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Sample records for human neutral ceramidase

  1. Identification of a functional hepatocyte nuclear factor 4 binding site in the neutral ceramidase promoter.

    PubMed

    Maltesen, Henrik R; Troelsen, Jesper T; Olsen, Jørgen

    2010-12-01

    The brush border membrane of the differentiated small intestinal epithelial cell is studded with membrane bound hydrolytic ectoenzymes involved in digestion. Previous studies of the regulation of genes encoding brush border enzymes have especially implicated the transcription factors hepatocyte nuclear factor HNF-1 and Cdx2. Recent genome-wide studies have, however, also identified HNF-4α as a transcription factor with a high number of target genes in the differentiated small intestinal epithelial cell. The Asah2 gene encodes neutral ceramidase, which is a hydrolytic brush border enzyme involved in ceramide digestion. It was the purpose of the present work to experimentally verify the functional importance of a HNF-4α binding site predicted by bioinformatic analysis to be present in the Asah2 promoter. Using supershift analysis, HNF-4α overexpression, and HNF-4α knockdown experiments it was confirmed that the predicted HNF-4α binding site identified in the Asah2 promoter is functional. The results support the hypothesis that HNF-4α might be important for intestinal glycolipid metabolism. PMID:20803549

  2. Pseudomonas-derived ceramidase induces production of inflammatory mediators from human keratinocytes via sphingosine-1-phosphate.

    PubMed

    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-α via S

  3. Aerobic training in rats increases skeletal muscle sphingomyelinase and serine palmitoyltransferase activity, while decreasing ceramidase activity.

    PubMed

    Błachnio-Zabielska, Agnieszka; Zabielski, Piotr; Baranowski, Marcin; Gorski, Jan

    2011-03-01

    Sphingolipids are important components of cell membranes that may also serve as cell signaling molecules; ceramide plays a central role in sphingolipid metabolism. The aim of this study was to examine the effect of 5 weeks of aerobic training on key enzymes and intermediates of ceramide metabolism in skeletal muscles. The experiments were carried out on rats divided into two groups: (1) sedentary and (2) trained for 5 weeks (on a treadmill). The activity of serine palmitoyltransferase (SPT), neutral and acid sphingomyelinase (nSMase and aSMase), neutral and alkaline ceramidases (nCDase and alCDase) and the content of sphingolipids was determined in three types of skeletal muscle. We also measured the fasting plasma insulin and glucose concentration for calculating HOMA-IR (homeostasis model assessment) for estimating insulin resistance. We found that the activities of aSMase and SPT increase in muscle in the trained group. These changes were followed by elevation in the content of sphinganine. The activities of both isoforms of ceramidase were reduced in muscle in the trained group. Although the activities of SPT and SMases increased and the activity of CDases decreased, the ceramide content did not change in any of the studied muscle. Although ceramide level did not change, we noticed increased insulin sensitivity in trained animals. It is concluded that training affects the activity of key enzymes of ceramide metabolism but also activates other metabolic pathways which affect ceramide metabolism in skeletal muscles.

  4. 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.

  5. Discovery of highly potent acid ceramidase inhibitors with in vitro tumor chemosensitizing activity

    PubMed Central

    Realini, Natalia; Solorzano, Carlos; Pagliuca, Chiara; Pizzirani, Daniela; Armirotti, Andrea; Luciani, Rosaria; Costi, Maria Paola; Bandiera, Tiziano; Piomelli, Daniele

    2013-01-01

    The expression of acid ceramidase (AC) – a cysteine amidase that hydrolyses the proapoptotic lipid ceramide – is abnormally high in several human tumors, which is suggestive of a role in chemoresistance. Available AC inhibitors lack, however, the potency and drug-likeness necessary to test this idea. Here we show that the antineoplastic drug carmofur, which is used in the clinic to treat colorectal cancers, is a potent AC inhibitor and that this property is essential to its anti-proliferative effects. Modifications in the chemical scaffold of carmofur yield new AC inhibitors that act synergistically with standard antitumoral drugs to prevent cancer cell proliferation. These findings identify AC as an unexpected target for carmofur, and suggest that this molecule can be used as starting point for the design of novel chemosensitizing agents. PMID:23301156

  6. 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

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

    PubMed

    Li, Lianwei; Ma, Zhanshan Sam

    2016-01-01

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

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

    PubMed

    Li, Lianwei; Ma, Zhanshan Sam

    2016-08-16

    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.

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

    PubMed

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

    2016-04-01

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

  10. Multivariate analysis of human immunodeficiency virus type 1 neutralization data.

    PubMed Central

    Nyambi, P N; Nkengasong, J; Lewi, P; Andries, K; Janssens, W; Fransen, K; Heyndrickx, L; Piot, P; van der Groen, G

    1996-01-01

    We report on the use of spectral map analysis of the inter- and intraclade neutralization data of 14 sera of human immunodeficiency virus type 1 (HIV-1)-infected individuals and 16 primary isolates, representing genetic clades A to H in group M and group O. This multivariate analysis has been used previously to study the interaction between drugs and receptors and between viruses and antiviral compounds. The analysis reveals the existence of neutralization clusters, not correlated with the known genetic clades. The structural factors that have been identified may correlate with the most important neutralization epitopes. Three key primary HIV-1 isolates, which allow discrimination of sera that are likely or unlikely to neutralize primary isolates from most of the genetic clades, were identified. Our method of analysis will facilitate the evaluation as well as the design of suitable HIV-1 vaccines, which induce high-titer interclade cross-neutralizing antibodies. PMID:8709250

  11. 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. PMID:25723164

  12. 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.

  13. Human genomic disease variants: A neutral evolutionary explanation

    PubMed Central

    Dudley, Joel T.; Kim, Yuseob; Liu, Li; Markov, Glenn J.; Gerold, Kristyn; Chen, Rong; Butte, Atul J.; Kumar, Sudhir

    2012-01-01

    Many perspectives on the role of evolution in human health include nonempirical assumptions concerning the adaptive evolutionary origins of human diseases. Evolutionary analyses of the increasing wealth of clinical and population genomic data have begun to challenge these presumptions. In order to systematically evaluate such claims, the time has come to build a common framework for an empirical and intellectual unification of evolution and modern medicine. We review the emerging evidence and provide a supporting conceptual framework that establishes the classical neutral theory of molecular evolution (NTME) as the basis for evaluating disease- associated genomic variations in health and medicine. For over a decade, the NTME has already explained the origins and distribution of variants implicated in diseases and has illuminated the power of evolutionary thinking in genomic medicine. We suggest that a majority of disease variants in modern populations will have neutral evolutionary origins (previously neutral), with a relatively smaller fraction exhibiting adaptive evolutionary origins (previously adaptive). This pattern is expected to hold true for common as well as rare disease variants. Ultimately, a neutral evolutionary perspective will provide medicine with an informative and actionable framework that enables objective clinical assessment beyond convenient tendencies to invoke past adaptive events in human history as a root cause of human disease. PMID:22665443

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

    PubMed

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

    2016-01-01

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

  15. 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

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

    PubMed

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

    2015-04-01

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

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

    PubMed

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

    2013-02-01

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

  18. Substrate specificity of human liver neutral alpha-mannosidase.

    PubMed Central

    al Daher, S; De Gasperi, R; Daniel, P; Hirani, S; Warren, C; Winchester, B

    1992-01-01

    The digestion of radiolabelled natural oligosaccharide substrates by human liver neutral alpha-mannosidase has been studied by h.p.l.c. and h.p.t.l.c. The high-mannose oligosaccharides Man9GlcNAc and Man8GlcNAc are hydrolysed by the enzyme by two distinct non-random routes to a common product of composition Man6GlcNAc, which is then slowly converted into a unique Man5GlcNAc oligosaccharide, Man alpha(1----2)Man alpha(1----2)Man alpha(1----3)[Man alpha (1----6)] Man beta(1----4)GlcNAc. These pathways are different from the processing and lysosomal catabolic pathways for these structures. In particular, the alpha(1----2)-linked mannose residues attached to the core alpha(1----3)-linked mannose residue are resistant to hydrolysis. The key processing intermediate, Man alpha(1----3)[Man alpha(1----6)]Man alpha(1----6)[Man alpha(1----3)] Man beta(1----4)GlcNAc, is not produced in the digestion of high-mannose glycans by the neutral alpha-mannosidase, but it is hydrolysed by the enzyme by a non-random route to Man beta(1----4)GlcNAc via the core structure Man alpha(1----3)[Man alpha(1----6)]Man beta(1----4)GlcNAc. In contrast with its ready hydrolysis by lysosomal alpha-mannosidase, the core alpha(1----3)-mannosidic linkage is quite resistant to hydrolysis by neutral alpha-mannosidase. The precise specificity of neutral alpha-mannosidase towards high-mannose oligosaccharides suggests that it has a role in the modification of such structures in the cytosol. Images Fig. 4. PMID:1520283

  19. Human Monoclonal Antibodies Broadly Neutralizing against Influenza B Virus

    PubMed Central

    Yasugi, Mayo; Kubota-Koketsu, Ritsuko; Yamashita, Akifumi; Kawashita, Norihito; Du, Anariwa; Sasaki, Tadahiro; Nishimura, Mitsuhiro; Misaki, Ryo; Kuhara, Motoki; Boonsathorn, Naphatsawan; Fujiyama, Kazuhito; Okuno, Yoshinobu; Nakaya, Takaaki; Ikuta, Kazuyoshi

    2013-01-01

    Influenza virus has the ability to evade host immune surveillance through rapid viral genetic drift and reassortment; therefore, it remains a continuous public health threat. The development of vaccines producing broadly reactive antibodies, as well as therapeutic strategies using human neutralizing monoclonal antibodies (HuMAbs) with global reactivity, has been gathering great interest recently. Here, three hybridoma clones producing HuMAbs against influenza B virus, designated 5A7, 3A2 and 10C4, were prepared using peripheral lymphocytes from vaccinated volunteers, and were investigated for broad cross-reactive neutralizing activity. Of these HuMAbs, 3A2 and 10C4, which recognize the readily mutable 190-helix region near the receptor binding site in the hemagglutinin (HA) protein, react only with the Yamagata lineage of influenza B virus. By contrast, HuMAb 5A7 broadly neutralizes influenza B strains that were isolated from 1985 to 2006, belonging to both Yamagata and Victoria lineages. Epitope mapping revealed that 5A7 recognizes 316G, 318C and 321W near the C terminal of HA1, a highly conserved region in influenza B virus. Indeed, no mutations in the amino acid residues of the epitope region were induced, even after the virus was passaged ten times in the presence of HuMAb 5A7. Moreover, 5A7 showed significant therapeutic efficacy in mice, even when it was administered 72 hours post-infection. These results indicate that 5A7 is a promising candidate for developing therapeutics, and provide insight for the development of a universal vaccine against influenza B virus. PMID:23408886

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

    PubMed Central

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

    2016-01-01

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

  1. Human Neutral Genetic Variation and Forensic STR Data

    PubMed Central

    Silva, Nuno M.; Pereira, Luísa; Poloni, Estella S.; Currat, Mathias

    2012-01-01

    The forensic genetics field is generating extensive population data on polymorphism of short tandem repeats (STR) markers in globally distributed samples. In this study we explored and quantified the informative power of these datasets to address issues related to human evolution and diversity, by using two online resources: an allele frequency dataset representing 141 populations summing up to almost 26 thousand individuals; a genotype dataset consisting of 42 populations and more than 11 thousand individuals. We show that the genetic relationships between populations based on forensic STRs are best explained by geography, as observed when analysing other worldwide datasets generated specifically to study human diversity. However, the global level of genetic differentiation between populations (as measured by a fixation index) is about half the value estimated with those other datasets, which contain a much higher number of markers but much less individuals. We suggest that the main factor explaining this difference is an ascertainment bias in forensics data resulting from the choice of markers for individual identification. We show that this choice results in average low variance of heterozygosity across world regions, and hence in low differentiation among populations. Thus, the forensic genetic markers currently produced for the purpose of individual assignment and identification allow the detection of the patterns of neutral genetic structure that characterize the human population but they do underestimate the levels of this genetic structure compared to the datasets of STRs (or other kinds of markers) generated specifically to study the diversity of human populations. PMID:23185401

  2. Human leucocyte neutral proteases, with special reference to collagen metabolism.

    PubMed

    Kobayashi, S; Nagai, Y

    1978-09-01

    Three different types of neutral proteases related to collagen metabolism have been found in the granule fraction of human leucocytes from normal adults, using collagen, gelatin, and synthetic peptides as substrates. These are collagenase, an enzyme showing a potent hydrolytic activity against gelatin but little against native collagen, and one splitting the cross-links region of collagen. Their molecular weights were estimated to be about 75,000 150,000, and 25,000, respectively, by gel chromatography. The former two enzymes were inhibited by a alpha2-macroglobulin and ethylenediaminetetraacetate, but not by alpha1-proteinase inhibitor (alpha1-antitrypsin) or phenylmethylsulfonylfluoride, while the latter enzyme, associated in behavior with an enzyme hydrolyzing succinyl-(l-alanyl)3-p-nitroanilide, was inhibited by alpha1-proteinase inhibitor, alpha2-macroglobulin, and phenylmethylsulfonylfluoride, but not by ethylenediaminetetraacetate. A possible cooperative function of these enzymes in collagen catabolism is discussed.

  3. The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator in plant cells.

    PubMed

    Chen, Li-Yu; Shi, Dong-Qiao; Zhang, Wen-Juan; Tang, Zuo-Shun; Liu, Jie; Yang, Wei-Cai

    2015-01-01

    Turgor pressure plays pivotal roles in the growth and movement of walled cells that make up plants and fungi. However, the molecular mechanisms regulating turgor pressure and the coordination between turgor pressure and cell wall remodelling for cell growth remain poorly understood. Here, we report the characterization of Arabidopsis TurgOr regulation Defect 1 (TOD1), which is preferentially expressed in pollen tubes and silique guard cells. We demonstrate that TOD1 is a Golgi-localized alkaline ceramidase. tod1 mutant pollen tubes have higher turgor than wild type and show growth retardation both in pistils and in agarose medium. In addition, tod1 guard cells are insensitive to abscisic acid (ABA)-induced stomatal closure, whereas sphingosine-1-phosphate, a putative downstream component of ABA signalling and product of alkaline ceramidases, promotes closure in both wild type and tod1. Our data suggest that TOD1 acts in turgor pressure regulation in both guard cells and pollen tubes.

  4. Deficiency of the alkaline ceramidase ACER3 manifests in early childhood by progressive leukodystrophy

    PubMed Central

    Edvardson, Simon; Yi, Jae Kyo; Jalas, Chaim; Xu, Ruijuan; Webb, Bryn D; Snider, Justin; Fedick, Anastasia; Kleinman, Elisheva; Treff, Nathan R; Mao, Cungui; Elpeleg, Orly

    2016-01-01

    Background/aims Leukodystrophies due to abnormal production of myelin cause extensive morbidity in early life; their genetic background is still largely unknown. We aimed at reaching a molecular diagnosis in Ashkenazi-Jewish patients who suffered from developmental regression at 6–13 months, leukodystrophy and peripheral neuropathy. Methods Exome analysis, determination of alkaline ceramidase activity catalysing the conversion of C18:1-ceramide to sphingosine and D-ribo-C12-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD)-phytoceramide to NBD-C12-fatty acid using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and thin layer chromatography, respectively, and sphingolipid analysis in patients’ blood by LC-MS/MS. Results The patients were homozygous for p.E33G in the ACER3, which encodes a C18:1-alkaline ceramidase and C20:1-alkaline ceramidase. The mutation abolished ACER3 catalytic activity in the patients’ cells and failed to restore alkaline ceramidase activity in yeast mutant strain. The levels of ACER3 substrates, C18:1-ceramides and dihydroceramides and C20:1-ceramides and dihydroceramides and other long-chain ceramides and dihydroceramides were markedly increased in the patients’ plasma, along with that of complex sphingolipids, including monohexosylceramides and lactosylceramides. Conclusions Homozygosity for the p.E33G mutation in the ACER3 gene results in inactivation of ACER3, leading to the accumulation of various sphingolipids in blood and probably in brain, likely accounting for this new form of childhood leukodystrophy. PMID:26792856

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

    PubMed

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

    2016-06-01

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

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

    PubMed

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

    2015-10-01

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

  7. 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

  8. A monoclonal antibody to human immunodeficiency virus type 1 which mediates cellular cytotoxicity and neutralization.

    PubMed Central

    Broliden, P A; Ljunggren, K; Hinkula, J; Norrby, E; Akerblom, L; Wahren, B

    1990-01-01

    Monoclonal antibodies (MAbs) were raised against human immunodeficiency virus type 1 gp120. One MAb, P4/D10, was found to mediate highly efficient antibody-dependent cellular cytotoxicity and virus neutralization. The reactivity was located to a major neutralizing region (amino acids 304 to 323) on gp120. Five other MAbs with a similar epitopic reactivity did not show any antibody-dependent cellulan cytotoxicity activity but had a virus-neutralizing capacity. PMID:2296090

  9. Neutralization of tetanus toxin by human and rabbit immunoglobulin classes and subunits.

    PubMed Central

    Ourth, D D; MacDonald, A B

    1977-01-01

    This investigation found that the human antibody class of importance in neutralizing tetanus toxin in mice was IgG, and that toxin neutralization was retained by the F(ab')2 and Fab' subunits of the human IgG class. Although human IgM and IgA classes appeared to neutralize tetanus toxin at very low levels, evidence was obtained that this neutralization was probably due to IgG contamination. Human Fabmu isolated from the IgM class did not neutralize tetanus toxin. Human antibodies of the IgG, IgM and IgA classes reacted with tetanus toxoid in the indirect haemagglutination (HA) test with IgG giving the highest HA titre. Rabbit antibodies of the IgG class also neutralized tetanus toxin, with neutralization being retained by the F(ab')2 and Fab' subunits of the rabbit IgG class. Absorption of several rabbit antisera to tetanus toxoid with goat-antirabbit Fc which is specific for absorption of IgG from antiserum, rendered them incapable of neutralizing tetanus toxin. Images Figure 1 PMID:590997

  10. Lack of MERS Coronavirus Neutralizing Antibodies in Humans, Eastern Province, Saudi Arabia

    PubMed Central

    Gierer, Stefanie; Hofmann-Winkler, Heike; Albuali, Waleed H.; Bertram, Stephanie; Al-Rubaish, Abdullah M.; Yousef, Abdullah A.; Al-Nafaie, Awatif N.; Al-Ali, Amein K.; Obeid, Obeid E.; Alkharsah, Khaled R.

    2013-01-01

    We used a lentiviral vector bearing the viral spike protein to detect neutralizing antibodies against Middle East respiratory syndrome coronavirus (MERS-CoV) in persons from the Eastern Province of Saudi Arabia. None of the 268 samples tested displayed neutralizing activity, which suggests that MERS-CoV infections in humans are infrequent in this province. PMID:24274664

  11. 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

  12. Lysosomal glycosphingolipid catabolism by acid ceramidase: formation of glycosphingoid bases during deficiency of glycosidases.

    PubMed

    Ferraz, Maria J; Marques, André R A; Appelman, Monique D; Verhoek, Marri; Strijland, Anneke; Mirzaian, Mina; Scheij, Saskia; Ouairy, Cécile M; Lahav, Daniel; Wisse, Patrick; Overkleeft, Herman S; Boot, Rolf G; Aerts, Johannes M

    2016-03-01

    Glycosphingoid bases are elevated in inherited lysosomal storage disorders with deficient activity of glycosphingolipid catabolizing glycosidases. We investigated the molecular basis of the formation of glucosylsphingosine and globotriaosylsphingosine during deficiency of glucocerebrosidase (Gaucher disease) and α-galactosidase A (Fabry disease). Independent genetic and pharmacological evidence is presented pointing to an active role of acid ceramidase in both processes through deacylation of lysosomal glycosphingolipids. The potential pathophysiological relevance of elevated glycosphingoid bases generated through this alternative metabolism in patients suffering from lysosomal glycosidase defects is discussed.

  13. Lysosomal glycosphingolipid catabolism by acid ceramidase: formation of glycosphingoid bases during deficiency of glycosidases.

    PubMed

    Ferraz, Maria J; Marques, André R A; Appelman, Monique D; Verhoek, Marri; Strijland, Anneke; Mirzaian, Mina; Scheij, Saskia; Ouairy, Cécile M; Lahav, Daniel; Wisse, Patrick; Overkleeft, Herman S; Boot, Rolf G; Aerts, Johannes M

    2016-03-01

    Glycosphingoid bases are elevated in inherited lysosomal storage disorders with deficient activity of glycosphingolipid catabolizing glycosidases. We investigated the molecular basis of the formation of glucosylsphingosine and globotriaosylsphingosine during deficiency of glucocerebrosidase (Gaucher disease) and α-galactosidase A (Fabry disease). Independent genetic and pharmacological evidence is presented pointing to an active role of acid ceramidase in both processes through deacylation of lysosomal glycosphingolipids. The potential pathophysiological relevance of elevated glycosphingoid bases generated through this alternative metabolism in patients suffering from lysosomal glycosidase defects is discussed. PMID:26898341

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

    PubMed

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

    2016-07-01

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

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

    PubMed

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

    2016-07-01

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

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

    PubMed Central

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

    2014-01-01

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

  17. Effective ex vivo neutralization of human immunodeficiency virus type 1 in plasma by recombinant immunoglobulin molecules.

    PubMed Central

    Gauduin, M C; Allaway, G P; Maddon, P J; Barbas, C F; Burton, D R; Koup, R A

    1996-01-01

    We tested the ability of human monoclonal antibodies (immunoglobulin G1b12 [IgG1b12] and 19b) and CD4-based molecules (CD4-IgG2 and soluble CD4 [sCD4]) to neutralize human immunodeficiency virus type 1 directly from the plasma of seropositive donors in an ex vivo neutralization assay. IgG1b12 and CD4-IgG2, at concentrations from 1 to 25 micrograms/ml, were found to be effective at reducing the HIV-1 titer in most plasma samples. When viruses recovered from plasma samples were expanded to produce virus stocks, no correlation between the neutralization sensitivities to IgG1b12 and CD4-IgG2 of the in vitro passaged stocks and those of the ex vivo neutralizations performed directly on the plasma was observed. These differences could be due to changes in neutralization sensitivity that occur after one passage of the virus in vitro, or they could be related to the presence of complement or antibodies in the plasma. Furthermore, differences in expression of adhesion molecules on plasma-derived and phytohemagglutinin-activated peripheral blood mononuclear cell-derived viruses could be involved. These studies suggest that IgG1b12 and CD4-IgG2 have broad and potent neutralizing activity in both in vitro and ex vivo neutralization assays and should be considered for use as potential immunoprophylactic or therapeutic agents. PMID:8642690

  18. Plaque assay of neonatal calf diarrhea virus and the neutralizing antibody in human sera.

    PubMed

    Matsuno, S; Inouye, S; Kono, R

    1977-01-01

    Neonatal calf diarrhea virus (a bovine rotavirus) formed distinct plaques in monolayers of MA-104 cells, an established macacus rhesus monkey kidney cell line, when diethylaminoethyl dextran and trypsin were included in the overlay medium. By using this plaque assay method, titration of neutralizing antibody to neonatal calf diarrhea virus was made feasible. It was demonstrated that some human sera contained neutralizing antibody to this agent.

  19. 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.

  20. Benzoxazolone Carboxamides: Potent and Systemically Active Inhibitors of Intracellular Acid Ceramidase**

    PubMed Central

    Pizzirani, Daniela; Bach, Anders; Realini, Natalia; Armirotti, Andrea; Mengatto, Luisa; Bauer, Inga; Girotto, Stefania; Pagliuca, Chiara; De Vivo, Marco; Summa, Maria; Ribeiro, Alison; Piomelli, Daniele

    2015-01-01

    The ceramides are a family of bioactive lipid-derived messengers involved in the control of cellular senescence, inflammation, and apoptosis. Ceramide hydrolysis by acid ceramidase (AC) stops the biological activity of these substances and influences survival and function of normal and neoplastic cells. Because of its central role in the ceramide metabolism, AC may offer a novel molecular target in disorders with dysfunctional ceramide-mediated signaling. Here, a class of benzoxazolone carboxamides is identified as the first potent and systemically active inhibitors of AC. Prototype members of this class inhibit AC with low nanomolar potency by covalent binding to the catalytic cysteine. Their metabolic stability and high in vivo efficacy suggest that these compounds may be used as probes to investigate the roles of ceramide in health and disease, and that this scaffold may represent a promising starting point for the development of novel therapeutic agents. PMID:25395373

  1. Serrumab: a novel human single chain-fragment antibody with multiple scorpion toxin-neutralizing capacities.

    PubMed

    Pucca, Manuela Berto; Cerni, Felipe Augusto; Peigneur, Steve; Arantes, Eliane Candiani; Tytgat, Jan; Barbosa, José Elpidio

    2014-01-01

    In Brazil, scorpion envenomation is an important public health problem. The yellow scorpion, Tityus serrulatus (Ts), is considered the most dangerous species in the country, being responsible for the most severe clinical cases of envenomation. Currently, the administration of serum produced in horses is recognized and used as a treatment for accidents with scorpions. However, horse herds' maintenance is costly and the antibodies are heterologous, which can cause anaphylaxis and Serum Sickness. In the present work, a human monoclonal fragment antibody, Serrumab, has been analysed. Toxin neutralizing effects of Serrumab were evaluated using a two-electrode voltage-clamp technique. The results show that Serrumab presented a high neutralizing effect against Ts β-toxins (Ts1, 43.2% and Ts2, 68.8%) and none or low neutralizing effect against α-toxins (Ts3, 0% and Ts5, 10%). Additional experiments demonstrated that Serrumab was also able to neutralize the action of toxins from other scorpion genus (Css II, 45.96% and Lqh III, 100%/β- and α-toxins, respectively). This work indicated that Serrumab is able to neutralize many toxins in Ts venom, and could being considered as a neutralizing antibody for formulating a human anti-scorpion serum in Brazil. Additionally, this work demonstrated that Serrumab could neutralize different toxins from distinct scorpion genus. All these results reinforce the idea that Serrumab is a scFv antibody with multiple neutralizing capacities and a promising candidate for inclusion in scorpion anti-venoms against different genera. PMID:24001307

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

    PubMed Central

    Tan, Yunrui; Ng, Qingyong; Jia, Qiang

    2015-01-01

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

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

    PubMed

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

    2000-08-01

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

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

    PubMed

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

    2000-08-01

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

  5. RELEASE OF CARTILAGE MUCOPOLYSACCHARIDE-DEGRADING NEUTRAL PROTEASE FROM HUMAN LEUKOCYTES

    PubMed Central

    Oronsky, A.; Ignarro, L.; Perper, R.

    1973-01-01

    The granule fraction of human leukocytes contains neutral protease capable of degrading the noncollagenous protein mucopolysaccharide matrix of cartilage at neutral pH in physiological salt solution. Cartilage degradation was monitored by quantitating the release of 35S from labeled rabbit ear cartilage. Degradation of cartilage matrix occurs when intact viable human leukocytes are incubated with cartilage opsonized with aggregated human gamma globulin (AHGG). During a similar 4 h incubation period cells did not degrade uncoated cartilage or cartilage coated with nonaggregated gamma globulin. Cells remain viable during the enzyme release process as evidenced by the absence of a cytoplasmic enzyme marker (lactic dehydrogenase) in the supernatant and dye exclusion studies. The release of 35S from labeled cartilage by human leukocytes in the presence of cartilage coated with AHGG (nonphagocytic enzyme release) was compared with the cartilage degrading activity of the supernatant from the same number of cells preincubated with a suspension of AHGG (phagocytic enzyme release). Nonphagocytic enzyme release by 5 x 106 cells provoked two to four times more 35S and β-glucuronidase (β-G) release from cartilage than phagocytic enzyme release conditions. β-glucuronidase was used as an indicator of the release of lysosomal granule enzymes. By the use of selected pharmacological agents it was possible to dissociate the enzyme release process from intrinsic enzyme (neutral protease) activity. Neutral protease and β-G release by human cells in the presence of AHGG-coated cartilage was inhibited by 10–5M colchicine, whereas the protease activity, but not the release process, was inhibited by 10–6M gold thiomalate and 10% human serum. It is suggested that the release of a cartilage degrading neutral protease by viable human cells when exposed to AHGG might be a relevant model for the study of cartilage destruction as it occurs in rheumatoid arthritis. PMID:4124211

  6. 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.

  7. Human IgG Subclasses against Enterovirus Type 71: Neutralization versus Antibody Dependent Enhancement of Infection

    PubMed Central

    Han, Jian-Feng; Wang, Guang-Chuan; Zhao, Hui; Li, Xiao-Feng; Deng, Yong-Qiang; Zhu, Shun-Ya; Wang, Xiao-Yu; Lin, Fang; Zhang, Fu-Jun; Chen, Wei; Qin, E-De; Qin, Cheng-Feng

    2013-01-01

    The emerging human enterovirus 71 (EV71) represents a growing threat to public health, and no vaccine or specific antiviral is currently available. Human intravenous immunoglobulin (IVIG) is clinical used in treating severe EV71 infections. However, the discovery of antibody dependent enhancement (ADE) of EV71 infection illustrates the complex roles of antibody in controlling EV71 infection. In this study, to identify the distinct role of each IgG subclass on neutralization and enhancement of EV71 infection, different lots of pharmaceutical IVIG preparations manufactured from Chinese donors were used for IgG subclass fractionation by pH gradient elution with the protein A-conjugated affinity column. The neutralization and ADE capacities on EV71 infection of each purified IgG subclass were then assayed, respectively. The neutralizing activity of human IVIG is mainly mediated by IgG1 subclass and to less extent by IgG2 subclass. Interestingly, IgG3 fraction did not have neutralizing activity but enhanced EV71 infection in vitro. These results revealed the different roles of human IgG subclasses on EV71 infection, which is of critical importance for the rational design of immunotherapy and vaccines against severe EV71 diseases. PMID:23700449

  8. Human monoclonal antibodies against West Nile virus induced by natural infection neutralize at a postattachment step.

    PubMed

    Vogt, Matthew R; Moesker, Bastiaan; Goudsmit, Jaap; Jongeneelen, Mandy; Austin, S Kyle; Oliphant, Theodore; Nelson, Steevenson; Pierson, Theodore C; Wilschut, Jan; Throsby, Mark; Diamond, Michael S

    2009-07-01

    West Nile virus (WNV) is a neurotropic flavivirus that is now a primary cause of epidemic encephalitis in North America. Studies of mice have demonstrated that the humoral immune response against WNV limits primary infection and protects against a secondary challenge. The most-potent neutralizing mouse monoclonal antibodies (MAbs) recognize an epitope on the lateral ridge of domain III (DIII-lr) of the envelope (E) protein. However, studies with serum from human patients show that antibodies against the DIII-lr epitope comprise, at best, a minor component of the human anti-WNV antibody response. Herein, we characterize in detail two WNV-specific human MAbs, CR4348 and CR4354, that were isolated from B-cell populations of convalescent patients. These MAbs strongly neutralize WNV infection of cultured cells, protect mice against lethal infection in vivo, and yet poorly recognize recombinant forms of the E protein. Instead, CR4348 and CR4354 bind determinants on intact WNV virions and subviral particles in a pH-sensitive manner, and neutralization is altered by mutations at the dimer interface in domain II and the hinge between domains I and II, respectively. CR4348 and CR4354 human MAbs neutralize infection at a postattachment step in the viral life cycle, likely by inhibiting acid-induced fusion within the endosome.

  9. Plaque-reduction assays for human and simian immunodeficiency virus neutralization.

    PubMed

    Nordqvist, Anna; Fenyö, Eva Maria

    2005-01-01

    Research on HIV vaccines, as well as studies on HIV pathogenesis in human and SIV in the macaque model, require the availability of simple and standardized assays for quantification of neutralizing antibodies to primary virus isolates. We have recently developed and standardized assays using human cell lines engineered to express CD4 and co-receptors for HIV and SIV entry. One cell line originated from a glioma (U87) and the other from an osteosarcoma (HOS). Both cell lines and their derivatives form monolayer cultures, a prerequisite for counting plaques. HIV-infected U87.CD4-CCR5 or -CXCR4 cells form syncytia, that is, plaques that can be stained with hematoxylin and enumerated by light microscopy. In addition to CD4 and co-receptors (most often used CCR5 and CXCR6 by SIV), GHOST(3) cells have been engineered to express the green fluorescent protein following virus infection. Infected cells show green fluorescence and can be enumerated by fluorescence microscopy. Neutralization is determined by the ability of a serum to reduce the number of plaque-forming units (PFU) relative to controls exposed to medium or negative serum. Both assays are run in microtiter format and neutralization is evaluated after 3 d. Intra-assay variation has been used for estimation of the cutoff for neutralization. Testing 15 serum-virus combinations in the U87.CD4 assay and four serum-virus combinations in the GHOST(3) assay revealed that standard deviation of differences ranged from 9.1% to 9.9% in the two assays. This allowed the use of a cutoff >3 SD; that is, 30% neutralization. Virus titration experiments showed that neutralization results were dependent on virus dose and therefore the neutralization assays should be performed with a virus dose of 10-100 PFU/well. The assays have high specificity and reproducibility, and are simple and sensitive high-throughput assays.

  10. Identification of a Neutralizing Epitope within Antigenic Domain 5 of Glycoprotein B of Human Cytomegalovirus

    PubMed Central

    Wiegers, Anna-Katharina; Sticht, Heinrich; Winkler, Thomas H.; Britt, William J.

    2014-01-01

    ABSTRACT Human cytomegalovirus (HCMV) is an important, ubiquitous pathogen that causes severe clinical disease in immunocompromised individuals, such as organ transplant recipients and infants infected in utero. The envelope glycoprotein B (gB) of HCMV is a major antigen for the induction of virus-neutralizing antibodies. We have begun to define target structures within gB that are recognized by virus-neutralizing antibodies. Antigenic domain 5 (AD-5) of gB has been identified as an important target for neutralizing antibodies in studies using human monoclonal antibodies (MAbs). Anti-AD-5 MAbs share a target site on gB, despite originating from different, healthy, HCMV-infected donors. Mutational analysis of AD-5 identified tyrosine 280 in combination with other surface-exposed residues (the YNND epitope) as critical for antibody binding. The YNND epitope is strictly conserved among different HCMV strains. Recombinant viruses carrying YNND mutations in AD-5 were resistant to virus-neutralizing MAbs. Competition enzyme-linked immunosorbent assays (ELISAs) with human HCMV-convalescent-phase sera from unselected donors confirmed the conserved antibody response for the YNND epitope in HCMV-infected individuals and, because a significant fraction of the gB AD-5 response was directed against the YNND epitope, further argued that this epitope is a major target of anti-AD-5 antibody responses. In addition, affinity-purified polyclonal anti-AD-5 antibodies prepared from individual sera showed reactivity to AD-5 and neutralization activity toward gB mutant viruses that were similar to those of AD-5-specific MAbs. Taken together, our data indicate that the YNND epitope represents an important target for anti-gB antibody responses as well as for anti-AD-5 virus-neutralizing antibodies. IMPORTANCE HCMV is a major global health concern, and a vaccine to prevent HCMV disease is a widely recognized medical need. Glycoprotein B of HCMV is an important target for neutralizing

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

    PubMed

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

    2016-01-01

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

  12. 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

  13. Oxidative Stress Kills Human Primary Oligodendrocytes Via Neutral Sphingomyelinase: Implications for Multiple Sclerosis

    PubMed Central

    Jana, Arundhati

    2007-01-01

    Multiple sclerosis (MS) is the most common human demyelinating disease of the central nervous system where oxidative stress has been proposed to play an important role in oligodendroglial death. However, molecular mechanisms that couple oxidative stress to the loss of oligodendrocytes are poorly understood. This study underlines the importance of neutral sphingomyelinase–ceramide pathway in mediating oxidative stress-induced apoptosis and cell death of human primary oligodendrocytes. Various oxidative stress-inducing agents, such as, superoxide radical produced by hypoxanthine and xanthine oxidase, hydrogen peroxide, aminotriazole capable of inhibiting catalase and increasing intracellular level of H2O2, or reduced glutathione-depleting diamide induced the activation of neutral sphingomyelinase and the production of ceramide. It is interesting to note that antisense knockdown of neutral but not acidic sphingomyelinase ablated oxidative stress-induced apoptosis and cell death in human primary oligodendrocytes. This study identifies neutral but not acidic sphingomyelinase as a target for possible therapeutic intervention in MS. PMID:18040843

  14. 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

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

    PubMed

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

    2016-01-01

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

  16. 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.

