neutralizing antibodies is kinetically restricted. This study thus reveals an important role of α-defensin in enhancing adaptive immune responses to HIV-1 infection and suggests future strategies to augment these responses.

  1. 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

  2. Animal and human antibodies to distinct Staphylococcus aureus antigens mutually neutralize opsonic killing and protection in mice

    PubMed Central

    Skurnik, David; Merighi, Massimo; Grout, Martha; Gadjeva, Mihaela; Maira-Litran, Tomas; Ericsson, Maria; Goldmann, Donald A.; Huang, Susan S.; Datta, Rupak; Lee, Jean C.; Pier, Gerald B.

    2010-01-01

    New prophylactic approaches are needed to control infection with the Gram-positive bacterium Staphylococcus aureus, which is a major cause of nosocomial and community-acquired infections. To develop these, greater understanding of protective immunity against S. aureus infection is needed. Human immunity to extracellular Gram-positive bacterial pathogens is primarily mediated by opsonic killing (OPK) via antibodies specific for surface polysaccharides. S. aureus expresses two such antigens, capsular polysaccharide (CP) and poly-N-acetyl glucosamine (PNAG). Here, we have shown that immunization-induced polyclonal animal antisera and monoclonal antibodies specific for either CP or PNAG antigens have excellent in vitro OPK activity in human blood but that when mixed together they show potent interference in OPK activity. In addition, reductions in antibody binding to the bacterial surface, complement deposition, and passive protection were seen in two mouse models of S. aureus infection. Electron microscopy, isothermal calorimetry, and surface plasmon resonance indicated that antibodies to CP and PNAG bound together via an apparent idiotype–anti-idiotype interaction. This interaction was also found in sera from humans with S. aureus bacteremia. These findings suggest that the lack of effective immunity to S. aureus infections in humans could be due, in part, to interference in OPK when antibodies to CP and PNAG antigens are both present. This information could be used to better design S. aureus vaccine components. PMID:20739753

  3. Potent and synergistic neutralization of human immunodeficiency virus (HIV) type 1 primary isolates by hyperimmune anti-HIV immunoglobulin combined with monoclonal antibodies 2F5 and 2G12.

    PubMed

    Mascola, J R; Louder, M K; VanCott, T C; Sapan, C V; Lambert, J S; Muenz, L R; Bunow, B; Birx, D L; Robb, M L

    1997-10-01

    Three antibody reagents that neutralize primary human immunodeficiency virus type 1 (HIV-1) isolates were tested for magnitude and breadth of neutralization when used alone or in double or triple combinations. Hyperimmune anti-HIV immunoglobulin (HIVIG) is derived from the plasma of HIV-1-infected donors, and monoclonal antibodies (MAbs) 2F5 and 2G12 bind to distinct regions of the HIV-1 envelope glycoprotein. The antibodies were initially tested against a panel of 15 clade B HIV-1 isolates, using a single concentration that is achievable in vivo (HIVIG, 2,500 microg/ml; MAbs, 25 microg/ml). Individual antibody reagents neutralized many of the viruses tested, but antibody potency varied substantially among the viruses. The virus neutralization produced by double combinations of HIVIG plus 2F5 or 2G12, the two MAbs together, or the triple combination of HIVIG, 2F5, and 2G12 was generally equal to or greater than that predicted by the effect of individual antibodies. Overall, the triple combination displayed the greatest magnitude and breadth of neutralization. Synergistic neutralization was evaluated by analyzing data from dose-response curves of each individual antibody reagent compared to the triple combination and was demonstrated against each of four viruses tested. Therefore, combinations of polyclonal and monoclonal anti-HIV antibodies can produce additive or synergistic neutralization of primary HIV-1 isolates. Passive immunotherapy for treatment or prophylaxis of HIV-1 should consider mixtures of potent neutralizing antibody reagents to expand the magnitude and breadth of virus neutralization.

  4. Potent and synergistic neutralization of human immunodeficiency virus (HIV) type 1 primary isolates by hyperimmune anti-HIV immunoglobulin combined with monoclonal antibodies 2F5 and 2G12.

    PubMed Central

    Mascola, J R; Louder, M K; VanCott, T C; Sapan, C V; Lambert, J S; Muenz, L R; Bunow, B; Birx, D L; Robb, M L

    1997-01-01

    Three antibody reagents that neutralize primary human immunodeficiency virus type 1 (HIV-1) isolates were tested for magnitude and breadth of neutralization when used alone or in double or triple combinations. Hyperimmune anti-HIV immunoglobulin (HIVIG) is derived from the plasma of HIV-1-infected donors, and monoclonal antibodies (MAbs) 2F5 and 2G12 bind to distinct regions of the HIV-1 envelope glycoprotein. The antibodies were initially tested against a panel of 15 clade B HIV-1 isolates, using a single concentration that is achievable in vivo (HIVIG, 2,500 microg/ml; MAbs, 25 microg/ml). Individual antibody reagents neutralized many of the viruses tested, but antibody potency varied substantially among the viruses. The virus neutralization produced by double combinations of HIVIG plus 2F5 or 2G12, the two MAbs together, or the triple combination of HIVIG, 2F5, and 2G12 was generally equal to or greater than that predicted by the effect of individual antibodies. Overall, the triple combination displayed the greatest magnitude and breadth of neutralization. Synergistic neutralization was evaluated by analyzing data from dose-response curves of each individual antibody reagent compared to the triple combination and was demonstrated against each of four viruses tested. Therefore, combinations of polyclonal and monoclonal anti-HIV antibodies can produce additive or synergistic neutralization of primary HIV-1 isolates. Passive immunotherapy for treatment or prophylaxis of HIV-1 should consider mixtures of potent neutralizing antibody reagents to expand the magnitude and breadth of virus neutralization. PMID:9311792

  5. 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.

  6. Toxicological screening of human plasma by on-line SPE-HPLC-DAD: identification and quantification of acidic and neutral drugs.

    PubMed

    Mut, Ludmila; Grobosch, Thomas; Binscheck-Domaß, Torsten; Frenzel, Wolfgang

    2016-03-01

    A multi-analyte screening method for the quantification of 50 acidic/neutral drugs in human plasma based on on-line solid-phase extraction (SPE)-HPLC with photodiode array detection (DAD) was developed, validated and applied for clinical investigation. Acetone and methanol for protein precipitation, three different SPE materials (two electro-neutral, one strong anion-exchange, one weak cation-exchange) for on-line extraction, five HPLC-columns [one C18 (GeminiNX), two phenyl-hexyl (Gemini C6 -Phenyl, Kinetex Phenyl-Hexyl) and two pentafluorophenyl (LunaPFP(2), KinetexPFP)] for analytical separation were tested. For sample pre-treatment, acetone in the ratio 1:2 (plasma:acetone) showed a better baseline and fewer matrix peaks in the chromatogram than methanol. Only the strong anion-exchanger SPE cartridge (StrataX-A, pH 6) allowed the extraction of salicylic acid. Analytical separation was carried out on a Gemini C6 -Phenyl column (150 × 4.6 mm, 3 µm) using gradient elution with acetonitrile-water 90:10 (v/v) and phosphate buffer (pH 2.3). Linear calibration curves with correlation coefficients r ≥ 0.9950/0.9910 were obtained for 46/four analytes. Additionally, this method allows the quantification of 23 analytes for therapeutic drug monitoring. Limits of quantitation ranged from 0.1 (amobarbital) to 23 mg/L (salicylic acid). Inter-/intra-day precisions of quality control samples (low/high) were better than 13% and accuracy (bias) ranged from -14 to 10%. A computer-assisted database was created for automated detection of 223 analytes of toxicological interests. Four cases of multi-drug intoxications are presented.

  7. 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.

  8. Phylogenetic analysis of the main neutralization and hemagglutination determinants of all human adenovirus prototypes as a basis for molecular classification and taxonomy.

    PubMed

    Madisch, Ijad; Harste, Gabi; Pommer, Heidi; Heim, Albert

    2005-12-01

    Human adenoviruses (HAdV) are responsible for a wide spectrum of diseases. The neutralization epsilon determinant (loops 1 and 2) and the hemagglutination gamma determinant are relevant for the taxonomy of HAdV. Precise type identification of HAdV prototypes is crucial for detection of infection chains and epidemiology. epsilon and gamma determinant sequences of all 51 HAdV were generated to propose molecular classification criteria. Phylogenetic analysis of epsilon determinant sequences demonstrated sufficient genetic divergence for molecular classification, with the exception of HAdV-15 and HAdV-29, which also cannot be differentiated by classical cross-neutralization. Precise sequence divergence criteria for typing (<2.5% from loop 2 prototype sequence and <2.4% from loop 1 sequence) were deduced from phylogenetic analysis. These criteria may also facilitate identification of new HAdV prototypes. Fiber knob (gamma determinant) phylogeny indicated a two-step model of species evolution and multiple intraspecies recombination events in the origin of HAdV prototypes. HAdV-29 was identified as a recombination variant of HAdV-15 (epsilon determinant) and a speculative, not-yet-isolated HAdV prototype (gamma determinant). Subanalysis of molecular evolution in hypervariable regions 1 to 6 of the epsilon determinant indicated different selective pressures in subclusters of species HAdV-D. Additionally, gamma determinant phylogenetic analysis demonstrated that HAdV-8 did not cluster with -19 and -37 in spite of their having the same tissue tropism. The phylogeny of HAdV-E4 suggested origination by interspecies recombination between HAdV-B (hexon) and HAdV-C (fiber), as in simian adenovirus 25, indicating additional zoonotic transfer. In conclusion, molecular classification by systematic sequence analysis of immunogenic determinants yields new insights into HAdV phylogeny and evolution.

  9. Crystallographic Definition of the Epitope Promiscuity of the Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 Antibody 2F5: Vaccine Design Implications▿

    PubMed Central

    Bryson, Steve; Julien, Jean-Philippe; Hynes, Rosemary C.; Pai, Emil F.

    2009-01-01

    The quest to create a human immunodeficiency virus type 1 (HIV-1) vaccine capable of eliciting broadly neutralizing antibodies against Env has been challenging. Among other problems, one difficulty in creating a potent immunogen resides in the substantial overall sequence variability of the HIV envelope protein. The membrane-proximal region (MPER) of gp41 is a particularly conserved tryptophan-rich region spanning residues 659 to 683, which is recognized by three broadly neutralizing monoclonal antibodies (bnMAbs), 2F5, Z13, and 4E10. In this study, we first describe the variability of residues in the gp41 MPER and report on the invariant nature of 15 out of 25 amino acids comprising this region. Subsequently, we evaluate the ability of the bnMAb 2F5 to recognize 31 varying sequences of the gp41 MPER at a molecular level. In 19 cases, resulting crystal structures show the various MPER peptides bound to the 2F5 Fab′. A variety of amino acid substitutions outside the 664DKW666 core epitope are tolerated. However, changes at the 664DKW666 motif itself are restricted to those residues that preserve the aspartate's negative charge, the hydrophobic alkyl-π stacking arrangement between the β-turn lysine and tryptophan, and the positive charge of the former. We also characterize a possible molecular mechanism of 2F5 escape by sequence variability at position 667, which is often observed in HIV-1 clade C isolates. Based on our results, we propose a somewhat more flexible molecular model of epitope recognition by bnMAb 2F5, which could guide future attempts at designing small-molecule MPER-like vaccines capable of eliciting 2F5-like antibodies. PMID:19740978

  10. The Inhibitory Effects of Anti-Oxidants on Ultraviolet-Induced Up-Regulation of the Wrinkling-Inducing Enzyme Neutral Endopeptidase in Human Fibroblasts

    PubMed Central

    Nakajima, Hiroaki; Terazawa, Shuko; Niwano, Takao; Yamamoto, Yorihiro; Imokawa, Genji

    2016-01-01

    We recently reported that the over-expression of skin fibroblast-derived neutral endopeptidase (NEP) plays a pivotal role in impairing the three-dimensional architecture of dermal elastic fibers during the biological mechanism of ultraviolet (UV)-induced skin wrinkling. In that process, a UVB-associated epithelial-mesenchymal cytokine interaction as well as a direct UVA-induced cellular stimulation are associated with the up-regulation of NEP in human fibroblasts. In this study, we characterized the mode of action of ubiquinol10 which may abrogate the up-regulation of NEP by dermal fibroblasts, resulting in a reported in vivo anti-wrinkling action, and compared that with 3 other anti-oxidants, astaxanthin (AX), riboflavin (RF) and flavin mononucleotide (FMN). Post-irradiation treatment with all 4 of those anti-oxidants elicited an interrupting effect on the UVB-associated epithelial-mesenchymal cytokine interaction leading to the up-regulation of NEP in human fibroblasts but with different modes of action. While AX mainly served as an inhibitor of the secretion of wrinkle-inducing cytokines, such as interleukin-1α (IL-1α) and granulocyte macrophage colony stimulatory factor (GM-CSF) in UVB-exposed epidermal keratinocytes, ubiquinol10, RF and FMN predominantly interrupted the IL-1α and GM-CSF-stimulated expression of NEP in dermal fibroblasts. On the other hand, as for the UVA-associated mechanism, similar to the abrogating effects reported for AX and FMN, ubiquinol10 but not RF had the potential to abrogate the increased expression of NEP and matrix-metalloproteinase-1 in UVA-exposed human fibroblasts. Our findings strongly support the in vivo anti-wrinkling effects of ubiquinol10 and AX on human and animal skin and provide convincing proof of the UV-induced wrinkling mechanism that essentially focuses on the over-expression of NEP by dermal fibroblasts as an intrinsic causative factor. PMID:27648570

  11. Human monoclonal antibodies derived from a patient infected with 2009 pandemic influenza A virus broadly cross-neutralize group 1 influenza viruses.

    PubMed

    Pan, Yang; Sasaki, Tadahiro; Kubota-Koketsu, Ritsuko; Inoue, Yuji; Yasugi, Mayo; Yamashita, Akifumi; Ramadhany, Ririn; Arai, Yasuha; Du, Anariwa; Boonsathorn, Naphatsawan; Ibrahim, Madiha S; Daidoji, Tomo; Nakaya, Takaaki; Ono, Ken-ichiro; Okuno, Yoshinobu; Ikuta, Kazuyoshi; Watanabe, Yohei

    2014-07-18

    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.

  12. Recombinant Receptor-Binding Domains of Multiple Middle East Respiratory Syndrome Coronaviruses (MERS-CoVs) Induce Cross-Neutralizing Antibodies against Divergent Human and Camel MERS-CoVs and Antibody Escape Mutants.

    PubMed

    Tai, Wanbo; Wang, Yufei; Fett, Craig A; Zhao, Guangyu; Li, Fang; Perlman, Stanley; Jiang, Shibo; Zhou, Yusen; Du, Lanying

    2017-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) binds to cellular receptor dipeptidyl peptidase 4 (DPP4) via the spike (S) protein receptor-binding domain (RBD). The RBD contains critical neutralizing epitopes and serves as an important vaccine target. Since RBD mutations occur in different MERS-CoV isolates and antibody escape mutants, cross-neutralization of divergent MERS-CoV strains by RBD-induced antibodies remains unknown. Here, we constructed four recombinant RBD (rRBD) proteins with single or multiple mutations detected in representative human MERS-CoV strains from the 2012, 2013, 2014, and 2015 outbreaks, respectively, and one rRBD protein with multiple changes derived from camel MERS-CoV strains. Like the RBD of prototype EMC2012 (EMC-RBD), all five RBDs maintained good antigenicity and functionality, the ability to bind RBD-specific neutralizing monoclonal antibodies (MAbs) and the DPP4 receptor, and high immunogenicity, able to elicit S-specific antibodies. They induced potent neutralizing antibodies cross-neutralizing 17 MERS pseudoviruses expressing S proteins of representative human and camel MERS-CoV strains identified during the 2012-2015 outbreaks, 5 MAb escape MERS-CoV mutants, and 2 live human MERS-CoV strains. We then constructed two RBDs mutated in multiple key residues in the receptor-binding motif (RBM) of RBD and demonstrated their strong cross-reactivity with anti-EMC-RBD antibodies. These RBD mutants with diminished DPP4 binding also led to virus attenuation, suggesting that immunoevasion after RBD immunization is accompanied by loss of viral fitness. Therefore, this study demonstrates that MERS-CoV RBD is an important vaccine target able to induce highly potent and broad-spectrum neutralizing antibodies against infection by divergent circulating human and camel MERS-CoV strains.

  13. 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.

  14. Human Circulating PD-1+CXCR3−CXCR5+ Memory Tfh Cells Are Highly Functional and Correlate with Broadly Neutralizing HIV Antibody Responses

    PubMed Central

    Locci, Michela; Havenar-Daughton, Colin; Landais, Elise; Wu, Jennifer; Kroenke, Mark A.; Arlehamn, Cecilia L.; Su, Laura F.; Cubas, Rafael; Davis, Mark M.; Sette, Alessandro; Haddad, Elias K.; Poignard, Pascal; Crotty, Shane

    2014-01-01

    SUMMARY The vast majority of currently licensed human vaccines work on the basis of long-term protective antibody responses. It is now conceivable that an antibody-dependent HIV vaccine might be possible, given the discovery of HIV broadly neutralizing antibodies (bnAbs) in some HIV-infected individuals. However, these antibodies are difficult to develop and have characteristics indicative of a high degree of affinity maturation in germinal centers (GCs). CD4+ T follicular helper (Tfh) cells are specialized for B cell help and necessary for GCs. Therefore, the development of HIV bnAbs might depend on Tfh cells. Here, we identified in normal individuals a subpopulation of circulating memory PD-1+CXCR5+ CD4+ T cells that are resting memory cells most related to bona fide GC Tfh cells by gene expression profile, cytokine profile, and functional properties. Importantly, the frequency of these cells correlated with the development of bnAbs against HIV in a large cohort of HIV+ individuals. PMID:24035365

  15. 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

  16. Human circulating PD-1+CXCR3-CXCR5+ memory Tfh cells are highly functional and correlate with broadly neutralizing HIV antibody responses.

    PubMed

    Locci, Michela; Havenar-Daughton, Colin; Landais, Elise; Wu, Jennifer; Kroenke, Mark A; Arlehamn, Cecilia L; Su, Laura F; Cubas, Rafael; Davis, Mark M; Sette, Alessandro; Haddad, Elias K; Poignard, Pascal; Crotty, Shane

    2013-10-17

    The vast majority of currently licensed human vaccines work on the basis of long-term protective antibody responses. It is now conceivable that an antibody-dependent HIV vaccine might be possible, given the discovery of HIV broadly neutralizing antibodies (bnAbs) in some HIV-infected individuals. However, these antibodies are difficult to develop and have characteristics indicative of a high degree of affinity maturation in germinal centers (GCs). CD4⁺ T follicular helper (Tfh) cells are specialized for B cell help and necessary for GCs. Therefore, the development of HIV bnAbs might depend on Tfh cells. Here, we identified in normal individuals a subpopulation of circulating memory PD-1⁺CXCR5⁺CD4⁺ T cells that are resting memory cells most related to bona fide GC Tfh cells by gene expression profile, cytokine profile, and functional properties. Importantly, the frequency of these cells correlated with the development of bnAbs against HIV in a large cohort of HIV⁺ individuals.

  17. Epitope mapping of rat neutralizing monoclonal antibody against human immunodeficiency virus type-1 by a phage peptide library: comparison with ELISA using synthetic peptides.