  17. Human cytomegalovirus neutralizing antibody-resistant phenotype is associated with reduced expression of glycoprotein H.

    PubMed Central

    Li, L; Coelingh, K L; Britt, W J

    1995-01-01

    We have characterized a neutralizing antibody-resistant mutant human cytomegalovirus (HCMV) obtained from a patient treated with a human monoclonal antiglycoprotein H (gH; unique long region 75) antibody. This virus exhibited resistance to several different neutralizing anti-gH murine monoclonal antibodies (MAbs), as well as to a polyvalent anti-gH serum. The resistant phenotype was unstable and could be maintained only by passage of plaque-purified virus under neutralizing MAb selection. In the absence of a MAb, the resistant phenotype reverted to a neutralizing antibody-sensitive phenotype within one passage. The predicted amino acid sequences of gH from the MAb-resistant and -susceptible parent viruses were identical. Biochemical analysis of the MAb-resistant and -susceptible parent viruses revealed a marked decrease of gH expression in the envelope of the MAb-resistant virus. Furthermore, propagation of the virus in various MAb concentrations resulted in the production of extracellular virions with various levels of resistance to the neutralizing activity of the MAb. These results suggest a mechanism for the generation of neutralizing antibody-resistant viruses which could evade host-derived antiviral antibody responses. In addition, our findings indicate that the stoichiometry of gH in the envelope of infectious HCMV virions is not rigidly fixed and therefore offer a simple explanation for production of phenotypic variants of HCMV through an assembly process in which the content of gH in the envelope of progeny virions varies randomly. PMID:7666509

  18. 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

  19. Generation of potent neutralizing human monoclonal antibodies against cytomegalovirus infection from immune B cells

    PubMed Central

    Funaro, Ada; Gribaudo, Giorgio; Luganini, Anna; Ortolan, Erika; Lo Buono, Nicola; Vicenzi, Elisa; Cassetta, Luca; Landolfo, Santo; Buick, Richard; Falciola, Luca; Murphy, Marianne; Garotta, Gianni; Malavasi, Fabio

    2008-01-01

    Background Human monoclonal antibodies (mAbs) generated as a result of the immune response are likely to be the most effective therapeutic antibodies, particularly in the case of infectious diseases against which the immune response is protective. Human cytomegalovirus (HCMV) is an ubiquitous opportunistic virus that is the most serious pathogenic agent in transplant patients. The available therapeutic armamentarium (e.g. HCMV hyperimmune globulins or antivirals) is associated with severe side effects and the emergence of drug-resistant strains; therefore, neutralizing human mAb may be a decisive alternative in the prevention of primary and re-activated HCMV infections in these patients. Results The purpose of this study was to generate neutralizing mAb against HCMV from the immunological repertoire of immune donors. To this aim, we designed an efficient technology relying on two discrete and sequential steps: first, human B-lymphocytes are stimulated with TLR9-agonists and IL-2; second, after both additives are removed, the cells are infected with EBV. Using this strategy we obtained 29 clones secreting IgG neutralizing the HCMV infectivity; four among these were further characterized. All of the mAbs neutralize the infection in different combinations of HCMV strains and target cells, with a potency ~20 fold higher than that of the HCMV hyperimmune globulins, currently used in transplant recipients. Recombinant human monoclonal IgG1 suitable as a prophylactic or therapeutic tool in clinical applications has been generated. Conclusion The technology described has proven to be more reproducible, efficient and rapid than previously reported techniques, and can be adopted at low overall costs by any cell biology laboratory for the development of fully human mAbs for immunotherapeutic uses. PMID:19014469

  20. 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

  1. Inter- and intraclade neutralization of human immunodeficiency virus type 1: genetic clades do not correspond to neutralization serotypes but partially correspond to gp120 antigenic serotypes.

    PubMed Central

    Moore, J P; Cao, Y; Leu, J; Qin, L; Korber, B; Ho, D D

    1996-01-01

    We have studied genetic variation among clades A through E of human immunodeficiency virus type 1 (HIV-1) at the levels of antibody binding to gp120 molecules and virus neutralization. We are unable to identify neutralization serotypes that correspond to the genetic clades. Instead, we observe that inter- and intraclade neutralization of primary isolates by HIV-1-positive sera is generally weak and sporadic; some sera show a reasonable degree of neutralization breadth and potency whereas others are relatively sensitive to neutralization, but no consistent pattern was found. However, a few sera were able to neutralize across clades with significant potency, an observation which may have implications for the feasibility of a broadly effective HIV-1 vaccine involving humoral immunity. Serological assays measuring anti-gp120 antibody binding also failed to identify serotypes that correspond precisely to the genetic clades, but some indications of clade-specific binding were observed, notably with sera from clades B and E. A representative protein for each clade (A through E) was selected on the basis of its specificity, defined as high seroreactivity with sera from individuals infected with virus of that clade and lower reactivity with sera from individuals infected with viruses from other clades. The seroreactivity patterns against these five proteins could be used to predict the genotype of the infecting virus with moderate success. PMID:8523556

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2015-09-01

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

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

    PubMed

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

    2015-09-01

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

  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. A rapid microneutralization assay for the measurement of neutralizing antibody reactive with human cytomegalovirus.

    PubMed

    Andreoni, M; Faircloth, M; Vugler, L; Britt, W J

    1989-02-01

    We have developed a murine monoclonal antibody reactive with a major immediate early 72,000 dalton protein of human cytomegalovirus and utilized this reagent in a rapid virus titration and microneutralization assay. Because of the early expression of this virus encoded protein, both assays could be accomplished within 16 h following virus inoculation. In addition, both assays resulted in considerable savings of reagents because the assays were carried out in 96-well microtiter plates. These assays should prove useful in the preparation and study of neutralizing antibodies directed against human cytomegalovirus.

  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. Preexisting human antibodies neutralize recently emerged H7N9 influenza strains

    PubMed Central

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

    2015-01-01

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

  11. 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.

  12. Prefusion F-specific antibodies determine the magnitude of RSV neutralizing activity in human sera.

    PubMed

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

    2015-10-14

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-06-23

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

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

    PubMed Central

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

    2015-01-01

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

  16. 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.

  17. 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

  18. 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

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

    PubMed

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

    2016-09-01

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

  20. Novel off-target effect of tamoxifen--inhibition of acid ceramidase activity in cancer cells.

    PubMed

    Morad, Samy A F; Levin, Jonathan C; Tan, Su-Fern; Fox, Todd E; Feith, David J; Cabot, Myles C

    2013-12-01

    Acid ceramidase (AC), EC 3.5.1.23, a lysosomal enzyme, catalyzes the hydrolysis of ceramide to constituent sphingoid base, sphingosine, and fatty acid. Because AC regulates the levels of pro-apoptotic ceramide and mitogenic sphingosine-1-phosphate, it is considered an apt target in cancer therapy. The present study reveals, for the first time, that the prominent antiestrogen, tamoxifen, is a pan-effective AC inhibitor in the low, single digit micromolar range, as demonstrated in a wide spectrum of cancer cell types, prostate, pancreatic, colorectal, and breast. Prostate cancer cells were chosen for the detailed investigations. Treatment of intact PC-3 cells with tamoxifen produced time- and dose-dependent inhibition of AC activity. Tamoxifen did not impact cell viability nor did it inhibit AC activity in cell-free assays. In pursuit of mechanism of action, we demonstrate that tamoxifen induced time-, as early as 5min, and dose-dependent, as low as 5μM, increases in lysosomal membrane permeability (LMP), and time- and dose-dependent downregulation of AC protein expression. Assessing various protease inhibitors revealed that a cathepsin B inhibitor blocked tamoxifen-elicited downregulation of AC protein; however, this action failed to restore AC activity unless assayed in a cell-free system at pH4.5. In addition, pretreatment with tamoxifen inhibited PC-3 cell migration. Toremifene, an antiestrogen structurally similar to tamoxifen, was also a potent inhibitor of AC activity. This study reveals a new, off-target action of tamoxifen that may be of benefit to enhance anticancer therapies that either incorporate ceramide or target ceramide metabolism. PMID:23939396

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

    SciTech Connect

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

    2009-09-11

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

  2. 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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  6. 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

  7. 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.

  8. Evaluation of an improved rapid neutralizing antibody detection test (RAPINA) for qualitative and semiquantitative detection of rabies neutralizing antibody in humans and dogs.

    PubMed

    Nishizono, Akira; Yamada, Kentaro; Khawplod, Pakamatz; Shiota, Seiji; Perera, Devika; Matsumoto, Takashi; Wimalaratne, Omala; Mitui, Marcelo Takahiro; Ahmed, Kamruddin

    2012-06-01

    Using the principle of immunochromatography, we previously developed a method called RAPINA (Rapid Neutralizing Antibody detection test) that can measure the level of rabies virus -neutralizing antibody (VNA) in serum samples [Shiota S, Mannen K, Matsumoto T, Yamada K, Yasui T, Takayama K, et al. Development and evaluation of a rapid neutralizing antibody test for rabies. J Virol Methods 2009;161:58-62]. RAPINA is faster, simpler, and easier to perform compared with a virus-neutralizing test or enzyme-linked immunosorbent assay (ELISA). The improved version of RAPINA has greater positive and negative predictive values corresponding to a VNA level of 0.5 IU/mL, as recommended by the World Health Organization and the World Organization for Animal Health. To verify the efficacy of this improved method, serum samples were collected from humans and dogs before and after immunization against rabies and were tested in Japan, Sri Lanka, and Thailand. The results were compared between RAPINA and the true VNA levels measured by the Rapid Fluorescent Focus Inhibition Test (RFFIT). The improved RAPINA accurately predicted seropositivity for 182 of 183 seropositive human samples as assessed by RFFIT (99.5%) and for 138 of 140 RFFIT-negative human samples (98.6%). In dog serum samples, the positive and negative predictive values were 99.7% (345/355) and 95.6% (174/182), respectively. RAPINA was also used to estimate VNA levels in a semiquantitative manner by using serial dilution of serum samples. Our results show that RAPINA is an easy and rapid method for measuring VNA levels before and after immunization with the rabies vaccine and does not need a high skill level or sophisticated equipment. RAPINA can be used to monitor the success of preexposure prophylaxis in at-risk persons, vaccine coverage, and animal control. It can also be used in laboratories with modest facilities and where a large number of samples are screened.

  9. 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

  10. Heterogeneity of VP4 neutralization epitopes among serotype P1A human rotavirus strains.

    PubMed Central

    Contreras, J F; Menchaca, G E; Padilla-Noriega, L; Tamez, R S; Greenberg, H B; López, S; Arias, C F

    1995-01-01

    We have used serotype-specific VP4 and VP7 neutralizing monoclonal antibodies (Nt-MAbs), as well as subgroup (SG)-specific MAbs, to characterize by enzyme immunoassay rotavirus strains isolated from diarrheic infants in the city of Monterrey, Mexico, from July 1993 to March 1994. Of a total of 465 children studied, 140 were rotavirus positive, including 3 patients infected with non-group A rotaviruses. The SG and VP7 (G) serotype specificities could be determined for 118 (84%) of the 140 rotavirus-positive stool specimens; 4 rotavirus strains were serotype G1 and SGII; 1 strain was serotype G2 and SGI+II; 112 strains were serotype G3 and SGII; 1 strain was serotype G3 and SGI; and none of the strains was serotype G4. Fifty-eight specimens, representing the 13 different group A rotavirus electropherotypes detected, were chosen for VP4 (P) serotyping. Of these, 48 (83%) strains reacted with the P1A serotype-specific Nt-MAb 1A10. None of the strains reacted with the serotype P2-specific Nt-MAbs tested. Not all viruses that reacted with Nt-MAb 1A10 were recognized by Nt-MAbs 2A3 and 2G1, which also recognize P1A strains, indicating heterogeneity of neutralization epitopes among serotype P1A human rotaviruses. This heterogeneity could be relevant for the specificity of the VP4-mediated neutralizing antibody immune response and indicates the need for antigenic characterization, in addition to genomic typing, of the VP4 proteins of circulating human rotavirus field strains. PMID:7583936

  11. Human monoclonal antibody that recognizes the V3 region of human immunodeficiency virus gp120 and neutralizes the human T-lymphotropic virus type IIIMN strain.

    PubMed Central

    Scott, C F; Silver, S; Profy, A T; Putney, S D; Langlois, A; Weinhold, K; Robinson, J E

    1990-01-01

    We describe a human IgG1 monoclonal antibody (N701.9b) derived by Epstein-Barr virus transformation of B cells from a human immunodeficiency virus-seropositive asymptomatic donor. This antibody was shown to recognize the principal neutralizing domain contained within the V3 region of gp120 of the MN strain of human immunodeficiency virus and MN-like strains, as determined by binding to the PB-1 fragment of MN gp120 and to synthetic peptides corresponding to the V3 region of MN and related virus strains. The epitope identified by monoclonal antibody N701.9b was mapped to a segment of V3 containing at least 7 amino acids (amino acids 316-322), which is located in the "tip" and "right" side of the V3 loop of the MN strain. Furthermore, this antibody manifested potent type-specific fusion-inhibitory activity against the MN strain but not against the IIIB or RF virus strains. This antibody also neutralized four virus isolates that had MN-like V3 region sequences and failed to neutralize three other strains containing unrelated V3 region sequences. Our findings confirm that the V3 region stimulates type-specific neutralizing antibody during natural human immunodeficiency virus infection in humans. The potential clinical use of this antibody is discussed. PMID:1700435

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

    SciTech Connect

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

    2008-10-17

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

  13. Neutralizing monoclonal antibodies against human immunodeficiency virus type 2 gp120.

    PubMed

    Matsushita, S; Matsumi, S; Yoshimura, K; Morikita, T; Murakami, T; Takatsuki, K

    1995-06-01

    Monoclonal antibodies (MAbs) were obtained by immunizing mice with synthetic peptides corresponding to the third variable (V3) or the third conserved (C3) domain of the external envelope protein (gp120) of human immunodeficiency virus type 2 (HIV-2ROD). One MAb, designated B2C, which was raised against V3 peptide NKI26, bound to the surface of HIV-2-infected cells but not to their uninfected counterparts. B2C was capable of neutralizing cell-free and cell-associated virus infection in an isolate-specific fashion. The antibody-binding epitope was mapped to a 6-amino-acid peptide in the V3 variable domain which had the core sequence His-Tyr-Gln. Two MAbs, 2H1B and 2F19C, which were raised against the C3 peptide TND27 reacted with gp120 of HIV-2ROD in a Western immunoblot assay. The C3 epitopes recognized by these two MAbs appeared inaccessible because of their poor reactivity in a surface immunofluorescence assay. Although partial inhibition of syncytium formation was observed in the presence of the anti-C3 MAbs, their neutralizing activity appeared weak. Finally, the effects of these MAbs against CD4-gp120 binding were assessed. Partial inhibition of CD4-gp120 binding was observed in the presence of high concentrations of B2C. On the other hand, no inhibition of CD4-gp120 binding was observed in the presence of anti-C3 MAbs. Since complete neutralization could be achieved at a concentration corresponding to that of partial binding inhibition by B2C, some different mechanisms may be involved in the B2C-mediated neutralization. These results, taken together, indicated that analogous to the function of the V3 region of HIV-1, the V3 region of HIV-2ROD contained at least a type-specific fusion-inhibiting neutralizing epitope. In this respect, the V3 sequence of HIV-2 may be a useful target in an animal model for HIV vaccine development.

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

    PubMed

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

    2015-06-12

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

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

    PubMed Central

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

    2004-01-01

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

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

    PubMed

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

    2015-01-01

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

  17. 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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2013-09-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2009-10-01

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

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

    PubMed

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

    2016-01-28

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

  3. A highly potent human antibody neutralizes dengue virus serotype 3 by binding across three surface proteins.

    PubMed

    Fibriansah, Guntur; Tan, Joanne L; Smith, Scott A; de Alwis, Ruklanthi; Ng, Thiam-Seng; Kostyuchenko, Victor A; Jadi, Ramesh S; Kukkaro, Petra; de Silva, Aravinda M; Crowe, James E; Lok, Shee-Mei

    2015-01-01

    Dengue virus (DENV) infects ~400 million people annually. There is no licensed vaccine or therapeutic drug. Only a small fraction of the total DENV-specific antibodies in a naturally occurring dengue infection consists of highly neutralizing antibodies. Here we show that the DENV-specific human monoclonal antibody 5J7 is exceptionally potent, neutralizing 50% of virus at nanogram-range antibody concentration. The 9 Å resolution cryo-electron microscopy structure of the Fab 5J7-DENV complex shows that a single Fab molecule binds across three envelope proteins and engages three functionally important domains, each from a different envelope protein. These domains are critical for receptor binding and fusion to the endosomal membrane. The ability to bind to multiple domains allows the antibody to fully coat the virus surface with only 60 copies of Fab, that is, half the amount compared with other potent antibodies. Our study reveals a highly efficient and unusual mechanism of molecular recognition by an antibody. PMID:25698059

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-04-01

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

  6. Genome-wide identification of human functional DNA using a neutral indel model.

    PubMed

    Lunter, Gerton; Ponting, Chris P; Hein, Jotun

    2006-01-01

    It has become clear that a large proportion of functional DNA in the human genome does not code for protein. Identification of this non-coding functional sequence using comparative approaches is proving difficult and has previously been thought to require deep sequencing of multiple vertebrates. Here we introduce a new model and comparative method that, instead of nucleotide substitutions, uses the evolutionary imprint of insertions and deletions (indels) to infer the past consequences of selection. The model predicts the distribution of indels under neutrality, and shows an excellent fit to human-mouse ancestral repeat data. Across the genome, many unusually long ungapped regions are detected that are unaccounted for by the neutral model, and which we predict to be highly enriched in functional DNA that has been subject to purifying selection with respect to indels. We use the model to determine the proportion under indel-purifying selection to be between 2.56% and 3.25% of human euchromatin. Since annotated protein-coding genes comprise only 1.2% of euchromatin, these results lend further weight to the proposition that more than half the functional complement of the human genome is non-protein-coding. The method is surprisingly powerful at identifying selected sequence using only two or three mammalian genomes. Applying the method to the human, mouse, and dog genomes, we identify 90 Mb of human sequence under indel-purifying selection, at a predicted 10% false-discovery rate and 75% sensitivity. As expected, most of the identified sequence represents unannotated material, while the recovered proportions of known protein-coding and microRNA genes closely match the predicted sensitivity of the method. The method's high sensitivity to functional sequence such as microRNAs suggest that as yet unannotated microRNA genes are enriched among the sequences identified. Furthermore, its independence of substitutions allowed us to identify sequence that has been subject to

  7. 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

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

    PubMed Central

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

    2013-01-01

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

  9. High-avidity and potently neutralizing cross-reactive human monoclonal antibodies derived from secondary dengue virus infection.

    PubMed

    Tsai, Wen-Yang; Lai, Chih-Yun; Wu, Yi-Chieh; Lin, Hong-En; Edwards, Carolyn; Jumnainsong, Amonrat; Kliks, Srisakul; Halstead, Scott; Mongkolsapaya, Juthathip; Screaton, Gavin R; Wang, Wei-Kung

    2013-12-01

    The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies (Abs) and vaccine development. Previous studies of human dengue-immune sera reported that a significant proportion of anti-E Abs, known as group-reactive (GR) Abs, were cross-reactive to all four DENV serotypes and to one or more other flaviviruses. Based on studies of mouse anti-E monoclonal antibodies (MAbs), GR MAbs were nonneutralizing or weakly neutralizing compared with type-specific MAbs; a GR response was thus not regarded as important for vaccine strategy. We investigated the epitopes, binding avidities, and neutralization potencies of 32 human GR anti-E MAbs. In addition to fusion loop (FL) residues in E protein domain II, human GR MAbs recognized an epitope involving both FL and bc loop residues in domain II. The neutralization potencies and binding avidities of GR MAbs derived from secondary DENV infection were stronger than those derived from primary infection. GR MAbs derived from primary DENV infection primarily blocked attachment, whereas those derived from secondary infection blocked DENV postattachment. Analysis of the repertoire of anti-E MAbs derived from patients with primary DENV infection revealed that the majority were GR, low-avidity, and weakly neutralizing MAbs, whereas those from secondary infection were primarily GR, high-avidity, and potently neutralizing MAbs. Our findings suggest that the weakly neutralizing GR anti-E Abs generated from primary DENV infection become potently neutralizing MAbs against the four serotypes after secondary infection. The observation that the dengue immune status of the host affects the quality of the cross-reactive Abs generated has implications for new strategies for DENV vaccination. PMID:24027331

  10. 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.

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

    PubMed

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

    2016-03-18

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

  12. 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.

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

    PubMed Central

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

    1998-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  16. Establishment of titration system for human herpesvirus 6 and evaluation of neutralizing antibody response to the virus.

    PubMed

    Asada, H; Yalcin, S; Balachandra, K; Higashi, K; Yamanishi, K

    1989-10-01

    The susceptibilities of seven T-cell lines to human herpesvirus 6 (HHV-6) infection were examined. MT-4 cells were the most susceptible of these lines to infection with this virus. Therefore, chemically adhered MT-4 cell monolayers were used for infectious HHV-6 assay by indirect immunofluorescent-antibody (IFA) staining. When cell monolayers were fixed 30 to 45 h postinfection, the foci stained with IFA were easy to count and a linear relationship was observed between the number of foci and the virus concentration. MT-4 cell monolayers were also used for a focus reduction neutralizing-antibody test. In this test, sera from patients in the convalescent stage of exanthem subitum all showed significant neutralizing activity (1:80 to 1:320), whereas sera from patients in the acute stage of disease showed no detectable neutralizing activity. The titers of neutralizing antibody correlated well with the levels of anti-HHV-6 antibodies detected by IFA.

  17. 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

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

    PubMed

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

    2011-08-01

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

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

    PubMed

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

    2016-01-01

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

  20. 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.

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

    PubMed Central

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

    2016-01-01

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

  2. Induced conformational change in human IL‐4 upon binding of a signal‐neutralizing DARPin

    PubMed Central

    Teplyakov, Alexey; Malia, Thomas J.; Keough, Edward; Luo, Jinquan; Sweet, Raymond; Jacobs, Steven A.; Yi, Fang; Hippensteel, Randi; O'Neil, Karyn T.

    2015-01-01

    ABSTRACT The crystal structure of DARPin 44C12V5 that neutralizes IL‐4 signaling has been determined alone and bound to human IL‐4. A significant conformational change occurs in the IL‐4 upon DARPin binding. The DARPin binds to the face of IL‐4 formed by the A and C α‐helices. The structure of the DARPin remains virtually unchanged. The conformational changes in IL‐4 include a reorientation of the C‐helix Trp91 side chain and repositioning of CD‐loop residue Leu96. Both side chains move by >9 Å, becoming buried in the central hydrophobic region of the IL‐4:DARPin interface. This hydrophobic region is surrounded by a ring of hydrophilic interactions comprised of hydrogen bonds and salt bridges and represents a classical “hotspot.” The structures also reveal how the DARPin neutralizes IL‐4 signaling. Comparing the IL‐4:DARPin complex structure with the structures of IL‐4 bound to its receptors (Hage et al., Cell 1999; 97, 271‐281; La Porte et al., Cell 2008, 132, 259‐272), it is found that the DARPin binds to the same IL‐4 face that interacts with the junction of the D1 and D2 domains of the IL‐4Rα receptors. Signaling is blocked since IL‐4 cannot bind to this receptor, which it must do first before initiating a productive receptor complex with either the IL‐13α1 or the γ c receptor. Proteins 2015; 83:1191–1197. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. PMID:25900776

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

    PubMed

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

    2016-04-01

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

  4. 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.

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

    PubMed

    Hosey, Geoff; Melfi, Vicky

    2015-01-01

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

  6. Purified envelope glycoproteins from human immunodeficiency virus type 1 variants induce individual, type-specific neutralizing antibodies.

    PubMed Central

    Nara, P L; Robey, W G; Pyle, S W; Hatch, W C; Dunlop, N M; Bess, J W; Kelliher, J C; Arthur, L O; Fischinger, P J

    1988-01-01

    Repeated immunizations of goats, horses, or chimpanzees with envelope glycoprotein gp120 isolated from human immunodeficiency virus type 1 (HIV-1) resulted in type-specific neutralizing-antibody responses, which began to decay approximately 20 days following the administration of antigen. This was true repeatedly for serum samples from animals hyperimmunized with gp120s from either the HTLV-IIIB (IIIB) or the envelope-divergent HTLV-IIIRF (RF) HIV-1 isolates. Animals previously immunized with the IIIB gp120 were then inoculated with purified RF gp120. The first response in these animals was an anamnestic resurgence of neutralizing antibody to IIIB without detectable neutralizing antibody for RF. However, with later RF gp120 boosts, the IIIB neutralizing-antibody titers fell and an RF type-specific neutralizing-antibody response developed. When assessed with other HIV-1 variants, no group-specific neutralizing antibody was seen in any of the vaccination protocols evaluated. These results will pose real obstacles in the development of an effective vaccine for HIV. PMID:3392769

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

    PubMed Central

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

    1993-01-01

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

  8. Profile and persistence of the virus-specific neutralizing humoral immune response in human survivors of Sudan ebolavirus (Gulu).

    PubMed

    Sobarzo, Ariel; Groseth, Allison; Dolnik, Olga; Becker, Stephan; Lutwama, Julius Julian; Perelman, Eddie; Yavelsky, Victoria; Muhammad, Majidat; Kuehne, Ana I; Marks, Robert S; Dye, John M; Lobel, Leslie

    2013-07-15

    To better understand humoral immunity following ebolavirus infection, a serological study of the humoral immune response against the individual viral proteins of Sudan ebolavirus (Gulu) in human survivors was performed. An enzyme-linked immunosorbent assay specific for full-length recombinant viral proteins NP, VP30, VP40, and GP1-649 (GP lacking the transmembrane domain) of Sudan ebolavirus (Gulu) was used as well as a plaque reduction neutralization test. Serum samples from human survivors, which were collected up to 10 years following recovery, were screened and analyzed. Results demonstrate that samples obtained 10 years following infection contain virus-specific antibodies that can neutralize virus. Neutralization correlates well with immunoreactivity against the viral proteins NP, VP30, and GP1-649. Sera from individuals who died or those with no documented infection but immunoreactive to ebolavirus did not neutralize. This work provides insight into the duration, profile of immunoreactivity, and neutralization capacity of the humoral immune response in ebolavirus survivors. PMID:23585686

  9. Frequency and domain specificity of toxin-neutralizing paratopes in the human antibody response to anthrax vaccine adsorbed.

    PubMed

    Reason, Donald; Liberato, Justine; Sun, Jinying; Keitel, Wendy; Zhou, Jianhui

    2009-05-01

    Protective antigen (PA) is the cell surface recognition unit of the binary anthrax toxin system and the primary immunogenic component in both the current and proposed "next-generation" anthrax vaccines. Several studies utilizing animal models have indicated that PA-specific antibodies, acquired by either active or passive immunization, are sufficient to protect against infection with Bacillus anthracis. To investigate the human antibody response to anthrax immunization, we have established a large panel of human PA-specific monoclonal antibodies derived from multiple individuals vaccinated with the currently approved anthrax vaccine BioThrax. We have determined that although these antibodies bind PA in standard binding assays such as enzyme-linked immunosorbent assay, Western blotting, capture assays, and dot blots, less than 25% are capable of neutralizing lethal toxin (LT) in vitro. Nonneutralizing antibodies also fail to neutralize toxin when present in combination with other nonneutralizing paratopes. Although neutralizing antibodies recognize determinants throughout the PA monomer, they are significantly less common among those paratopes that bind to the immunodominant amino-terminal portion of the molecule. These findings demonstrate that PA binding alone is not sufficient to neutralize LT and suggest that for an antibody to effectively block PA-mediated toxicity, it must bind to PA such that one of the requisite toxin functions is disrupted. A vaccine design strategy that directed a higher percentage of the antibody response toward neutralizing epitopes may result in a more efficacious vaccine for the prevention of anthrax infection.

  10. 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.

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

    PubMed Central

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

    2016-01-01

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

  12. 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

  13. Electrically neutral microheterogeneity of human plasma transthyretin (prealbumin) detected by isoelectric focusing in urea gradients.

    PubMed

    Altland, K; Winter, P; Sauerborn, M K

    1999-06-01

    Mutants of the human plasma transthyretin (TTR, prealbumin) have attracted interest due to their rather frequent association with the autosomal dominant disease familial amyloidotic polyneuropathy (FAP). Some three quarters of known TTR mutations produce electrically neutral amino acid substitutions undetectable via separation by charge. We have developed an electrophoretic procedure sensitive to differences in the stability of tetramers and monomers under partially denaturing conditions. The differential folding states were found to be fully reversible. Applying the procedure we found 14 electrically silent mutants of TTR among 2000 plasma samples from German donors. We demonstrate that the normal TTR monomer exists in different forms of variable stability and/or charge due to binding of sulfhydryls from plasma to the unique cysteine at position 10 of the primary structure as well as due to modification by treatment with an oxidant. We found that reduction of Cys10 increases the stability of the folded monomeric and tetrameric conformations. The conformational changes of TTR induced by isoelectric focusing in a urea gradient were found to be associated by a gain of three positive charge units. Using published crystallographic data we present structural sites in the TTR molecule which could explain the observed effects.

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

    PubMed

    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

    2015-09-01

    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

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

    PubMed

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

    2016-01-01

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

  16. Neutral endopeptidase-24.11 (enkephalinase). Biosynthesis and localization in human fibroblasts.

    PubMed Central

    Lorkowski, G; Zijderhand-Bleekemolen, J E; Erdös, E G; von Figura, K; Hasilik, A

    1987-01-01

    The biosynthesis, glycosylation and subcellular localization of the neutral endopeptidase-24.11 were studied in cultured human fibroblasts. The enzyme was synthesized as a precursor (Mr 88,000) containing four or five N-linked oligosaccharides. Within 1 h the synthesis-mature (Mr 94,000) endopeptidase-24.11 was formed and contained sialylated oligosaccharides. The half-life of endopeptidase-24.11 was 3.7 days and in the presence of 10 mM-NH4Cl it increased to 6 days. Mature endopeptidase-24.11 was solubilized with 0.2% saponin and partitioned into Triton X-114. In intact fibroblasts, endopeptidase-24.11 was accessible to antibodies and to neuraminidase even when the treatment was performed at 4 degrees C. The localization of endopeptidase-24.11 to the plasma membrane in cultured fibroblasts was further demonstrated by immunocytochemistry. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. Fig. 6. PMID:3481263

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

    PubMed Central

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

    2016-01-01

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

  18. Carnosine and neocuproine as neutralizing agents for copper overload-induced damages in cultured human cells.

    PubMed

    Arnal, Nathalie; de Alaniz, María J T; Marra, Carlos A

    2011-07-15

    Copper is dangerous when it is present in excess, mainly because it can participate in the Fenton reaction, which produces radical species. As a consequence of copper pollution, people are involuntarily exposed to a copper overload under sub-clinical and sub-symptomatological conditions, which may be very difficult to detect. Thus, we investigated (i) the possible use of the chelator molecules carnosine and neocuproine to prevent the Cu overload-induced damage on cellular lipids and proteins, as tested in human cell culture systems, and (ii) the differential response of these two chelating agents in relation to their protective action, and the type of copper ion involved in the process, by using two types of human cultured cells (HepG2 and A-549). Cu treatment clearly enhanced (p<0.01) the formation of protein carbonyls, thiobarbituric acid-reactive substances (TBARS) and the concentration of nitrate plus nitrites, with a concomitant decrease in cell survival, as estimated by the trypan dye exclusion test and lactate dehydrogenase leakage. Simultaneous treatment with Cu and carnosine or neocuproine indicated that carnosine is more efficient than neocuproine in protecting both types of cells from the effect of cupric ions on both the cell-associated damages and the decrease in the cellular viability. This observation was supported by the fact that carnosine is not only a complexing agent for Cu(II), but also an effective antioxidant that can dismutate superoxide radicals, scavenge hydroxyl radicals and neutralize TBARS formation. Carnosine should be investigated in more detail in order to establish its putative utility as an agent to prevent copper-associated damages in biological systems.

  19. 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.

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

    PubMed

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

    2015-09-01

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

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

    PubMed

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

    2015-09-01

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

  2. 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

  3. 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.

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

    PubMed

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

    2015-06-01

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

  5. Use of microplate cell culture and enzyme immunoassay in titration of serum neutralizing antibody against Hochi strain of serotype 4 human rotavirus.

    PubMed

    Urasawa, T; Morita, Y; Urasawa, S; Taniguchi, K

    1986-08-01

    The tube neutralization test read by enzyme immunoassay developed by Wyatt et al. (1983) for serotype determination of human rotavirus was modified so as to use stationary cultures of MA104 cells in a microtiter plate instead of roller tube cultures. Sera obtained from different age groups were titrated for neutralizing antibody against serotype 4 human rotavirus Hochi strain by this test and the results were compared with those obtained by the plaque neutralization test. There was a good correlation between the titers obtained by the two tests and the age distribution pattern of serotype 4 neutralizing antibody was similar to those of serotype 1 and 3 antibodies previously reported.

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

    PubMed Central

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

    2014-01-01

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

  7. 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.

  8. Establishment of titration system for human herpesvirus 6 and evaluation of neutralizing antibody response to the virus.

    PubMed Central

    Asada, H; Yalcin, S; Balachandra, K; Higashi, K; Yamanishi, K

    1989-01-01

    The susceptibilities of seven T-cell lines to human herpesvirus 6 (HHV-6) infection were examined. MT-4 cells were the most susceptible of these lines to infection with this virus. Therefore, chemically adhered MT-4 cell monolayers were used for infectious HHV-6 assay by indirect immunofluorescent-antibody (IFA) staining. When cell monolayers were fixed 30 to 45 h postinfection, the foci stained with IFA were easy to count and a linear relationship was observed between the number of foci and the virus concentration. MT-4 cell monolayers were also used for a focus reduction neutralizing-antibody test. In this test, sera from patients in the convalescent stage of exanthem subitum all showed significant neutralizing activity (1:80 to 1:320), whereas sera from patients in the acute stage of disease showed no detectable neutralizing activity. The titers of neutralizing antibody correlated well with the levels of anti-HHV-6 antibodies detected by IFA. Images PMID:2555389

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

    PubMed

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

    2016-09-15

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

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

    PubMed

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

    2016-09-15

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

  11. 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.

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

    PubMed Central

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

    2009-01-01

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

  13. 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

  14. Computational prediction of neutralization epitopes targeted by human anti-V3 HIV monoclonal antibodies.

    PubMed

    Shmelkov, Evgeny; Krachmarov, Chavdar; Grigoryan, Arsen V; Pinter, Abraham; Statnikov, Alexander; Cardozo, Timothy

    2014-01-01

    The extreme diversity of HIV-1 strains presents a formidable challenge for HIV-1 vaccine design. Although antibodies (Abs) can neutralize HIV-1 and potentially protect against infection, antibodies that target the immunogenic viral surface protein gp120 have widely variable and poorly predictable cross-strain reactivity. Here, we developed a novel computational approach, the Method of Dynamic Epitopes, for identification of neutralization epitopes targeted by anti-HIV-1 monoclonal antibodies (mAbs). Our data demonstrate that this approach, based purely on calculated energetics and 3D structural information, accurately predicts the presence of neutralization epitopes targeted by V3-specific mAbs 2219 and 447-52D in any HIV-1 strain. The method was used to calculate the range of conservation of these specific epitopes across all circulating HIV-1 viruses. Accurately identifying an Ab-targeted neutralization epitope in a virus by computational means enables easy prediction of the breadth of reactivity of specific mAbs across the diversity of thousands of different circulating HIV-1 variants and facilitates rational design and selection of immunogens mimicking specific mAb-targeted epitopes in a multivalent HIV-1 vaccine. The defined epitopes can also be used for the purpose of epitope-specific analyses of breakthrough sequences recorded in vaccine clinical trials. Thus, our study is a prototype for a valuable tool for rational HIV-1 vaccine design.