    PubMed

    Ichiyama, K; Ishikawa, D; Tanaka, Y; Kashiwa, T; Koyanagi, Y; Handa, S; Yamashita, A; Fukushi, M; Yamamoto, N; Taki, T

    1999-01-01

    We generated a rat monoclonal antibody (mAb W#10) with the ability to neutralize human immunodeficiency virus type 1IIIB (HIV-1IIIB) infection. The epitope recognized by mAb W#10 was defined as R-I-Q-R-G-P-G by enzyme-linked immunosorbent assay (ELISA) with the use of synthetic peptides. The filamentous phage clones displaying random 15-amino-acid peptides on the amino terminus of the pIII coat protein reacting with mAb W#10 were identified with affinity and immunological selection procedures. Thirteen out of 16 selected phage clones contained the G-X-G-R-X-F sequence in the coat protein region representing significant homology to a part of conserved G-P-G-R-A-F sequence in the V3 loop of various HIV-1 strains. In addition, the phage clones included the G-X-G sequence in the sequence detected by synthetic peptides as the recognition site. The selected phage clones were stained by mAb W#10 specifically and were able to compete with mAb binding to cells expressing viral antigens.

  18. 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

  19. Quantification of neutralizing antibodies to human type I interferons using division-arrested frozen cells carrying an interferon-regulated reporter-gene.

    PubMed

    Lallemand, C; Meritet, J-F; Erickson, R; Grossberg, S E; Roullet, E; Lyon-Caen, O; Lebon, P; Tovey, M G

    2008-06-01

    Development of neutralizing antibodies (NAbs) to interferons (IFNs) can reduce the clinical response to IFN therapy. As current cell-based assays for quantifying NAbs have limitations, a highly sensitive and reproducible assay was developed, using division-arrested frozen human U937 cells transfected with the luciferase reportergene controlled by an IFN-responsive chimeric promoter, which allows IFN activity to be determined with precision within hours. Assay-ready PIL5 cells can be stored frozen for >3 years without loss of IFN sensitivity or the need for cell propagation. The assay is highly IFN sensitive (detecting <1.0 IU/mL), reproducible (SE +/- 15%) over concentrations from <1.0 to 100 IU/mL and able to measure different IFN subtypes and their pegylated variants. The use of this assay has shown that NAbs from patients treated with IFN-alpha2 exhibited markedly lower titers against 10 LU/mL of low specific activity IFNs, namely, IFN-alpha1, PEG-Intron(TM) (Schering-Plough, Levallois-Perret,France), or Pegasys(TM) (Hoffmann-La Roche, Neuilly-sur-Seine, France, than against 10 LU/mL IFN-alpha2. Similarly, NAbs from patients treated with IFN-beta1a exhibit lower titers against 10 LU/mL of low specific activity IFN-beta1b than against IFN-beta1a. The combination of the use of division-arrested, IFN-responsive human cells transfected with the luciferase reporter-gene makes the rapid PIL5 assay for NAbs highly advantageous.

  20. 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

    2014-05-13

    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.

  1. Applying antibody-sensitive hypervariable region 1-deleted hepatitis C virus to the study of escape pathways of neutralizing human monoclonal antibody AR5A

    PubMed Central

    Velázquez-Moctezuma, Rodrigo; Bukh, Jens

    2017-01-01

    Hepatitis C virus (HCV) is a major cause of end-stage liver diseases. With 3–4 million new HCV infections yearly, a vaccine is urgently needed. A better understanding of virus escape from neutralizing antibodies and their corresponding epitopes are important for this effort. However, for viral isolates with high antibody resistance, or antibodies with moderate potency, it remains challenging to induce escape mutations in vitro. Here, as proof-of-concept, we used antibody-sensitive HVR1-deleted (ΔHVR1) viruses to generate escape mutants for a human monoclonal antibody, AR5A, targeting a rare cross-genotype conserved epitope. By analyzing the genotype 1a envelope proteins (E1/E2) of recovered Core-NS2 recombinant H77/JFH1ΔHVR1 and performing reverse genetic studies we found that resistance to AR5A was caused by substitution L665W, also conferring resistance to the parental H77/JFH1. The mutation did not induce viral fitness loss, but abrogated AR5A binding to HCV particles and intracellular E1/E2 complexes. Culturing J6/JFH1ΔHVR1 (genotype 2a), for which fitness was decreased by L665W, with AR5A generated AR5A-resistant viruses with the substitutions I345V, L665S, and S680T, which we introduced into J6/JFH1 and J6/JFH1ΔHVR1. I345V increased fitness but had no effect on AR5A resistance. L665S impaired fitness and decreased AR5A sensitivity, while S680T combined with L665S compensated for fitness loss and decreased AR5A sensitivity even further. Interestingly, S680T alone had no fitness effect but sensitized the virus to AR5A. Of note, H77/JFH1L665S was non-viable. The resistance mutations did not affect cell-to-cell spread or E1/E2 interactions. Finally, introducing L665W, identified in genotype 1, into genotypes 2–6 parental and HVR1-deleted variants (not available for genotype 4a) we observed diverse effects on viral fitness and a universally pronounced reduction in AR5A sensitivity. Thus, we were able to take advantage of the neutralization-sensitive HVR1

  2. 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.

  3. 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…

  4. Phytosphingosine-containing neutral glycosphingolipids and sulfatides in the human female genital tract: their association in the cervical epithelium and the uterine endometrium and their dissociation in the mucosa of fallopian tube with the menstrual cycle.

    PubMed

    Takamatsu, K

    1992-09-01

    In human cervical epithelium and uterine endometrium, globo-series neutral glycosphingolipids with N-alpha-hydroxy fatty acyl phytosphingosine (4-D-hydroxysphinganine) as the ceramide and sulfatide (I3SO3-GalCer), which were contained in trace amount at the follicular phase, significantly increased in concentration at the luteal phase, comprising about 20% of the individual neutral glycosphingolipids and about 15% of the total acidic glycosphingolipids, respectively. However, in the mucosa of fallopian tube, neutral glycosphingolipids with the same polarity as those in the cervical epithelium and uterine endometrium at the luteal phase and sulfatide remained at a constant and higher level independently of the menstrual cycle. The structures of neutral glycosphingolipids in the fallopian tube, having the same polarity as that of N-alpha-hydroxy fatty acyl phytosphingosine-containing molecules appeared in the cervical epithelium and uterine endometrium at the luteal phase, were determined to be N-alpha-hydroxy palmitoyl 4-sphingenine-containing ones by negative-ion FABMS. Also, laminin, but not collagen type IV, was found to be contained in the concentration correlated well with that of sulfatide in the genital tract, when determined by western blotting with monoclonal anti-laminin and anti-collagen type IV antibodies, indicating a possible function of sulfatide as a receptor for laminin in the human female genital tract.

  5. 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

  6. Antigenic analysis of divergent genotypes human Enterovirus 71 viruses by a panel of neutralizing monoclonal antibodies: current genotyping of EV71 does not reflect their antigenicity.

    PubMed

    Chen, Yixin; Li, Chuan; He, Delei; Cheng, Tong; Ge, Shengxiang; Shih, James Wai-Kuo; Zhao, Qinjian; Chen, Pei-Jer; Zhang, Jun; Xia, Ningshao

    2013-01-02

    In recent year, Enterovirus 71 (EV71)-associated hand, foot and mouth disease (HFMD) has become an important public health issue in China. EV71 has been classified into genotypes A, B1-B5 and C1-C5. With such genetic diversity, whether the convalescent or recovery antibody responses can cross-protect infections from other genotypes remains a question. Understanding of the antigenicity of such diverse genetic EV71 isolates is crucial for the EV71 vaccine development. Here, a total of 186 clones anti-EV71 MAbs was generated and characterized with Western blot and cell-based neutralization assay. Forty neutralizing anti-EV71 MAbs were further used to analyze the antigenic properties of 18 recent EV71 isolates representing seven genotypes in neutralization assay. We found that most neutralizing anti-EV71 MAbs are specific to conformational epitopes. We also classified the 40 neutralizing anti-EV71 MAbs into two classes according to their reactivity patterns with 18 EV71 isolates. Class I MAb can neutralize all isolates, suggesting conserved epitopes are present among EV71. Class II MAb includes four subclasses (IIa-IId) and neutralizes only subgroups of EV71 strains. Conversely, 18 EV71 strains were grouped into antigenic types 1 and four antigenic subtypes (2.1-2.4). These results suggest that the current genotyping of EV71 does not reflect their antigenicity which may be important in the selection of EV71 vaccine strains. This panel of neutralizing anti-EV71 MAbs may be useful for the recognition of emerging antigenic variants of EV71 and vaccine development.

  7. Cytoskeletal protein Flightless (Flii) is elevated in chronic and acute human wounds and wound fluid: neutralizing its activity in chronic but not acute wound fluid improves cellular proliferation.

    PubMed

    Ruzehaji, Nadira; Grose, Randall; Krumbiegel, Doreen; Zola, Heddy; Dasari, Pallave; Wallace, Hilary; Stacey, Michael; Fitridge, Robert; Cowin, Allison J

    2012-01-01

    Chronic non-healing wounds form a medical need which will expand as the population ages and the obesity epidemic grows. Whilst the complex mechanisms underlying wound repair are not fully understood, remodelling of the actin cytoskeleton plays a critical role. Elevated expression of the actin cytoskeletal protein Flightless I (Flii) is known to impair wound outcomes. To determine if Flii is involved in the impaired healing observed in chronic wounds, its expression in non-healing human wounds from patients with venous leg ulcers was determined and compared to its expression in acute wounds and unwounded skin. Increased expression of Flii was observed in both chronic and acute wounds with wound fluid and plasma also containing secreted Flii protein. Inflammation is a key aspect of wound repair and fluorescence-activated cell sorting (FACS) analysis revealed Flii was located in neutrophils within the blood and that it co-localised with CD16+ neutrophils in chronic wounds. The function of secreted Flii was investigated as both chronic wound fluid and Flii have previously been shown to inhibit fibroblast proliferation. To determine if the inhibitory effect of wound fluid was due in part to the presence of Flii, wound fluids were depleted of Flii using Flii-specific neutralizing antibodies (FnAb). Flii depleted chronic wound fluid no longer inhibited fibroblast proliferation, suggesting that Flii may contribute to the inhibitory effect of chronic wound fluid on fibroblast function. Application of FnAbs to chronic wounds may therefore be a novel approach used to improve the local environment of non-healing wounds and potentially improve healing outcomes.

  8. Automated facial coding software outperforms people in recognizing neutral faces as neutral from standardized datasets.

    PubMed

    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.

  9. 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

  10. Co-vaccination with adeno-associated virus vectors encoding human papillomavirus 16 L1 proteins and adenovirus encoding murine GM-CSF can elicit strong and prolonged neutralizing antibody.

    PubMed

    Liu, Dai-Wei; Chang, Junn-Liang; Tsao, Yeou-Ping; Huang, Chien-Wei; Kuo, Shu-Wen; Chen, Show-Li

    2005-01-01

    Non-infectious human papillomavirus-like particles (VLPs), encoded by the major capsid gene L1, have been shown to be effective as vaccines to prevent cervical cancer. We have developed the genetic immunization of the L1 gene to induce a neutralizing antibody. We constructed and generated a recombinant adeno-associated virus encoding human papillomavirus (HPV) 16 L1 protein that could form virus-like particles in transduced cells. Previous reports have demonstrated that the formation of VLP is necessary to induce high titers of neutralizing antibodies to protect an animal from viral challenge. Therefore, we carried out a single intramuscular (i.m.) injection with recombinant adeno-associated virus encoding HPV-16 L1 protein (rAAV-16L1) in BALB/c mice, which ultimately produced stronger and more prolonged neutralizing L1 antibodies, when compared to the DNA vaccine. Immunohistochemistry showed that the accumulation of antigen presenting cells, such as macrophages and dendritic cells, in rAAV-16L1 and L1 DNA-injected muscle fibers may be due to the L1 protein expression, but not to AAV infection. When compared to the L1 VLP vaccine, however, the titers of neutralizing L1 antibodies induced by VLP were higher than those induced by rAAV-16L1. Co-vaccinating with rAAV-16L1 and adenovirus encoding murine GM-CSF (rAAV-16L1/rAd-mGM-CSF) induced comparable higher levels of neutralizing L1 antibodies with those of VLP. This implies that a single i.m. co-injection with rAAV-16L1/rAd-mGM-CSF can achieve the same vaccine effect as a VLP vaccine requiring 3 booster injections.

  11. In vitro assay for neutralizing antibody to hepatitis C virus: evidence for broadly conserved neutralization epitopes.

    PubMed

    Bartosch, Birke; Bukh, Jens; Meunier, Jean-Christophe; Granier, Christelle; Engle, Ronald E; Blackwelder, William C; Emerson, Suzanne U; Cosset, François-Loïc; Purcell, Robert H

    2003-11-25

    Our understanding of the humoral immune response to hepatitis C virus (HCV) is limited because the virus can be studied only in humans and chimpanzees and because previously described neutralization assays have not been robust or simple to perform. Nevertheless, epidemiologic and laboratory studies suggested that neutralizing Ab to HCV might be important in preventing infection. We have recently described a neutralization assay based on the neutralization of pseudotyped murine retrovirus constructs bearing HCV envelope glycoproteins on their surface. We have applied the assay to well characterized clinical samples from HCV-infected patients and chimpanzees, confirmed the existence of neutralizing Ab to HCV, and validated most previously reported neutralizations of the virus. We did not find neutralizing anti-HCV in resolving infections but did find relatively high titers (>1:320) of such Ab in chronic infections. Neutralizing Ab was directed not only to epitope(s) in the hypervariable region of the E2 envelope protein but also to one or more epitopes elsewhere in the envelope of the virus. Neutralizing Ab was broadly reactive and could neutralize pseudotype particles bearing the envelope glycoproteins of two different subgenotypes (1a and 1b). The ability to assay neutralizing anti-HCV should permit an assessment of the prospects for successful Ab-mediated passive and active immunoprophylaxis against hepatitis C.

  12. In vitro assay for neutralizing antibody to hepatitis C virus: Evidence for broadly conserved neutralization epitopes

    PubMed Central

    Bartosch, Birke; Bukh, Jens; Meunier, Jean-Christophe; Granier, Christelle; Engle, Ronald E.; Blackwelder, William C.; Emerson, Suzanne U.; Cosset, François-Loïc; Purcell, Robert H.

    2003-01-01

    Our understanding of the humoral immune response to hepatitis C virus (HCV) is limited because the virus can be studied only in humans and chimpanzees and because previously described neutralization assays have not been robust or simple to perform. Nevertheless, epidemiologic and laboratory studies suggested that neutralizing Ab to HCV might be important in preventing infection. We have recently described a neutralization assay based on the neutralization of pseudotyped murine retrovirus constructs bearing HCV envelope glycoproteins on their surface. We have applied the assay to well characterized clinical samples from HCV-infected patients and chimpanzees, confirmed the existence of neutralizing Ab to HCV, and validated most previously reported neutralizations of the virus. We did not find neutralizing anti-HCV in resolving infections but did find relatively high titers (>1:320) of such Ab in chronic infections. Neutralizing Ab was directed not only to epitope(s) in the hypervariable region of the E2 envelope protein but also to one or more epitopes elsewhere in the envelope of the virus. Neutralizing Ab was broadly reactive and could neutralize pseudotype particles bearing the envelope glycoproteins of two different subgenotypes (1a and 1b). The ability to assay neutralizing anti-HCV should permit an assessment of the prospects for successful Ab-mediated passive and active immunoprophylaxis against hepatitis C. PMID:14617769

  13. Cross-neutralizing anti-HIV-1 human single chain variable fragments(scFvs) against CD4 binding site and N332 glycan identified from a recombinant phage library

    PubMed Central

    Khan, Lubina; Kumar, Rajesh; Thiruvengadam, Ramachandran; Parray, Hilal Ahmad; Makhdoomi, Muzamil Ashraf; Kumar, Sanjeev; Aggarwal, Heena; Mohata, Madhav; Hussain, Abdul Wahid; Das, Raksha; Varadarajan, Raghavan; Bhattacharya, Jayanta; Vajpayee, Madhu; Murugavel, K. G.; Solomon, Suniti; Sinha, Subrata; Luthra, Kalpana

    2017-01-01

    More than 50% of HIV-1 infection globally is caused by subtype_C viruses. Majority of the broadly neutralizing antibodies (bnAbs) targeting HIV-1 have been isolated from non-subtype_C infected donors. Mapping the epitope specificities of bnAbs provides useful information for vaccine design. Recombinant antibody technology enables generation of a large repertoire of monoclonals with diverse specificities. We constructed a phage recombinant single chain variable fragment (scFv) library with a diversity of 7.8 × 108 clones, using a novel strategy of pooling peripheral blood mononuclear cells (PBMCs) of six select HIV-1 chronically infected Indian donors whose plasma antibodies exhibited potent cross neutralization efficiency. The library was panned and screened by phage ELISA using trimeric recombinant proteins to identify viral envelope specific clones. Three scFv monoclonals D11, C11 and 1F6 selected from the library cross neutralized subtypes A, B and C viruses at concentrations ranging from 0.09 μg/mL to 100 μg/mL. The D11 and 1F6 scFvs competed with mAbs b12 and VRC01 demonstrating CD4bs specificity, while C11 demonstrated N332 specificity. This is the first study to identify cross neutralizing scFv monoclonals with CD4bs and N332 glycan specificities from India. Cross neutralizing anti-HIV-1 human scFv monoclonals can be potential candidates for passive immunotherapy and for guiding immunogen design. PMID:28332627

  14. 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

    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

    2015-06-26

    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.

  15. 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.

  16. 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.

  17. 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

  18. Neutralization Assay for Chikungunya Virus Infection: Plaque Reduction Neutralization Test.

    PubMed

    Azami, Nor Azila Muhammad; Moi, Meng Ling; Takasaki, Tomohiko

    2016-01-01

    Neutralization assay is a technique that detects and quantifies neutralizing antibody in serum samples by calculating the percentage of reduction of virus activity, as the concentration of virus used is usually constant. Neutralizing antibody titer is conventionally determined by calculating the percentage reduction in total virus infectivity by counting and comparing number of plaques (localized area of infection due to cytopathic effect) with a standard amount of virus. Conventional neutralizing test uses plaque-reduction neutralization test (PRNT) to determine neutralizing antibody titers against Chikungunya virus (CHIKV). Here we describe the plaque reduction neutralization assay (PRNT) using Vero cell lines to obtain neutralizing antibody titers.

  19. 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 ...

  20. Ions and neutralization

    NASA Astrophysics Data System (ADS)

    Poncet, A.

    After a short presentation of intensity limitations examples due to trapped ions, the processes of ionization and neutralization build up in particle accelerators and storage rings are briefly reviewed. The tolerable limits in neutralization are then assessed at the light of current theories of incoherent and coherent effects driven by ions. Finally the usual antidotes such as clearing electrodes, missing bunch schemes and beam shaking are presented.

  1. Use of monoclonal antibodies with neutralizing effects on toxic antigens from human bacterial plaque to detect specific bacteria by colony blotting.

    PubMed Central

    Levine, M; Miller, F C

    1991-01-01

    Inflammatory periodontal diseases are provoked by bacteria which adhere to teeth at the gingival margin and form plaques containing toxins detectable by their effect on mammalian cells in culture. The aim of this study was to make toxin-neutralizing monoclonal antibodies and determine whether they detect antigen in specific oral bacteria. Bacterial plaque was collected from teeth and homogenized, and the fluid phase (plaque extract) was boiled or first fractionated over Sephacryl S-300. Hybridomas from immunized mice secreted immunoglobulin M (IgM) antibodies which reacted to plaque antigens. Neutralization was detected by an increase in the growth of HL60 cells which were exposed to plaque toxins in the presence of IgM from hybridoma culture or ascitic fluids. However, the neutralization was obvious only when the plaque toxins reduced growth by 50% or less. Plaque toxin preparations were found to contain proteases which hydrolyzed all of the IgM in ascitic fluids within 24 h. Replenishing the IgM daily preserved protection compared with protection from IgM from other hybridomas or saline only. The decrease in the specific activity of plaque proteins caused by replenishing one such antibody (3hE5) was 2.5-fold compared with activity with unreplenished 3hE5, 3.8-fold compared with activity with saline only, and 10.7-fold compared with activity with replenished, unrelated antibody. The neutralizing IgM detected an array of 14,000- to 22,000-molecular-weight antigens. The native toxins may be aggregates of these antigens, or the array may indicate fragments of an undetected, larger antigen or a common, nonpeptide adduct. Only 0.5 to 0.8% of the bacteria from sites with periodontitis and grown on blood agar contained antigen. One group of reactive bacteria was identified as Actinomyces odontolyticus serotype I. Other isolates were identified as Staphylococcus epidermidis, but antigen disappeared from the these isolates within 6 weeks of subculture. Epitope

  2. 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.

  3. 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.

  4. A strategy for the generation of specific human antibodies by directed evolution and phage display. An example of a single-chain antibody fragment that neutralizes a major component of scorpion venom.

    PubMed

    Riaño-Umbarila, Lidia; Juárez-González, Victor Rivelino; Olamendi-Portugal, Timoteo; Ortíz-León, Mauricio; Possani, Lourival Domingos; Becerril, Baltazar

    2005-05-01

    This study describes the construction of a library of single-chain antibody fragments (scFvs) from a single human donor by individual amplification of all heavy and light variable domains (1.1 x 10(8) recombinants). The library was panned using the phage display technique, which allowed selection of specific scFvs (3F and C1) capable of recognizing Cn2, the major toxic component of Centruroides noxius scorpion venom. The scFv 3F was matured in vitro by three cycles of directed evolution. The use of stringent conditions in the third cycle allowed the selection of several improved clones. The best scFv obtained (6009F) was improved in terms of its affinity by 446-fold, from 183 nm (3F) to 410 pm. This scFv 6009F was able to neutralize 2 LD(50) of Cn2 toxin when a 1 : 10 molar ratio of toxin-to-antibody fragment was used. It was also able to neutralize 2 LD(50) of the whole venom. These results pave the way for the future generation of recombinant human antivenoms.

  5. Non-random escape pathways from a broadly neutralizing human monoclonal antibody map to a highly conserved region on the hepatitis C virus E2 glycoprotein encompassing amino acids 412-423.

    PubMed

    Keck, Zhen-yong; Angus, Allan G N; Wang, Wenyan; Lau, Patrick; Wang, Yong; Gatherer, Derek; Patel, Arvind H; Foung, Steven K H

    2014-08-01

    A challenge for hepatitis C virus (HCV) vaccine development is to define epitopes that are able to elicit protective antibodies against this highly diverse virus. The E2 glycoprotein region located at residues 412-423 is conserved and antibodies to 412-423 have broadly neutralizing activities. However, an adaptive mutation, N417S, is associated with a glycan shift in a variant that cannot be neutralized by a murine but by human monoclonal antibodies (HMAbs) against 412-423. To determine whether HCV escapes from these antibodies, we analyzed variants that emerged when cell culture infectious HCV virions (HCVcc) were passaged under increasing concentrations of a specific HMAb, HC33.1. Multiple nonrandom escape pathways were identified. Two pathways occurred in the context of an N-glycan shift mutation at N417T. At low antibody concentrations, substitutions of two residues outside of the epitope, N434D and K610R, led to variants having improved in vitro viral fitness and reduced sensitivity to HC33.1 binding and neutralization. At moderate concentrations, a S419N mutation occurred within 412-423 in escape variants that have greatly reduced sensitivity to HC33.1 but compromised viral fitness. Importantly, the variants generated from these pathways differed in their stability. N434D and K610R-associated variants were stable and became dominant as the virions were passaged. The S419N mutation reverted back to N419S when immune pressure was reduced by removing HC33.1. At high antibody concentrations, a mutation at L413I was observed in variants that were resistant to HC33.1 neutralization. Collectively, the combination of multiple escape pathways enabled the virus to persist under a wide range of antibody concentrations. Moreover, these findings pose a different challenge to vaccine development beyond the identification of highly conserved epitopes. It will be necessary for a vaccine to induce high potency antibodies that prevent the formation of escape variants, which

  6. Identification of 81LGxGxxIxW89 and 171EDRW174 Domains from Human Immunodeficiency Virus Type 1 Vif That Regulate APOBEC3G and APOBEC3F Neutralizing Activity▿

    PubMed Central

    Dang, Ying; Davis, Roderick W.; York, Ian A.; Zheng, Yong-Hui

    2010-01-01

    The human cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) potently restrict human immunodeficiency virus type 1 (HIV-1) replication, but they are neutralized by the viral protein Vif. Vif bridges A3G and A3F with a Cullin 5 (Cul5)-based E3 ubiquitin ligase and mediates their proteasomal degradation. This mechanism has been extensively studied, and several Vif domains have been identified that are critical for A3G and A3F neutralization. Here, we identified two additional domains. Via sequence analysis of more than 2,000 different HIV-1 Vif proteins, we identified two highly conserved amino acid sequences, 81LGxGxSIEW89 and 171EDRWN175. Within the 81LGxGxSIEW89 sequence, residues L81, G82, G84, and, to a lesser extent, I87 and W89 play very critical roles in A3G/A3F neutralization. In particular, residues L81 and G82 determine Vif binding to A3F, residue G84 determines Vif binding to both A3G and A3F, and residues 86SIEW89 affect Vif binding to A3F, A3G, and Cul5. Accordingly, this 81LGxGxSIEW89 sequence was designated the 81LGxGxxIxW89 domain. Within the 171EDRWN175 sequence, all residues except N175 are almost equally important for regulation of A3F neutralization, and consistently, they determine Vif binding only to A3F. Accordingly, this domain was designated 171EDRW174. The LGxGxxIxW domain is also partially conserved in simian immunodeficiency virus Vif from rhesus macaques (SIVmac239) and has a similar activity. Thus, 81LGxGxxIxW89 and 171EDRW174 are two novel functional domains that are very critical for Vif function. They could become new targets for inhibition of Vif activity during HIV replication. PMID:20335268

  7. Tautomerism in neutral histidine.

    PubMed

    Bermúdez, Celina; Mata, Santiago; Cabezas, Carlos; Alonso, José L

    2014-10-06

    Histidine is an important natural amino acid, involved in many relevant biological processes, which, because of its physical properties, proved difficult to characterize experimentally in its neutral form. In this work, neutral histidine has been generated in the gas phase by laser ablation of solid samples and its N(ε)H tautomeric form unraveled through its rotational spectrum. The quadrupole hyperfine structure, arising from the existing three (14)N nuclei, constituted a site-specifically probe for revealing the tautomeric form as well as the side chain configuration of this proteogenic amino acid.

  8. 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.

  9. 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.

  10. 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…

  11. An investigation of the potential effect of sperm nuclear vacuoles in human spermatozoa on DNA fragmentation using a neutral and alkaline Comet assay.

    PubMed

    Pastuszek, E; Kiewisz, J; Skowronska, P; Liss, J; Lukaszuk, M; Bruszczynska, A; Jakiel, G; Lukaszuk, K

    2017-03-01

    Presence of vacuoles and degree of sperm DNA damage are considered to be the basic factors used for the assessment of sperm fertilization capacity. We aimed to investigate the link between these two parameters. According to our knowledge, this is the first study where the Comet assay was used to assess the degree of DNA fragmentation of sperm categorized by Motile Sperm Organelle Morphology Examination (MSOME) Grades. Semen samples from 10 patients were assessed. Spermatozoa were graded into four MSOME groups according to the Vanderzwalmen's criteria. A total of 3930 motile spermatozoa were selected one-by-one using an inverted microscope and transferred onto two different slides. The degree of DNA fragmentation was analyzed by alkaline and neutral Comet assay. Results of the neutral Comet assay showed that Grade I spermatozoa (absence of vacuoles) presented significantly lower dsDNA fragmentation level (mean: 3.13 ± 1.17%) than Grade II (maximum of two small vacuoles; mean: 10.34 ± 2.65%), Grade III (more than two small vacuoles or at least one large vacuole; mean: 23.88 ± 8.37%), and Grade IV (large vacuoles associated with abnormal head shapes or other abnormalities; mean: 36.94 ± 7.78%; p < 0.05). Results of the alkaline Comet assay showed that Grade I spermatozoa had significantly lower DNA (ssDNA + dsDNA) fragmentation level (mean: 8.33 ± 3.62%) than Grade III (mean: 25.64 ± 9.15%) and Grade IV (mean: 40.10 ± 9.10%, p < 0.05), but not significantly lower than Grade II (mean: 12.73 ± 5.06%; p > 0.05). Probably, the vacuoles may be responsible for double strand DNA breaks rather than single strand DNA breaks (only 2.39% spermatozoa in MSOME Grade II, 1.76% in III, and 3.16% in IV has single strand breaks). The results demonstrate that lower MSOME grading correlates with lower sperm DNA fragmentation. Therefore, the observation of sperm nuclear vacuoles using real-time optical microscopy without precise DNA fragmentation

  12. An immunodominant epitope in a functional domain near the N-terminus of human granulocyte-macrophage colony-stimulating factor identified by cross-reaction of synthetic peptides with neutralizing anti-protein and anti-peptide antibodies.

    PubMed

    Beffy, P; Rovero, P; Di Bartolo, V; Laricchia Robbio, L; Dané, A; Pegoraro, S; Bertolero, F; Revoltella, R P

    1994-12-01

    We produced polyclonal and monoclonal antibodies (MAbs) against recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) and performed studies of epitope mapping by ELISA, using five synthetic peptides corresponding to sequences along this molecule. Additionally, anti-peptide MAbs were generated. The antibody ability to inhibit rhGM-CSF activity was determined using as bioassay the MO7e cell line, which is dependent on hGM-CSF for growth in vitro. An immunodominant epitope able to induce the highest neutralization antibody titers was identified near the N terminus of hGM-CSF. A synthetic peptide 14-24, homologous to a sequence including part of the first alpha-helix of the molecule, was recognized by neutralizing anti-protein antibodies. Similarly, MAbs anti- 14-24 cross-reacted with rhGM-CSF and specifically blocked its function. Replacement of Val16 or Asn17 with alanine greatly reduced the antibody-binding capacity to peptide 14-24, whereas substitution of Gln20 or Glu21 was less critical. Monoclonal antibodies generated against residues 30-41 (corresponding to an intrahelical loop) and 79-91 (homologous to a sequence including part of the third alpha-helix) or its analog [Ala88](79-91)beta Ala-Cys, were conformation dependent and nonneutralizing: they failed to react or bound poorly to rhGM-CSF in ELISA, but readily recognized the homologous sequence in the denatured protein, by Western blotting.

  13. Decreasing sensitivity to RANTES (regulated on activation, normally T cell-expressed and -secreted) neutralization of CC chemokine receptor 5-using, non-syncytium-inducing virus variants in the course of human immunodeficiency virus type 1 infection.

    PubMed

    Koning, Fransje A; Kwa, David; Boeser-Nunnink, Brigitte; Dekker, Jos; Vingerhoed, Jose; Hiemstra, Harry; Schuitemaker, Hanneke

    2003-09-15

    In approximately half of human immunodeficiency virus (HIV) type 1-infected individuals, the development of CXC chemokine receptor 4-using, syncytium-inducing (SI) virus variants precedes a rapid progression to acquired immunodeficiency syndrome (AIDS). In other individuals, only CC chemokine receptor 5-using (R5), non-SI (NSI) virus variants are present throughout infection. These individuals may be either long-term survivors (LTSs) or rapid progressors. The basis for this variable disease progression in individuals with only R5 virus variants is not yet fully understood. In this study, the beta-chemokine sensitivity of biological HIV-1 clones isolated from 13 individuals who harbored only R5, NSI virus variants (7 LTSs and 6 progressors) was investigated. We found a statistically significant decrease in sensitivity of virus variants to RANTES (regulated on activation, normally T cell-expressed and -secreted) neutralization during the course of progressive infection, but not during follow-up of LTSs. Our data suggest that a role exists for RANTES neutralization sensitivity of HIV-1 in AIDS pathogenesis.

  14. Rapid isolation of dengue-neutralizing antibodies from single cell-sorted human antigen-specific memory B-cell cultures.

    PubMed

    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.

  15. Study on the use of an enzyme-linked immunosorbent assay in determining human antibodies to diphtheria toxin as compared with a reference toxin neutralization assay.

    PubMed

    Skoura, L; Efstratiou, A; Tsakris, A; Pournaras, S; George, R C; Douboyas, J

    1999-07-01

    Serum samples from 156 Greek persons were assessed by an IgG-specific enzyme-linked immunosorbent assay (ELISA) and a reference tissue culture toxin-neutralization (TN) assay for the quantitation of diphtheria toxin antibodies. By the reference method, 7.7% of the persons were susceptible to diphtheria (antitoxin < 0.01 IU/ml), 28.8% had basic protection (antitoxin 0.01-0.09 IU/ml) and 63.5% were fully protective (antitoxin > or = 0.1 IU/ ml), while the corresponding figures were 17.9, 36.5 and 45.5% when they were tested by the immunoassay. None of the samples been susceptible by the TN assay were found to have some protection when tested by ELISA. However, three (6.7%) of the 45 samples showing a basic protection with TN, were fully protective when titrated by the immunoassay. In addition, 31 (31.3%) of the 99 samples been fully protective by the bioassay, were found to be either basically protective or susceptible by means of the ELISA. Overall, validity features of the immunoassay were: sensitivity 68.7%, specificity 94.7%, positive predictive value 95.8% and negative predictive value 63.5%. The ELISA tested in our study could be used to determine diphtheria antitoxin in individuals needed a booster immunization (susceptible or basic protective samples), although it might falsely include in the above categories samples that are within the fully protective levels of antibodies.

  16. 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

  17. Carbon neutral hydrocarbons.

    PubMed

    Zeman, Frank S; Keith, David W

    2008-11-13

    Reducing greenhouse gas emissions from the transportation sector may be the most difficult aspect of climate change mitigation. We suggest that carbon neutral hydrocarbons (CNHCs) offer an alternative pathway for deep emission cuts that complement the use of decarbonized energy carriers. Such fuels are synthesized from atmospheric carbon dioxide (CO2) and carbon neutral hydrogen. The result is a liquid fuel compatible with the existing transportation infrastructure and therefore capable of a gradual deployment with minimum supply disruption. Capturing the atmospheric CO2 can be accomplished using biomass or industrial methods referred to as air capture. The viability of biomass fuels is strongly dependent on the environmental impacts of biomass production. Strong constraints on land use may favour the use of air capture. We conclude that CNHCs may be a viable alternative to hydrogen or conventional biofuels and warrant a comparable level of research effort and support.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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…

  3. 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...

  4. 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...

  5. 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...

  6. 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...

  7. Neutralization Effects of Interleukin-6 (IL-6) Antibodies on Sulfur Mustard (HD)-Induced IL-6 Secretion on Human Epidermal Keratinocytes

    DTIC Science & Technology

    2004-01-01

    epidermal necrolysis. Their observations con- inflammatory blister induction. Rhodes et al. examined the firm that different secretion-related...blisters in the immune-based inflammatory disorders concluded that individual cytokines have different actions depending on the cytokine microenvironment...human epidermal keratinocytes are reported. Fi- stimulation. No significant differences were observed when nally, a discussion in the light of their

  8. 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

    Fujii, Hideki; Shimizu, Mamoru; Miyagi, Takuya; Kunihiro, Marie; Tanaka, Reiko; Takahashi, Yoshiaki; Tanaka, Yuetsu

    2016-02-03

    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.

  9. Neutral glycosphingolipid content of ovine milk.

    PubMed

    Zancada, L; Sánchez-Juanes, F; Alonso, J M; Hueso, P

    2010-01-01

    Milk glycosphingolipids (GSL) have been reported to participate in the newborn's defense against pathogens. Taking this into account, in this study we determined the neutral GSL content of ovine milk, including its fatty acid profile. Its role in bacterial adhesion was also addressed by immunodetection of separate GSL in a high-performance thin-layer chromatography overlay assay. Ovine milk has a neutral GSL pattern similar to human milk and includes lactosylceramide (LacCer; 45.7%), monohexosylceramide (glucosylceramide and galactosylceramide, 31.2%), globotriaosylceramide (Gb3; 19.1%), and globotetraosylceramide (Gb4; 3.5%). Globotriaosylceramide and Gb4 are present in human but not bovine milk. Neutral GSL contained C23:0 and C24:0 as the most abundant fatty acids, a finding consistent with its high content of very long chain fatty acids (longer than C20). Most fatty acids were saturated and had a low content of polyunsaturated fatty acids. Bovine enterotoxigenic Escherichia coli strains bound strongly to LacCer and showed a weak binding to monohexosylceramide. The K99 strain also bound strongly to Gb3, and F41 to Gb4. Lactosylceramide, monohexosylceramide, and Gb3 were also observed to bind to human uropathogenic E. coli strains. The results reported here show the ability of neutral GSL in ovine milk to bind to E. coli strains. These compounds could be used as an alternative and available source to supplement infant or bovine formulas with a view to preventing bacterial infections.

  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. Broadly Neutralizing Antibodies for HIV Eradication.

    PubMed

    Stephenson, Kathryn E; Barouch, Dan H

    2016-02-01

    Passive transfer of antibodies has long been considered a potential treatment modality for infectious diseases, including HIV. Early efforts to use antibodies to suppress HIV replication, however, were largely unsuccessful, as the antibodies that were studied neutralized only a relatively narrow spectrum of viral strains and were not very potent. Recent advances have led to the discovery of a large portfolio of human monoclonal antibodies that are broadly neutralizing across many HIV-1 subtypes and are also substantially more potent. These antibodies target multiple different epitopes on the HIV envelope, thus allowing for the development of antibody combinations. In this review, we discuss the application of broadly neutralizing antibodies (bNAbs) for HIV treatment and HIV eradication strategies. We highlight bNAbs that target key epitopes, such as the CD4 binding site and the V2/V3-glycan-dependent sites, and we discuss several bNAbs that are currently in the clinical development pipeline.

  12. 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…

  13. Ultracold neutral plasmas

    NASA Astrophysics Data System (ADS)

    Lyon, M.; Rolston, S. L.

    2017-01-01

    By photoionizing samples of laser-cooled atoms with laser light tuned just above the ionization limit, plasmas can be created with electron and ion temperatures below 10 K. These ultracold neutral plasmas have extended the temperature bounds of plasma physics by two orders of magnitude. Table-top experiments, using many of the tools from atomic physics, allow for the study of plasma phenomena in this new regime with independent control over the density and temperature of the plasma through the excitation process. Characteristic of these systems is an inhomogeneous density profile, inherited from the density distribution of the laser-cooled neutral atom sample. Most work has dealt with unconfined plasmas in vacuum, which expand outward at velocities of order 100 m/s, governed by electron pressure, and with lifetimes of order 100 μs, limited by stray electric fields. Using detection of charged particles and optical detection techniques, a wide variety of properties and phenomena have been observed, including expansion dynamics, collective excitations in both the electrons and ions, and collisional properties. Through three-body recombination collisions, the plasmas rapidly form Rydberg atoms, and clouds of cold Rydberg atoms have been observed to spontaneously avalanche ionize to form plasmas. Of particular interest is the possibility of the formation of strongly coupled plasmas, where Coulomb forces dominate thermal motion and correlations become important. The strongest impediment to strong coupling is disorder-induced heating, a process in which Coulomb energy from an initially disordered sample is converted into thermal energy. This restricts electrons to a weakly coupled regime and leaves the ions barely within the strongly coupled regime. This review will give an overview of the field of ultracold neutral plasmas, from its inception in 1999 to current work, including efforts to increase strong coupling and effects on plasma properties due to strong coupling.