  15. Computational Prediction of Neutralization Epitopes Targeted by Human Anti-V3 HIV Monoclonal Antibodies

    PubMed Central

    Shmelkov, Evgeny; Krachmarov, Chavdar; Grigoryan, Arsen V.; Pinter, Abraham; Statnikov, Alexander; Cardozo, Timothy

    2014-01-01

    The extreme diversity of HIV-1 strains presents a formidable challenge for HIV-1 vaccine design. Although antibodies (Abs) can neutralize HIV-1 and potentially protect against infection, antibodies that target the immunogenic viral surface protein gp120 have widely variable and poorly predictable cross-strain reactivity. Here, we developed a novel computational approach, the Method of Dynamic Epitopes, for identification of neutralization epitopes targeted by anti-HIV-1 monoclonal antibodies (mAbs). Our data demonstrate that this approach, based purely on calculated energetics and 3D structural information, accurately predicts the presence of neutralization epitopes targeted by V3-specific mAbs 2219 and 447-52D in any HIV-1 strain. The method was used to calculate the range of conservation of these specific epitopes across all circulating HIV-1 viruses. Accurately identifying an Ab-targeted neutralization epitope in a virus by computational means enables easy prediction of the breadth of reactivity of specific mAbs across the diversity of thousands of different circulating HIV-1 variants and facilitates rational design and selection of immunogens mimicking specific mAb-targeted epitopes in a multivalent HIV-1 vaccine. The defined epitopes can also be used for the purpose of epitope-specific analyses of breakthrough sequences recorded in vaccine clinical trials. Thus, our study is a prototype for a valuable tool for rational HIV-1 vaccine design. PMID:24587168

  16. Computational prediction of neutralization epitopes targeted by human anti-V3 HIV monoclonal antibodies.

    PubMed

    Shmelkov, Evgeny; Krachmarov, Chavdar; Grigoryan, Arsen V; Pinter, Abraham; Statnikov, Alexander; Cardozo, Timothy

    2014-01-01

    The extreme diversity of HIV-1 strains presents a formidable challenge for HIV-1 vaccine design. Although antibodies (Abs) can neutralize HIV-1 and potentially protect against infection, antibodies that target the immunogenic viral surface protein gp120 have widely variable and poorly predictable cross-strain reactivity. Here, we developed a novel computational approach, the Method of Dynamic Epitopes, for identification of neutralization epitopes targeted by anti-HIV-1 monoclonal antibodies (mAbs). Our data demonstrate that this approach, based purely on calculated energetics and 3D structural information, accurately predicts the presence of neutralization epitopes targeted by V3-specific mAbs 2219 and 447-52D in any HIV-1 strain. The method was used to calculate the range of conservation of these specific epitopes across all circulating HIV-1 viruses. Accurately identifying an Ab-targeted neutralization epitope in a virus by computational means enables easy prediction of the breadth of reactivity of specific mAbs across the diversity of thousands of different circulating HIV-1 variants and facilitates rational design and selection of immunogens mimicking specific mAb-targeted epitopes in a multivalent HIV-1 vaccine. The defined epitopes can also be used for the purpose of epitope-specific analyses of breakthrough sequences recorded in vaccine clinical trials. Thus, our study is a prototype for a valuable tool for rational HIV-1 vaccine design. PMID:24587168

  17. Immunization with a consensus epitope from Human Papillomavirus L2 induces antibodies that are broadly neutralizing

    PubMed Central

    Tyler, Mitchell; Tumban, Ebenezer; Dziduszko, Agnieszka; Ozbun, Michelle A.; Peabody, David S.; Chackerian, Bryce

    2014-01-01

    Vaccines targeting conserved epitopes in the HPV minor capsid protein, L2, can elicit antibodies that can protect against a broad spectrum of HPV types that are associated with cervical cancer and other HPV malignancies. Thus, L2 vaccines have been explored as alternatives to the current HPV vaccines, which are largely type-specific. In this study we assessed the immunogenicity of peptides spanning the N-terminal domain of L2 linked to the surface of a highly immunogenic bacteriophage virus-like particle (VLP) platform. Although all of the HPV16 L2 peptide-displaying VLPs elicited high-titer anti-peptide antibody responses, only a subset of the immunogens elicited antibody responses that were strongly protective from HPV16 pseudovirus (PsV) infection in a mouse genital challenge model. One of these peptides, mapping to HPV16 L2 amino acids 65–85, strongly neutralized HPV16 PsV but showed little ability to cross-neutralize other high-risk HPV types. In an attempt to broaden the protection generated through vaccination with this peptide, we immunized mice with VLPs displaying a peptide that represented a consensus sequence from high-risk and other HPV types. Vaccinated mice produced antibodies with broad, high-titer neutralizing activity against all of the HPV types that we tested. Therefore, immunization with virus-like particles displaying a consensus HPV sequence is an effective method to broaden neutralizing antibody responses against a type-specific epitope. PMID:24962748

  18. Cross-clade neutralization of primary isolates of human immunodeficiency virus type 1 by human monoclonal antibodies and tetrameric CD4-IgG.

    PubMed Central

    Trkola, A; Pomales, A B; Yuan, H; Korber, B; Maddon, P J; Allaway, G P; Katinger, H; Barbas, C F; Burton, D R; Ho, D D

    1995-01-01

    We have tested three human monoclonal antibodies (MAbs) IgG1b12, 2G12, and 2F5) to the envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1), and a tetrameric CD4-IgG molecule (CD4-IgG2), for the ability to neutralize primary HIV-1 isolates from the genetic clades A through F and from group O. Each of the reagents broadly and potently neutralized B-clade isolates. The 2F5 MAb and the CD4-IgG2 molecule also neutralized strains from outside the B clade, with the same breadth and potency that they showed against B-clade strains. The other two MAbs were able to neutralize a significant proportion of strains from outside the B clade, although there was a reduction in their efficacy compared with their activity against B-clade isolates. Neutralization of isolates by 2F5 correlated with their possession of the LDKW motif in a segment of gp41 near the membrane-spanning domain. The other two MAbs and CD4-IgG2 recognize discontinuous binding sites on gp120, and so no comparison between genetic sequence and virus neutralization was possible. Our data show that a vaccine based on the induction of humoral immunity that is broadly active across the genetic clades is not impossible if immunogens that express the epitopes for MAbs such as 2F5, 2G12, and IgG1b12 in immunogenic configurations can be created. Furthermore, if the three MAbs and CD4-IgG2 produce clinical benefit in immunotherapeutic trials in the United States or Europe, they may also do so elsewhere in the world. PMID:7474069

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

    PubMed

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

    2016-03-01

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

  20. 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

  1. 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.

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

    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

    2015-01-01

    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. PMID:26391698

  5. Ceramide metabolism is affected by obesity and diabetes in human adipose tissue.

    PubMed

    Błachnio-Zabielska, A U; Pułka, M; Baranowski, M; Nikołajuk, A; Zabielski, P; Górska, M; Górski, J

    2012-02-01

    Ceramide is involved in development of insulin resistance. However, there are no data on ceramide metabolism in human adipose tissue. The aim of our study was to examine sphingolipid metabolism in fat tissue from obese nondiabetic (n = 11), obese diabetic (n = 11), and lean nondiabetic (n = 8) subjects. The content of ceramide (Cer), dihydroceramide (dhCer), sphingosine (SPH), sphinganine (SPA), sphingosine-1-phosphate (S1P; pmol/mg of protein), the expression (mRNA) and activity of key enzymes responsible for Cer metabolism: serine palmitoyltransferase (SPT), neutral and acidic sphingomyelinase (nSMase and aSMase, respectively), and neutral and acidic ceramidase (nCDase and aCDase, respectively) were examined in human adipose tissue. The contents of SPA and Cer were significantly lower whereas the content of dhCer was higher in both obese groups than the respective values in the lean subjects. The expression of examined enzymes was elevated in both obese groups. The SPT and CDases activity increased whereas aSMase activity deceased in both obese groups. We have found correlation between adipose tissue Cer content and plasma adiponectin concentration (r = 0.69, P < 0.001) and negative correlation between total Cer content and HOMA-IR index (homeostasis model of insulin resistance) (r = -0.67, P < 0.001). We have found that both obesity and diabetes affected pathways of sphingolipid metabolism in the adipose tissue.

  6. 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.

  7. Immunization for HIV-1 Broadly Neutralizing Antibodies in Human Ig Knockin Mice.

    PubMed

    Dosenovic, Pia; von Boehmer, Lotta; Escolano, Amelia; Jardine, Joseph; Freund, Natalia T; Gitlin, Alexander D; McGuire, Andrew T; Kulp, Daniel W; Oliveira, Thiago; Scharf, Louise; Pietzsch, John; Gray, Matthew D; Cupo, Albert; van Gils, Marit J; Yao, Kai-Hui; Liu, Cassie; Gazumyan, Anna; Seaman, Michael S; Björkman, Pamela J; Sanders, Rogier W; Moore, John P; Stamatatos, Leonidas; Schief, William R; Nussenzweig, Michel C

    2015-06-18

    A subset of individuals infected with HIV-1 develops broadly neutralizing antibodies (bNAbs) that can prevent infection, but it has not yet been possible to elicit these antibodies by immunization. To systematically explore how immunization might be tailored to produce them, we generated mice expressing the predicted germline or mature heavy chains of a potent bNAb to the CD4 binding site (CD4bs) on the HIV-1 envelope glycoprotein (Env). Immunogens specifically designed to activate B cells bearing germline antibodies are required to initiate immune responses, but they do not elicit bNAbs. In contrast, native-like Env trimers fail to activate B cells expressing germline antibodies but elicit bNAbs by selecting for a restricted group of light chains bearing specific somatic mutations that enhance neutralizing activity. The data suggest that vaccination to elicit anti-HIV-1 antibodies will require immunization with a succession of related immunogens.

  8. Hansa: an automated method for discriminating disease and neutral human nsSNPs.

    PubMed

    Acharya, Vishal; Nagarajaram, Hampapathalu A

    2012-02-01

    Variations are mostly due to nonsynonymous single nucleotide polymorphisms (nsSNPs), some of which are associated with certain diseases. Phenotypic effects of a large number of nsSNPs have not been characterized. Although several methods have been developed to predict the effects of nsSNPs as "disease" or "neutral," there is still a need for development of methods with improved prediction accuracies. We, therefore, developed a support vector machine (SVM) based method named Hansa which uses a novel set of discriminatory features to classify nsSNPs into disease (pathogenic) and benign (neutral) types. Validation studies on a benchmark dataset and further on an independent dataset of well-characterized known disease and neutral mutations show that Hansa outperforms the other known methods. For example, fivefold cross-validation studies using the benchmark HumVar dataset reveal that at the false positive rate (FPR) of 20% Hansa yields a true positive rate (TPR) of 82% that is about 10% higher than the best-known method. Hansa is available in the form of a web server at http://hansa.cdfd.org.in:8080.

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-11-10

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

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

    PubMed Central

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

    2012-01-01

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

  17. Acid ceramidase (AC)--a key enzyme of sphingolipid metabolism--correlates with better prognosis in epithelial ovarian cancer.

    PubMed

    Hanker, Lars Christian; Karn, Thomas; Holtrich, Uwe; Gätje, Regine; Rody, Achim; Heinrich, Tomas; Ruckhäberle, Eugen; Engels, Knut

    2013-05-01

    Acid ceramidase (AC), a key enzyme of sphingolipid metabolism, seems to play an important role in cancer progression. The objective of this study was to explore the expression of AC in ovarian cancer and its impact on prognosis. Expression analysis of AC in n=112 ovarian cancer patients was performed by immunohistochemical analysis of primary paraffin-embedded tumor samples. The results were scored on the basis of the staining intensity and percentage of positive tumor cells, resulting in an immunoreactive score from 0 to 12. These results were correlated to clinical and pathologic characteristics and survival. AC expression correlated significantly only with FIGO stage (0.047). In serous carcinoma, low level of AC was independently associated with reduced progression-free survival and overall survival of 12.0 mo [95% confidence interval (CI), 5.78-18.23] versus 18.1 mo (95% CI, 11.61-24.59; P=0.008) and 35.7 mo (95% CI, 22.24-47.16) versus 58.7 mo (95% CI, 36.48-80.91; P=0.032), respectively. In multivariate analysis, AC presents as an independent prognostic factor for progression-free survival (hazard ratio 1.88; 95% CI, 1.13-3.11; P=0.015). AC is a prognostic factor in epithelial ovarian cancer. Low AC expression can be associated with tumor progression in carcinoma of the ovaries. These results are in contrast to the concept of AC as a promoter for cancer progression. Nevertheless, they are supported by the lately discovered tumor-suppressing function of sphingosine, the enzymatic product of AC.

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

    PubMed Central

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

    2016-01-01

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

  19. 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

  20. Neutral endopeptidase regulates neurogenic inflammatory responses induced by stimulation of human oral keratinocytes with bacterial lipopolysaccharide and nicotine.

    PubMed

    Nakata, Motoki; Awano, Shuji; Kinoshita, Naomasa; Yoshida, Akihiro; Ansai, Toshihiro

    2013-10-01

    Neutral endopeptidase (NEP) is present on various epithelial cells and inactivates numerous physiologically active peptides. Neutral endopeptidase may regulate proinflammatory signals in oral mucosal epithelium. However, the function of NEP in oral mucosal epithelium is unknown. The present study investigated the action of NEP upon proinflammatory signals on human oral keratinocytes and the influence of endothelin-converting enzyme (ECE)-1, an enzyme similar to NEP, on the functions of NEP. Oral keratinocytes were cultured in medium containing inflammatory inducers [lipopolysaccharide (LPS) and nicotine], NEP inhibitors, and ECE-1/NEP inhibitors, either alone or in combination. The concentrations of substance P (SP) and interleukin-1β (IL-1β) were measured in the supernatant. Additionally, the concentrations of SP and IL-1β were measured in the supernatant of cells incubated with LPS or nicotine after transfection with NEP small interfering RNA (siRNA). The concentrations of SP and IL-1β were significantly increased in cells incubated with NEP inhibitors and, to a lesser extent, in cells incubated with ECE-1/NEP inhibitors, compared with controls (cells incubated with LPS or nicotine alone). The concentrations of SP and IL-1β in cells transfected with NEP siRNA were significantly augmented compared with controls. In conclusion, the present study demonstrated that NEP down-regulated the levels of SP and IL-1β produced from human oral keratinocytes, although ECE-1 may be partly related to the down-regulation.

  1. In vivo hepatitis B virus-neutralizing activity of an anti-HBsAg humanized antibody in chimpanzees

    PubMed Central

    Kim, Se Ho; Oh, Han Kyu; Ryu, Chun Jeih; Park, Song Yong

    2008-01-01

    Previously, we constructed a humanized antibody (HuS10) that binds to the common a antigenic determinant on the S protein of HBV. In this study, we evaluated its HBV-neutralizing activity in chimpanzees. A study chimpanzee was intravenously administered with a single dose of HuS10, followed by intravenous challenge with the adr subtype of HBV, while a control chimpanzee was only challenged with the virus. The result showed that the control chimpanzee was infected by the virus, and thus serum HBV surface antigen (HBsAg) became positive from the 14th to 20th week and actively acquired serum anti-HBc and anti-HBs antibodies appeared from the 19th and 23rd week, respectively. However, in the case of the study chimpanzee, serum HBsAg became positive from the 34th to 37th week, while actively acquired serum anti-HBc and anti-HBs antibodies appeared from the 37th and 40th week, respectively, indicating that HuS10 neutralized the virus in vivo and thus delayed the HBV infection. This novel humanized antibody will be useful in the immunoprophylaxis of HBV infection. PMID:18305407

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

    PubMed

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

    2015-08-01

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

  3. 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.

  4. Human Rhinovirus Type 14:Human Immunodeficiency Virus Type 1 (HIV-1) V3 Loop Chimeras from a Combinatorial Library Induce Potent Neutralizing Antibody Responses against HIV-1

    PubMed Central

    Smith, Allen D.; Geisler, Sheila C.; Chen, Anne A.; Resnick, Dawn A.; Roy, Birgit M.; Lewi, Paul J.; Arnold, Edward; Arnold, Gail Ferstandig

    1998-01-01

    In an effort to develop a useful AIDS vaccine or vaccine component, we have generated a combinatorial library of chimeric viruses in which the sequence IGPGRAFYTTKN from the V3 loop of the MN strain of human immunodeficiency virus type 1 (HIV-1) is displayed in many conformations on the surface of human rhinovirus 14 (HRV14). The V3 loop sequence was inserted into a naturally immunogenic site of the cold-causing HRV14, bridged by linkers consisting of zero to three randomized amino acids on each side. The library of chimeric viruses obtained was subjected to a variety of immunoselection schemes to isolate viruses that provided the most useful presentations of the V3 loop sequence for potential use in a vaccine against HIV. The utility of the presentations was assessed by measures of antigenicity and immunogenicity. Most of the immunoselected chimeras examined were potently neutralized by each of the four different monoclonal anti-V3 loop antibodies tested. Seven of eight chimeric viruses were able to elicit neutralizing antibody responses in guinea pigs against the MN and ALA-1 strains of HIV-1. Three of the chimeras elicited HIV neutralization titers that exceeded those of all but a small number of previously described HIV immunogens. These results indicate that HRV14:HIV-1 chimeras may serve as useful immunogens for stimulating immunity against HIV-1. This method can be used to flexibly reconstruct varied immunogens on the surface of a safe and immunogenic vaccine vehicle. PMID:9420270

  5. A Monoclonal Fab Derived from a Human Nonimmune Phage Library Reveals a New Epitope on gp41 and Neutralizes Diverse Human Immunodeficiency Virus Type 1 Strains▿

    PubMed Central

    Gustchina, Elena; Louis, John M.; Lam, Son N.; Bewley, Carole A.; Clore, G. Marius

    2007-01-01

    A monoclonal Fab (Fab 3674) selected from a human nonimmune phage library by panning against the chimeric construct NCCG-gp41 (which comprises an exposed coiled-coil trimer of gp41 N helices fused in the helical phase onto the minimal thermostable ectodomain of gp41) is described. Fab 3674 is shown to neutralize diverse laboratory-adapted B strains of human immunodeficiency virus type 1 (HIV-1) and primary isolates of subtypes A, B, and C in an Env-pseudotyped-virus neutralization assay, albeit with reduced potency (approximately 25-fold) compared to that of 2F5 and 4E10. Alanine scanning mutagenesis maps a novel epitope to a shallow groove on the N helices of gp41 that is exposed between two C helices in the fusogenic six-helix bundle conformation of gp41. Bivalent Fab 3674 and the C34 peptide (a potent fusion inhibitor derived from the C helix of gp41) are shown to act at similar stages of the fusion reaction and to neutralize HIV-1 synergistically, providing additional evidence that the epitope of Fab 3674 is new and distinct from the binding site of C34. PMID:17898046

  6. Neutralizing IgG at the Portal of Infection Mediates Protection against Vaginal Simian/Human Immunodeficiency Virus Challenge

    PubMed Central

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

    2013-01-01

    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. PMID:23966410

  7. Association of Human Immunoglobulin G1 Heavy Chain Variants With Neutralization Capacity and Antibody-Dependent Cellular Cytotoxicity Against Human Cytomegalovirus.

    PubMed

    Vietzen, Hannes; Görzer, Irene; Puchhammer-Stöckl, Elisabeth

    2016-10-15

    Human cytomegalovirus (HCMV) infection is limited by HCMV-specific antibody functions. Here the association between the genetic marker (GM) 3/17 variants in the immunoglobulin G1 (IgG1) heavy chain constant region, virus neutralization, and natural killer (NK)-cell activation was investigated. In 100 HCMV-seropositive individuals, the GM3/17 polymorphism, serum 50% HCMV antibody neutralization titer (NT50), and in vitro HCMV-specific antibody NK-cell activation were assessed. The HCMV NT50 was higher in heterozygous GM3/17 persons than in GM3/3 persons (P = .0276). Furthermore, individuals expressing GM3/17 exhibited significantly higher NK-cell activation than persons carrying GM3/3 (P < .0001) or GM17/17 (P = .0095). Thus, persons expressing GM3/17 have potentially a selective advantage in HCMV defense.

  8. 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.

  9. Interactions between natural killer cells and antibody Fc result in enhanced antibody neutralization of human immunodeficiency virus type 1.

    PubMed

    Forthal, Donald N; Landucci, Gary; Phan, Tran B; Becerra, Juan

    2005-02-01

    Antibodies can prevent lentivirus infections in animals and may play a role in controlling viral burden in established infection. In preventing and particularly in controlling infection, antibodies likely function in the presence of large quantities of virus. In this study, we explored the mechanisms by which antibodies neutralize large inocula of human immunodeficiency virus type 1 (HIV-1) on different target cells. Immunoglobulin G (IgG) from HIV-infected patients was tested for neutralizing activity against primary R5 strains of HIV-1 at inocula ranging from 100 to 20,000 50% tissue culture infective doses. At all virus inocula, inhibition by antibody was enhanced when target cells for virus growth were monocyte-depleted, peripheral blood mononuclear cells (PBMCs) rather than CD4(+) lymphocytes. However, enhanced inhibition on PBMCs was greatest with larger amounts of virus. Depleting PBMCs of natural killer (NK) cells, which express Fc receptors for IgG (FcgammaRs), abrogated the enhanced antibody inhibition, whereas adding NK cells to CD4(+) lymphocytes restored inhibition. There was no enhanced inhibition on PBMCs when F(ab')(2) was used. Further experiments demonstrated that the release of beta-chemokines, most likely through FcgammaR triggering of NK cells, contributed modestly to the antiviral activity of antibody on PBMCs and that antibody-coated virus adsorbed to uninfected cells provided a target for NK cell-mediated inhibition of HIV-1. These results indicate that Fc-FcgammaR interactions enhance the ability of antibody to neutralize HIV-1. Since FcgammaR-bearing cells are always present in vivo, FcgammaR-mediated antibody function may play a role in the ability of antibody to control lentivirus infection.

  10. Interactions between Natural Killer Cells and Antibody Fc Result in Enhanced Antibody Neutralization of Human Immunodeficiency Virus Type 1

    PubMed Central

    Forthal, Donald N.; Landucci, Gary; Phan, Tran B.; Becerra, Juan

    2005-01-01

    Antibodies can prevent lentivirus infections in animals and may play a role in controlling viral burden in established infection. In preventing and particularly in controlling infection, antibodies likely function in the presence of large quantities of virus. In this study, we explored the mechanisms by which antibodies neutralize large inocula of human immunodeficiency virus type 1 (HIV-1) on different target cells. Immunoglobulin G (IgG) from HIV-infected patients was tested for neutralizing activity against primary R5 strains of HIV-1 at inocula ranging from 100 to 20,000 50% tissue culture infective doses. At all virus inocula, inhibition by antibody was enhanced when target cells for virus growth were monocyte-depleted, peripheral blood mononuclear cells (PBMCs) rather than CD4+ lymphocytes. However, enhanced inhibition on PBMCs was greatest with larger amounts of virus. Depleting PBMCs of natural killer (NK) cells, which express Fc receptors for IgG (FcγRs), abrogated the enhanced antibody inhibition, whereas adding NK cells to CD4+ lymphocytes restored inhibition. There was no enhanced inhibition on PBMCs when F(ab′)2 was used. Further experiments demonstrated that the release of β-chemokines, most likely through FcγR triggering of NK cells, contributed modestly to the antiviral activity of antibody on PBMCs and that antibody-coated virus adsorbed to uninfected cells provided a target for NK cell-mediated inhibition of HIV-1. These results indicate that Fc-FcγR interactions enhance the ability of antibody to neutralize HIV-1. Since FcγR-bearing cells are always present in vivo, FcγR-mediated antibody function may play a role in the ability of antibody to control lentivirus infection. PMID:15681406

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

    PubMed Central

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

    1986-01-01

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

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

    PubMed

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

    2015-09-23

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

  13. Mechanism of Lethal Toxin Neutralization by a Human Monoclonal Antibody Specific for the PA20 Region of Bacillus anthracis Protective Antigen

    PubMed Central

    Reason, Donald; Liberato, Justine; Sun, Jinying; Camacho, Jessica; Zhou, Jianhui

    2011-01-01

    The primary immunogenic component of the currently approved anthrax vaccine is the protective antigen (PA) unit of the binary toxin system. PA-specific antibodies neutralize anthrax toxins and protect against infection. Recent research has determined that in humans, only antibodies specific for particular determinants are capable of effecting toxin neutralization, and that the neutralizing epitopes recognized by these antibodies are distributed throughout the PA monomer. The mechanisms by which the majority of these epitopes effect neutralization remain unknown. In this report we investigate the process by which a human monoclonal antibody specific for the amino-terminal domain of PA neutralizes lethal toxin in an in vitro assay of cytotoxicity, and find that it neutralizes LT by blocking the requisite cleavage of the amino-terminal 20 kD portion of the molecule (PA20) from the remainder of the PA monomer. We also demonstrate that the epitope recognized by this human monoclonal does not encompass the 166RKKR169 furin recognition sequence in domain 1 of PA. PMID:22069752

  14. Neutralizer optimization

    NASA Technical Reports Server (NTRS)

    Patterson, Michael J.; Mohajeri, Kayhan

    1991-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  16. 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

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

    PubMed

    Skakauskas, Vladas; Katauskis, Pranas

    2016-06-01

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

  18. Variable epitope libraries: new vaccine immunogens capable of inducing broad human immunodeficiency virus type 1-neutralizing antibody response.

    PubMed

    Charles-Niño, Claudia; Pedroza-Roldan, Cesar; Viveros, Monica; Gevorkian, Goar; Manoutcharian, Karen

    2011-07-18

    The extreme antigenic variability of human immunodeficiency virus (HIV) leads to immune escape of the virus, representing a major challenge in the design of effective vaccine. We have developed a novel concept for immunogen construction based on introduction of massive mutations within the epitopes targeting antigenically variable pathogens and diseases. Previously, we showed that these immunogens carrying large combinatorial libraries of mutated epitope variants, termed as variable epitope libraries (VELs), induce potent, broad and long lasting CD8+IFN-γ+ T-cell response. Moreover, we demonstrated that these T cells recognize more than 50% of heavily mutated variants (5 out of 10 amino acid positions were mutated in each epitope variant) of HIV-1 gp120 V3 loop-derived cytotoxic T lymphocyte epitope (RGPGRAFVTI) in mice. The constructed VELs had complexities of 10000 and 12500 individual members, generated as plasmid DNA or as M13 phage display combinatorial libraries, respectively, and with structural composition RGPGXAXXXX or XGXGXAXVXI, where X is any of 20 natural amino acids. Here, we demonstrated that sera from mice immunized with these VELs are capable of neutralizing 5 out of 10 viral isolates from Tier 2 reference panel of subtype B envelope clones, including HIV-1 isolates which are known to be resistant to neutralization by several potent monoclonal antibodies, described previously. These data indicate the feasibility of the application of immunogens based on VEL concept as an alternative approach for the development of molecular vaccines against antigenically variable pathogens.

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

    PubMed Central

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

    2015-01-01

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

  20. 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.

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

    PubMed

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

    2016-03-29

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-03-29

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

  4. Monoclonal antibodies isolated from human B cells neutralize a broad range of H1 subtype influenza A viruses including swine-origin Influenza virus (S-OIV).

    PubMed

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

    2010-03-30

    The new H1N1 swine-origin influenza virus (S-OIV) strain is a global health problem. The elucidation of the virus-host relationship is crucial for the control of the new infection. Two human monoclonal antibody Fab fragments (HMab) neutralizing the novel H1N1 influenza strain at very low concentrations were cloned before the emergence of S-OIV from a patient who had a broad-range H1N1 serum neutralizing activity. The two HMabs neutralized all tested H1N1 strains, including S-OIV and a swine strain with IC(50) ranging from 2 to 7 microg/ml. Data demonstrate that infection with previously circulating H1N1 strains can elicit antibodies neutralizing S-OIV. Finally, the human genes coding for the neutralizing HMabs could be used for generating full human monoclonal IgGs that can be safely administered being potentially useful in the prophylaxis and the treatment of this human infection.

  5. Generation and characterization of human monoclonal neutralizing antibodies with distinct binding and sequence features against SARS coronavirus using XenoMouse.

    PubMed

    Coughlin, Melissa; Lou, Gin; Martinez, Osvaldo; Masterman, Stephanie K; Olsen, Ole A; Moksa, Angelica A; Farzan, Michael; Babcook, John S; Prabhakar, Bellur S

    2007-04-25

    Passive therapy with neutralizing human monoclonal antibodies (mAbs) could be an effective therapy against severe acute respiratory syndrome coronavirus (SARS-CoV). Utilizing the human immunoglobulin transgenic mouse, XenoMouse, we produced fully human SARS-CoV spike (S) protein specific antibodies. Antibodies were examined for reactivity against a recombinant S1 protein, to which 200 antibodies reacted. Twenty-seven antibodies neutralized 200TCID(50) SARS-CoV (Urbani). Additionally, 57 neutralizing antibodies were found that are likely specific to S2. Mapping of the binding region was achieved with several S1 recombinant proteins. Most S1 reactive neutralizing mAbs bound to the RBD, aa 318-510. However, two S1 specific mAbs reacted with a domain upstream of the RBD between aa 12 and 261. Immunoglobulin gene sequence analyses suggested at least 8 different binding specificities. Unique human mAbs could be used as a cocktail that would simultaneously target several neutralizing epitopes and prevent emergence of escape mutants. PMID:17161858

  6. Laser neutralization

    SciTech Connect

    Peterson, O.G.

    1986-06-17

    Laser photodetachment of the excess electron to neutralize relativistic ions offers many advantages over the more conventional collisional methods using gases or thin foils as the neutralization agents. Probably the two most important advantages of laser photodetachment are the generation of a compact and low divergence beam, and the production of intense neutral beams at very high efficiency (approximately 90%). The high intensities or high current densities of the neutral beam result from the fixed maximum divergence that can be added to the beam by photodetachment of the charge using laser intensity of fixed wavelength and incident angle. The high neutralization efficiency is possible because there is no theoretical maximum to the neutralization efficiency, although higher efficiencies require higher laser powers and, therefore, costs. Additional advantages include focusability of the laser light onto the ion beam to maximize its efficacy. There certainly is no residual gas left in the particle beam path as is typical with gas neutralizers. The photodetachment process leaves the neutral atoms in the ground state so there is no excited state fluorescence to interfere with the subsequent beam sensing. Finally, since the beams to be neutralized are very high powered, for a large range of neutralization efficiencies the neutral beam can be increased more by increasing the power to the laser neutralizer than by adding an equal amount of power to the primary accelerator. 26 figs.

  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. Chimeric gag-V3 virus-like particles of human immunodeficiency virus induce virus-neutralizing antibodies.

    PubMed Central

    Luo, L; Li, Y; Cannon, P M; Kim, S; Kang, C Y

    1992-01-01

    A 41-kDa unprocessed human immunodeficiency virus 2 (HIV-2) gag precursor protein that has a deletion of a portion of the viral protease assembles as virus-like particles by budding through the cytoplasmic membrane of recombinant baculovirus-infected insect cells. We have constructed six different combinations of chimeric genes by coupling the truncated HIV-2 gag gene to the neutralizing domain (V3) or the neutralizing and the CD4 binding domains (V3+CD4BD) of gp120 env gene sequences from HIV-1 or HIV-2. The env gene sequences were inserted either into the middle of the gag gene or at the 3' terminus of the gag gene. Virus-like particles were formed by chimeric gene products only when the env gene sequences were linked to the 3' terminus of the gag gene. Insertion of env gene sequence in the middle of the gag gene resulted in high-level chimeric gene expression but without the formation of virus-like particles. Three different chimeric genes [gag gene with HIV-1 V3 (1V3), gag gene with HIV-2 V3 (2V3), and gag gene with HIV-2 V3+CD4BD (2V3+CD4BD)] formed virus-like particles that were secreted into the cell culture medium. In contrast, the HIV-1 V3+CD4BD/HIV-2 gag construct did not form virus-like particles. The chimeric gag-env particles had spherical morphology and the size was slightly larger than that of the gag particles, but the chimeric particles were similar to the mature HIV particles. Western blot analysis showed that the gag-env chimeric proteins were recognized by antibodies in HIV-positive human serum and rabbit anti-gp120 serum. Rabbit anti-gag 1V3 and anti-gag 2V3 sera reacted with authentic gp120 of HIV-1 and HIV-2, respectively, and neutralized homologous HIV infectivity. Our results show that precursor gag protein has potential as a carrier for the presentation of foreign epitopes in good immunological context. The gag protein is highly immunogenic and has the ability to carry large foreign inserts; as such, it offers an attractive approach for

  9. 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

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

    PubMed

    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

    2015-10-01

    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. Can artificial techniques supply morally neutral human embryos for research? Part I. Creating novel categories of human embryos.

    PubMed

    Jones, Nancy L; Cheshire, William P

    2005-01-01

    Manipulations of the molecular composition and formation of human embryos are posing vital new challenges to traditional concepts of human identity and procreation. Current trends in embryology in particular are reshaping the ethical question of how scientific research should treat experimentally derived embryos. Some investigators have argued that embryos created through artificial means are technologically novel entities that should be exempt from ethical restraints placed on research involving human embryos that come into being through natural processes. These include uniparental embryos derived through cloning or parthenogenesis, as well as multiparental, hybrid-parental, and xenohybrid-parental embryos. If confined to natural means many of these genetic unions could not occur, but through the intervention of technology, it is becoming possible to design and grow strange and unusual forms of embryos, in some cases using human gametes. Regardless of the genetic contributors or the processes used to fertilize and stimulate egg activation, in each case the new embryo represents an individual organism that begins a process of development. We conclude that the prospect of creating or redesigning new human life should be held to a stringent ethical standard of precaution, even higher than that of deciding to destroy existing embryonic life. Accordingly, we urge cautious ethical reflection and broad public discussion prior to deciding whether to permit embryologic research into novel forms of procreative means in nonhuman animals, to be further extended to humans.

  12. Neutralizing and cross-neutralizing antibody titres induced by bivalent and quadrivalent human papillomavirus vaccines in the target population of organized vaccination programmes.