  14. 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.

  15. Neutral beam injection system

    SciTech Connect

    Duesing, G.; Altmann, H.; Falter, H.; Goede, A.; Haange, R.; Hemsworth, R.S.; Kupschus, P.; Stork, D.; Thompson, E.

    1987-01-01

    The development of the neutral injection (NI) system for the Joint European Torus and its status in 1985 are reported. First the system parameters are discussed and the layout is described, followed by a summary of the physics design calculations, the development, production, and testing of the components and the subsystem assembly. The system commissioning is presented, including a description of the function and the realization of the NI test bed. A summary of performance predictions for 80-keV beam heating experiments, and of the experimental evidence on balanced versus coinjection, is presented. The operational experience with the first injector and the plasma physics results obtained so far are summarized.

  16. Virions of primary human immunodeficiency virus type 1 isolates resistant to soluble CD4 (sCD4) neutralization differ in sCD4 binding and glycoprotein gp120 retention from sCD4-sensitive isolates.

    PubMed Central

    Moore, J P; McKeating, J A; Huang, Y X; Ashkenazi, A; Ho, D D

    1992-01-01

    Primary isolates of human immunodeficiency virus type 1 (HIV-1) are much less sensitive to neutralization by soluble CD4 (sCD4) and sCD4-immunoglobulin (Ig) chimeras (CD4-IgG) than are HIV-1 strains adapted to growth in cell culture. We demonstrated that there are significant reductions (10- to 30-fold) in the binding of sCD4 and CD4-IgG to intact virions of five primary isolates compared with sCD4-sensitive, cell culture-adapted isolates RF and IIIB. However, soluble envelope glycoproteins (gp120) derived from the primary isolate virions, directly by detergent solubilization or indirectly by recombinant DNA technology, differed in affinity from RF and IIIB gp120 by only one- to threefold. The reduced binding of sCD4 to these primary isolate virions must therefore be a consequence of the tertiary or quaternary structure of the envelope glycoproteins in their native, oligomeric form on the viral surface. In addition, the rate and extent of sCD4-induced gp120 shedding from these primary isolates was lower than that from RF. We suggest that reduced sCD4 binding and increased gp120 retention together account for the relative resistance of these primary isolates to neutralization by sCD4 and CD4-IgG and that virions of different HIV-1 isolates vary both in the mechanism of sCD4 binding and in subsequent conformational changes in their envelope glycoproteins. PMID:1727487

  17. 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

  18. 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

    2015-11-17

    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.

  19. Neutral polypropylene laser welding

    NASA Astrophysics Data System (ADS)

    Mandolfino, Chiara; Lertora, Enrico; Gambaro, Carla

    2016-10-01

    The joining of polymeric materials is a technology used in many industrial applications, from transport to telecommunications and the medical sector. A new technology for the joining of polymers is the laser welding process. In particular, fibre laser welding is a flexible technology which allows high process speed and the realization of good quality joints. Despite its application becoming more widespread in the production of assemblies of high precision, the application of laser technology for the welding of polymers has not been the subject of many studies up to now. This study focused on the welding of neutral polypropylene. The window process parameter was identified, without the use of additives to increase radiation absorption, and a mechanical characterization was conducted in order to evaluate the quality of the joints realized.

  20. 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.

  1. Transient Photochemistry of Neutral Red.

    DTIC Science & Technology

    1983-07-01

    ascorbic acid system to 50 successive flashes, indicating that no ground state neutral red is permanently converted to leuco dye . Since leuco neutral...complete regeneration of ground state neutral red in this pH range in the present study suggests that formation of leuco dye is not significant. The second...radical disproportionation step is followed by a slower step which converts leuco dye to semireduced radical. Because coupling the two steps

  2. 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.

  3. Constraining the Europa Neutral Torus

    NASA Astrophysics Data System (ADS)

    Smith, Howard T.; Mitchell, Donald; mauk, Barry; Johnson, Robert E.; clark, george

    2016-10-01

    "Neutral tori" consist of neutral particles that usually co-orbit along with their source forming a toroidal (or partial toroidal) feature around the planet. The distribution and composition of these features can often provide important, if not unique, insight into magnetospheric particles sources, mechanisms and dynamics. However, these features can often be difficult to directly detect. One innovative method for detecting neutral tori is by observing Energetic Neutral Atoms (ENAs) that are generally considered produced as a result of charge exchange interactions between charged and neutral particles.Mauk et al. (2003) reported the detection of a Europa neutral particle torus using ENA observations. The presence of a Europa torus has extremely large implications for upcoming missions to Jupiter as well as understanding possible activity at this moon and providing critical insight into what lies beneath the surface of this icy ocean world. However, ENAs can also be produced as a result of charge exchange interactions between two ionized particles and in that case cannot be used to infer the presence of neutral particle population. Thus, a detailed examination of all possible source interactions must be considered before one can confirm that likely original source population of these ENA images is actually a Europa neutral particle torus. For this talk, we examine the viability that the Mauk et al. (2003) observations were actually generated from a neutral torus emanating from Europa as opposed to charge particle interactions with plasma originating from Io. These results help constrain such a torus as well as Europa source processes.

  4. Implementation of design of experiments (DOE) in the development and validation of a cell-based bioassay for the detection of anti-drug neutralizing antibodies in human serum.

    PubMed

    Chen, Xinyi C; Zhou, Lei; Gupta, Shalini; Civoli, Francesca

    2012-02-28

    The administration of biological therapeutics can potentially elicit the development of neutralizing antibodies (NAbs) to the therapeutic drug in patients, which could have a significant impact on drug efficacy and safety. A rigorous in vitro cell-based assay for the detection of NAbs is critical for the assessment of the immunogenicity profile of the therapeutic drug. Conatumumab is a fully human monoclonal agonist antibody directed against the extracellular domain of human TRAIL receptor 2 (TR-2). It is being investigated as a cancer treatment because it is able to induce apoptosis in sensitive tumor cells. This report demonstrates how statistically designed experiments could be employed effectively in different stages of a NAb bioassay life cycle in order to characterize, optimize and stabilize the assay with added benefit of resource efficiency. By combining the approach of design of experiments (DOE) with subject matter expertise and experience, we were able to understand thoroughly how assay parameters affect the performance of the assay individually and interactively, identify the key assay parameters, define assay operating ranges and finally achieve a robust and sensitive cell-based assay for the detection of NAbs to Conatumumab. With the goal of developing a cell-based bioassay that is highly optimized for sensitivity, specificity, precision, and robustness, we performed 2 DOE experiments for assay optimization and 1 DOE experiment to validate assay robustness. We evaluated key operating parameters of the assay such as cell number, percentage of serum matrix, concentration of the therapeutic drug, concentration of the cross-linker, length of various incubation steps, cell age, interval between cell subculture and bioassay time, and detection equipment.

  5. Fluorescent Cell-Counting Neutralization Test for Psittacosis

    PubMed Central

    Hahon, Nicholas; Cooke, Kenneth O.

    1965-01-01

    Hahon, Nicholas (Fort Detrick, Frederick, Md.), and Kenneth O. Cooke. Fluorescent cell-counting neutralization test for psittacosis. J. Bacteriol. 89:1465–1471. 1965.—A sensitive, precise, and specific serological procedure, the fluorescent cell-counting neutralization test, was developed to detect and to measure quantitatively psittacosis serum-neutralizing antibodies within 24 hr. The test is based on the reduction of fluorescent cells in McCoy cell monolayers resulting from the neutralization of infective agent particles by specific antiserum. Small but significant rises in neutralizing titers were measured in serum specimens from monkeys previously exposed to the psittacosis agent and from humans with diagnoses of subclinical or established psittacosis infections. PMID:14291581

  6. 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

  7. 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

  8. 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.

  9. Weak neutral current chemistry

    SciTech Connect

    Mohan, R.

    1996-07-01

    Metal cluster organic complexes, neither atomic nor solid but in analogy to atomic nuclei and to mesoscopic systems, have unusual dynamics and catalytic properties. Organo-metal clusters as quintessence prebiotic enzymes could have originated the homochirality of the molecules from achiral precursors, controlled from the atomic-nucleus, with the initial product itself serving subsequently as chiral auxiliary transferring and amplifying the chirality in the autocatalytic process now. High resolution spectroscopic studies of diatomic molecules beginning now may lead to upper estimates of the interaction strength of weak neutral currents (WNG) with valence electrons of metal clusters and suggest kinetic pathways to dynamic symmetry breaking in the asymmetric synthesis of chiral molecules. An estimate of 10{sup {minus}5} kT (thousand times larger than for radiolysis) for the parity violating energy (PVE) could be sufficient to run an entropy driven spin-catalyzed asymmetric synthesis. Expect then, wherever there are metal clusters in interstellar dust or under the sea chiral molecular production. {copyright} {ital 1996 American Institute of Physics.}

  10. Weak neutral current chemistry

    NASA Astrophysics Data System (ADS)

    Mohan, R.

    1996-07-01

    Metal cluster organic complexes, neither atomic nor solid but in analogy to atomic nuclei and to mesoscopic systems, have unusual dynamics and catalytic properties. Organo-metal clusters as quintessence prebiotic enzymes could have originated the homochirality of the molecules from achiral precursors, controlled from the atomic-nucleus, with the initial product itself serving subsequently as chiral auxiliary transferring and amplifying the chirality in the autocatalytic process now. High resolution spectroscopic studies of diatomic molecules beginning now may lead to upper estimates of the interaction strength of weak neutral currents (WNG) with valence electrons of metal clusters and suggest kinetic pathways to dynamic symmetry breaking in the asymmetric synthesis of chiral molecules. An estimate of 10-5 kT (thousand times larger than for radiolysis) for the parity violating energy (PVE) could be sufficient to run an entropy driven spin-catalyzed asymmetric synthesis. Expect then, wherever there are metal clusters in interstellar dust or under the sea chiral molecular production.

  11. The neutral upper atmosphere

    NASA Astrophysics Data System (ADS)

    Ghosh, S. N.

    2002-07-01

    After World War II, Professor S.K. Mitra wrote a comprehensive book called The Upper Atmosphere, which dealt with information available from ground-based and balloon-borne experiments. As a result, topics such as day airglow were investigated and further ground-based experiments using incoherent back-scattering were carried out. These activities resulted in important new information on the ozonosphere. The dramatic discovery of ozone holes forms a new and exciting chapter in the discovery of atmospheric processes. While dealing with the limits of the atmosphere, reference may be made to interstellar molecules whose discovery has raised considerable scientific curiosity. Knowledge on the solar-terrestrial relationship advanced a great deal when more information on solar radiation became available by measuring higher energy photons in the UV, EUV, and even X-ray regime. All this information is incorporated in this volume and presented under the title The Neutral Upper Atmosphere. Link: http://www.wkap.nl/prod/b/0-7923-6434-1

  12. Neutralization Serotyping of BK Polyomavirus Infection in Kidney Transplant Recipients

    PubMed Central

    Pastrana, Diana V.; Brennan, Daniel C.; Çuburu, Nicolas; Storch, Gregory A.; Viscidi, Raphael P.; Randhawa, Parmjeet S.; Buck, Christopher B.

    2012-01-01

    BK polyomavirus (BKV or BKPyV) associated nephropathy affects up to 10% of kidney transplant recipients (KTRs). BKV isolates are categorized into four genotypes. It is currently unclear whether the four genotypes are also serotypes. To address this issue, we developed high-throughput serological assays based on antibody-mediated neutralization of BKV genotype I and IV reporter vectors (pseudoviruses). Neutralization-based testing of sera from mice immunized with BKV-I or BKV-IV virus-like particles (VLPs) or sera from naturally infected human subjects revealed that BKV-I specific serum antibodies are poorly neutralizing against BKV-IV and vice versa. The fact that BKV-I and BKV-IV are distinct serotypes was less evident in traditional VLP-based ELISAs. BKV-I and BKV-IV neutralization assays were used to examine BKV type-specific neutralizing antibody responses in KTRs at various time points after transplantation. At study entry, sera from 5% and 49% of KTRs showed no detectable neutralizing activity for BKV-I or BKV-IV neutralization, respectively. By one year after transplantation, all KTRs were neutralization seropositive for BKV-I, and 43% of the initially BKV-IV seronegative subjects showed evidence of acute seroconversion for BKV-IV neutralization. The results suggest a model in which BKV-IV-specific seroconversion reflects a de novo BKV-IV infection in KTRs who initially lack protective antibody responses capable of neutralizing genotype IV BKVs. If this model is correct, it suggests that pre-vaccinating prospective KTRs with a multivalent VLP-based vaccine against all BKV serotypes, or administration of BKV-neutralizing antibodies, might offer protection against graft loss or dysfunction due to BKV associated nephropathy. PMID:22511874

  13. 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

  14. 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.

  15. The Effects of Plasma-Neutral Interactions on Neutral Dynamics

    NASA Astrophysics Data System (ADS)

    Hsu, V.; Thayer, J. P.

    2014-12-01

    Plasma-neutral interactions are fundamental to the structure and behavior of the neutral thermosphere. This interaction, primarily through ion-neutral collisions, ties electrodynamics with hydrodynamics requiring a fully coupled ionosphere - thermosphere model to simulate and dissect the sequence of responses that occur in the neutral gas when a change occurs in the ionosphere. In particular, changes in the ion drag force prompt a hydrodynamic response that will alter several properties of the thermosphere, including neutral winds. Here, the fully coupled National Center for Atmospheric Research Thermosphere-Ionosphere Electrodynamics General Circulation Model (NCAR TIEGCM) is used to evaluate how changes in mechanical coupling, through the ion drag force, alter thermosphere properties, with a focus on thermospheric neutral winds. The equatorial thermosphere anomaly (ETA) produces a transient wind system, and a dissection of the hydrodynamic processes responsible for its formation will be used to demonstrate the causal structure in neutral gas response to a change in field-aligned ion drag force. This well-behaved response elucidates processes that must be occurring in other regions of the thermosphere where more significant changes in the ion drag force occur.

  16. 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.

  17. 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.

  18. Neutralization of reovirus: the gene responsible for the neutralization antigen

    PubMed Central

    1977-01-01

    The S1 genome segment of reovirus is linked to type specificity as determined by neutralization antibody. This gene segment codes for a minor outer capsid polypeptide (sigma1). Therefore, sigma1 is the peptide responsible for induction of neutralization antibody and confers type specificity. This biologic property of reovirus was defined using hybrid recombinants clones between reovirus types 1 and 3 and 2 and 3. PMID:925604

  19. 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.

  20. Vendor neutral archive in PACS.

    PubMed

    Agarwal, Tapesh Kumar; Sanjeev

    2012-10-01

    An archive is a location containing a collection of records, documents, or other materials of historical importance. An integral part of Picture Archiving and Communication System (PACS) is archiving. When a hospital needs to migrate a PACS vendor, the complete earlier data need to be migrated in the format of the newly procured PACS. It is both time and money consuming. To address this issue, the new concept of vendor neutral archive (VNA) has emerged. A VNA simply decouples the PACS and workstations at the archival layer. This is achieved by developing an application engine that receives, integrates, and transmits the data using the different syntax of a Digital Imaging and Communication in Medicine (DICOM) format. Transferring the data belonging to the old PACS to a new one is performed by a process called migration of data. In VNA, a number of different data migration techniques are available to facilitate transfer from the old PACS to the new one, the choice depending on the speed of migration and the importance of data. The techniques include simple DICOM migration, prefetch-based DICOM migration, medium migration, and the expensive non-DICOM migration. "Vendor neutral" may not be a suitable term, and "architecture neutral," "PACS neutral," "content neutral," or "third-party neutral" are probably better and preferred terms. Notwithstanding this, the VNA acronym has come to stay in both the medical IT user terminology and in vendor nomenclature, and radiologists need to be aware of its impact in PACS across the globe.

  1. Effectiveness of various methods of formaldehyde neutralization using monoethanolamine.

    PubMed

    Coskey, Andrew; Gest, Thomas R

    2015-05-01

    Formaldehyde is the most commonly used fixative chemical for the preservation of human cadavers used for educational purposes in the United States. Formaldehyde is also a known carcinogenic agent whose exposure level is regulated by guidelines of the Occupational Safety and Health Administration. Various methods for formaldehyde neutralization exist, yet many donations programs do not take any steps to neutralize the formaldehyde in embalmed donor bodies. The effectiveness of monoethanolamine (MEA) in neutralizing formaldehyde is well documented when used as a final injection during embalming. The purpose of this study is to report the effectiveness of several post-embalming techniques of formaldehyde neutralization. Twenty-four donor bodies were assigned to four experimental groups of six. For the three experimental groups, the techniques tested involve delivery of a 20:1 dilution of deionized water:MEA via recannulization and gravity flow infusion, compartment injection, and alternate wetting solution containing four percent MEA. Our results indicated that spray bottle delivery was not effective in neutralization of formaldehyde compared to the control group, but that formaldehyde levels decreased when recannulization or compartment injection were used. The most effective method of formaldehyde neutralization was compartment injection of MEA solution (P < 0.01). The results of this study indicate that, in situations where MEA is not used as a final infusion during embalming, compartment injection of MEA solution is an effective method of formaldehyde neutralization.

  2. 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.

  3. 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.

  4. 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.

  5. Post-learning psychosocial stress enhances consolidation of neutral stimuli.

    PubMed

    Preuss, Diana; Wolf, Oliver T

    2009-10-01

    Post-learning stress has been reported to enhance memory consolidation in humans. This effect was observed in studies using physical stressors or an anticipatory speech task. In the present study 58 participants (28 females and 30 males) were exposed to a psychosocial stressor (Trier Social Stress Test) or a control condition following the presentation of neutral and emotionally arousing positive and negative pictures, which were accompanied by a brief narrative. The stressor induced a significant neuroendocrine stress response in men and women. In a 24h delayed free recall test the stress group showed an enhanced memory for neutral but not for emotionally arousing positive and negative items. Additionally, a significant correlation between the cortisol stress response and memory for neutral items was evident. Thus, in contrast to previous studies, post-learning stress primarily enhanced consolidation of neutral material. Several theoretical and methodological explanations for the observed effects are discussed.

  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. Reduced neutral XLPE cable design

    SciTech Connect

    Valli, G.F.; Zawadzki, J.A.; Orton, H.E. )

    1990-04-01

    This paper describes the theoretical, laboratory and economic analyses undertaken to determine the optimum metallic concentric neutral design for its single conductor 750 and 500 kcmil aluminum XLPE 15 kV insulated concentric-neutral type feeder cables. The results suggest that reducing the cross-sectional area of this concentric neutral from the currently-recognized industry standard of 20 percent of the central conductor to 7% results in overall present-worth system cost saving of approximately $3 per conductor meter or approximately 22% of the cable first cost. The neutral configuration ultimately chosen to replace the previous standard 37 - number 14 AWG wires was 2 - 1 inch {times} 5 mil tinned copper tapes overlapped by 25%. Line voltage fault test were run in the high-power laboratory on samples with various neutral configurations to confirm they would successfully pass our worst-case fault duty of 10 kA for 20 cycles (i.e., .33 sec) with no reclosing.

  8. Selective neutrality and enzyme kinetics.

    PubMed

    Demetrius, L

    1997-10-01

    This article appeals to a recent theory of enzyme evolution to show that the properties, neutral or adaptive, which characterize the observed allelic variation in natural populations can be inferred from the functional parameters, substrate specificity, and reaction rate. This study delineates the following relations between activity variables, and the forces--adaptive or neutral--determining allelic variation: (1) Enzymes with broad substrate specificity: The observed polymorphism is adaptive; mutations in this class of enzymes can result in increased fitness of the organism and hence be relevant for positive selection. (2) Enzymes with absolute substrate specificity and diffusion-controlled rates: Observed allelic variation will be absolutely neutral; mutations in this class of enzymes will be either deleterious or have no effect on fitness. (3) Enzymes with absolute or group specificity and nondiffusion-controlled rates: Observed variation will be partially neutral; mutants which are selectively neutral may become advantageous under an appropriate environmental condition or different genetic background. We illustrate each of the relations between kinetic properties and evolutionary states with examples drawn from enzymes whose evolutionary dynamics have been intensively studied.

  9. Energetic Neutral Atom Precipitation (ENAP)

    NASA Technical Reports Server (NTRS)

    Tinsley, B. A.

    1988-01-01

    The Energetic Neutral Atom Precipitation experiment is scheduled to be flown on the Atmospheric Laboratory for Applications and Science (ATLAS 1) NASA mission. The objective of this experiment is to measure very faint emissions at nighttime arising from fluxes of energetic neutral atoms in the thermosphere. These energetic atoms have energies ranging up to about 50 keV, and arise from ions of hydrogen, helium, and oxygen trapped in the inner magnetosphere. Some of these ions become neutralized in charge exchange reactions with neutral hydrogen in the hydrogen geocorona that extends through the region. The ions are trapped on magnetic field lines which cross the equatorial plane at 2 to 6 earth radii distance, and they mirror at a range of heights on these field lines, extending down to the thermosphere at 500 km altitude. The ATLAS 1 measurements will not be of the neutral atoms themselves but of the optical emission produced by those on trajectories that intersect the thermosphere. The ENAP measurements are to be made using the Imaging Spectrometric Observatory (ISO) which is being flown on the ATLAS mission primarily for daytime spectral observations, and the ENAP measurements will all be nighttime measurements because of the faintness of the emissions and the relatively low level of magnetic activity expected.

  10. Recent Progress toward Engineering HIV-1-Specific Neutralizing Monoclonal Antibodies

    PubMed Central

    Sun, Ming; Li, Yue; Zheng, Huiwen; Shao, Yiming

    2016-01-01

    The recent discoveries of broadly potent neutralizing human monoclonal antibodies represent a new generation of antiretrovirals for the treatment and prophylaxis. Antibodies are generally considered more effective and safer and have been proved to provide passive protection against mucosal challenge in humanized mice and macaques. Several neutralizing Abs could protect animals against HIV-1 but are not effective when used in an established infected model for therapy. In order to overcome the limitation of antiviral activities, multiple antibody-engineering technologies have been explored to generate “the better” neutralizing antibodies against HIV-1 since bNAbs attack viral entry by various mechanisms. Thus, a promising direction of research is to discover and exploit rational antibody combination or engineered antibodies (eAbs) as potential candidate therapeutics against HIV-1. It has been reported that inclusion of fusion-neutralizing antibodies in a set of bNAbs could improve their overall activities and neutralizing spectrum. Here, we review several routes for engineering bNAbs, such as design and generation of bispecific antibodies, specific glycosylation of antibodies to enhance antiviral activity, and variable region-specific modification guided by structure and computer, as well as reviewing antibody-delivery technologies by non-viral vector, viral vector, and human hematopoietic stem/progenitor cells transduced with a lentiviral construct. We also discuss the optimized antiviral activities and benefits of these strategy and potential mechanisms. PMID:27746780

  11. 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.

  12. 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…

  13. Potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody

    PubMed Central

    Nowakowski, A.; Wang, C.; Powers, D. B.; Amersdorfer, P.; Smith, T. J.; Montgomery, V. A.; Sheridan, R.; Blake, R.; Smith, L. A.; Marks, J. D.

    2002-01-01

    The botulinum neurotoxins (BoNTs) cause the paralytic human disease botulism and are one of the highest-risk threat agents for bioterrorism. To generate a pharmaceutical to prevent or treat botulism, monoclonal antibodies (mAbs) were generated by phage display and evaluated for neutralization of BoNT serotype A (BoNT/A) in vivo. Although no single mAb significantly neutralized toxin, a combination of three mAbs (oligoclonal Ab) neutralized 450,000 50% lethal doses of BoNT/A, a potency 90 times greater than human hyperimmune globulin. The potency of oligoclonal Ab was primarily due to a large increase in functional Ab binding affinity. The results indicate that the potency of the polyclonal humoral immune response can be deconvoluted to a few mAbs binding nonoverlapping epitopes, providing a route to drugs for preventing and treating botulism and diseases caused by other pathogens and biologic threat agents. PMID:12177434

  14. Escape From Monoclonal Antibody Neutralization Affects Henipavirus Fitness In Vitro and In Vivo.

    PubMed

    Borisevich, Viktoriya; Lee, Benhur; Hickey, Andrew; DeBuysscher, Blair; Broder, Christopher C; Feldmann, Heinz; Rockx, Barry

    2016-02-01

    Henipaviruses are zoonotic viruses that can cause severe and acute respiratory diseases and encephalitis in humans. To date, no vaccine or treatments are approved for human use. The presence of neutralizing antibodies is a strong correlate of protection against lethal disease in animals. However, since RNA viruses are prone to high mutation rates, the possibility that these viruses will escape neutralization remains a potential concern. In the present study, we generated neutralization-escape mutants, using 6 different monoclonal antibodies, and studied the effect of these neutralization-escape mutations on in vitro and in vivo fitness. These data provide a mechanism for overcoming neutralization escape by use of cocktails of cross-neutralizing monoclonal antibodies that recognize residues within the glycoprotein that are important for virus replication and virulence.

  15. Escape From Monoclonal Antibody Neutralization Affects Henipavirus Fitness In Vitro and In Vivo

    PubMed Central

    Borisevich, Viktoriya; Lee, Benhur; Hickey, Andrew; DeBuysscher, Blair; Broder, Christopher C.; Feldmann, Heinz; Rockx, Barry

    2016-01-01

    Henipaviruses are zoonotic viruses that can cause severe and acute respiratory diseases and encephalitis in humans. To date, no vaccine or treatments are approved for human use. The presence of neutralizing antibodies is a strong correlate of protection against lethal disease in animals. However, since RNA viruses are prone to high mutation rates, the possibility that these viruses will escape neutralization remains a potential concern. In the present study, we generated neutralization-escape mutants, using 6 different monoclonal antibodies, and studied the effect of these neutralization-escape mutations on in vitro and in vivo fitness. These data provide a mechanism for overcoming neutralization escape by use of cocktails of cross-neutralizing monoclonal antibodies that recognize residues within the glycoprotein that are important for virus replication and virulence. PMID:26357909

  16. 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.

  17. 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.

  18. Low energy neutral atom imaging

    SciTech Connect

    McComas, D.J.; Funsten, H.O.; Gosling, J.T.; Moore, K.R.; Thomsen, M.F.

    1992-01-01

    Energetic neutral atom (ENA) and low energy neutral atom (LENA) imaging of space plasmas are emerging new technology which promises to revolutionize the way we view and understand large scale space plasma phenomena and dynamics. ENAs and LENAs are produced in the magnetosphere by charge exchange between energetic and plasma ions and cold geocoronal neutrals. While imaging techniques have been previously developed for observing ENAs, with energies above several tens of keV, most of the ions found in the terrestrial magnetosphere have lower energies. We recently suggested that LENAs could be imaged by first converting the neutrals to ions and then electrostatically analyzing them to reject the UV background. In this paper we extend this work to examine in detail the sensor elements needed to make an LENA imager. These elements are (1) a biased collimator to remove the ambient plasma ions and electrons and set the azimuthal field-of-view; (2) a charge modifier to convert a portion of the incident LENAs to ions; (3) an electrostatic analyzer to reject UV light and set the energy passband; and (4) a coincidence detector to measure converted LENAs while rejecting noise and penetrating radiation. We also examine the issue of LENA imager sensitivity and describe ways of optimizing sensitivity in the various sensor components. Finally, we demonstrate in detail how these general considerations are implemented by describing one relatively straightforward design based on a hemispherical electrostatic analyzer.

  19. 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.

  20. Immunoglobulins in Nasal Secretions of Healthy Humans: Structural Integrity of Secretory Immunoglobulin A1 (IgA1) and Occurrence of Neutralizing Antibodies to IgA1 Proteases of Nasal Bacteria

    PubMed Central

    Kirkeby, Line; Rasmussen, Trine Tang; Reinholdt, Jesper; Kilian, Mogens

    2000-01-01

    Certain bacteria, including overt pathogens as well as commensals, produce immunoglobulin A1 (IgA1) proteases. By cleaving IgA1, including secretory IgA1, in the hinge region, these enzymes may interfere with the barrier functions of mucosal IgA antibodies, as indicated by experiments in vitro. Previous studies have suggested that cleavage of IgA1 in nasal secretions may be associated with the development and perpetuation of atopic disease. To clarify the potential effect of IgA1 protease-producing bacteria in the nasal cavity, we have analyzed immunoglobulin isotypes in nasal secretions of 11 healthy humans, with a focus on IgA, and at the same time have characterized and quantified IgA1 protease-producing bacteria in the nasal flora of the subjects. Samples in the form of nasal wash were collected by using a washing liquid that contained lithium as an internal reference. Dilution factors and, subsequently, concentrations in undiluted secretions could thereby be calculated. IgA, mainly in the secretory form, was found by enzyme-linked immunosorbent assay to be the dominant isotype in all subjects, and the vast majority of IgA (median, 91%) was of the A1 subclass, corroborating results of previous analyses at the level of immunoglobulin-producing cells. Levels of serum-type immunoglobulins were low, except for four subjects in whom levels of IgG corresponded to 20 to 66% of total IgA. Cumulative levels of IgA, IgG, and IgM in undiluted secretions ranged from 260 to 2,494 (median, 777) μg ml−1. IgA1 protease-producing bacteria (Haemophilus influenzae, Streptococcus pneumoniae, or Streptococcus mitis biovar 1) were isolated from the nasal cavities of seven subjects at 2.1 × 103 to 7.2 × 106 CFU per ml of undiluted secretion, corresponding to 0.2 to 99.6% of the flora. Nevertheless, α-chain fragments characteristic of IgA1 protease activity were not detected in secretions from any subject by immunoblotting. Neutralizing antibodies to IgA1 proteases of autologous

  1. 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

  2. Optical neutrality: invisibility without cloaking.

    PubMed

    Hodges, Reed; Dean, Cleon; Durach, Maxim

    2017-02-15

    We show that it is possible to design an invisible wavelength-sized metal-dielectric metamaterial object without evoking cloaking. Our approach is an extension of the neutral inclusion concept by Zhou and Hu [Phys. Rev. E74, 026607 (2006)PLEEE81063-651X10.1103/PhysRevE.74.026607] to Mie scatterers. We demonstrate that an increase of metal fraction in the metamaterial leads to a transition from dielectric-like to metal-like scattering, which proceeds through invisibility or optical neutrality of the scatterer. Formally this is due to cancellation of multiple scattering orders, similarly to plasmonic cloaking introduced by Alù and Engheta [Phys. Rev. E72, 016623 (2005)PLEEE81063-651X10.1103/PhysRevE.72.016623], but without introduction of the separation of the scatterer into cloak and hidden regions.

  3. Neutral matter in planetary nebulae

    NASA Technical Reports Server (NTRS)

    Dinerstein, Harriet L.

    1991-01-01

    A review of current studies of neutral envelopes is presented with particular attention given to the use of the envelopes as test cases for understanding the ionization and thermal structure of photodissociation regions. The study of near-IR H2 emission is discussed with detailed spectra given for a few planetary nebulae, and airborne observations of far-IR atomic lines are discussed. These two methods can discern photodissociation regions with warm gas and UV flux is fairly prominent. The use of resonance-absorption-line spectroscopy is also reviewed with respect to the analysis of the Na D lines, and thereby allows the measurement of integrated columns of material through the shell. The methods provide evidence for the notion that planetary nebulae consist of more than just ionized material; large amounts of neutral and molecular material are being confirmed, which has important implications for the mass-loss episode of the nebulae.

  4. Neutral depletion versus repletion due to ionization

    SciTech Connect

    Fruchtman, A.; Makrinich, G.; Raimbault, J.-L.; Liard, L.; Rax, J.-M.; Chabert, P.

    2008-05-15

    Recent theoretical analyses which predicted unexpected effects of neutral depletion in both collisional and collisionless plasmas are reviewed. We focus on the depletion of collisionless neutrals induced by strong ionization of a collisionless plasma and contrast this depletion with the effect of strong ionization on thermalized neutrals. The collisionless plasma is analyzed employing a kinetic description. The collisionless neutrals and the plasma are coupled through volume ionization and wall recombination only. The profiles of density and pressure both of the plasma and of the neutral-gas and the profile of the ionization rate are calculated. It is shown that for collisionless neutrals the ionization results in neutral depletion, while when neutrals are thermalized the ionization induces a maximal neutral-density at the discharge center, which we call neutral repletion. The difference between the two cases stems from the relation between the neutral density and pressure. The pressure of the collisionless neutral-gas turns out to be maximal where its density is minimal, in contrast to the case of a thermalized neutral gas.

  5. 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.

  6. 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.

  7. Vendor neutral archive in PACS

    PubMed Central

    Agarwal, Tapesh Kumar; Sanjeev

    2012-01-01

    An archive is a location containing a collection of records, documents, or other materials of historical importance. An integral part of Picture Archiving and Communication System (PACS) is archiving. When a hospital needs to migrate a PACS vendor, the complete earlier data need to be migrated in the format of the newly procured PACS. It is both time and money consuming. To address this issue, the new concept of vendor neutral archive (VNA) has emerged. A VNA simply decouples the PACS and workstations at the archival layer. This is achieved by developing an application engine that receives, integrates, and transmits the data using the different syntax of a Digital Imaging and Communication in Medicine (DICOM) format. Transferring the data belonging to the old PACS to a new one is performed by a process called migration of data. In VNA, a number of different data migration techniques are available to facilitate transfer from the old PACS to the new one, the choice depending on the speed of migration and the importance of data. The techniques include simple DICOM migration, prefetch-based DICOM migration, medium migration, and the expensive non-DICOM migration. “Vendor neutral” may not be a suitable term, and “architecture neutral,” “PACS neutral,” “content neutral,” or “third-party neutral” are probably better and preferred terms. Notwithstanding this, the VNA acronym has come to stay in both the medical IT user terminology and in vendor nomenclature, and radiologists need to be aware of its impact in PACS across the globe. PMID:23833411

  8. Interstellar neutrals in interplanetary space

    SciTech Connect

    Hovestadt, D.; Moebius, E. )

    1989-03-01

    The solar system is moving through the interstellar medium with a velocity of about 20 km/s. The neutral interstellar gas, which thereby penetrates the heliosphere, is subject to ionization by solar UV radiation, charge exchange with the solar wind, and electron collisions. The newly created ions are then picked by the solar wind through interaction of interstellar neutrals with the interplanetary magnetic field. The pick-up ions with their peculiar elemental composition probably also constitute the source particles of the Anomalous Cosmic Ray Component (ACR). In this report descriptions of the interaction with the solar wind are reviewed. While most of the constituents are already ionized far beyond the orbit of the Earth, neutral helium (because of its high ionization potential) approaches the Sun to {lt}1 AU. The pick-up of interstellar He{sup +} ions has recently been directly observed for the first time. The observed velocity distribution of He{sup +} extending up to twice the solar wind velocity can be explained in terms of pitch angle scattering of the ions probably by interplanetary Alven waves and subsequent adiabatic cooling in the expanding solar wind. Thermal coupling of the He{sup +} to the solar wind is negligible in the inner heliosphere. Detailed studies of the pick-up distribution provide a method to investigate the interplanetary propagation parameters and the state of the local interstellar medium.

  9. Neutral naturalness with bifundamental gluinos

    NASA Astrophysics Data System (ADS)

    Gherghetta, Tony; Nguyen, Minh; Thomas, Zachary

    2016-12-01

    We study constraints on one-loop neutral naturalness at the LHC by considering gluon partners which are required to ameliorate the tuning in the Higgs mass-squared arising at two loops. This is done with a simple orbifold model of folded supersymmetry which not only contains color-neutral stops but also bifundamental gluinos that are charged under the Standard Model color group S U (3 )C and a separate S U (3 )C' group. The bifundamental gluinos reduce the Higgs mass tuning at two loops and maintain naturalness provided the gluinos are lighter than approximately 1.9 TeV for a 5 TeV cutoff scale. Limits from the LHC already forbid bifundamental gluinos below 1.4 TeV, and other noncolored states such as electroweakinos, Z' bosons and dark sector bound states may be probed at future colliders. The search for bifundamental gluinos therefore provides a direct probe of one-loop neutral naturalness that can be fully explored at the LHC.

  10. 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

  11. 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.

  12. 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.

  13. Biodiversity on island chains: Neutral model simulations

    NASA Astrophysics Data System (ADS)

    Warren, Patrick B.

    2010-11-01

    A neutral ecology model is simulated on an island chain, in which neighboring islands can exchange individuals but only the first island is able to receive immigrants from a metacommunity. It is found by several measures that α -diversity decreases along the chain. Subtle changes in taxon abundance distributions can be detected when islands in the chain are compared to diversity-matched single islands. The island chain is found to have unexpectedly rich dynamics. Significant β -diversity correlations are found between islands in the chain, which are absent between diversity-matched single islands. The results potentially apply to human microbial biodiversity and biogeography and suggest that measurements of interindividual and intraindividual β -diversity may give insights into microbial community assembly mechanisms.

  14. 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.

  15. 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.

  16. 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.

  17. Preferential Amygdala Reactivity to the Negative Assessment of Neutral Faces

    PubMed Central

    Blasi, Giuseppe; Hariri, Ahmad R.; Alce, Guilna; Taurisano, Paolo; Sambataro, Fabio; Das, Saumitra; Bertolino, Alessandro; Weinberger, Daniel R.; Mattay, Venkata S.

    2010-01-01

    Background Prior studies suggest that the amygdala shapes complex behavioral responses to socially ambiguous cues. We explored human amygdala function during explicit behavioral decision making about discrete emotional facial expressions that can represent socially unambiguous and ambiguous cues. Methods During functional magnetic resonance imaging, 43 healthy adults were required to make complex social decisions (i.e., approach or avoid) about either relatively unambiguous (i.e., angry, fearful, happy) or ambiguous (i.e., neutral) facial expressions. Amygdala activation during this task was compared with that elicited by simple, perceptual decisions (sex discrimination) about the identical facial stimuli. Results Angry and fearful expressions were more frequently judged as avoidable and happy expressions most often as approachable. Neutral expressions were equally judged as avoidable and approachable. Reaction times to neutral expressions were longer than those to angry, fearful, and happy expressions during social judgment only. Imaging data on stimuli judged to be avoided revealed a significant task by emotion interaction in the amygdala. Here, only neutral facial expressions elicited greater activity during social judgment than during sex discrimination. Furthermore, during social judgment only, neutral faces judged to be avoided were associated with greater amygdala activity relative to neutral faces that were judged as approachable. Moreover, functional coupling between the amygdala and both dorsolateral prefrontal (social judgment > sex discrimination) and cingulate (sex discrimination > social judgment) cortices was differentially modulated by task during processing of neutral faces. Conclusions Our results suggest that increased amygdala reactivity and differential functional coupling with prefrontal circuitries may shape complex decisions and behavioral responses to socially ambiguous cues. PMID:19709644

  18. 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.

  19. 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…

  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. Prebiotic potential of neutral oligo- and polysaccharides from seed mucilage of Hyptis suaveolens.