    PubMed

    Barzon, Luisa; Squarzon, Laura; Masiero, Serena; Pacenti, Monia; Marcati, Giorgia; Mantelli, Barbara; Gabrielli, Liliana; Pascucci, Maria Grazia; Lazzarotto, Tiziana; Caputo, Antonella; Palù, Giorgio

    2014-09-15

    Aim of this investigator-initiated study was to evaluate and compare the titres of neutralizing and cross-neutralizing antibodies (NAbs) induced by the bivalent (Cervarix(®)) and quadrivalent (Gardasil(®)) HPV vaccines in a cohort of girls aged 11-13 years from organized vaccination programmes. To this aim, HPV16 and HPV18 NAbs were measured by pseudovirion-based neutralization assays in serum collected at 1-6 months after the third vaccine dose in 107 girls vaccinated with Cervarix(®) and 126 vaccinated with Gardasil(®), while HPV31 and HPV45 cross-NAbs were tested in the first 50 consecutive girls of both vaccine groups. The results of this study demonstrated that all vaccinated girls developed HPV16 and HPV18 NAbs, with the exception of two Gardasil(®) vaccinees with undetectable HPV18 NAbs. Geometric mean titres (GMTs) of both HPV16 and HPV18 NAbs were significantly higher in Cervarix(®) than in Gardasil(®) vaccinees [HPV16 NAb GMT 22,136 (95% CI, 18,811-26,073) vs 5092 (4230-6151), respectively; P<0.0001; HPV18 NAb GMT 11,962 (9536-14,363) vs 1804 (1574-2110), respectively; P<0.0001]. Cross-NAbs to HPV31 and HPV45 were detected more frequently Cervarix(®) (HPV31 NAb positivity rates 92.7% and 36%, respectively; P<0.05) than in Gardasil(®) vaccinees (HPV45 NAb positivity rates 56% and 6%, respectively; P<0.0001). The titres of cross-NAbs against HPV31 and HPV45 were also significantly higher in Cervarix(®) than in Gardasil(®) vaccinees [HPV31 NAb GMT 157.2 (95% CI, 92-269) vs 13.0 (6.5-25.8), respectively; P<0.0001; HPV45 NAb GMT 4.7 (2.1-10.2) vs 1.3 (0.3-3.1), respectively; P<0.01]. In conclusion, in adolescent girls vaccinated within organized vaccination programmes, HPV vaccines drive the generation not only of NAbs to HPV vaccine types, but also of cross-NAbs. The bivalent vaccine induced significantly higher HPV16 and HPV18 NAb titres and more frequently and at higher titre HPV31 and HPV45 cross-NAbs than the quadrivalent vaccine.

  13. Chimpanzee Fab fragments and a derived humanized immunoglobulin G1 antibody that efficiently cross-neutralize dengue type 1 and type 2 viruses.

    PubMed

    Goncalvez, Ana P; Men, Ruhe; Wernly, Claire; Purcell, Robert H; Lai, Ching-Juh

    2004-12-01

    Passive immunization with monoclonal antibodies from humans or nonhuman primates represents an attractive alternative to vaccines for prevention of illness caused by dengue viruses (DENV) and other flaviviruses, including the West Nile virus. In a previous study, repertoire cloning to recover Fab fragments from bone marrow mRNA of chimpanzees infected with all four DENV serotypes (dengue virus serotype 1 [DENV-1] to DENV-4) was described. In that study, a humanized immunoglobulin G1 (IgG1) antibody that efficiently neutralized DENV-4 was recovered and characterized. In this study, the phage library constructed from the chimpanzees was used to recover Fab antibodies against the other three DENV serotypes. Serotype-specific neutralizing Fabs were not identified. Instead, we recovered DENV-neutralizing Fabs that specifically precipitated the envelope protein and were cross-reactive with all four DENV serotypes. Three of the Fabs competed with each other for binding to DENV-1 and DENV-2, although each of these Fabs contained a distinct complementarity determining region 3 (CDR3)-H sequence. Fabs that shared an identical or nearly identical CDR3-H sequences cross-neutralized DENV-1 and DENV-2 at a similar high 50% plaque reduction neutralization test (PRNT(50)) titer, ranging from 0.26 to 1.33 microg/ml, and neutralized DENV-3 and DENV-4 but at a titer 10- to 20-fold lower. One of these Fabs, 1A5, also neutralized the West Nile virus most efficiently among other flaviviruses tested. Fab 1A5 was converted to a full-length antibody in combination with human sequences for production in mammalian CHO cells. Humanized IgG1 1A5 proved to be as efficient as Fab 1A5 for cross-neutralization of DENV-1 and DENV-2 at a titer of 0.48 and 0.95 microg/ml, respectively. IgG1 1A5 also neutralized DENV-3, DENV-4, and the West Nile virus at a PRNT(50) titer of approximately 3.2 to 4.2 microg/ml. This humanized antibody represents an attractive candidate for further development of

  14. Molecular basis of acid ceramidase deficiency in a neonatal form of Farber disease: identification of the first large deletion in ASAH1 gene.

    PubMed

    Alves, Mariana Q; Le Trionnaire, Emmanuelle; Ribeiro, Isaura; Carpentier, Stéphane; Harzer, Klaus; Levade, Thierry; Ribeiro, M Gil

    2013-07-01

    Farber disease, also known as Farber's lipogranulomatosis, is a clinically heterogeneous autosomal recessive disease caused by mutations in the ASAH1 gene. This gene codes for acid ceramidase, a lysosomal heterodimeric enzyme that hydrolyzes ceramide into sphingosine and fatty acid. To date, less than 25 distinct mutations have been identified in Farber patients, but no large deletions have yet been reported. In this work, cultured fibroblasts from a Farber patient with the rare neonatal form of Farber disease were studied to elucidate the molecular basis of this extremely severe phenotype. Direct sequencing of ASAH1 genomic DNA revealed the causative heterozygous mutation in the donor splice site consensus sequence of intron 11, g.24491A > G (c.917 + 4A > G), that resulted in the absence of detectable mRNA. Subsequent analysis of ASAH1 mRNA showed total skipping of exons 3 to 5. Long-range PCR and sequencing led to the identification of a gross deletion of ASAH1 gene, g.8728_18197del (c.126-3941_382 + 1358del) predicting the synthesis of a truncated polypeptide, p.Tyr42_Leu127delinsArgfs*10. Accordingly, no molecular forms corresponding to precursor or proteolytically processed mature protein were observed. These findings indicate that any functionally active acid ceramidase is absent in patient cells, underscoring the severity of the clinical phenotype. Molecular findings in the non-consanguineous parents confirmed the compound heterozygous ASAH1 genotype identified in this Farber case. This work unravels for the first time the mutations underlying the neonatal form of Farber disease and represents the first report of a large deletion identified in the ASAH1 gene. Screening for gross deletions in other patients in whom the mutation present in the second allele had not yet been identified is required to elucidate further its overall contribution for the molecular pathogenesis of this devastating disease.

  15. Antibody germline characterization of cross-neutralizing human IgGs against 4 serotypes of dengue virus.

    PubMed

    Pitaksajjakul, Pannamthip; Benjathummarak, Surachet; Pipattanaboon, Chonlatip; Wongwit, Waranya; Okabayashi, Tamaki; Kuhara, Motoki; Misaki, Ryo; Fujiyama, Kazuhito; Ramasoota, Pongrama

    2014-04-01

    Dengue virus (DENV), a re-emerging virus, constitutes the largest vector-borne disease virus, with 50-100 million cases reported every year. Although DENV infection induces lifelong immunity against viruses of the same serotypes, the subsequent infection with the heterologous serotypes can cause more severe form of the disease, such as Dengue Haemorrhagic Fever (DHF) or Dengue Shock Syndrome (DSS). However, there is neither approved vaccine nor specific drugs available to treat this disease. In this study, previously developed 19 human monoclonal antibodies (HuMAbs) showing strong to moderate cross neutralizing activity were selected. Most of them (13/19) were targeted to domain II of envelop glycoprotein. To understand and clarify the recognition properties, the maturation mechanisms comprising Variable/Diversity/Joining (VDJ) recombination, Variable Heavy (VH)/Variable Light (VL) chain pairing, variability at junctional site, and somatic hypermutation (SHM) of those antibodies were studied and compared with their predecessor germline sequences. IMGT/V-QUEST database was applied to analyze the isolated VH and VL sequences. To confirm the correction of isolated VH/VL, 3 HuMAbs (1A10H7, 1B3B9, 1G7C2) was transiently expressed in HEK293T cell. All three clones of the expressed recombinant IgG (rIgG) showed the same binding and neutralizing activity as same as those from hybridomas. The data obtained in this study will elucidate the properties of those HuMAbs for further genetic modification, and its binding epitopes. PMID:24637211

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

    PubMed Central

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

    1998-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  18. 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.

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

    PubMed

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

    2016-05-13

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

  20. 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.

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

    PubMed

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

    2002-07-01

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

  2. Antibody-driven design of a human cytomegalovirus gHgLpUL128L subunit vaccine that selectively elicits potent neutralizing antibodies.

    PubMed

    Kabanova, Anna; Perez, Laurent; Lilleri, Daniele; Marcandalli, Jessica; Agatic, Gloria; Becattini, Simone; Preite, Silvia; Fuschillo, Dario; Percivalle, Elena; Sallusto, Federica; Gerna, Giuseppe; Corti, Davide; Lanzavecchia, Antonio

    2014-12-16

    The use of neutralizing antibodies to identify the most effective antigen has been proposed as a strategy to design vaccines capable of eliciting protective B-cell immunity. In this study, we analyzed the human antibody response to cytomegalovirus (human cytomegalovirus, HCMV) infection and found that antibodies to glycoprotein (g)B, a surface glycoprotein that has been developed as a HCMV vaccine, were primarily nonneutralizing. In contrast, most of the antibodies to the complex formed by gH, gL, protein (p)UL128, pUL130, and pUL131 (the gHgLpUL128L pentamer) neutralized HCMV infection with high potency. Based on this analysis, we developed a single polycistronic vector encoding the five pentamer genes separated by "self-cleaving" 2A peptides to generate a stably transfected CHO cell line constitutively secreting high levels of recombinant pentamer that displayed the functional antigenic sites targeted by human neutralizing antibodies. Immunization of mice with the pentamer formulated with different adjuvants elicited HCMV neutralizing antibody titers that persisted to high levels over time and that were a hundred- to thousand-fold higher than those found in individuals that recovered from primary HCMV infection. Sera from mice immunized with the pentamer vaccine neutralized infection of both epithelial cells and fibroblasts and prevented cell-to-cell spread and viral dissemination from endothelial cells to leukocytes. Neutralizing monoclonal antibodies from immunized mice showed the same potency as human antibodies and targeted the same as well as additional sites on the pentamer. These results illustrate with a relevant example a general and practical approach of analytic vaccinology for the development of subunit vaccines against complex pathogens.

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

    PubMed Central

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

    2016-01-01

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

  4. 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. PMID:25723165

  5. Pyruvate neutralizes peritoneal dialysate cytotoxicity: maintained integrity and proliferation of cultured human mesothelial cells.

    PubMed

    Brunkhorst, R; Mahiout, A

    1995-07-01

    Toxic effects of commercially available peritoneal dialysate (PD) fluid include damage to mesothelial cells (MC), causing a severely disturbed proliferation of cultured MC. We investigated the injury to the cell membrane (by release of lactate dehydrogenase, LDH), the proliferation (by cell counts and by 3H-thymidine incorporation), and optional the cytokine generation (by IL-1 receptor-antagonist production, IL-1 ra) of cultured human MC during the 48 hours after a 30 minute exposure to PD containing either 35 mmol/liter sodium lactate or sodium pyruvate. All solutions had a pH of 5.2 to 5.6 and were composed as standard PD. Glucose contents of 1.36 and 3.86 mmol/liter were tested. After exposure to the lactate-PD containing 1.36% glucose, LDH activity was increased by more than 30%, proliferation of MC was inhibited by more than 30%, and IL-1 ra production was reduced significantly when compared to pyruvate-PD and the control solution. After preincubation with 3.86% glucose containing PD, all negative effects became even more pronounced in the lactate group whereas the MC maintained their integrity, rate of proliferation and IL-1 ra release after pre-exposure to pyruvate containing PD. These results suggest that the acute toxic effects of commercially available PD on the integrity, proliferation and IL-1 ra production of MC can be avoided by the use of sodium pyruvate instead of sodium lactate.

  6. Neutralization activity and kinetics of two broad-range human monoclonal IgG1 derived from recombinant Fab fragments and directed against Hepatitis C virus E2 glycoprotein.

    PubMed

    Diotti, Roberta Antonia; Sautto, Giuseppe Andrea; Solforosi, Laura; Mancini, Nicasio; Clementi, Massimo; Burioni, Roberto

    2012-10-01

    Hepatitis C virus (HCV) is the major cause of chronic liver disease worldwide. There is evidence that neutralizing anti-HCV antibodies may find potential applications in novel prophylactic and therapeutic strategies. This paper describes the very high neutralization activity and unique biological features of two broadly cross-reactive and cross-neutralizing anti-HCV human monoclonal IgG1 derived from human monoclonal recombinant Fab fragments.

  7. Effect of aspartame-derived phenylalanine on neutral amino acid uptake in human brain: a positron emission tomography study.

    PubMed

    Koeppe, R A; Shulkin, B L; Rosenspire, K C; Shaw, L A; Betz, A L; Mangner, T; Price, J C; Agranoff, B W

    1991-05-01

    The possible effects of elevation of the plasma phenylalanine level secondary to the ingestion of aspartame on brain amino acid uptake in human subjects have been investigated by means of positron emission tomography (PET). 1-[11C]Aminocyclohexanecarboxylate [( 11C]ACHC) is a poorly metabolized synthetic amino acid that crosses the blood-brain barrier by the same carrier that transports naturally occurring large neutral amino acids. Quantitative test-retest PET studies were performed on 15 individuals. Seven received two identical baseline scans, whereas eight received a baseline scan followed by a scan performed approximately 40-45 min following ingestion of an orange-flavored beverage containing 34 mg/kg of body weight of the low-calorie sweetener aspartame, a dose equivalent to the amount in 5 L of diet soft drink consumed all at once by the study subjects, weighing an average of 76 kg. The 40-45-min interval was selected to maximize the detection of possible decreases in ACHC uptake resulting from increased competition for the carrier, because the plasma phenylalanine level is known to peak at this time. We observed an 11.5% decrease in the amino acid transport rate constant K1 and a smaller decrease in the tissue distribution volume of ACHC (6%). Under conditions of normal dietary use, aspartame is thus unlikely to cause changes in brain amino acid uptake that are measurable by PET.

  8. Procalcitonin neutralizes bacterial LPS and reduces LPS-induced cytokine release in human peripheral blood mononuclear cells

    PubMed Central

    2012-01-01

    Background Procalcitonin (PCT) is a polypeptide with several cationic aminoacids in its chemical structure and it is a well known marker of sepsis. It is now emerging that PCT might exhibit some anti-inflammatory effects. The present study, based on the evaluation of the in vitro interaction between PCT and bacterial lipopolisaccharide (LPS), reports new data supporting the interesting and potentially useful anti-inflammatory activity of PCT. Results PCT significantly decreased (p < 0.05) the limulus amoebocyte lysate (LAL) assay reactivity of LPS from both Salmonella typhimurium (rough chemotype) and Escherichia coli (smooth chemotype). Subsequently, the in vitro effects of PCT on LPS-induced cytokine release were studied in human peripheral blood mononuclear cells (PBMC). When LPS was pre-incubated for 30 minutes with different concentrations of PCT, the release of interleukin-10 (IL-10) and tumor necrosis factor alpha (TNFα) by PBMC decreased in a concentration-dependent manner after 24 hours for IL-10 and 4 hours for TNFα. The release of monocyte chemotactic protein-1 (MCP-1) exhibited a drastic reduction at 4 hours for all the PCT concentrations assessed, whereas such decrease was concentration-dependent after 24 hours. Conclusions This study provides the first evidence of the capability of PCT to directly neutralize bacterial LPS, thus leading to a reduction of its major inflammatory mediators. PMID:22568957

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

    PubMed Central

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

    2010-01-01

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

  10. 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

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

    PubMed Central

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

    2015-01-01

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

  12. 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.

  13. 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

    PubMed Central

    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

    2011-01-01

    Summary 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 >20Å 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. PMID:21565703

  14. Development of Broadly Neutralizing Antibodies and Their Mapping by Monomeric gp120 in Human Immunodeficiency Virus Type 1-Infected Humans and Simian-Human Immunodeficiency Virus SHIVSF162P3N-Infected Macaques

    PubMed Central

    Jia, Manxue; Lu, Hong; Markowitz, Martin; Cheng-Mayer, Cecilia

    2016-01-01

    ABSTRACT To improve our understanding of the similarities and differences between neutralizing antibodies elicited by simian-human immunodeficiency virus (SHIV)-infected rhesus macaques and human immunodeficiency virus type 1 (HIV-1)-infected humans, we examined the plasma of 13 viremic macaques infected with SHIVSF162P3N and 85 HIV-1-infected humans with known times of infection. We identified 5 macaques (38%) from 1 to 2 years postinfection (p.i.) with broadly neutralizing antibodies (bnAbs) against tier 2 HIV-1. In comparison, only 2 out of 42 (5%) human plasma samples collected in a similar time frame of 1 to 3 years p.i. exhibited comparable neutralizing breadths and potencies, with the number increasing to 7 out of 21 (30%) after 3 years p.i. Plasma mapping with monomeric gp120 identified only 2 out of 9 humans and 2 out of 4 macaques that contained gp120-reactive neutralizing antibodies, indicating distinct specificities in these plasma samples, with most of them recognizing the envelope trimer (including gp41) rather than the gp120 monomer. Indeed, a total of 20 gp120-directed monoclonal antibodies (MAbs) isolated from a human subject (AD358) and a Chinese rhesus macaque (GB40) displayed no or limited neutralizing activity against tier 2 strains. These isolated MAbs, mapped to the CD4-binding site, the V3 loop, the inner domain, and the C5 region of gp120, revealed genetic similarity between the human and macaque immunoglobulin genes used to encode some V3-directed MAbs. These results also support the use of envelope trimer probes for efficient isolation of HIV-1 bnAbs. IMPORTANCE HIV-1 vaccine research can benefit from understanding the development of broadly neutralizing antibodies (bnAbs) in rhesus macaques, commonly used to assess vaccine immunogenicity and efficacy. Here, we examined 85 HIV-1-infected humans and 13 SHIVSF162P3N-infected macaques for bnAbs and found that, similar to HIV-1-infected humans, bnAbs in SHIV-infected macaques are also rare

  15. 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.

  16. Reversion of somatic mutations of the respiratory syncytial virus-specific human monoclonal antibody Fab19 reveal a direct relationship between association rate and neutralizing potency.

    PubMed

    Bates, John T; Keefer, Christopher J; Utley, Thomas J; Correia, Bruno E; Schief, William R; Crowe, James E

    2013-04-01

    The role of affinity in determining neutralizing potency of mAbs directed against viruses is not well understood. We investigated the kinetic, structural, and functional advantage conferred by individual naturally occurring somatic mutations in the Ab H chain V region of Fab19, a well-described neutralizing human mAb directed to respiratory syncytial virus. Comparison of the affinity-matured Ab Fab19 with recombinant Fab19 Abs that were variants containing reverted amino acids from the inferred unmutated ancestor sequence revealed the molecular basis for affinity maturation of this Ab. Enhanced binding was achieved through mutations in the third H chain CDR (HCDR3) that conferred a markedly faster on-rate and a desirable increase in antiviral neutralizing activity. In contrast, most somatic mutations in the HCDR1 and HCDR2 regions did not significantly enhance Ag binding or antiviral activity. We observed a direct relationship between the measured association rate (Kon) for F protein and antiviral activity. Modeling studies of the structure of the Ag-Ab complex suggested the HCDR3 loop interacts with the antigenic site A surface loop of the respiratory syncytial virus F protein, previously shown to contain the epitope for this Ab by experimentation. These studies define a direct relationship of affinity and neutralizing activity for a viral glycoprotein-specific human mAb.

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

    PubMed Central

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

    1997-01-01

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

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

    PubMed

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

    2012-03-01

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

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  1. 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. PMID:24964084

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

    PubMed Central

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

    2014-01-01

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

  3. 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.

  4. 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

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

    PubMed

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

    2014-10-15

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

  6. X-ray absorption and resonance raman spectroscopy of human myeloperoxidase at neutral and acid pH.

    PubMed

    Yue, K T; Taylor, K L; Kinkade, J M; Sinclair, R B; Powers, L S

    1997-04-01

    Myeloperoxidase (MPO), an important enzyme in the oxygen-dependent host defense system of human polymorphonuclear leukocytes, utilizes hydrogen peroxide to catalyze the production of hypochlorous acid, an oxidizing bactericidal agent. While MPO shows significant sequence homology with other peroxidases and this homology is particularly striking among the active-site residues, MPO exhibits unusual spectral features and the unique ability to catalyze the oxidation of chloride ions. We have investigated the MPO active-site with X-ray absorption (XAS) and resonance Raman (RRS) spectroscopies at neutral pH and also at the physiological acidic pH (pH approximately 3) and have compared these results with those of horseradish peroxidase (HRP). At pH 7.5, XAS results show that the iron heme active site is 6-coordinate where the distal ligand is likely nitrogen or oxygen, but not sulfur. The heme is distorted compared to HRP, other peroxidases, and heme compounds, but at pH approximately 3, the distal ligand is lost and the heme is less distorted. RRS results under identical pH conditions show that the skeletal core-size sensitive modes and v3 are shifted to higher frequency at pH approximately 3 indicating a 6- to 5-coordination change of high spin ferric heme. In addition, a new band at 270 cm(-1) is observed at pH approximately 3 which is consistent with the loss of the sixth ligand. The higher symmetry of the heme at pH approximately 3 is reflected by a single v4 mode in the (RRS) spectrum. HRP also loses its loosely associated distal water at this pH, but little change in heme distortion is observed. This change suggests that loss of the distal ligand in MPO releases stress on the heme which may facilitate binding of chloride ion.

  7. 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.

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

    PubMed Central

    Côté, R H; Valet, J P

    1976-01-01

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

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

    PubMed

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

    2016-07-01

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

  10. 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

  11. IgG subclass antibodies to human cytomegalovirus (CMV) in normal human plasma samples and immune globulins and their neutralizing activities.

    PubMed

    Gupta, C K; Leszczynski, J; Gupta, R K; Siber, G R

    1996-06-01

    An enzyme-linked immunoabsorbent assay (ELISA) was developed for quantitation of IgG subclass antibodies to human cytomegalovirus (CMV) in human serum or plasma samples and in immune globulin (IG) preparations. The assay was based on the parallel titration of known concentrations of purified IgG subclass myeloma proteins and a specific CMV antiserum. The purified IgG subclass myeloma proteins were captured on an ELISA plate pre-coated with anti-human kappa, anti-human lambda or a mixture of anti-human kappa and lambda antibodies and the specific antiserum was titrated against CMV antigen coated on the plate. IgG subclass antibodies, captured or bound to antigen, were quantitated with IGG subclass heavy chain specific monoclonal antibodies. The method was highly reproducible, specific and sensitive. Using this method, 257 human plasma samples and 50 IG preparations were assayed for CMV specific IgG subclass and IgM antibodies. The major IgG subclass antibody to CMV was IgG1 which represented more than 96% of CMV IgG antibodies, followed by IgG3 (mean CMV IgG3 antibody content was 3% of IgG antibodies in IG preparations and 1.8% in plasma samples). A majority of the samples had low levels of IgG2 antibodies and a few samples exhibited low levels of IgG4 antibodies. IG preparations showed very low levels of CMV IgM antibodies whereas plasma samples had 14.2% of CMV antibodies (IgG and IgM) as IgM antibodies. Virus neutralizing (Nt) activity of these samples showed a significant correlation with CMV IgG1 antibodies. Nine samples of plasma and IGs were further evaluated for Nt activity of IgG1 and IgG3 antibodies by separating IgG3 from the rest of the antibodies with protein A agarose. IgG3 antibodies showed much higher Nt activity than IgG1 antibodies suggesting that enrichment of IgG3 antibodies in IG preparations may be useful in preparing CMV specific IG.

  12. 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.

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

    PubMed

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

    2016-09-01

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

  14. An enzyme immunoassay based micro-neutralization test for titration of antibodies to human cytomegalovirus (CMV) and its correlation with direct ELISA measuring CMV IgG antibodies.

    PubMed

    Gupta, C K; Leszczynski, J; Gupta, R K; Siber, G R

    1996-03-01

    An ELISA-based micro-neutralization (Nt) test in MRC-5 cells for titration of neutralizing antibodies against human cytomegalovirus (CMV) in human plasma and preparations of immune globulins was developed to eliminate microscopic reading of cytopathic effect (CPE), a process that is subjective and time consuming. Un-neutralized CMV from the Nt reaction and grown in MRC-5 cells as per the standard micro-Nt test was coated in the same plates by various methods and CMV antigen was quantified by polyclonal or monoclonal CMV antibodies. Optimal coating of plates with CMV antigen (100 TCID50 of virus grown on MRC-5 cells for 7 days) was obtained by freezing/thawing of virus infected MRC-5 cells in phosphate buffered saline, ph 7.2. The CMV antigen treated sequentially with CMV monoclonal antibody to late nuclear protein antigen, goat anti-mouse IgG3 alkaline phosphatase conjugate and phosphatase substrate gave an absorbance of 1 at 410 nm wavelength whereas uninfected MRC-5 cells treated under similar conditions did not show any absorbance. The optimal Nt reaction occurred at 37 degrees C for 1-2 h and was unaffected by complement. At 4 degrees C, CMV was inactivated in 1-2 h. The antibody titres were affected by the virus dose used in the Nt test over a range of 20 to 798 TCID50. When the titre was determined against a reference serum, the effect of virus dose on the Nt titre was reduced. Complete neutralization virus read microscopically correlated with ELISA absorbance of < 0.1. CPE produced by approximately 1 TCID50 of CMV showed an absorbance of 0.1 or more. The correlation coefficient (r) between Nt titres and CMV IgG antibodies determined by ELISA was 0.69 (P < 0.001) for 257 human plasma samples and 0.85 (P < 0.001) for 50 immune globulin preparations.

  15. Cross-neutralizing antibodies elicited by the Cervarix® human papillomavirus vaccine display a range of Alpha-9 inter-type specificities.

    PubMed

    Bissett, Sara L; Draper, Eve; Myers, Richard E; Godi, Anna; Beddows, Simon

    2014-02-26

    The highly efficacious human papillomavirus (HPV) vaccines contain virus-like particles (VLP) representing genotypes HPV16 and HPV18, which together account for approximately 70% of cervical cancer cases. Vaccine-type protection is thought to be mediated by high titer, type-specific neutralizing antibodies. The vaccines also confer a degree of cross-protection against some genetically-related types from the Alpha-9 (HPV16-like: HPV31, HPV33, HPV35, HPV52, HPV58) and Alpha-7 (HPV18-like: HPV39, HPV45, HPV59, HPV68) species groups. Cross-protection is coincident with the detection of low titer serum responses against non-vaccine types by vaccinees. Such antibodies may be the effectors of cross-protection or their detection may be useful as a correlate or surrogate. This study evaluated whether cross-neutralization of HPV types from the Alpha-9 species group is mediated by antibodies with a predominantly type-restricted specificity for HPV16 that nevertheless exhibit low affinity interactions with non-vaccine types, or by antibody specificities that demonstrate similar recognition of vaccine and non-vaccine types but are present at very low levels. Antibodies generated following Cervarix® vaccination of 13-14 year old girls were evaluated by pseudovirus neutralization, VLP ELISA and by enrichment of target antigen specificity using VLP-immobilized beads. Two-dimensional hierarchical clustering of serology data demonstrated that the antibody specificity profile generated by VLP ELISA was both quantitatively and qualitatively different from the neutralizing antibody specificity profile. Target-specific antibody enrichment demonstrated that cross-neutralization of non-vaccine types was due to a minority of antibodies rather than by the weak interactions of a predominantly type-restricted HPV16 antibody specificity. Furthermore, cross-neutralization of non-vaccine types appeared to be mediated by multiple antibody specificities, recognizing single and multiple non

  16. Use of a resin-bound synthetic peptide for identifying a neutralizing antigenic determinant associated with the human immunodeficiency virus envelope.

    PubMed

    Kennedy, R C; Dreesman, G R; Chanh, T C; Boswell, R N; Allan, J S; Lee, T H; Essex, M; Sparrow, J T; Ho, D D; Kanda, P

    1987-04-25

    A polyamide-based solid-phase support containing an acid-stable p-(oxymethyl)benzoic acid handle to anchor the COOH-terminal amino acid was utilized in the production of synthetic peptides analogous to amino acid sequences 503-532 from the human immunodeficiency virus (HIV) envelope glycoprotein. The resin-bound peptide was used to induce an antibody response to the native form of glycoprotein 120 in both rabbits and mice. This epitope was detected on the surface of HIV-infected cells and was capable of inducing an in vitro neutralizing HIV antibody response. In addition, sera from some individuals exposed to HIV react with this peptide bound to the resin in a solid-phase immunoassay. These data indicate that we have identified a neutralizing antigenic determinant present on the amino-terminal glycoprotein 120 subunits of HIV by utilizing resin-bound synthetic peptides.

  17. Augmentation of the Lipopolysaccharide-Neutralizing Activities of Human Cathelicidin CAP18/LL-37-Derived Antimicrobial Peptides by Replacement with Hydrophobic and Cationic Amino Acid Residues

    PubMed Central

    Nagaoka, Isao; Hirota, Satoko; Niyonsaba, François; Hirata, Michimasa; Adachi, Yoshiyuki; Tamura, Hiroshi; Tanaka, Shigenori; Heumann, Didier

    2002-01-01

    Mammalian myeloid and epithelial cells express various peptide antibiotics (such as defensins and cathelicidins) that contribute to the innate host defense against invading microorganisms. Among these peptides, human cathelicidin CAP18/LL-37 (L1 to S37) possesses not only potent antibacterial activity against gram-positive and gram-negative bacteria but also the ability to bind to gram-negative lipopolysaccharide (LPS) and neutralize its biological activities. In this study, to develop peptide derivatives with improved LPS-neutralizing activities, we utilized an 18-mer peptide (K15 to V32) of LL-37 as a template and evaluated the activities of modified peptides by using the CD14+ murine macrophage cell line RAW 264.7 and the murine endotoxin shock model. By replacement of E16 and K25 with two L residues, the hydrophobicity of the peptide (18-mer LL) was increased, and by further replacement of Q22, D26, and N30 with three K residues, the cationicity of the peptide (18-mer LLKKK) was enhanced. Among peptide derivatives, 18-mer LLKKK displayed the most powerful LPS-neutralizing activity: it was most potent at binding to LPS, inhibiting the interaction between LPS and LPS-binding protein, and attaching to the CD14 molecule, thereby suppressing the binding of LPS to CD14+ cells and attenuating production of tumor necrosis factor alpha (TNF-α) by these cells. Furthermore, in the murine endotoxin shock model, 18-mer LLKKK most effectively suppressed LPS-induced TNF-α production and protected mice from lethal endotoxin shock. Together, these observations indicate that the LPS-neutralizing activities of the amphipathic human CAP18/LL-37-derived 18-mer peptide can be augmented by modifying its hydrophobicity and cationicity, and that 18-mer LLKKK is the most potent of the peptide derivatives, with therapeutic potential for gram-negative bacterial endotoxin shock. PMID:12204946

  18. 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.

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

    PubMed

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

    2011-10-01

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

  20. A fully human monoclonal antibody with novel binding epitope and excellent neutralizing activity to multiple human IFN-α subtypes: A candidate therapy for systemic lupus erythematosus.

    PubMed

    Du, Peng; Xu, Lei; Qiu, Weiyi; Zeng, Dadi; Yue, Junjie; Wang, Shuang; Huang, Peitang; Sun, Zhiwei

    2015-01-01

    Systemic lupus erythematosus (SLE) is a chronic, heterogeneous autoimmune disease short of effective therapeutic agents. A multitude of studies of SLE in the last decade have accentuated a central role of the interferon alpha (IFN-α) pathway in SLE pathogenesis. We report here a candidate therapeutic neutralizing antibody, AIA22, with a different binding epitope and discrepant neutralizing profile from the anti-multiple IFN-α subtype antibodies currently in clinical trials. AIA22 specifically interacts with multiple IFN-α subtypes, binds to the type I IFN receptor 2 (IFNAR2) recognition region of IFN-α (considered a novel antigen epitope), and effectively neutralizes the activity of almost all of the IFN-α subtypes (with the exception of IFN-α7) both in vitro and in vivo. Concurrently, structural modeling and computational design yielded a mutational antibody of AIA22, AIAmut, which exhibited substantially improved neutralizing activity to multiple IFN-α subtypes.

  1. Neutral Atmospheres

    NASA Astrophysics Data System (ADS)

    Mueller-Wodarg, I. C. F.; Strobel, D. F.; Moses, J. I.; Waite, J. H.; Crovisier, J.; Yelle, R. V.; Bougher, S. W.; Roble, R. G.

    This paper summarizes the understanding of aeronomy of neutral atmospheres in the solar system, discussing most planets as well as Saturn's moon Titan and comets. The thermal structure and energy balance is compared, highlighting the principal reasons for discrepancies amongst the atmospheres, a combination of atmospheric composition, heliocentric distance and other external energy sources not common to all. The composition of atmospheres is discussed in terms of vertical structure, chemistry and evolution. The final section compares dynamics in the upper atmospheres of most planets and highlights the importance of vertical dynamical coupling as well as magnetospheric forcing in auroral regions, where present. It is shown that a first order understanding of neutral atmospheres has emerged over the past decades, thanks to the combined effects of spacecraft and Earth-based observations as well as advances in theoretical modeling capabilities. Key gaps in our understanding are highlighted which ultimately call for a more comprehensive programme of observation and laboratory measurements.

  2. Neutral Atmospheres

    NASA Astrophysics Data System (ADS)

    Mueller-Wodarg, I. C. F.; Strobel, D. F.; Moses, J. I.; Waite, J. H.; Crovisier, J.; Yelle, R. V.; Bougher, S. W.; Roble, R. G.

    2008-08-01

    This paper summarizes the understanding of aeronomy of neutral atmospheres in the solar system, discussing most planets as well as Saturn’s moon Titan and comets. The thermal structure and energy balance is compared, highlighting the principal reasons for discrepancies amongst the atmospheres, a combination of atmospheric composition, heliocentric distance and other external energy sources not common to all. The composition of atmospheres is discussed in terms of vertical structure, chemistry and evolution. The final section compares dynamics in the upper atmospheres of most planets and highlights the importance of vertical dynamical coupling as well as magnetospheric forcing in auroral regions, where present. It is shown that a first order understanding of neutral atmospheres has emerged over the past decades, thanks to the combined effects of spacecraft and Earth-based observations as well as advances in theoretical modeling capabilities. Key gaps in our understanding are highlighted which ultimately call for a more comprehensive programme of observation and laboratory measurements.

  3. Inhibition of in vivo HIV infection in humanized mice by gene therapy of human hematopoietic stem cells with a lentiviral vector encoding a broadly neutralizing anti-HIV antibody.

    PubMed

    Joseph, Aviva; Zheng, Jian Hua; Chen, Ken; Dutta, Monica; Chen, Cindy; Stiegler, Gabriela; Kunert, Renate; Follenzi, Antonia; Goldstein, Harris

    2010-07-01

    Due to the inherent immune evasion properties of the HIV envelope, broadly neutralizing HIV-specific antibodies capable of suppressing HIV infection are rarely produced by infected individuals. We examined the feasibility of utilizing genetic engineering to circumvent the restricted capacity of individuals to endogenously produce broadly neutralizing HIV-specific antibodies. We constructed a single lentiviral vector that encoded the heavy and light chains of 2G12, a broadly neutralizing anti-HIV human antibody, and that efficiently transduced and directed primary human B cells to secrete 2G12. To evaluate the capacity of this approach to provide protection from in vivo HIV infection, we used the humanized NOD/SCID/gamma(c)(null) mouse model, which becomes populated with human B cells, T cells, and macrophages after transplantation with human hematopoietic stem cells (hu-HSC) and develops in vivo infection after inoculation with HIV. The plasma of the irradiated NOD/SCID/gamma(c)(null) mice transplanted with hu-HSC transduced with the 2G12-encoding lentivirus contained 2G12 antibody, likely secreted by progeny human lymphoid and/or myeloid cells. After intraperitoneal inoculation with high-titer HIV-1(JR-CSF), mice engrafted with 2G12-transduced hu-HSC displayed marked inhibition of in vivo HIV infection as manifested by a profound 70-fold reduction in plasma HIV RNA levels and an almost 200-fold reduction in HIV-infected human cell numbers in mouse spleens, compared to control hu-HSC-transplanted NOD/SCID/gamma(c)(null) mice inoculated with equivalent high-titer HIV-1(JR-CSF). These results support the potential efficacy of this new gene therapy approach of using lentiviral vectors encoding a mixture of broadly neutralizing HIV antibodies for the treatment of HIV infection, particularly infection with multiple-drug-resistant isolates.

  4. 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.

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

    PubMed Central

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

    2011-01-01

    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. PMID:21939712

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

    PubMed

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

    2016-07-01

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

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

    PubMed

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

    2016-07-01

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

  8. 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.

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

    PubMed

    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

    2010-11-01

    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.

  10. 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.

  11. Neutralizing Polyclonal IgG Present during Acute Infection Prevents Rapid Disease Onset in Simian-Human Immunodeficiency Virus SHIVSF162P3-Infected Infant Rhesus Macaques

    PubMed Central

    Jaworski, J. Pablo; Kobie, James; Brower, Zachary; Malherbe, Delphine C.; Landucci, Gary; Sutton, William F.; Guo, Biwei; Reed, Jason S.; Leon, Enrique J.; Engelmann, Flora; Zheng, Bo; Legasse, Al; Park, Byung; Dickerson, Mary; Lewis, Anne D.; Colgin, Lois M. A.; Axthelm, Michael; Messaoudi, Ilhem; Sacha, Jonah B.; Burton, Dennis R.; Forthal, Donald N.; Hessell, Ann J.

    2013-01-01

    Simian-human immunodeficiency virus (SHIV) models for human immunodeficiency virus (HIV) infection have been widely used in passive studies with HIV neutralizing antibodies (NAbs) to test for protection against infection. However, because SHIV-infected adult macaques often rapidly control plasma viremia and any resulting pathogenesis is minor, the model has been unsuitable for studying the impact of antibodies on pathogenesis in infected animals. We found that SHIVSF162P3 infection in 1-month-old rhesus macaques not only results in high persistent plasma viremia but also leads to very rapid disease progression within 12 to 16 weeks. In this model, passive transfer of high doses of neutralizing IgG (SHIVIG) prevents infection. Here, we show that at lower doses, SHIVIG reduces both plasma and peripheral blood mononuclear cell (PBMC)-associated viremia and mitigates pathogenesis in infected animals. Moreover, production of endogenous NAbs correlated with lower set-point viremia and 100% survival of infected animals. New SHIV models are needed to investigate whether passively transferred antibodies or antibodies elicited by vaccination that fall short of providing sterilizing immunity impact disease progression or influence immune responses. The 1-month-old rhesus macaque SHIV model of infection provides a new tool to investigate the effects of antibodies on viral replication and clearance, mechanisms of B cell maintenance, and the induction of adaptive immunity in disease progression. PMID:23885083

  12. Strain specificity and binding affinity requirements of neutralizing monoclonal antibodies to the C4 domain of gp120 from human immunodeficiency virus type 1.

    PubMed Central

    Nakamura, G R; Byrn, R; Wilkes, D M; Fox, J A; Hobbs, M R; Hastings, R; Wessling, H C; Norcross, M A; Fendly, B M; Berman, P W

    1993-01-01

    The binding properties of seven CD4-blocking monoclonal antibodies raised against recombinant gp120 of human immunodeficiency virus type 1 strain MN (HIV-1MN) and two CD4-blocking monoclonal antibodies to recombinant envelope glycoproteins gp120 and gp160 of substrain IIIB of HIVLAI were analyzed. With a panel of recombinant gp120s from seven diverse HIV-1 isolates, eight of the nine antibodies were found to be strain specific and one was broadly cross-reactive. Epitope mapping revealed that all nine antibodies bound to epitopes located in the fourth conserved domain (C4) of gp120. Within this region, three distinct epitopes could be identified: two were polymorphic between HIV-1 strains, and one was highly conserved. Studies with synthetic peptides demonstrated that the conserved epitope, recognized by antibody 13H8, was located between residues 431 and 439. Site-directed mutagenesis of gp120 demonstrated that residue 429 and/or 432 was critical for the binding of the seven antibodies to gp120 from HIV-1MN. Similarly, residues 423 and 429 were essential for the binding of monoclonal antibody 5C2 raised against gp120 from HIV-1IIIB. The amino acids located at positions 423 and 429 were found to vary between strains of HIV-1 as well as between molecular clones derived from the MN and LAI isolates of HIV-1. Polymorphism at these positions prevented the binding of virus-neutralizing monoclonal antibodies and raised the possibility that HIV-1 neutralization serotypes may be defined on the basis of C4 domain sequences. Analysis of the binding characteristics of the CD4-blocking antibodies demonstrated that their virus-neutralizing activity was directly proportional to their gp120-binding affinity. These studies account for the strain specificity of antibodies to the C4 domain of gp120 and demonstrate for the first time that antibodies to this region can be as effective as those directed to the principal neutralizing determinant (V3 domain) in neutralizing HIV-1

  13. Characterization of neutralizing monoclonal antibodies to linear and conformation-dependent epitopes within the first and second variable domains of human immunodeficiency virus type 1 gp120.

    PubMed Central

    McKeating, J A; Shotton, C; Cordell, J; Graham, S; Balfe, P; Sullivan, N; Charles, M; Page, M; Bolmstedt, A; Olofsson, S

    1993-01-01

    A number of linear and conformation-dependent neutralizing monoclonal antibodies (MAbs) have been mapped to the first and second variable (V1 and V2) domains of human immunodeficiency virus type 1 (HIV-1) gp120. The majority of these MAbs are as effective at neutralizing HIV-1 infectivity as MAbs to the V3 domain and the CD4 binding site. The linear MAbs bind to amino acid residues 162 to 171, and changes at residues 183/184 (PI/SG) and 191/192/193 (YSL/GSS) within the V2 domain abrogate the binding of the two conformation-dependent MAbs, 11/68b and CRA-4, respectively. Surprisingly, a change at residue 435 (Y/H or Y/S), in a region of gp120 near the CD4 binding site (M. Kowalski, J. Potz, L. Basiripour, T. Dorfman, W. C. Goh, E. Terwilliger, A. Dayton, C. Rosen, W. Haseltine, and J. Sodroski, Science 237:1351-1355, 1987; L. A. Lasky, G. M. Nakamura, D. H. Smith, C. Fennie, C. Shimasaki, E. Patzer, P. Berman, T. Gregory, and D. Capon, Cell 50:975-985, 1987; and U. Olshevsky, E. Helseth, C. Furman, J. Li, W. Haseltine, and J. Sodroski, J. Virol. 64:5701-5707, 1990), abrogated gp120 recognition by both of the conformation-dependent MAbs. However, both MAbs 11/68b and CRA-4 were able to bind to HIV-1 V1V2 chimeric fusion proteins expressing the V1V2 domains in the absence of C4, suggesting that residues in C4 are not components of the epitopes but that amino acid changes in C4 may affect the structure of the V1V2 domains. This is consistent with the ability of soluble CD4 to block 11/68b and CRA-4 binding to both native cell surface-expressed gp120 and recombinant gp120 and suggests that the binding of the neutralizing MAbs to the virus occurs prior to receptor interaction. Since the reciprocal inhibition, i.e., antibody inhibition of CD4-gp120 binding, was not observed, the mechanism of neutralization is probably not a blockade of virus-receptor interaction. Finally, we demonstrate that linear sequences from the V2 region are immunogenic in HIV-1-infected individuals

  14. Cross-Reactive Human Immunodeficiency Virus Type 1-Neutralizing Human Monoclonal Antibody That Recognizes a Novel Conformational Epitope on gp41 and Lacks Reactivity against Self-Antigens ▿

    PubMed Central

    Zhang, Mei-Yun; Vu, Bang K.; Choudhary, Anil; Lu, Hong; Humbert, Michael; Ong, Helena; Alam, Munir; Ruprecht, Ruth M.; Quinnan, Gerald; Jiang, Shibo; Montefiori, David C.; Mascola, John R.; Broder, Christopher C.; Haynes, Barton F.; Dimitrov, Dimiter S.

    2008-01-01

    Broadly cross-reactive human immunodeficiency virus (HIV)-neutralizing antibodies are infrequently elicited in infected humans. The two best-characterized gp41-specific cross-reactive neutralizing human monoclonal antibodies, 4E10 and 2F5, target linear epitopes in the membrane-proximal external region (MPER) and bind to cardiolipin and several other autoantigens. It has been hypothesized that, because of such reactivity to self-antigens, elicitation of 2F5 and 4E10 and similar antibodies by vaccine immunogens based on the MPER could be affected by tolerance mechanisms. Here, we report the identification and characterization of a novel anti-gp41 monoclonal antibody, designated m44, which neutralized most of the 22 HIV type 1 (HIV-1) primary isolates from different clades tested in assays based on infection of peripheral blood mononuclear cells by replication-competent virus but did not bind to cardiolipin and phosphatidylserine in an enzyme-linked immunosorbent assay and a Biacore assay nor to any protein or DNA autoantigens tested in Luminex assays. m44 bound to membrane-associated HIV-1 envelope glycoproteins (Envs), to recombinant Envs lacking the transmembrane domain and cytoplasmic tail (gp140s), and to gp41 structures containing five-helix bundles and six-helix bundles, but not to N-heptad repeat trimers, suggesting that the C-heptad repeat is involved in m44 binding. In contrast to 2F5, 4E10, and Z13, m44 did not bind to any significant degree to denatured gp140 and linear peptides derived from gp41, suggesting a conformational nature of the epitope. This is the first report of a gp41-specific cross-reactive HIV-1-neutralizing human antibody that does not have detectable reactivity to autoantigens. Its novel conserved conformational epitope on gp41 could be helpful in the design of vaccine immunogens and as a target for therapeutics. PMID:18480433

  15. 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.

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

    PubMed Central

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

    2011-01-01

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

  17. 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.

  18. Antibody-Mediated Neutralization of Human Rhinovirus 14 Explored by Means of Cryoelectron Microscopy and X-Ray Crystallography of Virus-Fab Complexes

    PubMed Central

    Che, Zhiwei; Olson, Norman H.; Leippe, Donna; Lee, Wai-ming; Mosser, Anne G.; Rueckert, Roland R.; Baker, Timothy S.; Smith, Thomas J.

    1998-01-01

    The structures of three different human rhinovirus 14 (HRV14)-Fab complexes have been explored with X-ray crystallography and cryoelectron microscopy procedures. All three antibodies bind to the NIm-IA site of HRV14, which is the β-B–β-C loop of the viral capsid protein VP1. Two antibodies, Fab17-IA (Fab17) and Fab12-IA (Fab12), bind bivalently to the virion surface and strongly neutralize viral infectivity whereas Fab1-IA (Fab1) strongly aggregates and weakly neutralizes virions. The structures of the two classes of virion-Fab complexes clearly differ and correlate with observed binding neutralization differences. Fab17 and Fab12 bind in essentially identical, tangential orientations to the viral surface, which favors bidentate binding over icosahedral twofold axes. Fab1 binds in a more radial orientation that makes bidentate binding unlikely. Although the binding orientations of these two antibody groups differ, nearly identical charge interactions occur at all paratope-epitope interfaces. Nucleotide sequence comparisons suggest that Fab17 and Fab12 are from the same progenitor cell and that some of the differing residues contact the south wall of the receptor binding canyon that encircles each of the icosahedral fivefold vertices. All of the antibodies contact a significant proportion of the canyon region and directly overlap much of the receptor (intercellular adhesion molecule 1 [ICAM-1]) binding site. Fab1, however, does not contact the same residues on the upper south wall (the side facing away from fivefold axes) at the receptor binding region as do Fab12 and Fab17. All three antibodies cause some stabilization of HRV14 against pH-induced inactivation; thus, stabilization may be mediated by invariant contacts with the canyon. PMID:9573224

  19. H5N1 Whole-Virus Vaccine Induces Neutralizing Antibodies in Humans Which Are Protective in a Mouse Passive Transfer Model

    PubMed Central

    Howard, M. Keith; Sabarth, Nicolas; Savidis-Dacho, Helga; Portsmouth, Daniel; Kistner, Otfried; Kreil, Thomas R.; Ehrlich, Hartmut J.; Barrett, P. Noel

    2011-01-01

    Background Vero cell culture-derived whole-virus H5N1 vaccines have been extensively tested in clinical trials and consistently demonstrated to be safe and immunogenic; however, clinical efficacy is difficult to evaluate in the absence of wide-spread human disease. A lethal mouse model has been utilized which allows investigation of the protective efficacy of active vaccination or passive transfer of vaccine induced sera following lethal H5N1 challenge. Methods We used passive transfer of immune sera to investigate antibody-mediated protection elicited by a Vero cell-derived, non-adjuvanted inactivated whole-virus H5N1 vaccine. Mice were injected intravenously with H5N1 vaccine-induced rodent or human immune sera and subsequently challenged with a lethal dose of wild-type H5N1 virus. Results Passive transfer of H5N1 vaccine-induced mouse, guinea pig and human immune sera provided dose-dependent protection of recipient mice against lethal challenge with wild-type H5N1 virus. Protective dose fifty values for serum H5N1 neutralizing antibody titers were calculated to be ≤1∶11 for all immune sera, independently of source species. Conclusions These data underpin the confidence that the Vero cell culture-derived, whole-virus H5N1 vaccine will be effective in a pandemic situation and support the use of neutralizing serum antibody titers as a correlate of protection for H5N1 vaccines. PMID:21876771

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

    PubMed

    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

  1. 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

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

    PubMed

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

    2002-11-01

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

  3. On-line nano-HPLC/ESI QTOF MS and tandem MS for separation, detection, and structural elucidation of human erythrocytes neutral glycosphingolipid mixture.

    PubMed

    Kirsch, Stephan; Zarei, Mostafa; Cindrić, Mario; Müthing, Johannes; Bindila, Laura; Peter-Katalinić, Jasna

    2008-06-15

    A superior approach involving nano-high-performance liquid chromatography (nano-HPLC) in on-line conjunction to electrospray ionization quadrupole time-of-flight mass spectrometry (ESI QTOF MS) and tandem MS for screening and structural characterization of complex mixtures of neutral glycosphingolipids (GSLs) is here described. Neutral GSLs purified from human erythrocytes were efficiently separated according to the differences in carbohydrate chain length by an optimized nano-HPLC protocol and flow-through detected by ESI QTOF MS at the low femtomole level. Additionally, GSL species were accurately distinguished from the accompanying lipids in the mixture, thus permitting the determination of detailed structural characteristics by data-dependent analysis for identification of GSL constitution within single experiments. An alternative nano-HPLC/ESI QTOF MS approach was designed for dissection of unsaturation/saturation degree of the ceramide moieties defining the hydrophobic portion of GSLs and subsequent localization by nano-HPLC/ESI QTOF MS/MS of the -CH=CH- within the ceramide regions. The method is fast, highly sensitive, and high-throughput amenable and is highlighted as a new and valuable analytical dimension in glycolipidomics.

  4. Interspecies hybrid HbS: complete neutralization of Val6(beta)-dependent polymerization of human beta-chain by pig alpha-chains.

    PubMed

    Rao, M J; Malavalli, A; Manjula, B N; Kumar, R; Prabhakaran, M; Sun, D P; Ho, N T; Ho, C; Nagel, R L; Acharya, A S

    2000-07-28

    Interspecies hybrid HbS (alpha(2)(P)beta(2)(S)), has been assembled in vitro from pig alpha-globin and human beta(S)-chain. The alpha(2)(P)beta(2)(S) retains normal tetrameric structure (alpha(2)beta(2)) of human Hb and an O(2) affinity comparable to that of HbS in 50 mM Hepes buffer; but, its O(2) affinity is slightly higher than that of HbS in the presence of allosteric effectors (chloride, DPG and phosphate). The (1)H-NMR spectroscopy detected distinct differences between the heme environments and alpha(1)beta(1) interfaces of pig Hb and HbS, while their alpha(1)beta(2) interfaces appear very similar. The interspecies hybrid alpha(2)(H)beta(2)(P) resembles pig Hb; the pig beta-chain dictated the conformation of the heme environment of the human alpha-subunit, and to the alpha(1)beta(1) interfaces of the hybrid. In the alpha(2)(P)beta(2)(S) hybrid, beta(S)-chain dictated the conformation of human heme environment to the pig alpha-chain in the hybrid; but the conformation of alpha(1)beta(1) interface of this hybrid is close to, but not identical to that of HbS. On the other hand, the alpha(1)beta(2) interface conformation is identical to that of HbS. More important, the alpha(2)(P)beta(2)(S) does not polymerize when deoxygenated; pig alpha-chain completely neutralizes the beta(S)-chain dependent polymerization. The polymerization inhibitory propensity of pig alpha-chain is higher when it is present in the cis alpha(P)beta(S) dimer relative to that in a trans alpha(P)beta(A) dimer. The semisynthetically generated chimeric pig-human and human-pig alpha-chains by exchanging the alpha(1-30) segments of human and pig alpha-chains have established that the sequence differences of pig alpha(31-141) segment can also completely neutralize the polymerization. Comparison of the electrostatic potential energy landscape of the alpha-chain surfaces of HbS and alpha(2)(P)beta(2)(S) suggests that the differences in electrostatic potential energy surfaces on the alpha-chain of

  5. 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; Marcelino, José Maria; Bártolo, Inês; Rosado, Lino; Cavaco-Silva, Patrícia; Gomes, Perpétua; Família, Carlos; Quintas, Alexandre; et al

    2013-10-24

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

  6. 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

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

    PubMed Central

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

    2015-01-01

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

  8. 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.

  9. 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; Nicely, Nathan I.; Liao, Hua -Xin; Pollara, Justin; Liu, Pinghuang; Alam, S. Munir; Zhang, Ruijun; Cocklin, Sarah L.; et al

    2015-08-03

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

  10. 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

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

    PubMed Central

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

    2015-01-01

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

  12. 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.

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

    PubMed

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

    2012-07-01

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

  14. 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

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

    SciTech Connect

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

    1994-11-01

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

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

    PubMed

    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

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

    PubMed

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

    2012-07-01

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

  18. 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.

  19. A Rapid Immunization Strategy with a Live-Attenuated Tetravalent Dengue Vaccine Elicits Protective Neutralizing Antibody Responses in Non-Human Primates

    PubMed Central

    Ambuel, Yuping; Young, Ginger; Brewoo, Joseph N.; Paykel, Joanna; Weisgrau, Kim L.; Rakasz, Eva G.; Haller, Aurelia A.; Royals, Michael; Huang, Claire Y.-H.; Capuano, Saverio; Stinchcomb, Dan T.; Partidos, Charalambos D.; Osorio, Jorge E.

    2014-01-01

    Dengue viruses (DENVs) cause approximately 390 million cases of DENV infections annually and over 3 billion people worldwide are at risk of infection. No dengue vaccine is currently available nor is there an antiviral therapy for DENV infections. We have developed a tetravalent live-attenuated DENV vaccine tetravalent dengue vaccine (TDV) that consists of a molecularly characterized attenuated DENV-2 strain (TDV-2) and three chimeric viruses containing the pre-membrane and envelope genes of DENV-1, -3, and -4 expressed in the context of the TDV-2 genome. To impact dengue vaccine delivery in endemic areas and immunize travelers, a simple and rapid immunization strategy (RIS) is preferred. We investigated RIS consisting of two full vaccine doses being administered subcutaneously or intradermally on the initial vaccination visit (day 0) at two different anatomical locations with a needle-free disposable syringe jet injection delivery devices (PharmaJet) in non-human primates. This vaccination strategy resulted in efficient priming and induction of neutralizing antibody responses to all four DENV serotypes comparable to those elicited by the traditional prime and boost (2 months later) vaccination schedule. In addition, the vaccine induced CD4+ and CD8+ T cells producing IFN-γ, IL-2, and TNF-α, and targeting the DENV-2 NS1, NS3, and NS5 proteins. Moreover, vaccine-specific T cells were cross-reactive with the non-structural NS3 and NS5 proteins of DENV-4. When animals were challenged with DENV-2 they were protected with no detectable viremia, and exhibited sterilizing immunity (no increase of neutralizing titers post-challenge). RIS could decrease vaccination visits and provide quick immune response to all four DENV serotypes. This strategy could increase vaccination compliance and would be especially advantageous for travelers into endemic areas. PMID:24926294

  20. 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.

  1. Phylogenetic Analysis of the Main Neutralization and Hemagglutination Determinants of All Human Adenovirus Prototypes as a Basis for Molecular Classification and Taxonomy

    PubMed Central

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

    2005-01-01

    Human adenoviruses (HAdV) are responsible for a wide spectrum of diseases. The neutralization ɛ determinant (loops 1 and 2) and the hemagglutination γ determinant are relevant for the taxonomy of HAdV. Precise type identification of HAdV prototypes is crucial for detection of infection chains and epidemiology. ɛ and γ determinant sequences of all 51 HAdV were generated to propose molecular classification criteria. Phylogenetic analysis of ɛ 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 (γ 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 (ɛ determinant) and a speculative, not-yet-isolated HAdV prototype (γ determinant). Subanalysis of molecular evolution in hypervariable regions 1 to 6 of the ɛ determinant indicated different selective pressures in subclusters of species HAdV-D. Additionally, γ 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. PMID:16306598

  2. A non-VH1-69 heterosubtypic neutralizing human monoclonal antibody protects mice against H1N1 and H5N1 viruses.

    PubMed

    De Marco, Donata; Clementi, Nicola; Mancini, Nicasio; Solforosi, Laura; Moreno, Guisella J; Sun, Xiangjie; Tumpey, Terrence M; Gubareva, Larisa V; Mishin, Vasiliy; Clementi, Massimo; Burioni, Roberto

    2012-01-01

    Influenza viruses are among the most important human pathogens and are responsible for annual epidemics and sporadic, potentially devastating pandemics. The humoral immune response plays an important role in the defense against these viruses, providing protection mainly by producing antibodies directed against the hemagglutinin (HA) glycoprotein. However, their high genetic variability allows the virus to evade the host immune response and the potential protection offered by seasonal vaccines. The emergence of resistance to antiviral drugs in recent years further limits the options available for the control of influenza. The development of alternative strategies for influenza prophylaxis and therapy is therefore urgently needed. In this study, we describe a human monoclonal antibody (PN-SIA49) that recognizes a highly conserved epitope located on the stem region of the HA and able to neutralize a broad spectrum of influenza viruses belonging to different subtypes (H1, H2 and H5). Furthermore, we describe its protective activity in mice after lethal challenge with H1N1 and H5N1 viruses suggesting a potential application in the treatment of influenza virus infections. PMID:22496802

  3. Fine definition of the epitope on the gp41 glycoprotein of human immunodeficiency virus type 1 for the neutralizing monoclonal antibody 2F5.

    PubMed

    Parker, C E; Deterding, L J; Hager-Braun, C; Binley, J M; Schülke, N; Katinger, H; Moore, J P; Tomer, K B

    2001-11-01

    Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), in combination with proteolytic protection assays, has been used to identify the functional epitope on human immunodeficiency virus envelope glycoprotein gp41 for the broadly neutralizing anti-gp41 human monoclonal antibody 2F5. In this protection assay-based procedure, a soluble gp140 protein with a stabilizing intermolecular disulfide bond between the gp120 and gp41 subunits (SOS gp140) was affinity bound to immobilized 2F5 under physiological conditions. A combination of proteolytic enzymatic cleavages was then performed to remove unprotected residues. Residues of SOS gp140 protected by their binding to 2F5 were then identified based on their molecular weights as determined by direct MALDI-MS of the immobilized antibody beads. The epitope, NEQELLELDKWASLWN, determined by this MALDI-MS protection assay approach consists of 16 amino acid residues near the C terminus of gp41. It is significantly longer than the ELDKWA core epitope previously determined for 2F5 by peptide enzyme-linked immunosorbent assay. This new knowledge of the structure of the 2F5 epitope may facilitate the design of vaccine antigens intended to induce antibodies with the breadth and potency of action of the 2F5 monoclonal antibody. PMID:11602730

  4. Neutralization of Chlamydia trachomatis in cell culture.

    PubMed Central

    Howard, L V

    1975-01-01

    Neutralization of Chlamydia trachomatis was assayed by the decrease in inclusion-forming units in baby hamster kidney cells grown in culture. Five percent fresh guinea pig sera increased neutralization titers of rabbit antisera 100- to 1,000-fold but had no effect when normal rabbit sera were tested. Neutralization of a type A or B trachoma isolate was strain specific. Neutralization by human eye secretions and sera also was demonstrated when guinea pig sera were included in the test. All of the six human sera tested showed strain specificity against types A or B, in agreement with typing by the fluorescent antibody technique. PMID:1091549

  5. Five birds, one stone: neutralization of α-hemolysin and 4 bi-component leukocidins of Staphylococcus aureus with a single human monoclonal antibody.

    PubMed

    Rouha, Harald; Badarau, Adriana; Visram, Zehra C; Battles, Michael B; Prinz, Bianka; Magyarics, Zoltán; Nagy, Gábor; Mirkina, Irina; Stulik, Lukas; Zerbs, Manuel; Jägerhofer, Michaela; Maierhofer, Barbara; Teubenbacher, Astrid; Dolezilkova, Ivana; Gross, Karin; Banerjee, Srijib; Zauner, Gerhild; Malafa, Stefan; Zmajkovic, Jakub; Maier, Sabine; Mabry, Robert; Krauland, Eric; Wittrup, K Dane; Gerngross, Tillman U; Nagy, Eszter

    2015-01-01

    Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component leukocidins that share only ∼26% overall amino acid sequence identity. The molecular basis of cross-reactivity is the recognition of a conformational epitope shared by α-hemolysin and F-components of gamma-hemolysin (HlgAB and HlgCB), LukED and LukSF (Panton-Valentine Leukocidin). The amino acids predicted to form the epitope are conserved and known to be important for cytotoxic activity. We found that a single cross-reactive antibody prevented lysis of human phagocytes, epithelial and red blood cells induced by α-hemolysin and leukocidins in vitro, and therefore had superior effectiveness compared to α-hemolysin specific antibodies to protect from the combined cytolytic effect of secreted S. aureus toxins. Such mAb afforded high levels of protection in murine models of pneumonia and sepsis.

  6. Five birds, one stone: Neutralization of α-hemolysin and 4 bi-component leukocidins of Staphylococcus aureus with a single human monoclonal antibody

    PubMed Central

    Rouha, Harald; Badarau, Adriana; Visram, Zehra C; Battles, Michael B; Prinz, Bianka; Magyarics, Zoltán; Nagy, Gábor; Mirkina, Irina; Stulik, Lukas; Zerbs, Manuel; Jägerhofer, Michaela; Maierhofer, Barbara; Teubenbacher, Astrid; Dolezilkova, Ivana; Gross, Karin; Banerjee, Srijib; Zauner, Gerhild; Malafa, Stefan; Zmajkovic, Jakub; Maier, Sabine; Mabry, Robert; Krauland, Eric; Wittrup, K Dane; Gerngross, Tillman U; Nagy, Eszter

    2015-01-01

    Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component leukocidins that share only ∼26% overall amino acid sequence identity. The molecular basis of cross-reactivity is the recognition of a conformational epitope shared by α-hemolysin and F-components of gamma-hemolysin (HlgAB and HlgCB), LukED and LukSF (Panton-Valentine Leukocidin). The amino acids predicted to form the epitope are conserved and known to be important for cytotoxic activity. We found that a single cross-reactive antibody prevented lysis of human phagocytes, epithelial and red blood cells induced by α-hemolysin and leukocidins in vitro, and therefore had superior effectiveness compared to α-hemolysin specific antibodies to protect from the combined cytolytic effect of secreted S. aureus toxins. Such mAb afforded high levels of protection in murine models of pneumonia and sepsis. PMID:25523282

  7. 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.

  8. Five birds, one stone: neutralization of α-hemolysin and 4 bi-component leukocidins of Staphylococcus aureus with a single human monoclonal antibody.

    PubMed

    Rouha, Harald; Badarau, Adriana; Visram, Zehra C; Battles, Michael B; Prinz, Bianka; Magyarics, Zoltán; Nagy, Gábor; Mirkina, Irina; Stulik, Lukas; Zerbs, Manuel; Jägerhofer, Michaela; Maierhofer, Barbara; Teubenbacher, Astrid; Dolezilkova, Ivana; Gross, Karin; Banerjee, Srijib; Zauner, Gerhild; Malafa, Stefan; Zmajkovic, Jakub; Maier, Sabine; Mabry, Robert; Krauland, Eric; Wittrup, K Dane; Gerngross, Tillman U; Nagy, Eszter

    2015-01-01

    Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component leukocidins that share only ∼26% overall amino acid sequence identity. The molecular basis of cross-reactivity is the recognition of a conformational epitope shared by α-hemolysin and F-components of gamma-hemolysin (HlgAB and HlgCB), LukED and LukSF (Panton-Valentine Leukocidin). The amino acids predicted to form the epitope are conserved and known to be important for cytotoxic activity. We found that a single cross-reactive antibody prevented lysis of human phagocytes, epithelial and red blood cells induced by α-hemolysin and leukocidins in vitro, and therefore had superior effectiveness compared to α-hemolysin specific antibodies to protect from the combined cytolytic effect of secreted S. aureus toxins. Such mAb afforded high levels of protection in murine models of pneumonia and sepsis. PMID:25523282

  9. Mass spectrometric analysis of neutral and anionic N-glycans from a Dictyostelium discoideum model for human congenital disorder of glycosylation CDG IL.

    PubMed

    Hykollari, Alba; Balog, Crina I A; Rendić, Dubravko; Braulke, Thomas; Wilson, Iain B H; Paschinger, Katharina

    2013-03-01

    The HL241 mutant strain of the cellular slime mold Dictyostelium discoideum is a potential model for human congenital disorder of glycosylation type IL (ALG9-CDG) and has been previously predicted to possess a lower degree of modification of its N-glycans with anionic moieties than the parental wild-type. In this study, we first showed that this strain has a premature stop codon in its alg9 mannosyltransferase gene compatible with the occurrence of truncated N-glycans. These were subject to an optimized analytical workflow, considering that the mass spectrometry of acidic glycans often presents challenges due to neutral loss and suppression effects. Therefore, the protein-bound N-glycans were first fractionated, after serial enzymatic release, by solid phase extraction. Then primarily single glycan species were isolated by mixed hydrophilic-interaction/anion-exchange or reversed-phase HPLC and analyzed using chemical and enzymatic treatments and MS/MS. We show that protein-linked N-glycans of the mutant are of reduced size as compared to those of wild-type AX3, but still contain core α1,3-fucose, intersecting N-acetylglucosamine, bisecting N-acetylglucosamine, methylphosphate, phosphate, and sulfate residues. We observe that a single N-glycan can carry up to four of these six possible modifications. Due to the improved analytical procedures, we reveal fuller details regarding the N-glycomic potential of this fascinating model organism. PMID:23320427

  10. 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

  11. Effects of aspartame and carbohydrate administration on human and rat plasma large neutral amino acid levels and rat brain amino acid and monoamine levels.

    PubMed

    Romano, M; Casacci, F; De Marchi, F; Pacei, T; Esteve, A; Lomuscio, G; Mennini, T; Salmona, M

    1989-01-01

    Thirty fasted human volunteers were given 0.83 and 8.3 mg aspartame/kg body weight alone, as part of a basal low carbohydrate meal (648 kcal, 10% carbohydrate) or as part of a high energy carbohydrate-rich meal (1290 kcal, 34% carbohydrate). Amino acid concentrations in plasma were determined before and 30, 60 and 180 min after the consumption of aspartame. Under these conditions, which mimic realistic aspartame consumption, aspartame had no significant effect on plasma concentration of any amino acid. In addition, the effect of aspartame alone or with carbohydrates on plasma and brain amino acid levels was studied in rats after acute or subacute (14 d) oral treatment. In subacute dosing experiments aspartame was included in the diet. Brain monoamine concentrations were also measured in the same animals. Plasma concentrations of large neutral amino acids were modified under acute conditions. In contrast, after subacute treatment no significant differences in plasma or brain amino acid concentrations or in brain monoamine concentrations were observed.

  12. 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

  13. Ophiophagus hannah venom: proteome, components bound by Naja kaouthia antivenin and neutralization by N. kaouthia neurotoxin-specific human ScFv.

    PubMed

    Danpaiboon, Witchuda; Reamtong, Onrapak; Sookrung, Nitat; Seesuay, Watee; Sakolvaree, Yuwaporn; Thanongsaksrikul, Jeeraphong; Dong-din-on, Fonthip; Srimanote, Potjanee; Thueng-in, Kanyarat; Chaicumpa, Wanpen

    2014-05-01

    Venomous snakebites are an important health problem in tropical and subtropical countries. King cobra (Ophiophagus hannah) is the largest venomous snake found in South and Southeast Asia. In this study, the O. hannah venom proteome and the venom components cross-reactive to N. kaouthia monospecific antivenin were studied. O. hannah venom consisted of 14 different protein families, including three finger toxins, phospholipases, cysteine-rich secretory proteins, cobra venom factor, muscarinic toxin, L-amino acid oxidase, hypothetical proteins, low cysteine protein, phosphodiesterase, proteases, vespryn toxin, Kunitz, growth factor activators and others (coagulation factor, endonuclease, 5'-nucleotidase). N. kaouthia antivenin recognized several functionally different O. hannah venom proteins and mediated paratherapeutic efficacy by rescuing the O. hannah envenomed mice from lethality. An engineered human ScFv specific to N. kaouthia long neurotoxin (NkLN-HuScFv) cross-neutralized the O. hannah venom and extricated the O. hannah envenomed mice from death in a dose escalation manner. Homology modeling and molecular docking revealed that NkLN-HuScFv interacted with residues in loops 2 and 3 of the neurotoxins of both snake species, which are important for neuronal acetylcholine receptor binding. The data of this study are useful for snakebite treatment when and where the polyspecific antivenin is not available. Because the supply of horse-derived antivenin is limited and the preparation may cause some adverse effects in recipients, a cocktail of recombinant human ScFvs for various toxic venom components shared by different venomous snakes, exemplified by the in vitro produced NkLN-HuScFv in this study, should contribute to a possible future route for an improved alternative to the antivenins. PMID:24828754

  14. Ophiophagus hannah venom: proteome, components bound by Naja kaouthia antivenin and neutralization by N. kaouthia neurotoxin-specific human ScFv.

    PubMed

    Danpaiboon, Witchuda; Reamtong, Onrapak; Sookrung, Nitat; Seesuay, Watee; Sakolvaree, Yuwaporn; Thanongsaksrikul, Jeeraphong; Dong-din-on, Fonthip; Srimanote, Potjanee; Thueng-in, Kanyarat; Chaicumpa, Wanpen

    2014-05-01

    Venomous snakebites are an important health problem in tropical and subtropical countries. King cobra (Ophiophagus hannah) is the largest venomous snake found in South and Southeast Asia. In this study, the O. hannah venom proteome and the venom components cross-reactive to N. kaouthia monospecific antivenin were studied. O. hannah venom consisted of 14 different protein families, including three finger toxins, phospholipases, cysteine-rich secretory proteins, cobra venom factor, muscarinic toxin, L-amino acid oxidase, hypothetical proteins, low cysteine protein, phosphodiesterase, proteases, vespryn toxin, Kunitz, growth factor activators and others (coagulation factor, endonuclease, 5'-nucleotidase). N. kaouthia antivenin recognized several functionally different O. hannah venom proteins and mediated paratherapeutic efficacy by rescuing the O. hannah envenomed mice from lethality. An engineered human ScFv specific to N. kaouthia long neurotoxin (NkLN-HuScFv) cross-neutralized the O. hannah venom and extricated the O. hannah envenomed mice from death in a dose escalation manner. Homology modeling and molecular docking revealed that NkLN-HuScFv interacted with residues in loops 2 and 3 of the neurotoxins of both snake species, which are important for neuronal acetylcholine receptor binding. The data of this study are useful for snakebite treatment when and where the polyspecific antivenin is not available. Because the supply of horse-derived antivenin is limited and the preparation may cause some adverse effects in recipients, a cocktail of recombinant human ScFvs for various toxic venom components shared by different venomous snakes, exemplified by the in vitro produced NkLN-HuScFv in this study, should contribute to a possible future route for an improved alternative to the antivenins.