    PubMed

    Mueller, Monika; Čavarkapa, Andrea; Unger, Frank M; Viernstein, Helmut; Praznik, Werner

    2017-04-15

    Prebiotics are selectively fermented by the gastrointestinal microflora, resulting in benefits to human health. The seed mucilage of Hyptis suaveolens contains neutral and acidic polysaccharides in a ratio of 1:1. The neutral polysaccharides consist of galactose, glucose and mannose whereas the acidic polysaccharides contain fucose, xylose and 4-O-methylglucuronic acid -residues. The growth of probiotics in the presence of total, acidic or neutral polysaccharides and oligosaccharides was tested using turbidity measurements. The majority (11 out of 14) of the tested probiotic strains significantly grew in the neutral fraction. Growth occurred with some time delay, but may be longer lasting than with other lower molecular prebiotics. The extent of growth increased with neutral polysaccharides from H. suaveolens corresponding to the externally available galactose units (20%). In conclusion, neutral poly- and oligosaccharides from H. suaveolens have a prebiotic potential characterized by a delayed but long lasting effect.

  2. Naturally selected hepatitis C virus polymorphisms confer broad neutralizing antibody resistance

    PubMed Central

    Bailey, Justin R.; Wasilewski, Lisa N.; Snider, Anna E.; El-Diwany, Ramy; Osburn, William O.; Keck, Zhenyong; Foung, Steven K.H.; Ray, Stuart C.

    2014-01-01

    For hepatitis C virus (HCV) and other highly variable viruses, broadly neutralizing mAbs are an important guide for vaccine development. The development of resistance to anti-HCV mAbs is poorly understood, in part due to a lack of neutralization testing against diverse, representative panels of HCV variants. Here, we developed a neutralization panel expressing diverse, naturally occurring HCV envelopes (E1E2s) and used this panel to characterize neutralizing breadth and resistance mechanisms of 18 previously described broadly neutralizing anti-HCV human mAbs. The observed mAb resistance could not be attributed to polymorphisms in E1E2 at known mAb-binding residues. Additionally, hierarchical clustering analysis of neutralization resistance patterns revealed relationships between mAbs that were not predicted by prior epitope mapping, identifying 3 distinct neutralization clusters. Using this clustering analysis and envelope sequence data, we identified polymorphisms in E2 that confer resistance to multiple broadly neutralizing mAbs. These polymorphisms, which are not at mAb contact residues, also conferred resistance to neutralization by plasma from HCV-infected subjects. Together, our method of neutralization clustering with sequence analysis reveals that polymorphisms at noncontact residues may be a major immune evasion mechanism for HCV, facilitating viral persistence and presenting a challenge for HCV vaccine development. PMID:25500884

  3. Antiviral Therapy by HIV-1 Broadly Neutralizing and Inhibitory Antibodies.

    PubMed

    Zhang, Zhiqing; Li, Shaowei; Gu, Ying; Xia, Ningshao

    2016-11-18

    Human immunodeficiency virus type 1 (HIV-1) infection causes acquired immune deficiency syndrome (AIDS), a global epidemic for more than three decades. HIV-1 replication is primarily controlled through antiretroviral therapy (ART) but this treatment does not cure HIV-1 infection. Furthermore, there is increasing viral resistance to ART, and side effects associated with long-term therapy. Consequently, there is a need of alternative candidates for HIV-1 prevention and therapy. Recent advances have discovered multiple broadly neutralizing antibodies against HIV-1. In this review, we describe the key epitopes on the HIV-1 Env protein and the reciprocal broadly neutralizing antibodies, and discuss the ongoing clinical trials of broadly neutralizing and inhibitory antibody therapy as well as antibody combinations, bispecific antibodies, and methods that improve therapeutic efficacy by combining broadly neutralizing antibodies (bNAbs) with latency reversing agents. Compared with ART, HIV-1 therapeutics that incorporate these broadly neutralizing and inhibitory antibodies offer the advantage of decreasing virus load and clearing infected cells, which is a promising prospect in HIV-1 prevention and treatment.

  4. Lipopolysaccharide Neutralization by Cationic-Amphiphilic Polymers through Pseudoaggregate Formation.

    PubMed

    Uppu, Divakara S S M; Haldar, Jayanta

    2016-03-14

    Synthetic polymers incorporating the cationic charge and hydrophobicity to mimic the function of antimicrobial peptides (AMPs) have been developed. These cationic-amphiphilic polymers bind to bacterial membranes that generally contain negatively charged phospholipids and cause membrane disintegration resulting in cell death; however, cationic-amphiphilic antibacterial polymers with endotoxin neutralization properties, to the best of our knowledge, have not been reported. Bacterial endotoxins such as lipopolysaccharide (LPS) cause sepsis that is responsible for a great amount of mortality worldwide. These cationic-amphiphilic polymers can also bind to negatively charged and hydrophobic LPS and cause detoxification. Hence, we envisaged that cationic-amphiphilic polymers can have both antibacterial as well as LPS binding properties. Here we report synthetic amphiphilic polymers with both antibacterial as well as endotoxin neutralizing properties. Levels of proinflammatory cytokines in human monocytes caused by LPS stimulation were inhibited by >80% when coincubated with these polymers. These reductions were found to be dependent on concentration and, more importantly, on the side-chain chemical structure due to variations in the hydrophobicity profiles of these polymers. These cationic-amphiphilic polymers bind and cause LPS neutralization and detoxification. Investigations of polymer interaction with LPS using fluorescence spectroscopy and dynamic light scattering (DLS) showed that these polymers bind but neither dissociate nor promote LPS aggregation. We show that polymer binding to LPS leads to sort of a pseudoaggregate formation resulting in LPS neutralization/detoxification. These findings provide an unusual mechanism of LPS neutralization using novel synthetic cationic-amphiphilic polymers.

  5. Antiviral Therapy by HIV-1 Broadly Neutralizing and Inhibitory Antibodies

    PubMed Central

    Zhang, Zhiqing; Li, Shaowei; Gu, Ying; Xia, Ningshao

    2016-01-01

    Human immunodeficiency virus type 1 (HIV-1) infection causes acquired immune deficiency syndrome (AIDS), a global epidemic for more than three decades. HIV-1 replication is primarily controlled through antiretroviral therapy (ART) but this treatment does not cure HIV-1 infection. Furthermore, there is increasing viral resistance to ART, and side effects associated with long-term therapy. Consequently, there is a need of alternative candidates for HIV-1 prevention and therapy. Recent advances have discovered multiple broadly neutralizing antibodies against HIV-1. In this review, we describe the key epitopes on the HIV-1 Env protein and the reciprocal broadly neutralizing antibodies, and discuss the ongoing clinical trials of broadly neutralizing and inhibitory antibody therapy as well as antibody combinations, bispecific antibodies, and methods that improve therapeutic efficacy by combining broadly neutralizing antibodies (bNAbs) with latency reversing agents. Compared with ART, HIV-1 therapeutics that incorporate these broadly neutralizing and inhibitory antibodies offer the advantage of decreasing virus load and clearing infected cells, which is a promising prospect in HIV-1 prevention and treatment. PMID:27869733

  6. The neutralization of interferons by antibody. I. Quantitative and theoretical analyses of the neutralization reaction in different bioassay systems.

    PubMed

    Grossberg, S E; Kawade, Y; Kohase, M; Yokoyama, H; Finter, N

    2001-09-01

    The highly specific ability of antibodies to inhibit the biologic activity of cytokines or other therapeutic proteins is widely used in research and a subject of increasing clinical importance. The need exists for a standardized approach to the reporting of neutralizing antibody potency soundly based on theoretical and practical considerations and tested by experimental data. Pursuant to the original studies of Kawade on the theoretical and functional aspects of neutralization of interferons (IFN), experimental data were obtained by different laboratories employing varied methodology to address two hypotheses concerning the nature of IFN neutralization reactions, based on a derived formula that allows expression of neutralizing power as the reduction of 10 laboratory units (LU)/ml to 1 LU/ml, the end point of most bioassays. Two hypotheses are posed: (1) antibody acts to neutralize a fixed amount of biologically active IFN molecules, or (2) antibody reduces IFN activity in a set ratio of added/residual biologically active IFN. The first, or fixed amount, hypothesis relates to the reactivity of high-affinity antibodies neutralizing equimolar amounts of antigen, whereas the second, or constant proportion, hypothesis postulates a reduction in the ratio of total added IFN to residual active IFN molecules, such as a low-affinity antibody might exhibit. Analyses of data of the neutralization of IFN-alpha and IFN-beta are presented, employing human polyclonal antibodies and murine monoclonal antibodies (mAb). The theoretical constructs of Kawade are extended in the Appendix and correlated with new experimental data in the text. The data clearly indicate that the low-antibody affinity, constant proportion hypothesis, rather than the high-antibody affinity, fixed amount hypothesis, is applicable, if the bioassay is sensitive to IFN. The findings presented here and in the following paper (pp. 743-755, this issue) taken together provide the basis for a standardized method of

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. Broadening the neutralizing capacity of a family of antibody fragments against different toxins from Mexican scorpions.

    PubMed

    Rodríguez-Rodríguez, Everardo Remi; Olamendi-Portugal, Timoteo; Serrano-Posada, Hugo; Arredondo-López, Jonathan Noé; Gómez-Ramírez, Ilse; Fernández-Taboada, Guillermo; Possani, Lourival D; Anguiano-Vega, Gerardo Alfonso; Riaño-Umbarila, Lidia; Becerril, Baltazar

    2016-09-01

    New approaches aimed at neutralizing the primary toxic components present in scorpion venoms, represent a promising alternative to the use of antivenoms of equine origin in humans. New potential therapeutics developed by these approaches correspond to neutralizing antibody fragments obtained by selection and maturation processes from libraries of human origin. The high sequence identity shared among scorpion toxins is associated with an important level of cross reactivity exhibited by these antibody fragments. We have exploited the cross reactivity showed by single chain variable antibody fragments (scFvs) of human origin to re-direct the neutralizing capacity toward various other scorpion toxins. As expected, during these evolving processes several variants derived from a parental scFv exhibited the capacity to simultaneously recognize and neutralize different toxins from Centruroides scorpion venoms. A sequence analyses of the cross reacting scFvs revealed that specific mutations are responsible for broadening their neutralizing capacity. In this work, we generated a set of new scFvs that resulted from the combinatorial insertion of these point mutations. These scFvs are potential candidates to be part of a novel recombinant antivenom of human origin that could confer protection against scorpion stings. A remarkable property of one of these new scFvs (ER-5) is its capacity to neutralize at least three different toxins and its complementary capacity to neutralize the whole venom from Centruroides suffusus in combination with a second scFv (LR), which binds to a different epitope shared by Centruroides scorpion toxins.

  12. Correlations Between Neutral and Ionized Solar Wind

    NASA Astrophysics Data System (ADS)

    Collier, M.; Pilkerton, B.; Moore, T.

    The Low Energy Neutral Atom (LENA) Imager on the IMAGE spacecraft has observed the neutral component of the solar wind (JGR, 106, 24,893, 2001) independently adumbrated by Akasofu and Dessler about forty years ago. Neutral solar wind is formed by solar wind charge exchange with interstellar neutrals, dust and the Earth's exosphere, in addition to any intrinsically neutral component. Here we report the results of a statistical study correlating the solar wind fluxes observed by ACE during late 2000 and throughout 2001 with neutral solar wind fluxes observed by LENA. The average correlation coefficient between the neutral and ionized solar wind is 0.66 with "good" correlations (peak correlation coefficient above 0.80) occurring about 28% of the time. The results are similar to those obtained by in-situ multi-spacecraft correlation studies. In this study, however, IMAGE is almost never in the solar wind or magnetosheath. The slope of the relationship between the neutral solar wind flux and the solar wind flux shows a peak in the upstream direction, but shifted toward higher ecliptic longitudes than the interstellar neutral (ISN) flow direction by about 20 degrees. The estimated peak interstellar neutral upstream density is about 10-2 cm-3.

  13. 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.

  14. Determination of serum antibodies against swine-origin influenza A virus H1N1/09 by immunofluorescence, haemagglutination inhibition, and by neutralization tests: how is the prevalence rate of protecting antibodies in humans?

    PubMed

    Allwinn, Regina; Geiler, Janina; Berger, Annemarie; Cinatl, J; Doerr, H W

    2010-05-01

    In April 2009, a new variant of influenza A virus, subtype H1N1v emerged in Mexico and spread all over the world producing the H1N1 pandemic in mankind after 1918-1920 and 1978/1979. Obviously there was no herd immunity against this new virus variant. Mainly young people, but less elderly were affected and presented severe and even lethal courses of disease. Since virus-specific antibodies are commonly regarded as markers of partial or complete immunoprotection, we performed antibody determinations in serum samples obtained from people before and after the pandemic has arrived in our region (Frankfurt/M., Germany). The assays were done by indirect immunofluorescence, by neutralization test, and by a haemagglutination inhibition test (HI), which was established in a practical modification for general and easy use. Among 145 individuals, of whom serum specimens had been drawn before the onset of pandemic, 19 revealed humoral immunity, i.e. titres of H1N1v neutralizing antibodies (at least 1:64). Eleven were older than 60 years, one belonged to the age group 40-59 years, three to the age group 20-39 years, and two to the age group 15-19 years. After the onset of pandemic in Frankfurt, serum specimens drawn from n = 225 randomly selected patients of our local university hospital were investigated for antibodies against H1N1v by HI, which is generally recommended for routine check of immunity. Twenty-eight individuals revealed the protecting antibody titre of at least 1:40. The age distribution had moved to mean age groups. The results fit to the incidence of influenza A/H1N1(09) disease, as confirmed by RT-PCR in patients admitted to our hospital, peaking in the younger age groups up to 30 years (second affected group: 30-40 years). While commonly used solid-phase antibody tests (like immunofluorescence) are not suitable to diagnose passed H1N1(09) infection and acquired immunity, this can be easily done by HI. Expecting the next waves of influenza A/H1N1v infections

  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. Neutral-beam current drive in tokamaks

    SciTech Connect

    Devoto, R.S.

    1986-01-01

    The theory of neutral-beam current drive in tokamaks is reviewed. Experiments are discussed where neutral beams have been used to drive current directly and also indirectly through neoclassical effects. Application of the theory to an experimental test reactor is described. It is shown that neutral beams formed from negative ions accelerated to 500 to 700 keV are needed for this device.

  17. 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)

  18. Influenza virus antigenicity and broadly neutralizing epitopes.

    PubMed

    Air, Gillian M

    2015-04-01

    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.

  19. 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.

  20. EFFECTS OF LEAKAGE NEUTRAL PARTICLES ON SHOCKS

    SciTech Connect

    Ohira, Yutaka

    2012-10-20

    In this paper, we investigate effects of neutral particles on shocks propagating into the partially ionized medium. We find that for 120 km s{sup -1} < u {sub sh} < 3000 km s{sup -1} (u {sub sh} is the shock velocity), about 10% of upstream neutral particles leak into the upstream region from the downstream region. Moreover, we investigate how the leakage neutral particles affect the upstream structure of the shock and particle accelerations. Using four-fluid approximations (upstream ions, upstream neutral particles, leakage neutral particles, and pickup ions), we provide analytical solutions of the precursor structure due to leakage neutral particles. It is shown that the upstream flow is decelerated in the precursor region and the shock compression ratio becomes smaller than without leakage neutral particles, but the total compression ratio does not change. Even if leakage of neutral particles is small (a few percent of total upstream particles), this smaller compression ratio of the shock can explain steep gamma-ray spectra from young supernova remnants. Furthermore, leakage neutral particles could amplify the magnetic field and heat the upstream region.

  1. CELLULAR CHANGES THAT ACCOMPANY SHEDDING OF HUMAN CORNEOCYTES

    PubMed Central

    Lin, Tzu-Kai; Crumrine, Debra; Ackerman, Larry D.; Santiago, Juan-Luis; Roelandt, Truus; Uchida, Yoshikazu; Hupe, Melanie; Fabriàs, Gemma; Abad, Jose Luis; Rice, Robert H.; Elias, Peter M.

    2012-01-01

    Corneocyte desquamation has been ascribed to either: 1) proteolytic degradation of corneodesmosomes (CD); 2) disorganization of extracellular lamellar bilayers; and/or 3) ‘swell-shrinkage-slough’ (SSS) from hydration/dehydration. To address the cellular basis for normal exfoliation, we compared changes in lamellar bilayer architecture and CD structure in DSquame® strips from the 1st vs. 5th stripping (‘outer’ vs. ‘mid’-stratum corneum [SC], respectively) from 9 normal adult forearms. Strippings were either processed for standard EM or for ruthenium (Ru-V)- or osmium-tetroxide (Os-V) vapor fixation, followed by immediate epoxy embedment, an artifact-free protocol that to our knowledge is previously unreported. CDs are largely intact in the mid-SC, but replaced by electron-dense (hydrophilic) clefts (lacunae) that expand laterally, splitting lamellar arrays in the outer SC. Some undegraded DSG1/DSC1 redistribute uniformly into corneocyte envelopes (CEs) in the outer SC (shown by proteomics, Z-stack confocal imaging and immunoEM). CEs then thicken, likely facilitating exfoliation by increasing corneocyte rigidity. In vapor-fixed images, hydration only altered the volume of the extracellular compartment, expanding lacunae further separating membrane arrays. During dehydration, air replaced water, maintaining the expanded extracellular compartment. Hydration also provoked degradation of membranes by activating contiguous acidic ceramidase activity. Together, these studies identify several parallel mechanisms that orchestrate exfoliation from the surface of normal human skin. PMID:22739796

  2. 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.

  3. 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.

  4. 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.

  5. 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...

  6. 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...

  7. 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...

  8. 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...

  9. 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...

  10. Energetic neutral particles from Jupiter and Saturn

    NASA Technical Reports Server (NTRS)

    Cheng, A. F.

    1986-01-01

    The Voyager 1 spacecraft has detected energetic neutral particles escaping from the magnetospheres of Jupiter and Saturn. These energetic neutrals are created in charge exchange reactions between radiation belt ions and ambient atoms or molecules in the magnetosphere. If the Io torus is assumed to be the dominant Jovian source region for energetic neutrals, the Voyager observations can be used to infer upper limits to the average ion intensities there below about 200 keV. No readily interpretable in-situ measurements are available in the Io torus at these energies. The middle and outer Jovian magnetospheres may also be a significant source of energetic neutrals. At Saturn, the observed neutral particle count rates are too high to be explained by charge exchange between fast protons and H atoms of the Titan torus. Most of the energetic neutrals may be produced by charge exchanges between heavy ions and a neutral cloud containing H2O in Saturn's inner magnetosphere. If so, the Voyager measurements of energetic neutral fluxes would be the first detected emissions from this region of Saturn's magnetosphere.

  11. The Source of Saturn's Extended Neutral Cloud

    NASA Astrophysics Data System (ADS)

    Fleshman, B. L.; Delamere, P. A.; Bagenal, F.

    2010-12-01

    The Enceladus plumes are largely responsible for the water and its by-products found throughout Saturn's Enceladus plasma-neutral torus. Flybys of Enceladus by the Cassini spacecraft from 2005 to present have revealed a dynamic system, from which varying estimates have been made of the release rate of neutral H2O from Enceladus at 75-1600 kg/s. The rate of fresh ion pick-up near Enceladus has also been observed to vary between flybys. In Fleshman et al. (2010a), we showed that the magnitude of fresh ion pick-up on respective flybys requires a source (possibly in beams) of `hot' electrons at Enceladus. In this work, we build on our understanding of the local interaction by quantifying the degree to which neutrals produced as a by-product of charge exchange contribute to the Saturn's neutral cloud extending from 3 to 10 RS (see also Johnson et al., (2006)). We follow individual ion and neutral trajectories, while accounting for velocity-dependent cross sections. Our aim is to compare the neutral cloud resulting from charge exchange at Enceladus to that resulting from both (1) photodissociation and (2) electron impact dissociation throughout the Enceladus torus. The goal of the study, when compared with neutral cloud observations, is to shed light on which of these mechanisms dominates the neutral cloud morphology at Saturn.

  12. 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...