  15. Structural Characterization of Humanized Nanobodies with Neutralizing Activity against the Bordetella pertussis CyaA-Hemolysin: Implications for a Potential Epitope of Toxin-Protective Antigen.

    PubMed

    Malik, Aijaz Ahmad; Imtong, Chompounoot; Sookrung, Nitat; Katzenmeier, Gerd; Chaicumpa, Wanpen; Angsuthanasombat, Chanan

    2016-04-01

    Previously, the 126-kDa CyaA-hemolysin (CyaA-Hly) fragment cloned from Bordetella pertussis--the causative agent of whooping cough--and functionally expressed in Escherichia coli was revealed as a key determinant for CyaA-mediated hemolysis against target erythrocytes. Here, phagemid-transfected E. coli clones producing nanobodies capable of binding to CyaA-Hly were selected from a humanized-camel VH/VHH phage-display library. Subsequently verified for binding activities by indirect ELISA and Western blotting, four CyaA-Hly-specific nanobodies were obtained and designated according to the presence/absence of VHH-hallmark amino acids as VHH2, VH5, VH18 and VHH37. In vitro neutralization assay revealed that all four ~17-kDa His-tagged VH/VHH nanobodies, in particular VHH37, which were over-expressed as inclusions and successfully unfolded-refolded, were able to effectively inhibit CyaA-Hly-mediated hemolysis. Phage-mimotope searching revealed that only peptides with sequence homologous to Linker 1 connecting Blocks I and II within the CyaA-RTX subdomain were able to bind to these four CyaA-Hly-specific nanobodies. Structural analysis of VHH37 via homology modeling and intermolecular docking confirmed that this humanized nanobody directly interacts with CyaA-RTX/Linker 1 through multiple hydrogen and ionic bonds. Altogether, our present data demonstrate that CyaA-RTX/Linker 1 could serve as a potential epitope of CyaA-protective antigen that may be useful for development of peptide-based pertussis vaccines. Additionally, such toxin-specific nanobodies have a potential for test-driven development of a ready-to-use therapeutic in passive immunization for mitigation of disease severity.

  16. Ophiophagus hannah Venom: Proteome, Components Bound by Naja kaouthia Antivenin and Neutralization by N. kaouthia Neurotoxin-Specific Human ScFv

    PubMed Central

    Danpaiboon, Witchuda; Reamtong, Onrapak; Sookrung, Nitat; Seesuay, Watee; Sakolvaree, Yuwaporn; Thanongsaksrikul, Jeeraphong; Dong-din-on, Fonthip; Srimanote, Potjanee; Thueng-in, Kanyarat; Chaicumpa, Wanpen

    2014-01-01

    Venomous snakebites are an important health problem in tropical and subtropical countries. King cobra (Ophiophagus hannah) is the largest venomous snake found in South and Southeast Asia. In this study, the O. hannah venom proteome and the venom components cross-reactive to N. kaouthia monospecific antivenin were studied. O. hannah venom consisted of 14 different protein families, including three finger toxins, phospholipases, cysteine-rich secretory proteins, cobra venom factor, muscarinic toxin, L-amino acid oxidase, hypothetical proteins, low cysteine protein, phosphodiesterase, proteases, vespryn toxin, Kunitz, growth factor activators and others (coagulation factor, endonuclease, 5’-nucleotidase). N. kaouthia antivenin recognized several functionally different O. hannah venom proteins and mediated paratherapeutic efficacy by rescuing the O. hannah envenomed mice from lethality. An engineered human ScFv specific to N. kaouthia long neurotoxin (NkLN-HuScFv) cross-neutralized the O. hannah venom and extricated the O. hannah envenomed mice from death in a dose escalation manner. Homology modeling and molecular docking revealed that NkLN-HuScFv interacted with residues in loops 2 and 3 of the neurotoxins of both snake species, which are important for neuronal acetylcholine receptor binding. The data of this study are useful for snakebite treatment when and where the polyspecific antivenin is not available. Because the supply of horse-derived antivenin is limited and the preparation may cause some adverse effects in recipients, a cocktail of recombinant human ScFvs for various toxic venom components shared by different venomous snakes, exemplified by the in vitro produced NkLN-HuScFv in this study, should contribute to a possible future route for an improved alternative to the antivenins. PMID:24828754

  17. Structural Characterization of Humanized Nanobodies with Neutralizing Activity against the Bordetella pertussis CyaA-Hemolysin: Implications for a Potential Epitope of Toxin-Protective Antigen.

    PubMed

    Malik, Aijaz Ahmad; Imtong, Chompounoot; Sookrung, Nitat; Katzenmeier, Gerd; Chaicumpa, Wanpen; Angsuthanasombat, Chanan

    2016-04-01

    Previously, the 126-kDa CyaA-hemolysin (CyaA-Hly) fragment cloned from Bordetella pertussis--the causative agent of whooping cough--and functionally expressed in Escherichia coli was revealed as a key determinant for CyaA-mediated hemolysis against target erythrocytes. Here, phagemid-transfected E. coli clones producing nanobodies capable of binding to CyaA-Hly were selected from a humanized-camel VH/VHH phage-display library. Subsequently verified for binding activities by indirect ELISA and Western blotting, four CyaA-Hly-specific nanobodies were obtained and designated according to the presence/absence of VHH-hallmark amino acids as VHH2, VH5, VH18 and VHH37. In vitro neutralization assay revealed that all four ~17-kDa His-tagged VH/VHH nanobodies, in particular VHH37, which were over-expressed as inclusions and successfully unfolded-refolded, were able to effectively inhibit CyaA-Hly-mediated hemolysis. Phage-mimotope searching revealed that only peptides with sequence homologous to Linker 1 connecting Blocks I and II within the CyaA-RTX subdomain were able to bind to these four CyaA-Hly-specific nanobodies. Structural analysis of VHH37 via homology modeling and intermolecular docking confirmed that this humanized nanobody directly interacts with CyaA-RTX/Linker 1 through multiple hydrogen and ionic bonds. Altogether, our present data demonstrate that CyaA-RTX/Linker 1 could serve as a potential epitope of CyaA-protective antigen that may be useful for development of peptide-based pertussis vaccines. Additionally, such toxin-specific nanobodies have a potential for test-driven development of a ready-to-use therapeutic in passive immunization for mitigation of disease severity. PMID:27043627

  18. 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.

  19. 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…

  20. Elimination of human T cell leukemia virus type-1-infected cells by neutralizing and antibody-dependent cellular cytotoxicity-inducing antibodies against human t cell leukemia virus type-1 envelope gp46.

    PubMed

    Tanaka, Yuetsu; Takahashi, Yoshiaki; Tanaka, Reiko; Kodama, Akira; Fujii, Hideki; Hasegawa, Atsuhiko; Kannagi, Mari; Ansari, Aftab A; Saito, Mineki

    2014-06-01

    Human T cell leukemia virus type-1 (HTLV-1) is prevalent worldwide with foci of high prevalence. However, to date no effective vaccine or drug against HTLV-1 infection has been developed. In efforts to define the role of antibodies in the control of HTLV-1 infection, we capitalized on the use of our previously defined anti-gp46 neutralizing monoclonal antibody (mAb) (clone LAT-27) and high titers of human anti-HTLV-1 IgG purified from HAM/TSP patients (HAM-IgG). LAT-27 and HAM-IgG completely blocked syncytium formation and T cell immortalization mediated by HTLV-1 in vitro. The addition of these antibodies to cultures of CD8(+) T cell-depleted peripheral blood mononuclear cells (PBMCs) from HAM/TSP patients at the initiation of culture not only decreased the numbers of Tax-expressing cells and the production of HTLV-1 p24 but also inhibited the spontaneous immortalization of T cells. Coculture of in vitro-HTLV-1-immortalized T cell lines with autologous PBMCs in the presence of LAT-27 or HAM-IgG, but not an F(ab')2 fragment of LAT-27 or nonneutralizing anti-gp46 mAbs, resulted in depletion of HTLV-1-infected cells. A 24-h (51)Cr release assay showed the presence of significant antibody-dependent cellular cytotoxicity (ADCC) activity in LAT-27 and HAM-IgG, but not F(ab')2 of LAT-27, resulting in the depletion of HTLV-1-infected T cells by autologous PBMCs. The depletion of natural killer (NK) cells from the effector PBMCs reduced this ADCC activity. Altogether, the present data demonstrate that the neutralizing and ADCC-inducing activities of anti-HTLV-1 antibodies are capable of reducing infection and eliminating HTLV-1-infected cells in the presence of autologous PBMCs. PMID:24524420

  1. Selection of affinity-improved neutralizing human scFv against HBV PreS1 from CDR3 VH/VL mutant library.

    PubMed

    Chen, YanMin; Bai, Yin; Guo, XiaoChen; Wang, WenFei; Zheng, Qi; Wang, FuXiang; Sun, Dejun; Li, DeShan; Ren, GuiPing; Yin, JieChao

    2016-07-01

    A CDR3 mutant library was constructed from a previously isolated anti-HBV neutralizing Homo sapiens scFv-31 template by random mutant primers PCR. Then the library was displayed on the inner membrane surface in Escherichia coli periplasmic space. Seven scFv clones were isolated from the mutant library through three rounds of screening by flow cytometry. Competition ELISA assay indicates that isolated scFv fragments show more efficient binding ability to HBV PreS1 compared with parental scFv-31. HBV neutralization assay indicated that two clones (scFv-3 and 59) show higher neutralizing activity by blocking the HBV infection to Chang liver cells. Our method provides a new strategy for rapid screening of mutant antibody library for affinity-enhanced scFv clones and the neutralizing scFvs obtained from this study provide a potential alternative of Hepatitis B immune globulin.

  2. 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

    2013-01-01

    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.

  3. Neutral beam monitoring

    DOEpatents

    Fink, Joel H.

    1981-08-18

    Method and apparatus for monitoring characteristics of a high energy neutral beam. A neutral beam is generated by passing accelerated ions through a walled cell containing a low energy neutral gas, such that charge exchange neutralizes the high energy ion beam. The neutral beam is monitored by detecting the current flowing through the cell wall produced by low energy ions which drift to the wall after the charge exchange. By segmenting the wall into radial and longitudinal segments various beam conditions are further identified.

  4. 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

  5. Identifying possible sites for antibody neutralization escape: Implications for unique functional properties of the C-terminal tail of Human Immunodeficiency Virus Type 1 gp41.

    PubMed

    Lu, Zhifeng; Huang, Yushen; Tan, Yue; Yu, Yang; Wang, Junyi; Chen, Ying-Hua

    2016-07-01

    A previous amino acid sequence analyses from our laboratory reported nine potential sites in gp41 glycoprotein of HIV-1 that may contribute to virus escape from antibody neutralization. Besides four sites found outside the membrane of HIV-1 virus, five located in the C-terminal tail of gp41 specifically in the lentivirus lytic peptides motifs (LLPs). To further study the bioinformatical results, the virus infectivity assay and the standard neutralization assay were conducted on conservatively mutated virus. Two sites in the LLP3 domain stood out with the ability to alter the resistance of HIV-1 virus to certain broadly neutralizing antibodies (bNAbs). While the glycoprotein incorporation on the viral membrane and the interaction of the LLP3 domain with the lipid membrane remained unaltered, the increase in neutralization resistance of the mutant virus was associated with the changes on Env conformation. Our findings demonstrate different sensibility of bNAbs to mutations in the C-terminal tail and indicate an unrecognized potential role for even minor sequence variation in the C-terminal tail in modulating the antigenicity of the ectodomain of HIV-1 envelope glycoprotein complex.

  6. Neutralizing activities of human immunoglobulin derived from donors in Japan against mosquito-borne flaviviruses, Japanese encephalitis virus, West Nile virus, and dengue virus

    PubMed Central

    Yunoki, Mikihiro; Kurosu, Takeshi; Koketsu, Ritsuko Kubota; Takahashi, Kazuo; Okuno, Yoshinobu; Ikuta, Kazuyoshi

    2016-01-01

    Japanese encephalitis virus (JEV), West Nile virus (WNV), and dengue virus (DenV) are causal agents of Japanese encephalitis, West Nile fever, and dengue fever, respectively. JEV is considered to be indigenized and widespread in Japan, whereas WNV and DenV are not indigenized in Japan. Globulin products seem to reflect the status of the donor population according to antivirus neutralization activity. However, the anti-JEV, -WNV, and -DenV neutralization activities of globulin products derived from donors in Japan have not been clarified. Furthermore, potential candidates for the development of an effective immunotherapeutic drug for encephalitis caused by JEV, WNV, or DenV have also not been identified. Therefore, the aim of this study was to determine the overall status of the donor population in Japan based on globulin products by evaluating anti-JEV, -WNV, and -DenV neutralizing activities of intravenous immunoglobulin. Overall, intravenous immunoglobulin products showed stable neutralizing activity against JEV but showed no or only weak activity against WNV or DenV. These results suggest that the epidemiological level against WNV and DenV in the donor population of Japan is still low, suggesting that these viruses are not yet indigenized. In addition, JEV vaccinations and/or infections in the donor population do not induce a cross-reactive antibody against WNV. PMID:27462140

  7. Neutralizing activities of human immunoglobulin derived from donors in Japan against mosquito-borne flaviviruses, Japanese encephalitis virus, West Nile virus, and dengue virus.

    PubMed

    Yunoki, Mikihiro; Kurosu, Takeshi; Koketsu, Ritsuko Kubota; Takahashi, Kazuo; Okuno, Yoshinobu; Ikuta, Kazuyoshi

    2016-01-01

    Japanese encephalitis virus (JEV), West Nile virus (WNV), and dengue virus (DenV) are causal agents of Japanese encephalitis, West Nile fever, and dengue fever, respectively. JEV is considered to be indigenized and widespread in Japan, whereas WNV and DenV are not indigenized in Japan. Globulin products seem to reflect the status of the donor population according to antivirus neutralization activity. However, the anti-JEV, -WNV, and -DenV neutralization activities of globulin products derived from donors in Japan have not been clarified. Furthermore, potential candidates for the development of an effective immunotherapeutic drug for encephalitis caused by JEV, WNV, or DenV have also not been identified. Therefore, the aim of this study was to determine the overall status of the donor population in Japan based on globulin products by evaluating anti-JEV, -WNV, and -DenV neutralizing activities of intravenous immunoglobulin. Overall, intravenous immunoglobulin products showed stable neutralizing activity against JEV but showed no or only weak activity against WNV or DenV. These results suggest that the epidemiological level against WNV and DenV in the donor population of Japan is still low, suggesting that these viruses are not yet indigenized. In addition, JEV vaccinations and/or infections in the donor population do not induce a cross-reactive antibody against WNV. PMID:27462140

  8. On neutral plasma oscillations

    SciTech Connect

    Shadwick, B.A.; Morrison, P.J.

    1993-06-01

    We examine the conditions for the existence of spectrally stable neutral modes in a Vlasov-Poisson plasma and show that for stable equilibria of systems that have unbounded spatial domain, the only possible neutral modes are those with phase velocities that correspond to stationary inflection points of the equilibrium distribution function. It is seen that these neutral modes can possess positive or negative free energy.

  9. ALEX neutral beam probe

    SciTech Connect

    Pourrezaei, K.

    1982-01-01

    A neutral beam probe capable of measuring plasma space potential in a fully 3-dimensional magnetic field geometry has been developed. This neutral beam was successfully used to measure an arc target plasma contained within the ALEX baseball magnetic coil. A computer simulation of the experiment was performed to refine the experimental design and to develop a numerical model for scaling the ALEX neutral beam probe to other cases of fully 3-dimensional magnetic field. Based on this scaling a 30 to 50 keV neutral cesium beam probe capable of measuring space potential in the thermal barrier region of TMX Upgrade was designed.

  10. Search for neutral leptons

    SciTech Connect

    Perl, M.L.

    1984-12-01

    At present we know of three kinds of neutral leptons: the electron neutrino, the muon neutrino, and the tau neutrino. This paper reviews the search for additional neutral leptons. The method and significance of a search depends upon the model used for the neutral lepton being sought. Some models for the properties and decay modes of proposed neutral leptons are described. Past and present searches are reviewed. The limits obtained by some completed searches are given, and the methods of searches in progress are described. Future searches are discussed. 41 references.

  11. 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.

  12. Neutrality in Language Policy

    ERIC Educational Resources Information Center

    Wee, Lionel

    2010-01-01

    The unavoidability of language makes it critical that language policies appeal to some notion of language neutrality as part of their rationale, in order to assuage concerns that the policies might otherwise be unduly discriminatory. However, the idea of language neutrality is deeply ideological in nature, since it is not only an attempt to treat…

  13. 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.

  14. A Mixture of Functionally Oligoclonal Humanized Monoclonal Antibodies That Neutralize Clostridium difficile TcdA and TcdB with High Levels of In Vitro Potency Shows In Vivo Protection in a Hamster Infection Model

    PubMed Central

    Davies, Nicola L.; Compson, Joanne E.; MacKenzie, Brendon; O'Dowd, Victoria L.; Oxbrow, Amanda K. F.; Heads, James T.; Turner, Alison; Sarkar, Kaushik; Dugdale, Sarah L.; Jairaj, Mark; Christodoulou, Louis; Knight, David E. O.; Cross, Amanda S.; Hervé, Karine J. M.; Tyson, Kerry L.; Hailu, Hanna; Doyle, Carl B.; Ellis, Mark; Kriek, Marco; Cox, Matthew; Page, Matthew J. T.; Moore, Adrian R.; Lightwood, Daniel J.

    2013-01-01

    Clostridium difficile infections are a major cause of antibiotic-associated diarrhea in hospital and care facility patients. In spite of the availability of effective antibiotic treatments, C. difficile infection (CDI) is still a major cause of patient suffering, death, and substantial health care costs. Clostridium difficile exerts its major pathological effects through the actions of two protein exotoxins, TcdA and TcdB, which bind to and disrupt gut tissue. Antibiotics target the infecting bacteria but not the exotoxins. Administering neutralizing antibodies against TcdA and TcdB to patients receiving antibiotic treatment might modulate the effects of the exotoxins directly. We have developed a mixture of three humanized IgG1 monoclonal antibodies (MAbs) which neutralize TcdA and TcdB to address three clinical needs: reduction of the severity and duration of diarrhea, reduction of death rates, and reduction of the rate of recurrence. The UCB MAb mixture showed higher potency in a variety of in vitro binding and neutralization assays (∼10-fold improvements), higher levels of protection in a hamster model of CDI (82% versus 18% at 28 days), and higher valencies of toxin binding (12 versus 2 for TcdA and 3 versus 2 for TcdB) than other agents in clinical development. Comparisons of the MAb properties also offered some insight into the potential relative importance of TcdA and TcdB in the disease process. PMID:23324518

  15. 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

  16. 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

    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

  17. Conformational Changes in the VP1-Unique Region of Native Human Parvovirus B19 Lead to Exposure of Internal Sequences That Play a Role in Virus Neutralization and Infectivity▿

    PubMed Central

    Ros, Carlos; Gerber, Marco; Kempf, Christoph

    2006-01-01

    The unique region of the capsid protein VP1 (VP1u) of human parvovirus B19 (B19) elicits a dominant immune response and has a phospholipase A2 (PLA2) activity, which is necessary for the infection. In contrast to the rest of the parvoviruses, the VP1u of B19 is thought to occupy an external position in the virion, making this region a promising candidate for vaccine development. By using a monoclonal antibody against the most-N-terminal portion of VP1u, we revealed that this region rich in neutralizing epitopes is not accessible in native capsids. However, exposure of capsids to increasing temperatures or low pH led to its progressive accessibility without particle disassembly. Although unable to bind free virus or to block virus attachment to the cell, the anti-VP1u antibody was neutralizing, suggesting that the exposure of the epitope and the subsequent virus neutralization occur only after receptor attachment. The measurement of the VP1u-associated PLA2 activity of B19 capsids revealed that this region is also internal but becomes exposed in heat- and in low-pH-treated particles. In sharp contrast to native virions, the VP1u of baculovirus-derived B19 capsids was readily accessible in the absence of any treatment. These results indicate that stretches of VP1u of native B19 capsids harboring neutralizing epitopes and essential functional motifs are not external to the capsid. However, a conformational change renders these regions accessible and triggers the PLA2 potential of the virus. The results also emphasize major differences in the VP1u conformation between natural and recombinant particles. PMID:17020940

  18. Conformational changes in the VP1-unique region of native human parvovirus B19 lead to exposure of internal sequences that play a role in virus neutralization and infectivity.

    PubMed

    Ros, Carlos; Gerber, Marco; Kempf, Christoph

    2006-12-01

    The unique region of the capsid protein VP1 (VP1u) of human parvovirus B19 (B19) elicits a dominant immune response and has a phospholipase A(2) (PLA(2)) activity, which is necessary for the infection. In contrast to the rest of the parvoviruses, the VP1u of B19 is thought to occupy an external position in the virion, making this region a promising candidate for vaccine development. By using a monoclonal antibody against the most-N-terminal portion of VP1u, we revealed that this region rich in neutralizing epitopes is not accessible in native capsids. However, exposure of capsids to increasing temperatures or low pH led to its progressive accessibility without particle disassembly. Although unable to bind free virus or to block virus attachment to the cell, the anti-VP1u antibody was neutralizing, suggesting that the exposure of the epitope and the subsequent virus neutralization occur only after receptor attachment. The measurement of the VP1u-associated PLA(2) activity of B19 capsids revealed that this region is also internal but becomes exposed in heat- and in low-pH-treated particles. In sharp contrast to native virions, the VP1u of baculovirus-derived B19 capsids was readily accessible in the absence of any treatment. These results indicate that stretches of VP1u of native B19 capsids harboring neutralizing epitopes and essential functional motifs are not external to the capsid. However, a conformational change renders these regions accessible and triggers the PLA(2) potential of the virus. The results also emphasize major differences in the VP1u conformation between natural and recombinant particles.

  19. 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.

  20. Solar Neutral Particles

    NASA Video Gallery

    This animation shows a neutral solar particle's path leaving the sun, following the magnetic field lines out to the heliosheath. The solar particle hits a hydrogen atom, stealing its electron, and ...

  1. Neutralizing antibodies against West Nile virus identified directly from human B cells by single-cell analysis and next generation sequencing.

    PubMed

    Tsioris, Konstantinos; Gupta, Namita T; Ogunniyi, Adebola O; Zimnisky, Ross M; Qian, Feng; Yao, Yi; Wang, Xiaomei; Stern, Joel N H; Chari, Raj; Briggs, Adrian W; Clouser, Christopher R; Vigneault, Francois; Church, George M; Garcia, Melissa N; Murray, Kristy O; Montgomery, Ruth R; Kleinstein, Steven H; Love, J Christopher

    2015-12-01

    West Nile virus (WNV) infection is an emerging mosquito-borne disease that can lead to severe neurological illness and currently has no available treatment or vaccine. Using microengraving, an integrated single-cell analysis method, we analyzed a cohort of subjects infected with WNV - recently infected and post-convalescent subjects - and efficiently identified four novel WNV neutralizing antibodies. We also assessed the humoral response to WNV on a single-cell and repertoire level by integrating next generation sequencing (NGS) into our analysis. The results from single-cell analysis indicate persistence of WNV-specific memory B cells and antibody-secreting cells in post-convalescent subjects. These cells exhibited class-switched antibody isotypes. Furthermore, the results suggest that the antibody response itself does not predict the clinical severity of the disease (asymptomatic or symptomatic). Using the nucleotide coding sequences for WNV-specific antibodies derived from single cells, we revealed the ontogeny of expanded WNV-specific clones in the repertoires of recently infected subjects through NGS and bioinformatic analysis. This analysis also indicated that the humoral response to WNV did not depend on an anamnestic response, due to an unlikely previous exposure to the virus. The innovative and integrative approach presented here to analyze the evolution of neutralizing antibodies from natural infection on a single-cell and repertoire level can also be applied to vaccine studies, and could potentially aid the development of therapeutic antibodies and our basic understanding of other infectious diseases. PMID:26481611

  2. 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.

  3. 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.

  4. Isolation, full genomic characterization and neutralization-based human seroprevalence of Medjerda Valley virus, a novel sandfly-borne phlebovirus belonging to the Salehabad virus complex in northern Tunisia.

    PubMed

    Bichaud, Laurence; Dachraoui, Khalil; Alwassouf, Sulaf; Alkan, Cigdem; Mensi, Mohamed; Piorkowski, Géraldine; Sakhria, Sonia; Seston, Morgan; Fares, Wasfi; De Lamballerie, Xavier; Zhioua, Elyes; Charrel, Rémi N

    2016-03-01

    A new phlebovirus, Medjerda Valley virus (MVV), was isolated from one pool of Phlebotomus sp. (Diptera; Psychodidae) sandflies trapped in the vicinity of the Utique site, northern Tunisia. Genetic analysis based on complete coding of genomic sequences of the three RNA segments indicated that MVV is most closely related to members of the Salehabad virus species, where it is the fourth virus for which the complete sequence is available. A seroprevalence study was performed to search for neutralizing antibodies in human sera in the same region. The results demonstrate that in this area, MVV can readily infect humans despite low seroprevalence rates. Salehabad species viruses have generally been considered to be a group of viruses with little medical or veterinary interest. This view deserves to be revisited according to our human seroprevalence results, together with high animal infection rate of Adana virus and recent evidence of human infection with Adria virus in Greece. Further studies are needed to investigate the capacity of each specific member of the Salehabad virus species to cause human or animal diseases. PMID:26704069

  5. Env-2dCD4 S60C complexes act as super immunogens and elicit potent, broadly neutralizing antibodies against clinically relevant human immunodeficiency virus type 1 (HIV-1).

    PubMed

    Killick, Mark A; Grant, Michelle L; Cerutti, Nichole M; Capovilla, Alexio; Papathanasopoulos, Maria A

    2015-11-17

    The ability to induce a broadly neutralizing antibody (bNAb) response following vaccination is regarded as a crucial aspect in developing an effective vaccine against human immunodeficiency virus type 1 (HIV-1). The bNAbs target the HIV-1 envelope glycoprotein (Env) which is exposed on the virus surface, thereby preventing cell entry. To date, conventional vaccine approaches such as the use of Env-based immunogens have been unsuccessful. We expressed, purified, characterized and evaluated the immunogenicity of several unique HIV-1 subtype C Env immunogens in small animals. Here we report that vaccine immunogens based on Env liganded to a two domain CD4 variant, 2dCD4(S60C) are capable of consistently eliciting potent, broadly neutralizing antibody responses in New Zealand white rabbits against a panel of clinically relevant HIV-1 pseudoviruses. This was irrespective of the Env protein subtype and context. Importantly, depletion of the anti-CD4 antibodies appeared to abrogate the neutralization activity in the rabbit sera. Taken together, this data suggests that the Env-2dCD4(S60C) complexes described here are "super" immunogens, and potentially immunofocus antibody responses to a unique epitope spanning the 2dCD4(60C). Recent data from the two available anti-CD4 monoclonal antibodies, Ibalizumab and CD4-Ig (and bispecific variants thereof) have highlighted that the use of these broad and potent entry inhibitors could circumvent the need for a conventional vaccine targeting HIV-1. Overall, the ability of the unique Env-2dCD4(S60C) complexes to elicit potent bNAb responses has not been described previously, reinforcing that further investigation for their utility in preventing and controlling HIV-1/SIV infection is warranted. PMID:26432912

  6. Tautomerism in neutral histidine.

    PubMed

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

    2014-10-01

    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.

  7. Neutralization activity in a geographically diverse East London cohort of human immunodeficiency virus type 1-infected patients: clade C infection results in a stronger and broader humoral immune response than clade B infection.

    PubMed

    Dreja, Hanna; O'Sullivan, Eithne; Pade, Corinna; Greene, Kelli M; Gao, Hongmei; Aubin, Keith; Hand, James; Isaksen, Are; D'Souza, Carl; Leber, Werner; Montefiori, David; Seaman, Michael S; Anderson, Jane; Orkin, Chloe; McKnight, Aine

    2010-11-01

    The array of human immunodeficiency virus (HIV) subtypes encountered in East London, an area long associated with migration, is unusually heterogeneous, reflecting the diverse geographical origins of the population. In this study it was shown that viral subtypes or clades infecting a sample of HIV type 1 (HIV-1)-positive individuals in East London reflect the global pandemic. The authors studied the humoral response in 210 treatment-naïve chronically HIV-1-infected (>1 year) adult subjects against a panel of 12 viruses from six different clades. Plasmas from individuals infected with clade C, but also plasmas from clade A, and to a lesser degree clade CRF02_AG and CRF01_AE, were significantly more potent at neutralizing the tested viruses compared with plasmas from individuals infected with clade B. The difference in humoral robustness between clade C- and B-infected patients was confirmed in titration studies with an extended panel of clade B and C viruses. These results support the approach to develop an HIV-1 vaccine that includes clade C or A envelope protein (Env) immunogens for the induction of a potent neutralizing humoral response.

  8. 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.

  9. Frozen translational and rotational motion of human immunodeficiency virus transacting activator of transcription peptide-modified nanocargo on neutral lipid bilayer.

    PubMed

    Wei, Lin; Zhao, Xin; Chen, Bo; Li, Hongchang; Xiao, Lehui; Yeung, Edward S

    2013-05-21

    With time-resolved high-precision single-particle tracking methodologies, we explored the adsorption and thermal motion of transacting activator of transcription (TAT) peptide-modified nanocargo on a model lipid bilayer in the nonelectrostatic domain. We found that the lateral and rotational motion of TAT peptide-modified nanocargo could be effectively suppressed on the surface of neutral lipid membrane, a feature that cannot be explained by existing hypotheses. A semiquantitative association activation energy analysis revealed that multiple weak bonds were required for the initial adsorption process. As a result, the localized multiple TAT peptides on the surface of the nanocargo can provide a pathway for the creation of a net of peptide-lipid complexes (e.g., lipid domain). The dragging forces caused by these complexes effectively confined the thermal motion of the nanocargo on the fluid membrane that cannot be achieved by individual peptides with random spatial and conformational distributions. These interesting findings could provide insightful information for the better understanding of the intracellular internalization mechanism of TAT peptide-modified nanocargo and might shed new light on the development of highly efficient intracellular carriers for site-specific delivery of drugs and genes.

  10. 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

  11. 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. PMID:26491897

  12. 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.

  13. 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…

  14. Comparison of primary human fibroblasts and keratinocytes with immortalized cell lines regarding their sensitivity to sodium dodecyl sulfate in a neutral red uptake cytotoxicity assay.

    PubMed

    Olschläger, Veronika; Schrader, Andreas; Hockertz, Stefan

    2009-01-01

    Cell lines present a valuable tool for in vitro assessment of skin damage caused by application of cosmeticals or pharmaceuticals. They form a reproducible test system under controllable test conditions and, in many cases, can be used as alternatives to animal testing in order to assess the compatibility of drugs or cosmetics and human skin. Yet, it can not necessarily be assumed that the behavior of cultured cells, when treated with different substances, is exactly consistent with the behavior of cells being part of a live organism. Becoming immortal, cells exhibit changes in genotype and/or phenotype, possibly resulting in modified reactions to external influences. Therefore, to obtain results close to in vivo studies, it seems apparent to use primary cells for testing that have not yet undergone any modifications. To compare the properties of primary fibroblasts (Normal Human Dermal Fibroblasts, NHDF) and primary keratinocytes (Normal Human Epidermal Keratinocytes, NHEK) with those of immortal cell lines (3T3 (ACC 173) Swiss albino mouse fibroblasts and HaCaT (human, adult, low calcium, high temperature, human adult skin keratinocytes) cells), their sensitivities in cytotoxicity assays have been assessed. While both fibroblast cell cultures showed similar sensitivities towards sodium dodecyl sulfate (SDS), primary keratinocytes died at SDS concentrations about three times lower than the immortal HaCaT cells. PMID:19402346

  15. Vaccination of Rhesus Macaques with Recombinant Mycobacterium bovis Bacillus Calmette-Guérin Env V3 Elicits Neutralizing Antibody-Mediated Protection against Simian-Human Immunodeficiency Virus with a Homologous but Not a Heterologous V3 Motif

    PubMed Central

    Someya, Kenji; Cecilia, Dayaraj; Ami, Yasushi; Nakasone, Tadashi; Matsuo, Kazuhiro; Burda, Sherri; Yamamoto, Hiroshi; Yoshino, Naoto; Kaizu, Masahiko; Ando, Shuji; Okuda, Kenji; Zolla-Pazner, Susan; Yamazaki, Shudo; Yamamoto, Naoki; Honda, Mitsuo

    2005-01-01

    Although the correlates of vaccine-induced protection against human immunodeficiency virus type 1 (HIV-1) are not fully known, it is presumed that neutralizing antibodies (NAb) play a role in controlling virus infection. In this study, we examined immune responses elicited in rhesus macaques following vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing an HIV-1 Env V3 antigen (rBCG Env V3). We also determined the effect of vaccination on protection against challenge with either a simian-human immunodeficiency virus (SHIV-MN) or a highly pathogenic SHIV strain (SHIV-89.6PD). Immunization with rBCG Env V3 elicited significant levels of NAb for the 24 weeks tested that were predominantly HIV-1 type specific. Sera from the immunized macaques neutralized primary HIV-1 isolates in vitro, including HIV-1BZ167/X4, HIV-1SF2/X4, HIV-1CI2/X4, and, to a lesser extent, HIV-1MNp/X4, all of which contain a V3 sequence homologous to that of rBCG Env V3. In contrast, neutralization was not observed against HIV-1SF33/X4, which has a heterologous V3 sequence, nor was it found against primary HIV-1 R5 isolates from either clade A or B. Furthermore, the viral load in the vaccinated macaques was significantly reduced following low-dose challenge with SHIV-MN, and early plasma viremia was markedly decreased after high-dose SHIV-MN challenge. In contrast, replication of pathogenic SHIV-89.6PD was not affected by vaccination in any of the macaques. Thus, we have shown that immunization with an rBCG Env V3 vaccine elicits a strong, type-specific V3 NAb response in rhesus macaques. While this response was not sufficient to provide protection against a pathogenic SHIV challenge, it was able to significantly reduce the viral load in macaques following challenge with a nonpathogenic SHIV. These observations suggest that rBCG vectors have the potential to deliver an appropriate virus immunogen for desirable immune elicitations. PMID:15650171

  16. Gangliosides in human, cow and goat milk, and their abilities as to neutralization of cholera toxin and botulinum type A neurotoxin.

    PubMed

    Iwamori, Masao; Takamizawa, Kotarou; Momoeda, Mikio; Iwamori, Yuriko; Taketani, Yuji

    2008-10-01

    To elucidate the potential of mammalian milk as to protection of infants from infections, we determined the ganglioside compositions of human, cow and goat milk in relation with cholera toxin and botulinum type A neurotoxin-receptors. Gangliosides accounted for 1 to 2 micromol of lipid-bound sialic acid (LSA) in 100 ml of milk, and GD3 comprised about 69% of LSA in all milk samples. Among the milk samples examined, goat milk was found to contain an amount of gangliosides belonging to the b-pathway representing 15.8% of the total LSA. Accordingly, botulinum neurotoxin bound to GT1b and GQ1b in goat milk, but not to any gangliosides in human or cow milk. On the other hand, GM1, the cholera toxin receptor, was found to be present in all milk samples at concentrations of 0.02% to 0.77% of the total LSA and to be maintained at a relatively constant level in human milk during the postpartum period. Gangliosides from 1 ml of pooled human milk exhibited the ability to attenuate the binding of cholera toxin (30 ng) to GM1 by 93%, and those from 500 microl of goat milk completely inhibited the binding of botulinum type A neurotoxin 1.5 microg to GT1b.