  13. 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…

  14. Implications of tritium in neutral beam injectors

    SciTech Connect

    Kim, J; Stewart, L D

    1980-01-01

    Neutral injectors for heating plasmas of D-T burning fusion reactors are subject to tritium contamination. This paper discusses relevant questions and problem areas pertinent to tritium environment, including calculations of tritium contaminations in different neutral injectors, gas handling and pumping systems, and implications on beam line components.

  15. Neutral-current x-distributions

    SciTech Connect

    Bogert, D.; Burnstein, R.; Fisk, R.; Fuess, S.; Bofill, J.; Busza, W.; Eldridge, T.; Friedman, J.I.; Abolins, M.; Brock, R.

    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. 4 references.

  16. 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...

  17. 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...

  18. 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...

  19. A New Age of Constructivism: "Mode Neutral"

    ERIC Educational Resources Information Center

    Reed, Peter; Smith, Brian; Sherratt, Cathy

    2008-01-01

    This article presents work in progress exploring social constructivism within Mode Neutral, and how various conditions impact upon the student experience. Mode Neutral's three dimensions--curriculum design, the role of the tutor and communication for learning--are affected by the conditions that can vary in any given context. The authors realise…

  20. Neutral red staining for plant vacuoles.

    PubMed

    Schwab, Birgit; Hülskamp, Martin

    2010-06-01

    For almost 100 years, neutral red has been used to stain living cells and fixed tissue. It can be used as a general-purpose stain, a pH indicator (turning from red to yellow, as the medium becomes alkaline), or a nuclear stain. In this protocol, neutral red is used to stain plant vacuoles.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Sensitive neutralization test for rubella antibody.

    PubMed Central

    Sato, H; Albrecht, P; Krugman, S; Ennis, F A

    1979-01-01

    A modified rubella virus plaque neutralization test for measuring rubella antibody was developed based on the potentiation of the virus-antibody complex by heterologous anti-immunoglobulin. The test is highly sensitive, yielding titers on the average 50 to 100 times higher than the haemagglutination inhibition test or the conventional plaque neutralization test. The sensitivity of this enhanced neutralization test is somewhat limited by the existence of a prozone phenomenon which precludes testing of low-titered sera below a dilution of 1:16. No prozone effect was observed with cerebrospinal fluids. The specificity of the enhanced neutralization test was determined by seroconversion of individuals receiving rubella vaccine. Although the rubella hemagglutination inhibition test remains the test of choice in routine diagnostic and surveillance work, the enhanced rubella neutralization test is particularly useful in monitoring low-level antibody in the cerebrospinal fluid in patients with neurological disorders and in certain instances of vaccine failure. PMID:107192

  7. Broadly Neutralizing Anti-Influenza Virus Antibodies: Enhancement of Neutralizing Potency in Polyclonal Mixtures and IgA Backbones

    PubMed Central

    He, Wenqian; Mullarkey, Caitlin E.; Duty, J. Andrew; Moran, Thomas M.; Palese, Peter

    2015-01-01

    ABSTRACT Current influenza virus vaccines rely upon the accurate prediction of circulating virus strains months in advance of the actual influenza season in order to allow time for vaccine manufacture. Unfortunately, mismatches occur frequently, and even when perfect matches are achieved, suboptimal vaccine efficacy leaves several high-risk populations vulnerable to infection. However, the recent discovery of broadly neutralizing antibodies that target the hemagglutinin (HA) stalk domain has renewed hope that the development of “universal” influenza virus vaccines may be within reach. Here, we examine the functions of influenza A virus hemagglutinin stalk-binding antibodies in an endogenous setting, i.e., as polyclonal preparations isolated from human sera. Relative to monoclonal antibodies that bind to the HA head domain, the neutralization potency of monoclonal stalk-binding antibodies was vastly inferior in vitro but was enhanced by several orders of magnitude in the polyclonal context. Furthermore, we demonstrated a surprising enhancement in IgA-mediated HA stalk neutralization relative to that achieved by antibodies of IgG isotypes. Mechanistically, this could be explained in two ways. Identical variable regions consistently neutralized virus more potently when in an IgA backbone compared to an IgG backbone. In addition, HA-specific memory B cells isolated from human peripheral blood were more likely to be stalk specific when secreting antibodies of IgA isotypes compared to those secreting IgG. Taken together, our data provide strong evidence that HA stalk-binding antibodies perform optimally when in a polyclonal context and that the targeted elicitation of HA stalk-specific IgA should be an important consideration during “universal” influenza virus vaccine design. IMPORTANCE Influenza viruses remain one of the most worrisome global public health threats due to their capacity to cause pandemics. While seasonal vaccines fail to protect against the

  8. Estimating the stoichiometry of HIV neutralization.

    PubMed

    Magnus, Carsten; Regoes, Roland R

    2010-03-19

    HIV-1 virions infect target cells by first establishing contact between envelope glycoprotein trimers on the virion's surface and CD4 receptors on a target cell, recruiting co-receptors, fusing with the cell membrane and finally releasing the genetic material into the target cell. Specific experimental setups allow the study of the number of trimer-receptor-interactions needed for infection, i.e., the stoichiometry of entry and also the number of antibodies needed to prevent one trimer from engaging successfully in the entry process, i.e., the stoichiometry of (trimer) neutralization. Mathematical models are required to infer the stoichiometric parameters from these experimental data. Recently, we developed mathematical models for the estimations of the stoichiometry of entry [1]. In this article, we show how our models can be extended to investigate the stoichiometry of trimer neutralization. We study how various biological parameters affect the estimate of the stoichiometry of neutralization. We find that the distribution of trimer numbers-which is also an important determinant of the stoichiometry of entry-influences the estimated value of the stoichiometry of neutralization. In contrast, other parameters, which characterize the experimental system, diminish the information we can extract from the data about the stoichiometry of neutralization, and thus reduce our confidence in the estimate. We illustrate the use of our models by re-analyzing previously published data on the neutralization sensitivity [2], which contains measurements of neutralization sensitivity of viruses with different envelope proteins to antibodies with various specificities. Our mathematical framework represents the formal basis for the estimation of the stoichiometry of neutralization. Together with the stoichiometry of entry, the stoichiometry of trimer neutralization will allow one to calculate how many antibodies are required to neutralize a virion or even an entire population of

  9. 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.

  10. Neutralization of Plasmodium falciparum merozoites by antibodies against PfRH5

    PubMed Central

    Douglas, Alexander D.; Williams, Andrew R.; Knuepfer, Ellen; Illingworth, Joseph J.; Furze, Julie M.; Crosnier, Cécile; Choudhary, Prateek; Bustamante, Leyla Y.; Zakutansky, Sara E.; Awuah, Dennis K.; Alanine, Daniel G. W.; Theron, Michel; Worth, Andrew; Shimkets, Richard; Rayner, Julian C.; Holder, Anthony A.; Wright, Gavin J.; Draper, Simon J.

    2013-01-01

    There is intense interest in induction and characterization of strain-transcending neutralizing antibody against antigenically variable human pathogens. We have recently identified the human malaria parasite Plasmodium falciparum reticulocyte-binding protein homologue 5 (PfRH5) as a target of broadly-neutralizing antibodies, but there is little information regarding the functional mechanism(s) of antibody-mediated neutralization. Here, we report that vaccine-induced polyclonal anti-PfRH5 antibodies inhibit the tight attachment of merozoites to erythrocytes, and are capable of blocking the interaction of PfRH5 with its receptor basigin. Furthermore, by developing anti-PfRH5 monoclonal antibodies (mAbs), we provide evidence that i) the ability to block the PfRH5-basigin interaction in vitro is predictive of functional activity, but absence of blockade does not predict absence of functional activity; ii) neutralizing mAbs bind spatially-related epitopes on the folded protein, involving at least two defined regions of the PfRH5 primary sequence; iii) a brief exposure window of PfRH5 is likely to necessitate rapid binding of antibody to neutralize parasites; and iv) intact bivalent IgG contributes to but is not necessary for parasite neutralization. These data provide important insight into the mechanisms of broadly-neutralizing anti-malaria antibodies and further encourage anti-PfRH5 based malaria prevention efforts. PMID:24293631

  11. Cross-Serotype Neutralization of Dengue Virus in Aotus nancymae Monkeys

    DTIC Science & Technology

    2005-01-31

    1000 • JID 2005:191 (15 March) • BRIEF REPORT B R I E F R E P O R T Cross-Serotype Neutralization of Dengue Virus in Aotus nancymae Monkeys Tadeusz J...Previously, we observed that serum from humans immune to dengue serotype 1 ( dengue -1) neutralized the American genotype of dengue serotype 2 (American...2) to a greater ex- tent than it neutralized the Asian genotype of dengue se- rotype 2 (Asian-2). To determine if this activity is protective, Aotus

  12. Molecular Classification of Enteroviruses Not Identified by Neutralization Tests

    PubMed Central

    Iritani, Nobuhiro; Seto, Yoshiyuki

    2002-01-01

    We isolated six viruses from patients diagnosed with aseptic meningitis or hand, foot, and mouth disease. The cytopathic effect of these viruses on cultured cells was like that of enteroviruses. However, viral neutralization tests against standard antisera were negative. Phylogenetic analysis with the complete VP4 nucleotide sequences of these 6 viruses and 29 serotypes of enteroviruses classified 3 of the viruses as serotype echovirus type 18 (EV18) and 3 as serotype human enterovirus 71 (HEV71). These results were confirmed by remicroneutralization tests with HEV-monospecific antisera or an additional phylogenetic analysis with the complete VP4 nucleotide sequences. Phylogenetic analysis with complete VP4 genes is more useful than neutralization tests with enterovirus serotype-specific antisera in identifying enterovirus serotypes. PMID:11927028

  13. 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 cross-neutralizing distantly related simian immunodeficiency viruses (SIVs) infecting central (Pan troglodytes troglodytes) (SIVcpzPtt) and eastern (Pan troglodytes schweinfurthii) (SIVcpzPts) chimpanzees (n = 11) as well as western gorillas (Gorilla gorilla gorilla) (SIVgor) (n = 1). We found that bNabs directed against the CD4 binding site (n = 10), peptidoglycans at the base of variable loop 3 (V3) (n = 5), and epitopes at the interface of surface (gp120) and membrane-bound (gp41) envelope glycoproteins (n = 5) failed to neutralize SIVcpz and SIVgor strains. In addition, apex V2-directed bNabs (n = 3) as well as llama-derived (heavy chain only) antibodies (n = 6) recognizing both the CD4 binding site and gp41 epitopes were either completely inactive or neutralized only a fraction of SIVcpzPtt strains. In contrast, one antibody targeting the membrane-proximal external region (MPER) of gp41 (10E8), functional CD4 and CCR5 receptor mimetics (eCD4-Ig, eCD4-Igmim2, CD4-218.3-E51, and CD4-218.3-E51-mim2), as well as mono- and bispecific anti-human CD4 (iMab and LM52) and CCR5 (PRO140, PRO140-10E8) receptor antibodies neutralized >90% of SIVcpz and SIVgor strains with low-nanomolar (0.13 to 8.4 nM) potency. Importantly, the latter antibodies blocked virus entry not only in TZM-bl cells but also in Cf2Th cells expressing chimpanzee CD4 and CCR5 and neutralized SIVcpz in chimpanzee CD4+ T cells, with 50% inhibitory concentrations (IC50s) ranging from 3.6 to 40.5 nM. These findings provide new insight into the protective capacity of anti-HIV-1 bNabs and identify candidates for further development to combat SIVcpz infection. PMID:25900654

  14. Neutralizing antibodies against rotavirus produced in transgenically labelled purple tomatoes.

    PubMed

    Juárez, Paloma; Presa, Silvia; Espí, Joaquín; Pineda, Benito; Antón, María T; Moreno, Vicente; Buesa, Javier; Granell, Antonio; Orzaez, Diego

    2012-04-01

    Edible fruits are inexpensive biofactories for human health-promoting molecules that can be ingested as crude extracts or partially purified formulations. We show here the production of a model human antibody for passive protection against the enteric pathogen rotavirus in transgenically labelled tomato fruits. Transgenic tomato plants expressing a recombinant human immunoglobulin A (hIgA_2A1) selected against the VP8* peptide of rotavirus SA11 strain were obtained. The amount of hIgA_2A1 protein reached 3.6 ± 0.8% of the total soluble protein in the fruit of the transformed plants. Minimally processed fruit-derived products suitable for oral intake showed anti-VP8* binding activity and strongly inhibited virus infection in an in vitro virus neutralization assay. In order to make tomatoes expressing hIgA_2A1 easily distinguishable from wild-type tomatoes, lines expressing hIgA_2A1 transgenes were sexually crossed with a transgenic tomato line expressing the genes encoding Antirrhinum majus Rosea1 and Delila transcription factors, which confer purple colour to the fruit. Consequently, transgenically labelled purple tomato fruits expressing hIgA_2A1 have been developed. The resulting purple-coloured extracts from these fruits contain high levels of recombinant anti-rotavirus neutralizing human IgA in combination with increased amounts of health-promoting anthocyanins.

  15. Neutral Supersymmetric Higgs Boson Searches

    SciTech Connect

    Robinson, Stephen Luke

    2008-07-01

    In some Supersymmetric extensions of the Standard Model, including the Minimal Supersymmetric Standard Model (MSSM), the coupling of Higgs bosons to b-quarks is enhanced. This enhancement makes the associated production of the Higgs with b-quarks an interesting search channel for the Higgs and Supersymmetry at D0. The identification of b-quarks, both online and offline, is essential to this search effort. This thesis describes the author's involvement in the development of both types of b-tagging and in the application of these techniques to the MSSM Higgs search. Work was carried out on the Level-3 trigger b-tagging algorithms. The impact parameter (IP) b-tagger was retuned and the effects of increased instantaneous luminosity on the tagger were studied. An extension of the IP-tagger to use the z-tracking information was developed. A new b-tagger using secondary vertices was developed and commissioned. A tool was developed to allow the use of large multi-run samples for trigger studies involving b-quarks. Offline, a neural network (NN) b-tagger was trained combining the existing offline lifetime based b-tagging tools. The efficiency and fake rate of the NN b-tagger were measured in data and MC. This b-tagger was internally reviewed and certified by the Collaboration and now provides the official b-tagging for all analyses using the Run IIa dataset at D0. A search was performed for neutral MSSM Higgs bosons decaying to a b{bar b} pair and produced in association with one or more b-quarks. Limits are set on the cross-section times the branching ratio for such a process. The limits were interpreted in various MSSM scenarios. This analysis uses the NN b-tagger and was the first to use this tool. The analysis also relies on triggers using the Level-3 IP b-tagging tool described previously. A likelihood discriminant was used to improve the analysis and a neural network was developed to cross-check this technique. The result of the analysis has been submitted to PRL and

  16. Evaluation of smallpox vaccines using variola neutralization.

    PubMed

    Damon, Inger K; Davidson, Whitni B; Hughes, Christine M; Olson, Victoria A; Smith, Scott K; Holman, Robert C; Frey, Sharon E; Newman, Frances; Belshe, Robert B; Yan, Lihan; Karem, Kevin

    2009-08-01

    The search for a 'third'-generation smallpox vaccine has resulted in the development and characterization of several vaccine candidates. A significant barrier to acceptance is the absence of challenge models showing induction of correlates of protective immunity against variola virus. In this light, virus neutralization provides one of few experimental methods to show specific 'in vitro' activity of vaccines against variola virus. Here, we provide characterization of the ability of a modified vaccinia virus Ankara vaccine to induce variola virus-neutralizing antibodies, and we provide comparison with the neutralization elicited by standard Dryvax vaccination.

  17. On the neutral points in Rayleigh transfer

    NASA Astrophysics Data System (ADS)

    Viik, T.

    2017-03-01

    In this paper we consider the dependence of the existence and position of the neutral points on the albedo of single scattering and the optical thickness in a Rayleigh scattering plane-parallel homogeneous atmospheres. We use the Chandrasekhar method of discrete ordinates and the method of approximating the Sobolev resolvent function to solve the vector equation of transfer in l- and r-representation. On the basis of many different models of Rayleigh atmospheres we show the behaviour of the neutral points while the parallel incident flux can be both unpolarized or polarized. Our calculations show with high probability that the maximum number of neutral points in a Rayleigh atmosphere is four.

  18. Structural basis for the antibody neutralization of Herpes simplex virus

    SciTech Connect

    Lee, Cheng-Chung; Lin, Li-Ling; Chan, Woan-Eng; Ko, Tzu-Ping; Lai, Jiann-Shiun; Wang, Andrew H.-J.

    2013-10-01

    The gD–E317-Fab complex crystal revealed the conformational epitope of human mAb E317 on HSV gD, providing a molecular basis for understanding the viral neutralization mechanism. Glycoprotein D (gD) of Herpes simplex virus (HSV) binds to a host cell surface receptor, which is required to trigger membrane fusion for virion entry into the host cell. gD has become a validated anti-HSV target for therapeutic antibody development. The highly inhibitory human monoclonal antibody E317 (mAb E317) was previously raised against HSV gD for viral neutralization. To understand the structural basis of antibody neutralization, crystals of the gD ectodomain bound to the E317 Fab domain were obtained. The structure of the complex reveals that E317 interacts with gD mainly through the heavy chain, which covers a large area for epitope recognition on gD, with a flexible N-terminal and C-terminal conformation. The epitope core structure maps to the external surface of gD, corresponding to the binding sites of two receptors, herpesvirus entry mediator (HVEM) and nectin-1, which mediate HSV infection. E317 directly recognizes the gD–nectin-1 interface and occludes the HVEM contact site of gD to block its binding to either receptor. The binding of E317 to gD also prohibits the formation of the N-terminal hairpin of gD for HVEM recognition. The major E317-binding site on gD overlaps with either the nectin-1-binding residues or the neutralizing antigenic sites identified thus far (Tyr38, Asp215, Arg222 and Phe223). The epitopes of gD for E317 binding are highly conserved between two types of human herpesvirus (HSV-1 and HSV-2). This study enables the virus-neutralizing epitopes to be correlated with the receptor-binding regions. The results further strengthen the previously demonstrated therapeutic and diagnostic potential of the E317 antibody.

  19. Antibody to gp41 MPER alters functional properties of HIV-1 Env without complete neutralization.

    PubMed

    Kim, Arthur S; Leaman, Daniel P; Zwick, Michael B

    2014-07-01

    Human antibody 10E8 targets the conserved membrane proximal external region (MPER) of envelope glycoprotein (Env) subunit gp41 and neutralizes HIV-1 with exceptional potency. Remarkably, HIV-1 containing mutations that reportedly knockout 10E8 binding to linear MPER peptides are partially neutralized by 10E8, producing a local plateau in the dose response curve. Here, we found that virus partially neutralized by 10E8 becomes significantly less neutralization sensitive to various MPER antibodies and to soluble CD4 while becoming significantly more sensitive to antibodies and fusion inhibitors against the heptad repeats of gp41. Thus, 10E8 modulates sensitivity of Env to ligands both pre- and post-receptor engagement without complete neutralization. Partial neutralization by 10E8 was influenced at least in part by perturbing Env glycosylation. With unliganded Env, 10E8 bound with lower apparent affinity and lower subunit occupancy to MPER mutant compared to wild type trimers. However, 10E8 decreased functional stability of wild type Env while it had an opposite, stabilizing effect on MPER mutant Envs. Clade C isolates with natural MPER polymorphisms also showed partial neutralization by 10E8 with altered sensitivity to various gp41-targeted ligands. Our findings suggest a novel mechanism of virus neutralization by demonstrating how antibody binding to the base of a trimeric spike cross talks with adjacent subunits to modulate Env structure and function. The ability of an antibody to stabilize, destabilize, partially neutralize as well as alter neutralization sensitivity of a virion spike pre- and post-receptor engagement may have implications for immunotherapy and vaccine design.