  17. 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…

  18. Between detection and neutralization.

    SciTech Connect

    Snell, Mark Kamerer; Green, Mary Wilson; Adams, Douglas Glenn; Pritchard, Daniel Allison

    2005-08-01

    Security system analytical performance analysis is generally based on the probability of system effectiveness. The probability of effectiveness is a function of the probabilities of interruption and neutralization. Interruption occurs if the response forces are notified in sufficient time to engage the adversary. Neutralization occurs if the adversary attack is defeated after the security forces have actively engaged the adversary. Both depend upon communications of data. This paper explores details of embedded communications functions that are often assumed to be inconsequential. It is the intent of the authors to bring focus to an issue in security system modeling that, if not well understood, has the potential to be a deciding factor in the overall system failure or effectiveness.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. Neutral Buoyancy Simulator Test

    NASA Technical Reports Server (NTRS)

    1995-01-01

    A diver tests a secondary camera and maneuvering platform in Marshall's Neutral Buoyancy Simulator (NBS).The secondary camera will be beneficial for recording repairs and other extra vehicular activities (EVA) the astronuats will perform while making repairs on the Hubble Space Telescope (HST). The maneuvering platform was developed to give the astronauts something to stand on while performing maintenance tasks. These platforms were developed to be mobile so that the astronauts could move them to accommadate different sites.

  4. 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.

  5. Chimpanzee/human mAbs to vaccinia virus B5 protein neutralize vaccinia and smallpox viruses and protect mice against vaccinia virus.

    PubMed

    Chen, Zhaochun; Earl, Patricia; Americo, Jeffrey; Damon, Inger; Smith, Scott K; Zhou, Yi-Hua; Yu, Fujuan; Sebrell, Andrew; Emerson, Suzanne; Cohen, Gary; Eisenberg, Roselyn J; Svitel, Juraj; Schuck, Peter; Satterfield, William; Moss, Bernard; Purcell, Robert

    2006-02-01

    Chimpanzee Fabs against the B5 envelope glycoprotein of vaccinia virus were isolated and converted into complete mAbs with human gamma 1 heavy chain constant regions. The two mAbs (8AH8AL and 8AH7AL) displayed high binding affinities to B5 (Kd of 0.2 and 0.7 nM). The mAb 8AH8AL inhibited the spread of vaccinia virus as well as variola virus (the causative agent of smallpox) in vitro, protected mice from subsequent intranasal challenge with virulent vaccinia virus, protected mice when administered 2 days after challenge, and provided significantly greater protection than that afforded by a previously isolated rat anti-B5 mAb (19C2) or by vaccinia immune globulin. The mAb bound to a conformational epitope between amino acids 20 and 130 of B5. These chimpanzee/human anti-B5 mAbs may be useful in the prevention and treatment of vaccinia virus-induced complications of vaccination against smallpox and may also be effective in the immunoprophylaxis and immunotherapy of smallpox.

  6. Neutrality between Government and Religion.

    ERIC Educational Resources Information Center

    Mawdsley, Ralph D.

    1996-01-01

    The overall guiding principle of neutrality between government and religion masks a tension that exists between free exercise of religion and establishment of religion. Reviews the development and current status of "Lemon" as a test for neutrality; proposes a new test for neutrality, evenhandedness, that is common to both the Free Exercise and…

  7. 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

  8. Therapeutic implications of a human neutralizing antibody to the macrophage-stimulating protein receptor tyrosine kinase (RON), a c-MET family member.

    PubMed

    O'Toole, Jennifer M; Rabenau, Karen E; Burns, Kerri; Lu, Dan; Mangalampalli, Venkat; Balderes, Paul; Covino, Nicole; Bassi, Rajiv; Prewett, Marie; Gottfredsen, Kimberly J; Thobe, Megan N; Cheng, Yuan; Li, Yiwen; Hicklin, Daniel J; Zhu, Zhenping; Waltz, Susan E; Hayman, Michael J; Ludwig, Dale L; Pereira, Daniel S

    2006-09-15

    RON is a member of the c-MET receptor tyrosine kinase family. Like c-MET, RON is expressed by a variety of epithelial-derived tumors and cancer cell lines and it is thought to play a functional role in tumorigenesis. To date, antagonists of RON activity have not been tested in vivo to validate RON as a potential cancer target. In this report, we used an antibody phage display library to generate IMC-41A10, a human immunoglobulin G1 (IgG1) antibody that binds with high affinity (ED50 = 0.15 nmol/L) to RON and effectively blocks interaction with its ligand, macrophage-stimulating protein (MSP; IC50 = 2 nmol/L). We found IMC-41A10 to be a potent inhibitor of receptor and downstream signaling, cell migration, and tumorigenesis. It antagonized MSP-induced phosphorylation of RON, mitogen-activated protein kinase (MAPK), and AKT in several cancer cell lines. In HT-29 colon, NCI-H292 lung, and BXPC-3 pancreatic cancer xenograft tumor models, IMC-41A10 inhibited tumor growth by 50% to 60% as a single agent, and in BXPC-3 xenografts, it led to tumor regressions when combined with Erbitux. Western blot analyses of HT-29 and NCI-H292 xenograft tumors treated with IMC-41A10 revealed a decrease in MAPK phosphorylation compared with control IgG-treated tumors, suggesting that inhibition of MAPK activity may be required for the antitumor activity of IMC-41A10. To our knowledge, this is the first demonstration that a RON antagonist and specifically an inhibitory antibody of RON negatively affects tumorigenesis. Another major contribution of this report is an extensive analysis of RON expression in approximately 100 cancer cell lines and approximately 300 patient tumor samples representing 10 major cancer types. Taken together, our results highlight the potential therapeutic usefulness of RON activity inhibition in human cancers.

  9. 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.

  10. Conformational studies of a short linear peptide corresponding to a major conserved neutralizing epitope of human respiratory syncytial virus fusion glycoprotein.

    PubMed

    Toiron, C; López, J A; Rivas, G; Andreu, D; Melero, J A; Bruix, M

    1996-10-01

    The conformational properties of a 21-residue peptide, corresponding to amino acids 255 to 275 (F255-275) of the human respiratory syncytial virus fusion (F) glycoprotein have been studied by CD and nmr spectroscopy. This peptide includes residues 262, 268, and 272 of the F polypeptide that are essential for integrity of most epitopes that mapped into a major antigenic site of the F molecule. CD data indicate that F255-275 adopts a random coil conformation in aqueous solution at low peptide concentrations. However, as the concentration of peptide is increased, a higher percentage of peptide molecules adopts an organized structure. This effect can be more easily observed when trifluoroethanol (30%) is added to peptide solutions, giving rise to CD spectra that resemble those of alpha-helix structures. These conformational changes were confirmed by nmr spectroscopy. The nuclear Overhauser effects observed in 30% trifluoroethanol/ water together with the conformational H alpha chemical shift data allowed us to propose a structural model of helix-loop-helix for the peptide in solution. In addition, these helical regions contain the amino acid residues essential for epitope integrity in the native F molecule. These results give new insights into the antigenic structure of the respiratory syncytical virus F glycoprotein. PMID:8837519

  11. Molecular events involved in up-regulating human Na+-independent neutral amino acid transporter LAT1 during T-cell activation.

    PubMed

    Nii, T; Segawa, H; Taketani, Y; Tani, Y; Ohkido, M; Kishida, S; Ito, M; Endou, H; Kanai, Y; Takeda, E; Miyamoto Ki

    2001-09-15

    We investigated the regulation of system-L amino acid transporter (LAT1) during T-cell activation. In quiescent T-cells, L-leucine transport is mediated mainly by the system-L amino acid transport system and is increased significantly during T-cell activation by PMA and ionomycin. In quiescent T-cells, the LAT1 protein was heterocomplexed with 4F2 heavy chain (4F2hc) in the plasma membrane. During T-cell activation, the amounts of 4F2hc and LAT1 heterocomplex were significantly elevated compared with those in quiescent T-cells. In addition, by Northern-blot analysis, these increments were found to be due to elevated levels of LAT1 and 4F2hc mRNA. Transient expression of constructs comprising various LAT1 gene promoter fragments, which contained all three of the GC boxes, was sufficient for promoting luciferase expression in Jurkat T-cells, but the promoter of the LAT1 gene did not respond to PMA and ionomycin. Similar observations were observed in the human 4F2hc gene promoter. In nuclear run-on assay, the LAT1 and 4F2hc genes were actively transcribed even in quiescent T-cells, but the low levels of both transcripts were shown to be the result of a block to transcription elongation within the exon 1 intron 1 regions. These findings indicated that a removal of the block to mRNA elongation stimulates the induction of system-L amino acid transporter gene transcripts (LAT1 and 4F2hc) in activated T-cells. PMID:11535130

  12. 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.

  13. Producing human ceramide-NS by metabolic engineering using yeast Saccharomyces cerevisiae.

    PubMed

    Murakami, Suguru; Shimamoto, Toshi; Nagano, Hideaki; Tsuruno, Masahiro; Okuhara, Hiroaki; Hatanaka, Haruyo; Tojo, Hiromasa; Kodama, Yukiko; Funato, Kouichi

    2015-01-01

    Ceramide is one of the most important intercellular components responsible for the barrier and moisture retention functions of the skin. Because of the risks involved with using products of animal origin and the low productivity of plants, the availability of ceramides is currently limited. In this study, we successfully developed a system that produces sphingosine-containing human ceramide-NS in the yeast Saccharomyces cerevisiae by eliminating the genes for yeast sphingolipid hydroxylases (encoded by SUR2 and SCS7) and introducing the gene for a human sphingolipid desaturase (encoded by DES1). The inactivation of the ceramidase gene YDC1, overexpression of the inositol phosphosphingolipid phospholipase C gene ISC1, and endoplasmic reticulum localization of the DES1 gene product resulted in enhanced production of ceramide-NS. The engineered yeast strains can serve as hosts not only for providing a sustainable source of ceramide-NS but also for developing further systems to produce sphingosine-containing sphingolipids. PMID:26573460

  14. 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

  15. Antihypertensive neutral lipid

    SciTech Connect

    Snyder, F.L.; Blank, M.L.

    1986-06-17

    A method is described for treating a warm-blooded animal comprising administering to the animal a neutral glycerolipid with a 12 to 20 carbon alkyl group at the sn-1 position, a short carbon chain acyl group at the sn-2 position and a hydroxyl group at the sn-3 position in an amount sufficient to lower the arterial blood pressure of the animal. A method is also described for treating a warm-blooded animal comprising administering a composition consisting essentially of a 1-alkyl-2-acetyl (or propionyl)-sn glycerol in combination with a 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, wherein the 1-alkyl groups contain 12 to 20 carbon atoms, dissolved in an inert pharmaceutically acceptable solvent in amounts sufficient to lower the arterial blood pressure of the animal.

  16. Antibody neutralization of retargeted measles viruses.

    PubMed

    Lech, Patrycja J; Pappoe, Roland; Nakamura, Takafumi; Tobin, Gregory J; Nara, Peter L; Russell, Stephen J

    2014-04-01

    The measles virus (MV) vaccine lineage is a promising oncolytic but prior exposure to the measles vaccine or wild-type MV strains limits treatment utility due to the presence of anti-measles antibodies. MV entry can be redirected by displaying a polypeptide ligand on the Hemagglutinin (H) C-terminus. We hypothesized that retargeted MV would escape neutralization by monoclonal antibodies (mAbs) recognizing the H receptor-binding surface and be less susceptible to neutralization by human antisera. Using chimeric H proteins, with and without mutations that ablate MV receptor binding, we show that retargeted MVs escape mAbs that target the H receptor-binding surface by virtue of mutations that ablate infection via SLAM and CD46. However, C-terminally displayed domains do not mediate virus entry in the presence of human antibodies that bind to the underlying H domain. In conclusion, utility of retargeted oncolytic measles viruses does not extend to evasion of human serum neutralization.

  17. 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.

  18. Chemistry of carotenoid neutral radicals.

    PubMed

    Ligia Focsan, A; Magyar, Adam; Kispert, Lowell D

    2015-04-15

    Proton loss from the carotenoid radical cations (Car(+)) to form neutral radicals (#Car) was investigated by numerous electrochemical, EPR, ENDOR and DFT studies described herein. The radical cation and neutral radicals were formed in solution electrochemically and stabilized on solid silica-alumina and MCM-41 matrices. Carotenoid neutral radicals were recently identified in Arabidopsis thaliana plant and photosystem II samples. Deprotonation at the terminal ends of a zeaxanthin radical cation could provide a secondary photoprotection pathway which involves quenching excited state chlorophyll by the long-lived zeaxanthin neutral radicals formed. PMID:25687648

  19. 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.

  20. A proposed neutral line signature

    NASA Technical Reports Server (NTRS)

    Doxas, I.; Speiser, T. W.; Dusenbery, P. B.; Horton, W.

    1992-01-01

    An identifying signature is proposed for the existence and location of the neutral line in the magnetotail. The signature, abrupt density, and temperature changes in the Earthtail direction, was first discovered in test particle simulations. Such temperature variations have been observed in ISEE data (Huang et. al. 1992), but their connection to the possible existence of a neutral line in the tail has not yet been established. The proposed signature develops earlier than the ion velocity space ridge of Martin and Speiser (1988), but can only be seen by spacecraft in the vicinity of the neutral line, while the latter can locate a neutral line remotely.

  1. Fecal neutral sterols in omnivorous and vegetarian women.

    PubMed

    Korpela, J T; Adlercreutz, H

    1985-12-01

    The purpose of this study was to investigate the effect of a vegetarian diet on human fecal neutral sterol excretion. Free and esterified fecal neutral sterols were analyzed by capillary gas-chromatography in healthy North-American white women who were consuming either a mixed Western diet (n = 19) or a vegetarian diet (n = 20). Vegetarians had lower mean concentrations of bacterial metabolites of cholesterol, coprostanol, and coprostanone, and their relative amounts of esterified neutral sterol metabolism in both populations. Most of the subjects in both groups excreted their neutral sterols mainly as metabolites. However, 25% of the omnivores and 21% of the vegetarians had exceptionally low amounts of these metabolites in their feces. The vegetarians in this study differed only slightly from omnivores with regard to intestinal bacterial metabolism of neutral sterols.

  2. 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

  3. 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

  4. Emotionally Neutral Stimuli Are Not Neutral in Schizophrenia: A Mini Review of Functional Neuroimaging Studies

    PubMed Central

    Potvin, Stéphane; Tikàsz, Andràs; Mendrek, Adrianna

    2016-01-01

    Reliable evidence shows that schizophrenia patients tend to experience negative emotions when presented with emotionally neutral stimuli. Similarly, several functional neuroimaging studies show that schizophrenia patients have increased activations in response to neutral material. However, results are heterogeneous. Here, we review the functional neuroimaging studies that have addressed this research question. Based on the 36 functional neuroimaging studies that we retrieved, it seems that the increased brain reactivity to neutral stimuli is fairly common in schizophrenia, but that the regions involved vary considerably, apart from the amygdala. Prefrontal and cingulate sub-regions and the hippocampus may also be involved. By contrasts, results in individuals at risk for psychosis are less consistent. In schizophrenia patients, results are less consistent in the case of studies using non-facial stimuli, explicit processing paradigms, and/or event-related designs. This means that human faces may convey subtle information (e.g., trustworthiness) other than basic emotional expressions. It also means that the aberrant brain reactivity to neutral stimuli is less likely to occur when experimental paradigms are too cognitively demanding as well as in studies lacking statistical power. The main hypothesis proposed to account for this increased brain reactivity to neutral stimuli is the aberrant salience hypothesis of psychosis. Other investigators propose that the aberrant brain reactivity to neutral stimuli in schizophrenia results from abnormal associative learning, untrustworthiness judgments, priming effects, and/or reduced habituation to neutral stimuli. In the future, the effects of antipsychotics on this aberrant brain reactivity will need to be determined, as well as the potential implication of sex/gender. PMID:27445871

  5. Emotionally Neutral Stimuli Are Not Neutral in Schizophrenia: A Mini Review of Functional Neuroimaging Studies.

    PubMed

    Potvin, Stéphane; Tikàsz, Andràs; Mendrek, Adrianna

    2016-01-01

    Reliable evidence shows that schizophrenia patients tend to experience negative emotions when presented with emotionally neutral stimuli. Similarly, several functional neuroimaging studies show that schizophrenia patients have increased activations in response to neutral material. However, results are heterogeneous. Here, we review the functional neuroimaging studies that have addressed this research question. Based on the 36 functional neuroimaging studies that we retrieved, it seems that the increased brain reactivity to neutral stimuli is fairly common in schizophrenia, but that the regions involved vary considerably, apart from the amygdala. Prefrontal and cingulate sub-regions and the hippocampus may also be involved. By contrasts, results in individuals at risk for psychosis are less consistent. In schizophrenia patients, results are less consistent in the case of studies using non-facial stimuli, explicit processing paradigms, and/or event-related designs. This means that human faces may convey subtle information (e.g., trustworthiness) other than basic emotional expressions. It also means that the aberrant brain reactivity to neutral stimuli is less likely to occur when experimental paradigms are too cognitively demanding as well as in studies lacking statistical power. The main hypothesis proposed to account for this increased brain reactivity to neutral stimuli is the aberrant salience hypothesis of psychosis. Other investigators propose that the aberrant brain reactivity to neutral stimuli in schizophrenia results from abnormal associative learning, untrustworthiness judgments, priming effects, and/or reduced habituation to neutral stimuli. In the future, the effects of antipsychotics on this aberrant brain reactivity will need to be determined, as well as the potential implication of sex/gender. PMID:27445871

  6. Neutral Buoyancy Simulator (NBS)

    NASA Technical Reports Server (NTRS)

    1972-01-01

    This is a cutaway illustration of the Neutral Buoyancy Simulator (NBS) at the Marshall Space Flight Center (MSFC ). The MSFC NBS provided an excellent environment for testing hardware to examine how it would operate in space and for evaluating techniques for space construction and spacecraft servicing. Here, engineers, designers, and astronauts performed various tests to develop basic concepts, preliminary designs, final designs, and crew procedures. The NBS was constructed of welded steel with polyester-resin coating. The water tank was 75-feet (22.9- meters) in diameter, 40-feet (12.2-meters) deep, and held 1.32 million gallons of water. Since it opened for operation in 1968, the NBS had supported a number of successful space missions, such as the Skylab, Solar Maximum Mission Satellite, Marned Maneuvering Unit, Experimental Assembly of Structures in Extravehicular Activity/Assembly Concept for Construction of Erectable Space Structures (EASE/ACCESS), the Hubble Space Telescope, and the Space Station. The function of the MSFC NBS was moved to the larger simulator at the Johnson Space Center and is no longer operational.

  7. Europa's Neutral Gas Torus

    NASA Astrophysics Data System (ADS)

    Mauk, B. H.; Mitchell, D. G.; McEntire, R. W.; Paranicas, C. P.; Roelof, E. C.; Williams, D. J.; Krimigis, S. M.; Lagg, A.

    2004-05-01

    In-situ energetic ion measurements from the Galileo spacecraft and remote energetic neutral atom (ENA) images from the Cassini spacecraft have been previously interpreted as revealing an unexpectedly massive torus of gas co-orbiting with Jupiter's moon Europa (Lagg et al., 2003; Mauk et al., 2003). Here we report on the results of detailed modeling of the ENA emission process from the Europa regions. Updates to the distribution and composition of the trapped energetic ion populations are included in the models, as are considerations of the partitioning of the gas products into multiple atomic and molecular species. Comparisons between the models and the Cassini observations reveal a torus with a total gas content equal to (0.5 +/- 0.2) E34 atoms plus molecules. This value is higher than, but within a factor of 3 of, an estimate inferred from a prediction of gas densities derived from Voyager plasma measurements and modeling of the interaction between the plasmas and the gases assumed to be emanating from Europa (Schreier et al., 1993). Lagg, A., N. Krupp, J. Woch, and D. J. Williams, Geophys. Res. Lett., 30, DOI 10.1029/2003GL017214, 2003. Mauk, B. H., D. G. Mitchell, S. M. Krimigs, E. C. Roelof, and C. P. Paranicas, Nature, 241, 920, 2003. Schreier, S., A. Eviatar, V. M. Vasyliunas, and J. D. Richardson, J. Geophys. Res., 98, 21231, 1993.

  8. 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

  9. 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.

  10. Neutral red supravital staining for cellular elements in the semen.

    PubMed

    Phadke, A M

    1978-01-01

    Human seminal fluid besides spermatozoa often contains other cellular elements. A supravital staining method designed to differentiate the above mentioned cellular elements was described. Amongst the spermatogenic cells only spermatocytes were stained with Neutral Red. They displayed two peculiar structures designated as "Y" granules and "Enigmatic Body". Neutral Red was absorbed by the spermiophage cells and was concentrated by them in the form of cytoplasmic granules. In addition the coarse granules of leukocytes and the gigestive vacuoles of Balantidium Coli and Trichomonads were stained with Neutral Red. PMID:75699

  11. Variation and infectivity neutralization in influenza

    PubMed Central

    Knossow, Marcel; Skehel, John J

    2006-01-01

    Worldwide epidemics of influenza are caused by viruses that normally infect other species, particularly waterfowl, and that contain haemagglutinin membrane glycoproteins (HAs) to which the human population has no immunity. Anti-HA immunoglobulins neutralize influenza virus infectivity. In this review we outline structural differences that distinguish the HAs of the 16 antigenic subtypes (H1–16) found in viruses from avian species. We also describe structural changes in HA required for the effective transfer to humans of viruses containing three of them, H1, H2 and H3, in the 1918 (Spanish), the 1957 (Asian) and the 1968 (Hong Kong) pandemics, respectively. In addition, we consider changes that may be required before the current avian H5 viruses could pass from human to human. PMID:16925526

  12. Neutral point detection by satellites. [magnetospheric neutral sheets

    NASA Technical Reports Server (NTRS)

    Schindler, K.; Ness, N. F.

    1974-01-01

    The concept of a neutral point depends on the physical phenomena described. The regions with B less than about 1 gamma detected by Schindler and Ness may be interpreted as neutral regions for the ion-tearing process. The assumption of the presence of a multiple neutral point structure (with temporal variations) is still the most promising interpretation of the Explorer 34 data. Alternatives suggested by Russell lead to difficulties. Nevertheless, the final answer can come only from multiple satellite systems. A 1-day displacement of the day count in the data discussed by Schindler and Ness is corrected.

  13. The molecular mechanism of the neutral-to-base transition of human serum albumin. Acid/base titration and proton nuclear magnetic resonance studies on a large peptic and a large tryptic fragment of albumin.

    PubMed

    Bos, O J; Labro, J F; Fischer, M J; Wilting, J; Janssen, L H

    1989-01-15

    In order to obtain a better understanding of the neutral-to-base (N-B) transition of human serum albumin, we performed acid/base titration experiments and 500-MHz 1H NMR experiments on albumin and on a large peptic (residues 1-387) and large tryptic (residues 198-585) fragment of albumin. The acid/base titration experiments revealed that Ca2+ ions induce a downward pK shift of several histidine residues of the peptic (P46) fragment and of albumin. By contrast, Ca2+ has very little influence on the pK of histidine residues of the tryptic (T45) fragment. In albumin, the pH-dependent His C-2 proton resonances, observed with 1H NMR experiments, have been allotted the numbers 1-17. It proved possible to locate these resonances in the P46 and the T45 fragments. A correspondence was found between the number of histidines detected by the acid/base titration and by the 1H NMR experiments. The results of the experiments lead us to conclude that in domain 1 at least the histidines corresponding to the His C-2 proton resonances 1-5 play a dominant role in the N-B transition. The Cu2+-binding histidine residue 3 (resonance 8) of the albumin molecule is not involved in the N-B transition. In addition, we were able to assign His C-2 proton resonance 9 to histidine 464 of the albumin molecule. The role of the N-B transition in the transport and cellular uptake mechanisms of endogenous and exogenous compounds is discussed.

  14. 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.

  15. Environmental neutralization of polonium-218

    SciTech Connect

    Goldstein, S.D.; Hopke, P.K.

    1985-01-01

    Previous work has indicated that two mechanisms of neutralization of the singly charged polonium ion exist. Charged Polonium-218 can be neutralized by reacting with oxygen to form a polonium oxide ion with a higher ionization potential than that of the polonium metal and then accepting an electron transferred from a lower ionization potential gas. In this present work, this mechanism has been verified by determining that the polonium oxide has an ionization potential in the range 10.35-10.53 eV. It was also previously reported that /sup 218/Po can be neutralized, in the absence of oxygen, by the scavenging of electrons by a trace gas such as water or nitrogen dioxide and their diffusion to the polonium ion. To verify this second neutralization mechanism, concentrations of nitrogen dioxide in nitrogen in the range of 50 ppb-1 ppm were examined for their ability to neutralize the polonium ion. Complete neutralization of /sup 218/Po was observed at nitrogen dioxide concentrations greater than 700 ppb. For concentrations below 700 ppb, the degree of neutralization was found to increase smoothly with the nitrogen dioxide concentration.

  16. Positional nystagmus showing neutral points.

    PubMed

    Hiruma, Kiyoshi; Numata, Tsutomu

    2004-01-01

    We encountered patients who had their static direction-changing positional nystagmus canceled at about 20-30 degrees yaw head rotation from the supine position. This nystagmus was also canceled when the head was rotated 180 degrees from this position. We call these head positions neutral points. At the neutral points, the cupula of the horizontal semicircular canal of the affected ear is positioned vertical to the gravitational plane and no deflection of the cupula occurs. The positional nystagmus observed (except the neutral points) was thought to occur due to a "heavy cupula" or "light cupula", which may be determined by the specific gravity of its endolymph.

  17. Neutral current interactions in MINOS

    SciTech Connect

    Sousa, Alexandre; /Oxford U.

    2007-07-01

    The Main Injector Neutrino Oscillation Search (MINOS) long-baseline experiment has been actively collecting beam data since 2005, having already accumulated 3 x 10{sup 20} protons-on-target (POT). The several million neutrinos per year observed at the Near detector may improve the existing body of knowledge of neutrino cross-sections and the Near-Far comparison of the observed energy spectrum neutral current events constrains oscillations into sterile neutrinos. MINOS capabilities of observing neutral current neutrino events are described and the employed methodology for event selection is discussed, along with preliminary results obtained. An outlook on the expected neutral current related contributions from MINOS is also presented.

  18. [Neutral Medical Claim Management Committee].

    PubMed

    Komatsu, Mitsuru

    2013-03-01

    The Ibaraki Medical Association established the Committee for Alternative Dispute Resolution called the Neutral Medical Claim Management Committee in 2006. Among 64 claims presented to the committee, 29 were settled through mediation or consultation. Patients were generally satisfied that their claims were considered fairly by the committee and that they were able to talk directly with healthcare professionals. However, some did not consider the committee to be completely neutral. The healthcare professionals involved rated the committee highly because they felt that the processes were neutral and no emotional aspects were involved. PMID:23617190

  19. Mechanism of Neutralization by the Broadly Neutralizing HIV-1 Monoclonal Antibody VRC01▿†

    PubMed Central

    Li, Yuxing; O'Dell, Sijy; Walker, Laura M.; Wu, Xueling; Guenaga, Javier; Feng, Yu; Schmidt, Stephen D.; McKee, Krisha; Louder, Mark K.; Ledgerwood, Julie E.; Graham, Barney S.; Haynes, Barton F.; Burton, Dennis R.; Wyatt, Richard T.; Mascola, John R.

    2011-01-01

    The structure of VRC01 in complex with the HIV-1 gp120 core reveals that this broadly neutralizing CD4 binding site (CD4bs) antibody partially mimics the interaction of the primary virus receptor, CD4, with gp120. Here, we extended the investigation of the VRC01-gp120 core interaction to the biologically relevant viral spike to better understand the mechanism of VRC01-mediated neutralization and to define viral elements associated with neutralization resistance. In contrast to the interaction of CD4 or the CD4bs monoclonal antibody (MAb) b12 with the HIV-1 envelope glycoprotein (Env), occlusion of the VRC01 epitope by quaternary constraints was not a major factor limiting neutralization. Mutagenesis studies indicated that VRC01 contacts within the gp120 loop D, the CD4 binding loop, and the V5 region were necessary for optimal VRC01 neutralization, as suggested by the crystal structure. In contrast to interactions with the soluble gp120 monomer, VRC01 interaction with the native viral spike did not occur in a CD4-like manner; VRC01 did not induce gp120 shedding from the Env spike or enhance gp41 membrane proximal external region (MPER)-directed antibody binding to the Env spike. Finally, VRC01 did not display significant reactivity with human antigens, boding well for potential in vivo applications. The data indicate that VRC01 interacts with gp120 in the context of the functional spike in a manner distinct from that of CD4. It achieves potent neutralization by precisely targeting the CD4bs without requiring alterations of Env spike configuration and by avoiding steric constraints imposed by the quaternary structure of the functional Env spike. PMID:21715490

  20. Incomplete Neutralization and Deviation from Sigmoidal Neutralization Curves for HIV Broadly Neutralizing Monoclonal Antibodies.

    PubMed

    McCoy, Laura E; Falkowska, Emilia; Doores, Katie J; Le, Khoa; Sok, Devin; van Gils, Marit J; Euler, Zelda; Burger, Judith A; Seaman, Michael S; Sanders, Rogier W; Schuitemaker, Hanneke; Poignard, Pascal; Wrin, Terri; Burton, Dennis R

    2015-08-01

    The broadly neutralizing HIV monoclonal antibodies (bnMAbs) PG9, PG16, PGT151, and PGT152 have been shown earlier to occasionally display an unusual virus neutralization profile with a non-sigmoidal slope and a plateau at <100% neutralization. In the current study, we were interested in determining the extent of non-sigmoidal slopes and plateaus at <100% for HIV bnMAbs more generally. Using both a 278 panel of pseudoviruses in a CD4 T-cell (U87.CCR5.CXCR4) assay and a panel of 117 viruses in the TZM-bl assay, we found that bnMAbs targeting many neutralizing epitopes of the spike had neutralization profiles for at least one virus that plateaued at <90%. Across both panels the bnMAbs targeting the V2 apex of Env and gp41 were most likely to show neutralization curves that plateaued <100%. Conversely, bnMAbs targeting the high-mannose patch epitopes were less likely to show such behavior. Two CD4 binding site (CD4bs) Abs also showed this behavior relatively infrequently. The phenomenon of incomplete neutralization was also observed in a large peripheral blood mononuclear cells (PBMC)-grown molecular virus clone panel derived from patient viral swarms. In addition, five bnMAbs were compared against an 18-virus panel of molecular clones produced in 293T cells and PBMCs and assayed in TZM-bl cells. Examples of plateaus <90% were seen with both types of virus production with no consistent patterns observed. In conclusion, incomplete neutralization and non-sigmoidal neutralization curves are possible for all HIV bnMAbs against a wide range of viruses produced and assayed in both cell lines and primary cells with implications for the use of antibodies in therapy and as tools for vaccine design. PMID:26267277

  1. Incomplete Neutralization and Deviation from Sigmoidal Neutralization Curves for HIV Broadly Neutralizing Monoclonal Antibodies.

    PubMed

    McCoy, Laura E; Falkowska, Emilia; Doores, Katie J; Le, Khoa; Sok, Devin; van Gils, Marit J; Euler, Zelda; Burger, Judith A; Seaman, Michael S; Sanders, Rogier W; Schuitemaker, Hanneke; Poignard, Pascal; Wrin, Terri; Burton, Dennis R

    2015-08-01

    The broadly neutralizing HIV monoclonal antibodies (bnMAbs) PG9, PG16, PGT151, and PGT152 have been shown earlier to occasionally display an unusual virus neutralization profile with a non-sigmoidal slope and a plateau at <100% neutralization. In the current study, we were interested in determining the extent of non-sigmoidal slopes and plateaus at <100% for HIV bnMAbs more generally. Using both a 278 panel of pseudoviruses in a CD4 T-cell (U87.CCR5.CXCR4) assay and a panel of 117 viruses in the TZM-bl assay, we found that bnMAbs targeting many neutralizing epitopes of the spike had neutralization profiles for at least one virus that plateaued at <90%. Across both panels the bnMAbs targeting the V2 apex of Env and gp41 were most likely to show neutralization curves that plateaued <100%. Conversely, bnMAbs targeting the high-mannose patch epitopes were less likely to show such behavior. Two CD4 binding site (CD4bs) Abs also showed this behavior relatively infrequently. The phenomenon of incomplete neutralization was also observed in a large peripheral blood mononuclear cells (PBMC)-grown molecular virus clone panel derived from patient viral swarms. In addition, five bnMAbs were compared against an 18-virus panel of molecular clones produced in 293T cells and PBMCs and assayed in TZM-bl cells. Examples of plateaus <90% were seen with both types of virus production with no consistent patterns observed. In conclusion, incomplete neutralization and non-sigmoidal neutralization curves are possible for all HIV bnMAbs against a wide range of viruses produced and assayed in both cell lines and primary cells with implications for the use of antibodies in therapy and as tools for vaccine design.

  2. 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.

  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. Neutralization of HIV by Milk Expressed Antibody

    PubMed Central

    Yu, Xiaocong; Pollock, Daniel; Duval, Mark; Lewis, Christopher; Joseph, Kristin; Meade, Harry; Cavacini, Lisa

    2012-01-01

    Background In some areas of the world mother-to-child transmission of HIV remains a significant problem in part due to widespread breastfeeding which is essential due to scarce supply of a safe replacement, protection conferred by breast milk against many enteric illnesses, and cultural norms. We propose that sustained, adequate levels of protective antibodies in breast milk will prevent transmission of HIV. Methods The HIV neutralizing human monoclonal antibody b12 (IgG1) has been expressed as an IgA2 in CHO cells and shown to retain full immunoreactivity and neutralizing activity as the parental IgG1. The expression plasmids containing the b12 heavy and light chains were also used to construct milk specific expression vectors using the GTC goat β-casein expression vector to direct expression of linked genes to the mammary gland with subsequent secretion into the milk. Female transgenic mice were generated and following parturition, their milk was tested for antibody immunoreactivity with gp120 and neutralization of HIV. Results When compared to CHO derived b12 IgA2 (or IgG1), immunoreactivity was retained. When tested for neutralization, milk derived b12 IgA2 was at least comparable to CHO derived antibody and in some cases superior to CHO derived antibody. Furthermore, milk that expressed b12 IgA2 was significantly more effective at mediating antibody dependent cell killing. Conclusions These results suggest it is possible to achieve functional HIV-specific mAb in the milk of transgenic mice and further investigations are warranted to explore ways for inducing this type of antibody response in the breast milk of HIV infected women. PMID:23269241

  5. 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.

  6. Neutral and Non-Neutral Evolution of Drosophila Mitochondrial DNA

    PubMed Central

    Rand, D. M.; Dorfsman, M.; Kann, L. M.