  20. Role of Ceramide from Glycosphingolipids and Its Metabolites in Immunological and Inflammatory Responses in Humans

    PubMed Central

    Iwabuchi, Kazuhisa; Nakayama, Hitoshi; Oizumi, Ami; Suga, Yasushi; Ogawa, Hideoki; Takamori, Kenji

    2015-01-01

    Glycosphingolipids (GSLs) are composed of hydrophobic ceramide and hydrophilic sugar chains. GSLs cluster to form membrane microdomains (lipid rafts) on plasma membranes, along with several kinds of transducer molecules, including Src family kinases and small G proteins. However, GSL-mediated biological functions remain unclear. Lactosylceramide (LacCer, CDw17) is highly expressed on the plasma membranes of human phagocytes and mediates several immunological and inflammatory reactions, including phagocytosis, chemotaxis, and superoxide generation. LacCer forms membrane microdomains with the Src family tyrosine kinase Lyn and the Gαi subunit of heterotrimeric G proteins. The very long fatty acids C24:0 and C24:1 are the main ceramide components of LacCer in neutrophil plasma membranes and are directly connected with the fatty acids of Lyn and Gαi. These observations suggest that the very long fatty acid chains of ceramide are critical for GSL-mediated outside-in signaling. Sphingosine is another component of ceramide, with the hydrolysis of ceramide by ceramidase producing sphingosine and fatty acids. Sphingosine is phosphorylated by sphingosine kinase to sphingosine-1-phosphate, which is involved in a wide range of cellular functions, including growth, differentiation, survival, chemotaxis, angiogenesis, and embryogenesis, in various types of cells. This review describes the role of ceramide moiety of GSLs and its metabolites in immunological and inflammatory reactions in human. PMID:26609196

  1. Neutralizing Antibodies Against AAV Serotypes 1, 2, 6, and 9 in Sera of Commonly Used Animal Models

    PubMed Central

    Rapti, Kleopatra; Louis-Jeune, Vedell; Kohlbrenner, Erik; Ishikawa, Kiyotake; Ladage, Dennis; Zolotukhin, Sergei; Hajjar, Roger J; Weber, Thomas

    2012-01-01

    Adeno-associated virus (AAV)-based vectors are promising gene delivery vehicles for human gene transfer. One significant obstacle to AAV-based gene therapy is the high prevalence of neutralizing antibodies in humans. Until now, it was thought that, except for nonhuman primates, pre-existing neutralizing antibodies are not a problem in small or large animal models for gene therapy. Here, we demonstrate that sera of several animal models of cardiovascular diseases harbor pre-existing antibodies against the cardiotropic AAV serotypes AAV1, AAV6, and AAV9 and against AAV2. The neutralizing antibody titers vary widely both between species and between serotypes. Of all species tested, rats displayed the lowest levels of neutralizing antibodies. Surprisingly, naive mice obtained directly from commercial vendors harbored neutralizing antibodies. Of the large animal models tested, the neutralization of AAV6 transduction by dog sera was especially pronounced. Sera of sheep and rabbits showed modest neutralization of AAV transduction whereas porcine sera strongly inhibited transduction by all AAV serotypes and displayed the largest variation between individual animals. Importantly, neutralizing antibody titers as low as 1/4 completely prevented in vivo transduction by AAV9 in rats. Our results suggest that prescreening of animals for neutralizing antibodies will be important for future gene transfer experiments in these animal models. PMID:21915102

  2. Mechanism and Significance of Cell Type-Dependent Neutralization of Flaviviruses

    PubMed Central

    Mukherjee, Swati; Dowd, Kimberly A.; Manhart, Carolyn J.; Ledgerwood, Julie E.; Durbin, Anna P.; Whitehead, Stephen S.

    2014-01-01

    ABSTRACT The production of neutralizing antibodies (NAbs) is a correlate of protection for many human vaccines, including currently licensed vaccines against flaviviruses. NAbs are typically measured using a plaque reduction neutralization test (PRNT). Despite its extensive use, parameters that impact the performance of the PRNT have not been investigated from a mechanistic perspective. The results of a recent phase IIb clinical trial of a tetravalent dengue virus (DENV) vaccine suggest that NAbs, as measured using a PRNT performed with Vero cells, do not correlate with protection. This surprising finding highlights the importance of understanding how well the PRNT captures the complexity of the NAb response to DENV. In this study, we demonstrated that the structural heterogeneity of flaviviruses arising from inefficient virion maturation impacts the results of neutralization assays in a cell type-dependent manner. Neutralization titers of several monoclonal antibodies were significantly reduced when assayed on Vero cells compared to Raji cells expressing DC-SIGNR. This pattern can be explained by differences in the efficiency with which partially mature flaviviruses attach to each cell type, rather than a differential capacity of antibody to block infection. Vero cells are poorly permissive to the fraction of virions that are most sensitive to neutralization. Analysis of sera from recipients of live-attenuated monovalent DENV vaccine candidates revealed a strong correlation between the sensitivity of serum antibodies to the maturation state of DENV and cell type-dependent patterns of neutralization. Cross-reactive patterns of neutralization may be underrepresented by the “gold-standard” PRNT that employs Vero cells. IMPORTANCE Cell type-dependent patterns of neutralization describe a differential capacity of antibodies to inhibit virus infection when assayed on multiple cellular substrates. In this study, we established a link between antibodies that

  3. Kinetics of neutralization of Po-218

    SciTech Connect

    Chu, K.D.

    1987-01-01

    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 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 potential of PoO/sub 2//sup +/ is also determined to be 10.44 +/- 0.05 eV.

  4. An autoneutralizing neutral molecular beam gun

    NASA Astrophysics Data System (ADS)

    Delmore, J. E.; Appelhans, A. D.; Dahl, D. A.

    1990-01-01

    A high-energy (up to 28 keV) neutral molecular beam gun has been developed and put into routine use that takes advantage of the autoneutralization properties of the sulfur hexafluoride anion for the production of high-energy sulfur hexafluoride neutral molecules. The anions are produced in an electron-capture source, accelerated, and focused in a lens assembly designed to minimize residence time, allowed to drift at their terminal velocity for a suitable distance during which up to 30% auto-eject an electron, and all remaining charged particles are electrostatically skimmed, resulting in a focused neutral beam. Rasterable neutral beams focused to a 5-mm spot size up to 3 m from the source have been produced with beam currents up to 40 pA equivalent. Spot sizes of 1 mm can be produced with intensity levels of a few picoamperes equivalent.

  5. Neutrality tests for sequences with missing data.

    PubMed

    Ferretti, Luca; Raineri, Emanuele; Ramos-Onsins, Sebastian

    2012-08-01

    Missing data are common in DNA sequences obtained through high-throughput sequencing. Furthermore, samples of low quality or problems in the experimental protocol often cause a loss of data even with traditional sequencing technologies. Here we propose modified estimators of variability and neutrality tests that can be naturally applied to sequences with missing data, without the need to remove bases or individuals from the analysis. Modified statistics include the Watterson estimator θW, Tajima's D, Fay and Wu's H, and HKA. We develop a general framework to take missing data into account in frequency spectrum-based neutrality tests and we derive the exact expression for the variance of these statistics under the neutral model. The neutrality tests proposed here can also be used as summary statistics to describe the information contained in other classes of data like DNA microarrays.

  6. Ion-Neutral Coupling in Solar Prominence

    NASA Technical Reports Server (NTRS)

    Gilbert, H.; DeVore, C. R.; Karpen, J.; Kucera, T.; Antiochos, S.; Kawashima, R.

    2011-01-01

    Coupling between ions and neutrals in magnetized plasmas is fundamentally important to many aspects of heliophysics, including our ionosphere, the solar chromosphere, the solar wind interaction with planetary atmospheres, and the interface between the heliosphere and the interstellar medium. Ion-neutral coupling also plays a major role in the physics of solar prominences. By combining theory, modeling, and observations we are working toward a better understanding of the structure and dynamics of partially ionized prominence plasma. Two key questions are addressed in the present work: 1) what physical mechanism(s) sets the cross-field scale of prominence threads? 2) Are ion-neutral interactions responsible for the vertical flows and structure in prominences? We present initial results from a study investigating what role ion-neutral interactions play in prominence dynamics and structure. This research was supported by NASA.

  7. ITER neutral beam system US conceptual design

    SciTech Connect

    Purgalis, P.

    1990-09-01

    In this document we present the US conceptual design of a neutral beam system for International Thermonuclear Experimental Reactor (ITER). The design incorporates a barium surface conversion D{sup {minus}} source feeding a linear array of accelerator channels. The system uses a dc accelerator with electrostatic quadrupoles for strong focusing. A high voltage power supply that is integrated with the accelerator is presented as an attractive option. A gas neutralizer is used and residual ions exiting the neutralizer are deflected to water-cooled dumps. Cryopanels are located at the accelerator exit to pump excess gas from the source and the neutralizer, and in the ion dump cavity to pump re-neutralized ions and neutralizer gas. All the above components are packaged in compact identical, independent modules which can be removed for remote maintenance. The neutral beam system delivers 75 MW of DO at 1.3 MeV, into three ports with a total of 9 modules arranged in stacks of three modules per port . To increase reliability each module is designed to deliver up to 10 MW; this allows eight modules operating at partial capacity to deliver the required power in the event one module is out of service, and provides 20% excess capacity to improve availability. Radiation protection is provided by shielding and by locating critical components in the source and accelerator 46.5 m from the torus centerline. Neutron shielding in the drift duct and neutralizer provides the added feature of limiting conductance and thus reducing gas flow to and from the torus.

  8. Nitrogen-neutrality: a step towards sustainability

    NASA Astrophysics Data System (ADS)

    Leip, Adrian; Leach, Allison; Musinguzi, Patrick; Tumwesigye, Trust; Olupot, Giregon; Tenywa, John Stephen; Mudiope, Joseph; Hutton, Olivia; Cordovil, Claudia M. d. S.; Bekunda, Mateete; Galloway, James

    2014-11-01

    We propose a novel indicator measuring one dimension of the sustainability of an entity in modern societies: Nitrogen-neutrality. N-neutrality strives to offset Nr releases an entity exerts on the environment from the release of reactive nitrogen (Nr) to the environment by reducing it and by offsetting the Nr releases elsewhere. N-neutrality also aims to increase awareness about the consequences of unintentional releases of nitrogen to the environment. N-neutrality is composed of two quantified elements: Nr released by an entity (e.g. on the basis of the N footprint) and Nr reduction from management and offset projects (N offset). It includes management strategies to reduce nitrogen losses before they occur (e.g., through energy conservation). Each of those elements faces specific challenges with regard to data availability and conceptual development. Impacts of Nr releases to the environment are manifold, and the impact profile of one unit of Nr release depends strongly on the compound released and the local susceptibility to Nr. As such, N-neutrality is more difficult to conceptualize and calculate than C-neutrality. We developed a workable conceptual framework for N-neutrality which was adapted for the 6th International Nitrogen Conference (N2013, Kampala, November 2013). Total N footprint of the surveyed meals at N2013 was 66 kg N. A total of US 3050 was collected from the participants and used to offset the conference’s N footprint by supporting the UN Millennium Village cluster Ruhiira in South-Western Uganda. The concept needs further development in particular to better incorporate the spatio-temporal variability of impacts and to standardize the methods to quantify the required N offset to neutralize the Nr releases impact. Criteria for compensation projects need to be sharply defined to allow the development of a market for N offset certificates.

  9. Neutral thermospheric temperature from ion concentration measurements

    NASA Technical Reports Server (NTRS)

    Breig, E. L.; Donaldson, J. S.; Hanson, W. B.; Hoffman, J. H.; Power, R. A.; Kayser, D. C.; Spencer, N. W.; Wharton, L. E.

    1981-01-01

    A technique for extracting information on neutral temperature from in situ F region measurements of O(+) and H(+) ion concentrations is analyzed and evaluated. Advantage is taken of the condition of charge-exchange equilibrium of these species in the neighborhood of 320 km to infer the associated relative abundances of neutral oxygen and hydrogen. Results are shown to be generally consistent with other concurrent in situ measurements.

  10. A class of neutral functional differential equations.

    NASA Technical Reports Server (NTRS)

    Melvin, W. R.

    1972-01-01

    Formulation and study of the initial value problem for neutral functional differential equations. The existence, uniqueness, and continuation of solutions to this problem are investigated, and an analysis is made of the dependence of the solutions on the initial conditions and parameters, resulting in the derivation of a continuous dependence theorem in which the fundamental mathematical principles underlying the continuous dependence problem for a very general system of nonlinear neutral functional differential equations are separated out.

  11. The Phobos neutral and ionized torus

    NASA Astrophysics Data System (ADS)

    Poppe, A. R.; Curry, S. M.; Fatemi, S.

    2016-05-01

    Charged particle sputtering, micrometeoroid impact vaporization, and photon-stimulated desorption are fundamental processes operating at airless surfaces throughout the solar system. At larger bodies, such as Earth's Moon and several of the outer planet moons, these processes generate tenuous surface-bound exospheres that have been observed by a variety of methods. Phobos and Deimos, in contrast, are too gravitationally weak to keep ejected neutrals bound and, thus, are suspected to generate neutral tori in orbit around Mars. While these tori have not yet been detected, the distribution and density of both the neutral and ionized components are of fundamental interest. We combine a neutral Monte Carlo model and a hybrid plasma model to investigate both the neutral and ionized components of the Phobos torus. We show that the spatial distribution of the neutral torus is highly dependent on each individual species (due to ionization rates that span nearly 4 orders of magnitude) and on the location of Phobos with respect to Mars. Additionally, we present the flux distribution of torus pickup ions throughout the Martian system and estimate typical pickup ion fluxes. We find that the predicted pickup ion fluxes are too low to perturb the ambient plasma, consistent with previous null detections by spacecraft around Mars.

  12. Protection in Macaques Immunized with HIV-1 Candidate Vaccines Can Be Predicted Using the Kinetics of Their Neutralizing Antibodies

    PubMed Central

    Davis, David; Koornstra, Wim; Mortier, Daniella; Fagrouch, Zahra; Verschoor, Ernst J.; Heeney, Jonathan L.; Bogers, Willy M. J. M.

    2011-01-01

    Background A vaccine is needed to control the spread of human immunodeficiency virus type 1 (HIV-1). An in vitro assay that can predict the protection induced by a vaccine would facilitate the development of such a vaccine. A potential candidate would be an assay to quantify neutralization of HIV-1. Methods and Findings We have used sera from rhesus macaques that have been immunized with HIV candidate vaccines and subsequently challenged with simian human immunodeficiency virus (SHIV). We compared neutralization assays with different formats. In experiments with the standardized and validated TZMbl assay, neutralizing antibody titers against homologous SHIVSF162P4 pseudovirus gave a variable correlation with reductions in plasma viremia levels. The target cells used in the assays are not just passive indicators of virus infection but are actively involved in the neutralization process. When replicating virus was used with GHOST cell assays, events during the absorption phase, as well as the incubation phase, determine the level of neutralization. Sera that are associated with protection have properties that are closest to the traditional concept of neutralization: the concentration of antibody present during the absorption phase has no effect on the inactivation rate. In GHOST assays, events during the absorption phase may inactivate a fixed number, rather than a proportion, of virus so that while complete neutralization can be obtained, it can only be found at low doses particularly with isolates that are relatively resistant to neutralization. Conclusions Two scenarios have the potential to predict protection by neutralizing antibodies at concentrations that can be induced by vaccination: antibodies that have properties close to the traditional concept of neutralization may protect against a range of challenge doses of neutralization sensitive HIV isolates; a window of opportunity also exists for protection against isolates that are more resistant to

  13. Comparison of JEV neutralization assay using pseudotyped JEV with the conventional plaque-reduction neutralization test.

    PubMed

    Lee, Hee-Jung; Min, Kyung-Il; Park, Ki Hoon; Choi, Hyo Jung; Kim, Min-Kyoung; Ahn, Chi-Young; Hong, Young-Jin; Kim, Young Bong

    2014-05-01

    We previously reported the development of a neutralization assay system for evaluating Japanese Encephalitis Virus (JEV) neutralizing antibody (NAb) using pseudotyped-JEV (JEV-PV). JEV-PV-based neutralization assay offers several advantages compared with the current standard plaque-reduction neutralization test (PRNT), including simplicity, safety, and speed. To evaluate the suitability of the JEV-PV assay as new replacement neutralization assay, we compared its repeatability, reproducibility, specificity, and correlated its results with those obtained using the PRNT. These analyses showed a close correlation between the results obtained with the JEV-PV assay and the PRNT, using the 50% plaque reduction method as a standard for measuring NAb titers to JEV. The validation results met all analytical acceptance criteria. These results suggest that the JEV-PV assay could serve as a safe and simple method for measuring NAb titer against JEV and could be used as an alternative approach for assaying the potency of JEV neutralization.

  14. The neutralizing role of IgM during early Chikungunya virus infection

    PubMed Central

    Chua, Chong-Long; Chiam, Chun-Wei; Chan, Yoke-Fun

    2017-01-01

    The antibody isotype IgM appears earlier than IgG, within days of onset of symptoms, and is important during the early stages of the adaptive immune response. Little is known about the functional role of IgM during infection with chikungunya virus (CHIKV), a recently reemerging arbovirus that has caused large global outbreaks. In this study, we studied antibody responses in 102 serum samples collected during CHIKV outbreaks in Malaysia. We described the neutralizing role of IgM at different times post-infection and examined the independent contributions of IgM and IgG towards the neutralizing capacity of human immune sera during the early phase of infection, including the differences in targets of neutralizing epitopes. Neutralizing IgM starts to appear as early as day 4 of symptoms, and their appearance from day 6 is associated with a reduction in viremia. IgM acts in a complementary manner with the early IgG, but plays the main neutralizing role up to a point between days 4 and 10 which varies between individuals. After this point, total neutralizing capacity is attributable almost entirely to the robust neutralizing IgG response. IgM preferentially binds and targets epitopes on the CHIKV surface E1-E2 glycoproteins, rather than individual E1 or E2. These findings provide insight into the early antibody responses to CHIKV, and have implications for design of diagnostic serological assays. PMID:28182795

  15. Molecular evolution of broadly neutralizing Llama antibodies to the CD4-binding site of HIV-1.

    PubMed

    McCoy, Laura E; Rutten, Lucy; Frampton, Dan; Anderson, Ian; Granger, Luke; Bashford-Rogers, Rachael; Dekkers, Gillian; Strokappe, Nika M; Seaman, Michael S; Koh, Willie; Grippo, Vanina; Kliche, Alexander; Verrips, Theo; Kellam, Paul; Fassati, Ariberto; Weiss, Robin A

    2014-12-01

    To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols.

  16. Molecular Evolution of Broadly Neutralizing Llama Antibodies to the CD4-Binding Site of HIV-1

    PubMed Central

    McCoy, Laura E.; Rutten, Lucy; Frampton, Dan; Anderson, Ian; Granger, Luke; Bashford-Rogers, Rachael; Dekkers, Gillian; Strokappe, Nika M.; Seaman, Michael S.; Koh, Willie; Grippo, Vanina; Kliche, Alexander; Verrips, Theo; Kellam, Paul; Fassati, Ariberto; Weiss, Robin A.

    2014-01-01

    To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols. PMID:25522326

  17. Structural Basis for the Binding of the Neutralizing Antibody, 7D11, to the Poxvirus L1 Protein

    DTIC Science & Technology

    2007-08-01

    enveloped particles, such as the humanized chimpanzee B5 antibodies (Chen et al., 2006), may serve as a replacement for vaccinia immune globulin and...Svitel, J., Schuck, P., Satterfield, W., Moss, B., Purcell, R., 2006. Chimpanzee /