    1994-01-01

    To test hypotheses of neutral evolution of mitochondrial DNA (mtDNA), nucleotide sequences were determined for 1515 base pairs of the NADH dehydrogenase subunit 5 (ND5) gene in the mitochondrial DNA of 29 lines of Drosophila melanogaster and 9 lines of its sibling species Drosophila simulans. In contrast to the patterns for nuclear genes, where D. melanogaster generally exhibits much less nucleotide polymorphism, the number of segregating sites was slightly higher in a global sample of nine ND5 sequences in D. melanogaster (s = 8) than in the nine lines of D. simulans (s = 6). When compared to variation at nuclear loci, the mtDNA variation in D. melanogaster does not depart from neutral expectations. The ND5 sequences in D. simulans, however, show fewer than half the number of variable sites expected under neutrality when compared to sequences from the period locus. While this reduction in variation is not significant at the 5% level, HKA tests with published restriction data for mtDNA in D. simulans do show a significant reduction of variation suggesting a selective sweep of variation in the mtDNA in this species. Tests of neutral evolution based on the ratios of synonymous and replacement polymorphism and divergence are generally consistent with neutral expectations, although a significant excess of amino acid polymorphism within both species is localized in one region of the protein. The rate of mtDNA evolution has been faster in D. melanogaster than in D. simulans and the population structure of mtDNA is distinct in these species. The data reveal how different rates of mtDNA evolution between species and different histories of neutral and adaptive evolution within species can compromise historical inferences in population and evolutionary biology. PMID:7851771

  7. Affinity maturation by targeted diversification of the CDR-H2 loop of a monoclonal Fab derived from a synthetic naïve human antibody library and directed against the internal trimeric coiled-coil of gp41 yields a set of Fabs with improved HIV-1 neutralization potency and breadth

    PubMed Central

    Gustchina, Elena; Louis, John M.; Frisch, Christian; Ylera, Francisco; Lechner, Annette; Bewley, Carole A.; Clore, G. Marius

    2009-01-01

    Previously we reported a broadly HIV-1 neutralizing mini-antibody (Fab 3674) of modest potency that was derived from a human non-immune phage library by panning against the chimeric gp41-derived construct NCCG-gp41. This construct presents the N-heptad repeat of the gp41 ectodomain as a stable, helical, disulfide-linked trimer that extends in helical phase from the six-helix bundle of gp41. In this paper, Fab 3674 was subjected to affinity maturation against the NCCG-gp41 antigen by targeted diversification of the CDR-H2 loop to generate a panel of Fabs with diverse neutralization activity. Three affinity-matured Fabs selected for further study, Fabs 8060, 8066 and 8068, showed significant increases in both potency and breadth of neutralization against HIV-1 pseudotyped with envelopes of primary isolates from the standard subtypes B and C HIV-1 reference panels. The parental Fab 3674 is 10-20 fold less potent in monovalent than bivalent format over the entire B and C panels of HIV-1 pseudotypes. Of note is that the improved neutralization activity of the affinity-matured Fabs relative to the parental Fab 3674 was, on average, significantly greater for the Fabs in monovalent than bivalent format. This suggests that the increased avidity of the Fabs for the target antigen in bivalent format can be partially offset by kinetic and/or steric advantages afforded by the smaller monovalent Fabs. Indeed, the best affinity-matured Fab (8066) in monovalent format (∼50 kDa) was comparable in HIV-1 neutralization potency to the parental Fab 3674 in bivalent format (∼120 kDa) across the subtypes B and C reference panels. PMID:19695655

  8. 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.

  9. 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

  10. 46 CFR 502.404 - Neutrals.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... resolution proceeding. A neutral shall have no official, financial, or personal conflict of interest with... Dispute Resolution § 502.404 Neutrals. (a) A neutral may be a permanent or temporary officer or employee... Maritime Commission Dispute Resolution Specialist will seek to provide a neutral in dispute...

  11. 46 CFR 502.404 - Neutrals.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... resolution proceeding. A neutral shall have no official, financial, or personal conflict of interest with... Dispute Resolution § 502.404 Neutrals. (a) A neutral may be a permanent or temporary officer or employee... Maritime Commission Dispute Resolution Specialist will seek to provide a neutral in dispute...

  12. 46 CFR 502.404 - Neutrals.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... resolution proceeding. A neutral shall have no official, financial, or personal conflict of interest with... Dispute Resolution § 502.404 Neutrals. (a) A neutral may be a permanent or temporary officer or employee... Maritime Commission Dispute Resolution Specialist will seek to provide a neutral in dispute...

  13. 46 CFR 502.404 - Neutrals.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... resolution proceeding. A neutral shall have no official, financial, or personal conflict of interest with... Dispute Resolution § 502.404 Neutrals. (a) A neutral may be a permanent or temporary officer or employee... Maritime Commission Dispute Resolution Specialist will seek to provide a neutral in dispute...

  14. 46 CFR 502.404 - Neutrals.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... resolution proceeding. A neutral shall have no official, financial, or personal conflict of interest with... Dispute Resolution § 502.404 Neutrals. (a) A neutral may be a permanent or temporary officer or employee... Maritime Commission Dispute Resolution Specialist will seek to provide a neutral in dispute...

  15. 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…

  16. Acid neutralization of precipitation in Northern China.

    PubMed

    Wang, Yuesi; Yu, Wenpeng; Pan, Yuepeng; Wu, Dan

    2012-02-01

    There is an increasing concern over the impact of human-related emissions on the acid precipitation in China. However, few measurements have been conducted so far to clarify the acid-neutralization of precipitation on a regional scale. Under a network of 10 sites across Northern China operated during a 3-year period from December 2007 to November 2010, a total of 1118 rain and snow samples were collected. Of this total, 28% was acid precipitation with pH < 5.6. Out of these acid samples, 53% were found heavily acidic with pH value below 5.0, indicating significantly high levels of acidification of precipitation. Most of the acidity of precipitation was caused by H2SO4 and HNO3, their relative contribution being 72% and 28%, respectively. However; the contribution of HNO3 to precipitation acidity will be enhanced due to the increasing NO(x) and stable SO2 emissions in future. Neutralization factors for K+, NH4+, Ca2+, Na+, and Mg2+ were estimated as 0.06, 0.71, 0.72, 0.15, and 0.13, respectively. The application of multiple regression analysis further quantified higher NH4+ and Ca2+ contribution to the neutralization process, but the dominant neutralizing agent varied from site to site. The neutralization was less pronounced in the rural than urban areas, probably due to different levels of alkaline species, which strongly buffered the acidity. Presence of high concentrations of basic ions was mainly responsible for high pH of precipitation with annual volume-weighted mean (VWM) values larger than 5.6 at several sites. It was estimated that in the absence of buffering ions, for the given concentration of SO4(2-) and NO3-, the annual VWM pH of precipitation would have been recorded around 3.5 across Northern China. This feature suggested that emissions of particles and gaseous NH3 played very important role in controlling the spatial variations of pH of precipitation in the target areas.

  17. PDX neutral beam reionization losses

    SciTech Connect

    Kugel, H.W.; Dylla, H.F.; Eubank, H.P.; Kozub, T.A.; Moore, R.; Schilling, G.; Stuart, L.D.; Von Halle, A.; Williams, M.D.

    1982-04-01

    Reionization losses for 1.5 MW H /sup 0/ and 2 MW D /sup 0/ neutral beams injected into the PDX tokamak were studied using pressure gauges, phototransistors, thermocouples, surface shielding, and surface sample analysis. Considerable outgassing of conventionally prepared 304 SS ducts occurred during initial injections and gradually decreased with the cumulative absorption of beam power. Reionization power losses are presently about 5% in the ducts and about 12% total for a beamline including the duct. Present duct pressures are attributed primarily to gas from the ion source and neutralizer with much smaller contributions from residual wall desorption. Physical mechanisms for the observed duct outgassing are discussed.

  18. ATF neutral beam injection system

    SciTech Connect

    Menon, M.M.; Morris, R.N.; Edmonds, P.H.

    1985-01-01

    The Advanced Toroidal Facility is a stellarator torsatron being built at Oak Ridge National Laboratory to investigate improved plasma confinement schemes. Plasmas heating will be carried out predominantly by means of neutral beam injection. This paper describes the basic parameters of the injection system. Numerical calculations were done to optimize the aiming of the injectors. The results of these calculations and their implications on the neutral power to the machine are elaborated. The effects of improving the beam optics and altering the focal length on the power transmitted to the plasma are discussed.

  19. Additive Effect of Neutralizing Antibody and Antiviral Drug Treatment in Preventing Virus Escape and Persistence

    PubMed Central

    Seiler, Peter; Senn, Beatrice M.; Klenerman, Paul; Kalinke, Ulrich; Hengartner, Hans; Zinkernagel, Rolf M.

    2000-01-01

    Poorly cytopathic or noncytopathic viruses can escape immune surveillance and establish a chronic infection. Here we exploited the strategy of combining antiviral drug treatment with the induction of a neutralizing antibody response to avoid the appearance of neutralization-resistant virus variants. Despite the fact that H25 immunoglobulin transgenic mice infected with lymphocytic choriomeningitis virus mounted an early neutralizing antibody response, the virus escaped from neutralization and persisted. After ribavirin treatment of H25 transgenic mice, the appearance of neutralization-resistant virus was prevented and virus was cleared. Thus, the combination of virus-neutralizing antibodies and chemotherapy efficiently controlled the infection, whereas each defense line alone did not. Similar additive effects may be unexpectedly efficient and beneficial in humans after infections with persistent viruses such as hepatitis C virus and hepatitis B virus and possibly human immunodeficiency virus. PMID:10846070

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. RE: Pedagogy--After Neutrality

    ERIC Educational Resources Information Center

    I'Anson, John

    2010-01-01

    Within the UK and in many parts of the world, official accounts of what it is to make sense of religion are framed within a rhetorics of neutrality in which such study is premised upon the possibility of dispassionate engagement and analysis. This paper, which is largely theoretical in scope, explores both the affordances and the costs of such an…

  5. Self-neutralized ion beam

    SciTech Connect

    Salvadori, M. C.; Teixeira, F. S.; Nikolaev, A.; Savkin, K. P.; Oks, E. M.; Spaedtke, P.; Yu, K. M.; Brown, I. G.

    2011-10-15

    A vacuum arc ion source provides high current beams of metal ions that have been used both for accelerator injection and for ion implantation, and in both of these applications the degree of space charge neutralization of the beam is important. In accelerator injection application, the beam from the ion source may be accelerated further (post-acceleration), redirected by a bending magnet(s), or focused with magnetic or electrostatic lenses, and knowledge of the beam space charge is needed for optimal design of the optical elements. In ion implantation application, any build-up of positive charge in the insulating targets must be compensated by a simultaneous flux of cold electrons so as to provide overall charge neutrality of the target. We show that in line-of-sight ion implantation using a vacuum arc ion source, the high current ion beam carries along its own background sea of cold electrons, and this copious source of electrons provides a ''self-neutralizing'' feature to the beam. Here we describe experiments carried out in order to demonstrate this effect, and we provide an analysis showing that the beam is space-charge-neutralized to a very high degree.

  6. Measurement of plasma production and neutralization in gas neutralizers

    SciTech Connect

    Maor, D.; Meron, M.; Johnson, B.; Jones, K.; Agagu, A.; Hu, B.

    1986-06-17

    In order to satisfy the need of experimental data for the designing of gas neutralizers we have started a project aimed at measuring all relevant cross sections for the charge exchange of H/sup -/, H/sup 0/ and H/sup +/ projectiles, as well as the cross sections for the production of ions in the target. The expected results of these latter measurements are shown schematically.

  7. Cellular uptake of neutral phosphorodiamidate morpholino oligomers.

    PubMed

    Iversen, Patrick L; Aird, Katherine M; Wu, Rebecca; Morse, Michael M; Devi, Gayathri R

    2009-09-01

    Phosphorodiamidate morpholino oligomers (PMO), which have a neutral chemistry, are extensively being used as tools for selective inhibition of gene expression in cell culture models and are currently in human clinical trials. Unlike phosphorothioates (PS ODN) and other charged oligonucleotides, little is known about the uptake characteristics of neutral oligomers. The purpose of this study was to understand the kinetics of PMO transport in cells and correlate with antisense activity. In contrast to primary cells and some transformed cell lines which were uptake permissive, established cancer cell lines showed very poor uptake with an occasional diffuse intracellular pattern. Differential PMO uptake was also observed in immune cells, with dendritic cells and monocytes showing highest uptake compared to T and B cells. In addition, PMO localization was observed to be heterogeneous within a population of uptake permissive cells. Unassisted PMO delivery targeting specific genes was correlated with functional antisense efficacy in experiments showing correction of pre-mRNA missplicing and inhibition of target enzyme activity in cells in culture. PMO internalization in uptake-permissive cells was identified to be specific, saturable, and energy-dependent, suggesting a receptor mediated uptake mechanism. Understanding PMO transport should facilitate the design of more effective synthetic antisense oligomers as therapeutic agents.

  8. 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

  9. Rocket Experiment For Neutral Upwelling

    NASA Astrophysics Data System (ADS)

    Kenward, D. R.; Lessard, M.

    2015-12-01

    Observations from the CHAMP satellite from 2004 show relatively small scale heating in the thermosphere. Several different mechanisms have been proposed to explain this phenomenon. The RENU 2 rocket mission includes a suite of 14 instruments which will acquire data to help understand processes involved in neutral upwelling in the cusp. Neutral, ion, and electron measurements will be made to provide an assessment of the upwelling process. SUPERDarn measurements of large- scale Joule heating in the cusp during overflight will also be acquired. Small-scale data which could possibly be associated with Alfvén waves, will be acquired using onboard electric field measurements. In-situ measurement of precipitating electrons and all other measurements will be used in thermodynamic and electrodynamic models for comparison to the observed upwelling.

  10. Sq Currents and Neutral Winds

    NASA Astrophysics Data System (ADS)

    Yamazaki, Y.

    2015-12-01

    The relationship between ionospheric dynamo currents and neutral winds is examined using the Thermosphere Ionosphere Mesosphere Electrodynamic General Circulation Model (TIME-GCM). The simulation is run for May and June 2009 with variable neutral winds but with constant solar and magnetospheric energy inputs, which ensures that day-to-day changes in the solar quiet (Sq) current system arise only from lower atmospheric forcing. The intensity and focus position of the simulated Sq current system exhibit large day-to-day variability, as is also seen in ground magnetometer data. We show how the day-to-day variation of the Sq current system relate to variable winds at various altitudes, latitudes, and longitudes.

  11. 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.

  12. 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.

  13. 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.

  14. Arenavirus Glycan Shield Promotes Neutralizing Antibody Evasion and Protracted Infection.

    PubMed

    Sommerstein, Rami; Flatz, Lukas; Remy, Melissa M; Malinge, Pauline; Magistrelli, Giovanni; Fischer, Nicolas; Sahin, Mehmet; Bergthaler, Andreas; Igonet, Sebastien; Ter Meulen, Jan; Rigo, Dorothée; Meda, Paolo; Rabah, Nadia; Coutard, Bruno; Bowden, Thomas A; Lambert, Paul-Henri; Siegrist, Claire-Anne; Pinschewer, Daniel D

    2015-11-01

    Arenaviruses such as Lassa virus (LASV) can cause severe hemorrhagic fever in humans. As a major impediment to vaccine development, delayed and weak neutralizing antibody (nAb) responses represent a unifying characteristic of both natural infection and all vaccine candidates tested to date. To investigate the mechanisms underlying arenavirus nAb evasion we engineered several arenavirus envelope-chimeric viruses and glycan-deficient variants thereof. We performed neutralization tests with sera from experimentally infected mice and from LASV-convalescent human patients. NAb response kinetics in mice correlated inversely with the N-linked glycan density in the arenavirus envelope protein's globular head. Additionally and most intriguingly, infection with fully glycosylated viruses elicited antibodies, which neutralized predominantly their glycan-deficient variants, both in mice and humans. Binding studies with monoclonal antibodies indicated that envelope glycans reduced nAb on-rate, occupancy and thereby counteracted virus neutralization. In infected mice, the envelope glycan shield promoted protracted viral infection by preventing its timely elimination by the ensuing antibody response. Thus, arenavirus envelope glycosylation impairs the protective efficacy rather than the induction of nAbs, and thereby prevents efficient antibody-mediated virus control. This immune evasion mechanism imposes limitations on antibody-based vaccination and convalescent serum therapy. PMID:26587982

  15. 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

  16. Passive neutralizing antibody controls SHIV viremia and enhances B cell responses in infant macaques

    PubMed Central

    Ng, Cherie T.; Jaworski, J. Pablo; Jayaraman, Pushpa; Sutton, William F.; Delio, Patrick; Kuller, LaRene; Anderson, David; Landucci, Gary; Richardson, Barbra A.; Burton, Dennis R.; Forthal, Donald N.; Haigwood, Nancy L.

    2010-01-01

    Maternal HIV-1-specific antibodies are efficiently transferred to newborns; their role in disease control is unknown. We administered non-sterilizing levels of neutralizing IgG, including the human neutralizing monoclonal IgG1b12, to six newborn macaques before oral challenge with SHIVSF612P3. All rapidly developed neutralizing antibodies and had significantly reduced plasma viremia for 6 months. These studies support the use of neutralizing antibodies in enhancing B cell responses and viral control in perinatal settings. PMID:20890292

  17. 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.

  18. The detergent octylglucoside neutralizes lipopolysaccharide in a specific manner.

    PubMed

    Henrich, B; Guadarrama, R; Zähringer, U; MacKenzie, C R

    2001-04-01

    The stimulatory effect of lipopolysaccharide (LPS) on human macrophages was found to be neutralized by the detergent octylglucoside (OG). Both macrophage stimulation and reactivity in a limulus amebocyte lysate test were suppressed by suspension of LPS in OG at concentrations between 0.25 and 2.5 mM, whereas other stimulatory lipopeptides and lipid containing stimulants were unaffected by OG. LPS at concentrations causing maximal stimulation of macrophages could be completely neutralized by non-toxic concentrations of OG. In addition, it was found that the neutralization in complex mixtures of macromolecules, such as bacterial cell lysate, was specific for LPS and that the stimulatory activity of the other substances in the mixture was not affected by the OG.

  19. 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.

  20. 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.

  1. The genetics of neutral lipid biosynthesis: an evolutionary perspective

    PubMed Central

    Turkish, Aaron R.; Sturley, Stephen L.

    2009-01-01

    The storage of fatty acids and fatty alcohols in the form of neutral lipids such as triacylglycerol (TAG), cholesteryl ester (CE), and wax ester (WE) serves to provide reservoirs for membrane formation and maintenance, lipoprotein trafficking, lipid detoxification, evaporation barriers, and fuel in times of stress or nutrient deprivation. This ancient process likely originated in actinomycetes and has persisted in eukaryotes, albeit by different molecular mechanisms. A surfeit of neutral lipids is strongly, perhaps causally, related to several human diseases such as diabetes mellitus, obesity, atherosclerosis and nonalcoholic fatty liver disease. Therefore, understanding the metabolic pathways of neutral lipid synthesis and the roles of the enzymes involved may facilitate the development of new therapeutic interventions for these syndromes. PMID:19116372

  2. HIV Neutralizing Antibodies Induced by Native-like Envelope Trimers

    PubMed Central

    Sanders, Rogier W.; van Gils, Marit J.; Derking, Ronald; Sok, Devin; Ketas, Thomas J.; Burger, Judith A.; Ozorowski, Gabriel; Cupo, Albert; Simonich, Cassandra; Goo, Leslie; Arendt, Heather; Kim, Helen J.; Lee, Jeong Hyun; Pugach, Pavel; Williams, Melissa; Debnath, Gargi; Moldt, Brian; van Breemen, Mariëlle J.; Isik, Gözde; Medina-Ramírez, Max; Back, Jaap Willem; Koff, Wayne; Julien, Jean-Philippe; Rakasz, Eva G.; Seaman, Michael S.; Guttman, Miklos; Lee, Kelly K.; Klasse, Per Johan; LaBranche, Celia; Schief, William R.; Wilson, Ian A.; Overbaugh, Julie; Burton, Dennis R.; Ward, Andrew B.; Montefiori, David C.; Dean, Hansi; Moore, John P.

    2015-01-01

    A challenge for HIV-1 immunogen design is inducing neutralizing antibodies (NAbs) against neutralization-resistant (Tier-2) viruses that dominate human transmissions. We show that a soluble recombinant HIV-1 envelope glycoprotein trimer that adopts a native conformation (BG505 SOSIP.664) induced NAbs potently against the sequence-matched Tier-2 virus in rabbits and similar but weaker responses in macaques. The trimer also consistently induced cross-reactive NAbs against more sensitive (Tier-1) viruses. Tier-2 NAbs recognized conformational epitopes that differed between animals and in some cases overlapped with those recognized by broadly neutralizing antibodies (bNAbs), whereas Tier-1 responses targeted linear V3 epitopes. A second trimer, B41 SOSIP.664, also induced a strong autologous Tier-2 NAb response in rabbits. Thus, native-like trimers represent a promising starting point for developing HIV-1 vaccines aimed at inducing bNAbs. PMID:26089353

  3. 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.

  4. 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…

  5. 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.

  6. Ion-beam Plasma Neutralization Interaction Images

    SciTech Connect

    Igor D. Kaganovich; Edward Startsev; S. Klasky; Ronald C. Davidson

    2002-04-09

    Neutralization of the ion beam charge and current is an important scientific issue for many practical applications. The process of ion beam charge and current neutralization is complex because the excitation of nonlinear plasma waves may occur. Computer simulation images of plasma neutralization of the ion beam pulse are presented.

  7. On abstract degenerate neutral differential equations

    NASA Astrophysics Data System (ADS)

    Hernández, Eduardo; O'Regan, Donal

    2016-10-01

    We introduce a new abstract model of functional differential equations, which we call abstract degenerate neutral differential equations, and we study the existence of strict solutions. The class of problems and the technical approach introduced in this paper allow us to generalize and extend recent results on abstract neutral differential equations. Some examples on nonlinear partial neutral differential equations are presented.

  8. The Net Neutrality Debate: The Basics

    ERIC Educational Resources Information Center

    Greenfield, Rich

    2006-01-01

    Rich Greenfield examines the basics of today's net neutrality debate that is likely to be an ongoing issue for society. Greenfield states the problems inherent in the definition of "net neutrality" used by Common Cause: "Network neutrality is the principle that Internet users should be able to access any web content they choose and use any…

  9. Broadly Neutralizing Activity of Zika Virus-Immune Sera Identifies a Single Viral Serotype.

    PubMed

    Dowd, Kimberly A; DeMaso, Christina R; Pelc, Rebecca S; Speer, Scott D; Smith, Alexander R Y; Goo, Leslie; Platt, Derek J; Mascola, John R; Graham, Barney S; Mulligan, Mark J; Diamond, Michael S; Ledgerwood, Julie E; Pierson, Theodore C

    2016-08-01

    Recent epidemics of Zika virus (ZIKV) have been associated with congenital malformation during pregnancy and Guillain-Barré syndrome. There are two ZIKV lineages (African and Asian) that share >95% amino acid identity. Little is known regarding the ability of neutralizing antibodies elicited against one lineage to protect against the other. We investigated the breadth of the neutralizing antibody response following ZIKV infection by measuring the sensitivity of six ZIKV strains to neutralization by ZIKV-confirmed convalescent human serum or plasma samples. Contemporary Asian and early African ZIKV strains were similarly sensitive to neutralization regardless of the cellular source of virus. Furthermore, mouse immune serum generated after infection with African or Asian ZIKV strains was capable of neutralizing homologous and heterologous ZIKV strains equivalently. Because our study only defines a single ZIKV serotype, vaccine candidates eliciting robust neutralizing antibody responses should inhibit infection of both ZIKV lineages, including strains circulating in the Americas. PMID:27481466

  10. Broadly Neutralizing Activity of Zika Virus-Immune Sera Identifies a Single Viral Serotype.

    PubMed

    Dowd, Kimberly A; DeMaso, Christina R; Pelc, Rebecca S; Speer, Scott D; Smith, Alexander R Y; Goo, Leslie; Platt, Derek J; Mascola, John R; Graham, Barney S; Mulligan, Mark J; Diamond, Michael S; Ledgerwood, Julie E; Pierson, Theodore C

    2016-08-01

    Recent epidemics of Zika virus (ZIKV) have been associated with congenital malformation during pregnancy and Guillain-Barré syndrome. There are two ZIKV lineages (African and Asian) that share >95% amino acid identity. Little is known regarding the ability of neutralizing antibodies elicited against one lineage to protect against the other. We investigated the breadth of the neutralizing antibody response following ZIKV infection by measuring the sensitivity of six ZIKV strains to neutralization by ZIKV-confirmed convalescent human serum or plasma samples. Contemporary Asian and early African ZIKV strains were similarly sensitive to neutralization regardless of the cellular source of virus. Furthermore, mouse immune serum generated after infection with African or Asian ZIKV strains was capable of neutralizing homologous and heterologous ZIKV strains equivalently. Because our study only defines a single ZIKV serotype, vaccine candidates eliciting robust neutralizing antibody responses should inhibit infection of both ZIKV lineages, including strains circulating in the Americas.

  11. 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.

  12. A novel linear neutralizing epitope of hepatitis E virus.

    PubMed

    Tang, Zi-Min; Tang, Ming; Zhao, Min; Wen, Gui-Ping; Yang, Fan; Cai, Wei; Wang, Si-Ling; Zheng, Zi-Zheng; Xia, Ning-Shao

    2015-07-01

    Hepatitis E virus (HEV) is a serious public health problem that causes acute hepatitis in humans and is primarily transmitted through fecal and oral routes. The major anti-HEV antibody responses are against conformational epitopes located in a.a. 459-606 of HEV pORF2. All reported neutralization epitopes are present on the dimer domain constructed by this peptide. While looking for a neutralizing monoclonal antibody (MAb)-recognized linear epitope, we found a novel neutralizing linear epitope (L2) located in a.a. 423-437 of pORF2. Moreover, epitope L2 is proved non-immunodominant in the HEV-infection process. Using the hepatitis B virus core protein (HBc) as a carrier to display this novel linear epitope, we show herein that this epitope could induce a neutralizing antibody response against HEV in mice and could protect rhesus monkeys from HEV infection. Collectively, our results showed a novel non-immunodominant linear neutralizing epitope of hepatitis E virus, which provided additional insight of HEV vaccine. PMID:26051517

  13. Nonlinear neutral inclusions: assemblages of coated ellipsoids

    PubMed Central

    Bolaños, Silvia Jiménez; Vernescu, Bogdan

    2015-01-01

    The problem of determining nonlinear neutral inclusions in (electrical or thermal) conductivity is considered. Neutral inclusions, inserted in a matrix containing a uniform applied electric field, do not disturb the field outside the inclusions. The well-known Hashin-coated sphere construction is an example of a neutral inclusion. In this paper, we consider the problem of constructing neutral inclusions from nonlinear materials. In particular, we discuss assemblages of coated ellipsoids. The proposed construction is neutral for a given applied field. PMID:26064633

  14. 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.

  15. 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.

  16. 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.

  17. Advanced neutral-beam technology

    SciTech Connect

    Berkner, K.H.

    1980-09-01

    Extensive development will be required to achieve the 50- to 75-MW, 175- to 200-keV, 5- to 10-sec pulses of deuterium atoms envisioned for ETF and INTOR. Multi-megawatt injector systems are large (and expansive); they consist of large vacuum tanks with many square meters of cryogenic pumping panels, beam dumps capable of dissipating several megawatts of un-neutralized beam, bending magnets, electrical power systems capable of fast turnoff with low (capacity) stored energy, and, of course, the injector modules (ion sources and accelerators). The technology requirements associated with these components are described.

  18. 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.

  19. 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.

  20. Boosting Heterosubtypic Neutralization Antibodies in Recipients of 2009 Pandemic H1N1 Influenza Vaccine

    PubMed Central

    Qiu, Chao; Huang, Yang; Wang, Qian; Tian, Di; Zhang, Wanju; Hu, Yunwen; Yuan, Zhenghong; Zhang, Xiaoyan

    2012-01-01

    Background. A mass vaccination has been implemented to prevent the spread of 2009 pandemic influenza virus in China. Highly limited information is available on whether this vaccine induces cross-reactive neutralization antibodies against other subtypes of influenza viruses. Methods. We employed pseudovirus-based assays to analyze heterosubtypic neutralization responses in serum samples of 23 recipients of 2009 pandemic influenza vaccine. Results. One dose of pandemic vaccine not only stimulated good neutralization antibodies against cognate influenza virus 2009 influenza A (H1N1), but also raised broad cross-reactive neutralization activities against seasonal H3N2 and highly pathogenic avian influenza virus H5N1 and lesser to H2N2. The cross-reactive neutralization activities were completely abolished after the removal of immunoglobin G (IgG). In contrast, H1N1 vaccination alone in influenza-naive mice elicited only vigorous homologous neutralizing activities but not cross-reactive neutralization activities. Conclusions. Our data suggest that the cross-reactive neutralization epitopes do exist in this vaccine and could elicit significant cross-reactive neutralizing IgG antibodies in the presence of preexisting responses. The exposure to H1N1 vaccine is likely to modify the hierarchical order of preexisting immune responses to influenza viruses. These findings provide insights into the evolution of human immunity to influenza viruses after experiencing multiple influenza virus infections and vaccinations. PMID:22052887

  1. Directed evolution of mutator adenoviruses resistant to antibody neutralization.

    PubMed

    Myers, Nicolle D; Skorohodova, Ksenia V; Gounder, Anshu P; Smith, Jason G

    2013-05-01

    We incorporated a previously identified mutation that reduces the fidelity of the DNA polymerase into a human adenovirus vector. Using this mutator vector, we demonstrate rapid selection of resistance to a neutralizing anti-hexon monoclonal antibody due to a G434D mutation in hexon that precludes antibody binding. Since mutator adenoviruses can accumulate compound mutations that are unattainable using traditional random mutagenesis techniques, this approach will be valuable to the study of antivirals and host factor interactions.

  2. 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.

  3. Neutral Hydrogen in Arp 158

    NASA Astrophysics Data System (ADS)

    Iyer, Mansie G.; Simpson, Caroline E.; Gottesman, Stephen T.; Malphrus, Benjamin K.

    2004-09-01

    We present 21 cm observations of Arp 158. We have performed a study of the neutral hydrogen (H I) to help us understand the overall formation and evolution of this system. This is a disturbed system with distinct optical knots connected by a linear structure embedded in luminous material. There is also a diffuse spray to the southeast. The H I seems to be made up of three distinct, kinematically separate systems. Arp 158 bears a certain optical resemblance to NGC 520 (Arp 157), which has been identified as a mid-stage merger. From our 21 cm observations of Arp 158, we also see a comparable H I content with NGC 520. These similarities suggest that Arp 158 is also an intermediate-stage merger.

  4. After treatment ends: neutral time.

    PubMed

    Hurt, G J; McQuellon, R P; Barrett, R J

    1994-01-01

    For persons diagnosed with cancer, the remission period may be marked by increased anxiety and distress. While the medical team may view remission as an eagerly anticipated milestone, the decreased medical surveillance during this time can cause a heightened fear of recurrence for the patient. One author has called this period of remission "neutral time," a time characterized by uncertainty. The safety signal hypothesis, developed by Martin Seligman, may help to explain the anxiety experienced by some patients during the remission period. Because cancer is frequently a silent disease with no overt symptoms, patients in remission often have no safety signal to indicate that the disease will not return. A case study is presented and discussed in light of these two concepts.

  5. An accessible heavy neutral lepton

    NASA Astrophysics Data System (ADS)

    Chang, Chao-Hsi

    1982-09-01

    In the SUL(2) ⊗ SUR(2) ⊗ UB-L(1) model, an accessible heavy neutral lepton may exist owing to the mixing of generations. Based on a rough estimate, it is pointed out that the most hopeful experiments to observe this lepton are ν-production in an emulsion (because the track of a particle with lifetime 10-11-10-13 s could be seen) and e-production. The author thanks Professor J.D. Bjorken, Professor He Zuo-Xiu, Professor R.E. Mashark, Professor S.J. Chang, Professor Zhu Cong-Yuan and Professor M. Dine for helpful discussions and comments. He would like to thank the referee for valuable comments.

  6. High Resolution Neutral Atom Microscope

    NASA Astrophysics Data System (ADS)

    Bucay, Igal; Castillo-Garza, Rodrigo; Stratis, Georgios; Raizen, Mark

    2015-03-01

    We are developing a high resolution neutral atom microscope based on metastable atom electron spectroscopy (MAES). When a metastable atom of a noble gas is near a solid, a surface electron will tunnel to an empty energy level of the metastable atom, thereby ejecting the excited electron from the atom. The emitted electrons carry information regarding the local topography and electronic, magnetic, and chemical structures of most hard materials. Furthermore, using a chromatic aberration corrected magnetic hexapole lens we expect to attain a spatial resolution below 10 nm. We will use this microscope to investigate how local phenomena can give rise to macroscopic effects in materials that cannot be probed using a scanning tunneling microscope, namely insulating transition metal oxides.

  7. Phenomenology of neutral heavy leptons

    SciTech Connect

    Kalyniak, P.; Melo, I.

    1997-02-01

    We continue our previous work on the flavor-conserving leptonic decays of the Z boson with neutral heavy leptons (NHL`s) in the loops by considering box, vertex, and self-energy diagrams for the muon decay. By inclusion of these loops (they contribute to the input parameter M{sub W}), we can probe the full parameter space spanned by the so-called flavor-conserving mixing parameters ee{sub mix},{mu}{mu}{sub mix},{tau}{tau}{sub mix}. We show that only two diagrams from each class (box, vertex, and self-energy) are important; further, after renormalization only two box diagrams {open_quotes}survive{close_quotes} as dominant. We compare the results of our analysis with the existing work in this field and conclude that flavor-conserving decays have certain advantages over traditionally considered flavor-violating ones. {copyright} {ital 1997} {ital The American Physical Society}

  8. Phenomenology of neutral heavy leptons

    NASA Astrophysics Data System (ADS)

    Kalyniak, Pat; Melo, I.

    1997-02-01

    We continue our previous work on the flavor-conserving leptonic decays of the Z boson with neutral heavy leptons (NHL's) in the loops by considering box, vertex, and self-energy diagrams for the muon decay. By inclusion of these loops (they contribute to the input parameter MW), we can probe the full parameter space spanned by the so-called flavor-conserving mixing parameters eemix,μμmix,ττmix. We show that only two diagrams from each class (box, vertex, and self-energy) are important; further, after renormalization only two box diagrams ``survive'' as dominant. We compare the results of our analysis with the existing work in this field and conclude that flavor-conserving decays have certain advantages over traditionally considered flavor-violating ones.

  9. 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.

  10. Molecular clock on a neutral network.

    PubMed

    Raval, Alpan

    2007-09-28

    The number of fixed mutations accumulated in an evolving population often displays a variance that is significantly larger than the mean (the overdispersed molecular clock). By examining a generic evolutionary process on a neutral network of high-fitness genotypes, we establish a formalism for computing all cumulants of the full probability distribution of accumulated mutations in terms of graph properties of the neutral network, and use the formalism to prove overdispersion of the molecular clock. We further show that significant overdispersion arises naturally in evolution when the neutral network is highly sparse, exhibits large global fluctuations in neutrality, and small local fluctuations in neutrality. The results are also relevant for elucidating aspects of neutral network topology from empirical measurements of the substitution process.

  11. Neutral Buoyancy Test - NB23 - Space Telescope

    NASA Technical Reports Server (NTRS)

    1979-01-01

    Once the United States' space program had progressed from Earth's orbit into outerspace, the prospect of building and maintaining a permanent presence in space was realized. To accomplish this feat, NASA launched a temporary workstation, Skylab, to discover the effects of low gravity and weightlessness on the human body, and also to develop tools and equipment that would be needed in the future to build and maintain a more permanent space station. The structures, techniques, and work schedules had to be carefully designed to fit this unique construction site. The components had to be lightweight for transport into orbit, yet durable. The station also had to be made with removable parts for easy servicing and repairs by astronauts. All of the tools necessary for service and repairs had to be designed for easy manipulation by a suited astronaut. And construction methods had to be efficient due to limited time the astronauts could remain outside their controlled environment. In lieu of all the specific needs for this project, an environment on Earth had to be developed that could simulate a low gravity atmosphere. A Neutral Buoyancy Simulator (NBS) was constructed by NASA Marshall Space Flight Center (MSFC) in 1968. Since then, NASA scientists have used this facility to understand how humans work best in low gravity and also provide information about the different kinds of structures that can be built. Included in the plans for the space station was a space telescope. This telescope would be attached to the space station and directed towards outerspace. Astronomers hoped that the space telescope would provide a look at space that is impossible to see from Earth because of Earth's atmosphere and other man made influences. In an effort to make replacement and repairs easier on astronauts the space telescope was designed to be modular. Practice makes perfect as demonstrated in this photo: an astronaut practices moving modular pieces of the space telescope in the Neutral

  12. 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.

  13. 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 re